Thèse Présenté Par Matthias

Georg Haberl

Melanie Ginger

Matthias Georg Haberl

Karl-Klaus Conzelmann

Martin K Schwarz

Andreas Frick
email: andreas.frick@inserm.fr

Silvia Viana Da Silva

Jason M Guest

Alexander Ghanem

Christophe Mulle

Marcel Oberlaender

Karl-Klaus Conzelmann

Valerio Zerbi

Andor Veltien

Arend Heerschap

Kevan Martin

Circuitry Melanie Ginger

Guillaume Bony

Karl-Klaus Conzelmann

Charles F Stevens

Edward M Callaway

Karl-Klaus Conzelmann

Á M Ginger

Á A Frick

Á C Mulle

Á M Oberlaender

K.-K Conzelmann

Studying Neuronal Connectivity in the Mouse Brain in Normal Condition and Fragile X Syndrome L'étude de la connectivité neuronale dans le cerveau de la souris dans des conditions normales et le syndrome de l'X fragile

Keywords: eines retrograden Tollwutvirus konnten wir die Anzahl von Eingangsverbindung von Neuronen zu einem dieser Bereiche, dem primären visuellen Kortex, quantifizieren. Wir entdeckten insgesamt eine Reduktion der anatomischen und funktionellen Langstrecken-Verbindungen zwischen mehreren Hirnarealen eine Pathologie der neuronalen Konnektivität, und bietet eine Erklärungsmöglichkeit für die Schwierigkeiten der zur Zeit getesteten Medikamente im FXS Virales Tracing, Fragiles-X-Syndrom, Konnektivität im Gehirn connectivity, wiring diagram, neuronal tracer, synapses, monosynaptic Neuronal morphology, Connectivity, Sparse labeling, Circuit reconstruction, Neuron-type classification, Alzheimer's disease

dans des conditions normales et le syndrome de l'X fragile

I am very grateful to my supervisor Andreas Frick for giving me the opportunity to work in his lab, for helping, guiding and supporting me throughout my PhD and I am very happy that I had the chance to be there during the establishing and growth of the Cortical plasticity lab. I would like to specially thank all the team members of the Frick lab: Melanie Ginger, Elisabetta Aloisi, Maria Szlapczynska, Guillaume Bony, Katy LeCorf, Virginie Labrousse, Vladimir Kouskoff, Elian Botelho, Audrey Bonnan, Yu Zhang -for their help, for providing a stimulating and fun environment for me to learn and grow. It has been a great pleasure to work with you.

I want to thank my co-supervisor Kevan Martin (Institute for NeuroInformatics Zurich) and all his lab members, in particular Nuno da Costa, for the time I could spend with them at the INI, for their thoughtful insights and the support they provided.

I wish to thank Karl-Klaus Conzelmann (LMU Munich) and the members of his team for the fruitful long-term collaboration and their expertise.

I would like to thank Arend Heerschap, Valerio Zerbi and Andor Veltien (UMC Radboud) for working with me for the magnetic resonance imaging measurements, it was a great pleasure and I really enjoyed my stay in Nijmegen and special thanks to Andor for all the time you spent.

I wish to thank also Marcel Oberlander and Mike Guest for the successful collaboration on the computational reconstruction of long-ranging axons.

I also want to thank Christophe Mulle for his support during the last years and for his advice, which was indispensable for the ENCODS organization.

I am grateful to all my friends in-and outside the lab for their every-day support and encouragement, Filipa, Sam, Nelson, Jo, Mario, Adam, Marilena, Joana, the soccer guys and many others, thank you for your moral support. Above all, I would like to thank my parents for the constant support they gave me on this journey and for their constant inspiration that led me towards science.

Without them I would not have achieved any of this. I would also like to thank my brothers and my entire family for being there for me anytime.

For your time, for your dedication, for your support, for being there during joy and misery, for being the kind person you are, everyday, I thank you, Silvia!

Summary

The goal of this work was the investigation of the anatomical and functional connectivity of neuronal networks and the development of novel tools for this purpose. Since the latter aspect is a major focus of current neuroscience, we first sought a novel viral tracer enabling sparse neuronal reconstruction and neuron classification. We then applied this and other techniques to probe neuronal connectivity defects in Fragile X Syndrome.

In the first part we discussed the merits and drawbacks of a emerging technique using a new type of viral vector that allows in a unique manner mapping of the input of a given brain area.

In the second part we developed, departing from this viral vector, a new variant to facilitate the tracing and reconstructing of morphologic features of neurons.

We showed the strength of this anterograde variant of the recombinant glycoproteindeleted rabies virus for computational reconstruction of all key morphological features of neurons: dendrites, spines, long-ranging axons throughout the brain and bouton terminals.

In the third part we examined alterations in the wiring of brain structures in the Fragile X Syndrome (FXS). FXS is the most common inherited mental retardation and most frequent genetic form of autism, leading to learning and memory deficits, repetitive behavior, seizures and hypersensitivity to sensory (e.g. visual) stimuli. One of the eminent hypotheses in the autism field assumes a local hyper-connectivity phenotype but hypo-connectivity for long-ranging connections. To test this hypothesis in a FXS mouse model we used magnetic resonance imaging, to scan the entire brain and measure the anatomical and functional connectivity. This allowed us to identify connectivity alterations in several areas that we further explored using viral tracers.

Using retrograde rabies virus to count the number of neurons projecting to such areas we confirmed an altered input connectivity to the primary visual cortex, which could contribute to the altered visual information processing. We discovered an overall reduced anatomical and functional long-range connectivity between several brain areas, identifying FXS as pathology of neuronal connectivity, which might explain the difficulties several rescue strategies aiming at molecular targets are currently facing.

Keywords: Viral tracing, Fragile X Syndrome, Connectivity 7

Titre en français:

Neuronale connectivité dans le cerveau de souris en condition normale et en syndrome du X fragile

Résumé en français:

Le but de ce travail est l'étude de la connectivité anatomique et fonctionnelle des réseaux neuronaux et le développement des nouveaux outils à cet effet. Car le dernier aspect est une préoccupation majeure de la neuroscience actuelle, nous avons developpé d'abord un nouveau traceur virale permettant la reconstruction neuronale.

Nous avons ensuite appliqué cet et d'autres techniques pour sonder les défauts de connectivité neuronale dans le syndrome de l'X fragile.

Dans la première partie, nous avons discuté les avantages et inconvénients d'une technique émergente en utilisant un nouveau type de vecteur viral qui permet une unique application pour l'étude du cerveau.

Dans la deuxième partie, nous avons développé, au départ de ce vecteur viral, une nouvelle variante de faciliter le traçage et reconstruction des caractéristiques morphologiques de neurones. Nous avons montré la force de cette variante antérograde du virus de la rage recombinant glycoprotéine supprimé pour la reconstruction de calcul de toutes les caractéristiques morphologiques clés de neurones: les dendrites, épines, les axones longs envergure dans tous les terminaux du cerveau et les boutons.

Dans la troisième partie, nous avons examiné les modifications dans la connectivité des structures cérébrales dans le syndrome du X fragile (FXS). FXS est le retard mental héréditaire la plus fréquente et la forme génétique la plus fréquente de l'autisme, ce qui conduit à l'apprentissage et de la mémoire des déficits, les comportements répétitifs, des convulsions et une hypersensibilité à des stimuli sensoriels (visuels). Une des hypothèses éminents dans le domaine de l'autisme suppose une phénotype de hyper-connectivité locale mais de hypo-connectivité pour les connexions longue portée. Pour tester cette hypothèse dans un modèle de souris FXS nous avons utilisé l'imagerie par résonance magnétique, pour balayer la totalité du cerveau et de mesurer la connectivité anatomique et fonctionnel. Cela nous a permis d'identifier des altérations de connectivité dans plusieurs domains. Après nous avons utilisé des traceurs viraux pour explorer un de ceux domains plus detaillé. En utilisant le virus de la rage rétrograde à quantifier le nombre de neurones projetant vers ces zones, nous avons confirmé une connectivité d'entrée modifié pour le cortex visuel primaire, ce qui pourrait contribuer au traitement visuel altéré de l'information.

Nous avons découvert une connectivité réduite à longue portée globale anatomique et fonctionnelle entre plusieurs régions du cerveau, l'identification FXS comme une pathologie de la connectivité neuronale, ce qui pourrait expliquer les difficultés de plusieurs stratégies de sauvetage visant des cibles moléculaires sont actuellement confrontés.

Mots clés en français

Résumé en français

L'objectif de cette thèse était l'étude de la connectivité anatomique et 10 Dans la revue comprise dans la première partie, nous avons expliqués 5 les principes de base de cette technologie. Nous avons ensuite montrés comment, dans des études récentes, c'était adapté pour révéler des aspects spécifiques de la structure des circuits neuronaux et éclaircir leur principe de fonctionnement. Nous avons aussi discutés les avantages considérables et le potentiel. On a également présente de ce que on croit sont les insuffisances 10 majeurs de cette nouveaux vecteur. Enfin, nous donnons un aperçu de la façon dont cette approche complémenté par d'autres récents développements technologiques dans le domaine de l'analyse des circuits neuronaux et le genre de questions qui peuvent être traitées en utilisant une combinaison de ces approches. Cet type modifié du virus de la rage est un outil puissant pour 15 l'analyse des circuits neuronaux. Il ouvre de nouvelles possibilités avec uniques applications pour l'étude du cerveau.

MBP

Myelin Basic Protein

MD

General Introduction

The brain is the most complex organ in our body conversely it is the least understood. But what is it that makes our brain so complicated?

The human brain contains a large number of cells -with an estimated of 10 11 neurons and 3x10 12 glial cells -almost 10 times greater than the number of hepatocytes and similar to the number of dermal cells but in comparison only 1/10 th of the number of erythrocytes that we have in our blood [START_REF] Bianconi | An estimation of the number of cells in the human body[END_REF]. We have a good understanding of how red blood cells or most organs of our body work. In contrast, neuroscience cannot, to date, give a clear answer to the question of how many cell types comprises the brain. For example it has been shown in the last years that glial cells are not a single group of cells but in fact arise from different origins and are classified at least into three broad groups: microglia, macroglia (like astrocytes) and others. These groups should probably be further subdivided (Cahoy et al., 2008), based on additional properties, such as gene expression patterns. On the other hand, neurons have a single common feature, they are all electrically excitable, but at the same time they are also highly diverse. The criteria for classifying neurons in subgroups are still highly debated but good candidates could be gene profiles, neurotransmitters expression or the pattern of their processes [START_REF] Fishell | The neuron identity problem: form meets function[END_REF]. The cell types problem is tightly linked to a large diversity of cell connections and interactions happening in the brain. Both neurons and glial cells can possess numerous processes, which can either be extremely ramified and fine [START_REF] Bushong | Protoplasmic astrocytes in CA1 stratum radiatum occupy separate anatomical domains[END_REF], e.g. astrocytes for removing neurotransmitters from the synaptic cleft, or in the case of neurons develop over several millimeters to connect one to another. The extreme complexity of the anatomical properties of brain cells is one of the main reasons why the brain is difficult to study. In addition, although not discussed further, the multitude of plasticity mechanisms that can also alter communication between neurons, without changing the anatomy. The anatomy however provides a frame of possibilities and limitations, showing us which brain areas and cells are directly interacting.

Camillo Golgi (1844-1926) and Santiago Ramón y Cajal (1852-1906) were pioneers in the field of labeling and 'reconstructing' the neuron morphology and presented us the first impressive portraits of brain cells (Figure 1.1; [START_REF] Sotelo | Viewing the brain through the master hand of Ramón y Cajal[END_REF]).

Both researchers worked with a silver-chrome staining method that Golgi had developed, leading to a black staining of random cells ('la reazione nera'; see Figure 1.1) and permitting the visualization of neural networks [START_REF] Golgi | Sulla struttura della sostanza grigia del cervello[END_REF]. Since these early days of neuroanatomy our knowledge of the neuronal wiring has increased drastically and numerous diagrams of isolated brain circuits within, or between brain areas have been developed (e.g. Figure 1.2). Before we just storm out looking for a full description of all circuits in the brain, which we (unfortunately?) do not possess and also will not have in any time soon, it would be important to define what is it that we are looking for? For one, drawing maps of the neuronal network is a cartographic endeavor, describing in a way similar to what is being done with electrical circuits, a sequence of (electrical) units, arranged in series or parallel. Having circuit maps of the brain bears the advantage that we can compare them, find patterns, common schemes and/or alterations in disease. However, when looking at single cell resolution we will either have to reduce the maps to the important parts, otherwise it will become too complicated (see the simplified diagram of the hippocampus Figure 1.2), or even better annotate a factor of relevance to each link (see Figure 1.3). Such an annotation could reflect how commonly the connection occurs, how strong it typically is or even if it is functionally relevant. Important features beyond the connection/non-connection of the circuit are which cell types are involved, and information about the synapse types and locations.

Among the distinct brain areas in the human brain the one that sticks out as the most puzzling is the neocortex. This layered structure that has colonized around 85% in the human brain and is crucial for all our higher cognitive function. Structurally, it is tremendously intertwined in a complex fashion and many of its neurons seem to not respect their layer boundaries, with input and output from/to countless areas. In fact, regarding neuronal contacts in the neocortex, only a few general rules are applicable, which help us to understand the information flow through the cortex. Those common cortical denominators, called canonical circuits (Figure 1.3), have been discussed

recently together with the course that has been taken for finding those paths of the neocortex (Douglas and Martin, 2007). We demonstrate the capacity and potential of this anterograde variant of the recombinant glycoprotein-deleted rabies virus for sparse labeling and computational reconstruction of all key morphological features of neurons: dendrites, spines, long-ranging axons throughout the brain and bouton terminals [START_REF] Haberl | An anterograde rabies virus vector for high-resolution large-scale reconstruction of 3D neuron morphology[END_REF].

In the last years new tools have been introduced and used to study the brain wiring in healthy brains and cognitive disorders and it has become evident that several brain diseases exhibit alterations in the neuronal wiring. Most prominent among those are neurodegenerative diseases [START_REF] Greicius | Neuroimaging insights into network-based neurodegeneration[END_REF] but also schizophrenia [START_REF] Uhlhaas | Dysconnectivity, large-scale networks and neuronal dynamics in schizophrenia[END_REF] and developmental disorders like autism spectrum disorders (ASD) [START_REF] Geschwind | Autism spectrum disorders: developmental disconnection syndromes[END_REF]. Fragile X Syndrome (FXS) is the most frequent genetic cause of ASD and most common inherited mental retardation, leading to learning and memory deficits, repetitive behavior, seizures and hypersensitivity to sensory stimuli [START_REF] O'donnell | A decade of molecular studies of fragile X syndrome[END_REF]. ASDs and FXS are both thought to have a wiring component as a feature of their deficits [START_REF] Geschwind | Autism spectrum disorders: developmental disconnection syndromes[END_REF][START_REF] He | The trouble with spines in fragile X syndrome: density, maturity and plasticity[END_REF]. As described in Chapter 2.3 we applied a combination of neuroanatomical tools to define the how the circuits are changed in FXS on the large-and small-scale. We used magnetic resonance imaging, to scan the entire brain and measure the anatomical and functional connectivity. This allowed us to identify connectivity alterations in several areas, which we further explored using viral tracers. Using retrograde rabies virus to visualize and quantify the number of neurons projecting to such an area we confirmed an altered input connectivity to the primary visual cortex, which could contribute to the altered visual information processing found in FXS.

Novel Tools for Studying Brain Wiring

Brains are unique in the complexity with which individual cells are connected to one another. Untangling these interconnections is a tremendous challenge given that e.g. the human brain consists of ~85 billion neurons most of which form over 1000 contacts with other neurons. Large efforts in neuroscience are devoted to delineate these connections for the purpose of creating maps of the brain structure, with the hope it will be helpful to understand the function of the brain. The extraordinary thing about the structure of neurons is their shape, usually having many extensions, which we know now serve as wires for the physical transmission of their communication signals. With this in mind we can also describe the questions about the neuronal anatomy differently -in terms of communication -and it becomes more evident why structure is an important feature. It contains the knowledge about where the neurons receive signals from and where they transmit to. The list of techniques used for these questions is long. Such techniques can reveal connections between brain-areas [START_REF] Goñi | Resting-brain functional connectivity predicted by analytic measures of network communication[END_REF] or even of between single-cell [START_REF] Vélez-Fort | The Stimulus Selectivity and Connectivity of Layer Six Principal Cells Reveals Cortical Microcircuits Underlying Visual Processing[END_REF], some methods are able to proof functional connections of defined cells [START_REF] Debanne | Paired-recordings from synaptically coupled cortical and hippocampal neurons in acute and cultured brain slices[END_REF], others are able to provide quantitative measures of synaptic connectivity and synapse location [START_REF] Binzegger | A quantitative map of the circuit of cat primary visual cortex[END_REF]. None of the techniques currently available is sufficient alone, to provide us a complete description of all connections and the connection strength in a complex brain, but most of the techniques can be used in a complementary fashion and will advance our knowledge when well combined. Neural circuits consist of local connections -where pre-and postsynaptic partners reside within the same brain area -and long-distance connections, which link different areas. Connectivity studies can therefore address short-and/or long-ranging connections. In this chapter I will introduce the concepts of two powerful techniques, which were used in this work: (i) the tracing of neuronal networks with viral vectors, which is invasive, but provides cellular resolution about the structure; and (ii) the non-invasive magnetic resonance imaging, limited to the macroscale, but which allows us to glean a structural and functional overview of connections of the whole brain.

Viral Tracing Tools to Unravel Neuronal Circuits

Due to several important features, viral vectors are widely used for studying the structure and function of neuronal circuits and more recently to achieve cellular precision. One major advantage of viral vectors is that they are ideal for expressing transgenes, allowing us to label circuits and monitor or manipulate the activity within these circuits (Osakada and Callaway, 2013). In transneuronal viruses, this feature is further combined with their ability to cross multisynaptic pathways and still achieve sufficient protein expression after several steps, making them ideal for the study of neuronal circuits (reviewed (Callaway, 2008)).

A number of different viruses are commonly used to study neuronal circuits and for each question at hand the best vector should be identified beforehand. Those vectors differ in a number of properties the most important among those are ease of handling, ease of production, packaging size, diffusion in the tissue, cellular uptake point, cell tropism, expression levels, expression speed and cytotoxicity.

Gold standards for the type of viral tracer that infects cells at the cell body/dendrite and fills their axonal projections anterogradely -which are called anterograde tracers (Figure 1.4 A) -are lentivirus (LV) and adeno-associated virus (AAV). LVs provide the advantages of a relatively strong and fast protein expression (e.g. fluorescence labeling) and large packaging size [START_REF] Jakobsson | Lentiviral Vectors for Use in the Central Nervous System[END_REF], whereas AAVs exhibit basically no cytotoxicity, can infect large brain areas and provide a different tropism, targeting preferentially inhibitory and excitatory cells [START_REF] Nathanson | Preferential labeling of inhibitory and excitatory cortical neurons by endogenous tropism of adeno-associated virus and lentivirus vectors[END_REF] but suffering from a lower packaging capacity [START_REF] Aschauer | Analysis of transduction efficiency, tropism and axonal transport of AAV serotypes 1, 2, 5, 6, 8 and 9 in the mouse brain[END_REF][START_REF] Wu | Effect of genome size on AAV vector packaging[END_REF]. AAVs can be thoroughly purified and concentrated to extremely high titers, further reducing side effects that are often introduced to the injected brain tissue by impurities coming from the production process [START_REF] Mcclure | Production and titering of recombinant adeno-associated viral vectors[END_REF].

Anterograde tracers are commonly used to label the projections and thereby target regions of a particular brain area or to reconstruct features of neuronal morphology. Retrograde tracers, on the other hand, are mainly used to identify the, classify and study neurons projecting to a certain target area. Anterograde and retrograde tracers alone or together have been used over decades to identify brain pathways. Recently three different large-scale mapping projects injected such tracers into hundreds of target areas in the mouse brain [START_REF] Hunnicutt | A comprehensive thalamocortical projection map at the mesoscopic level[END_REF][START_REF] Oh | A mesoscale connectome of the mouse brain[END_REF][START_REF] Zingg | Fmr1 knockout mice: a model to study fragile X mental retardation. The Dutch-Belgian Fragile X Consortium[END_REF] providing us now with a large online repository about the neuronal circuits.

Retrograde tracers get taken up by axon terminals of neuronal projections and label also the cell bodies of those projecting neurons (Figure 1 Such information can be gathered using transsynaptic viruses, which label chains of synaptically connected neurons. They cross from one neuron to another and replicate at each step, therefore amplifying their signal, which chemical tracers will not do.

Commonly used transneuronal tracers (reviewed in (Callaway, 2008)) are herpes viruses and different types of rabies strains (reviewed in (Kelly and Strick, 2000)).

Recently, a genetically modified rabies virus variant has been engineered that allows monosynaptic trans-synaptic tracing in a retrograde manner, defining the presynaptic 'input map' into neurons (Figure 1.4 C). Mono-trans-synaptic retrograde tracer, label specifically the pre-synaptic connections of a defined subset of cells, thereby labeling the input of the starter cells. Modified from (Ginger et al., 2013) The next section provides a description/discussion of how a new recombinant glycoprotein-deleted variant of the SAD-strain of rabies virus has been created and further how the new vector is now being widely used for two experimental settings (i) as retrograde tracer (not trans-synaptic) and (ii) as retrograde mono-trans-synaptic tracer for mapping the input of specific brain regions or neurons.

Rabies Virus Based Mono-Trans Synaptic Input Mapping

Unfortunately trans-neuronal viral tracers cross multiple synapses and it is suspected that they cross some (e.g. stronger) synapses faster than others making the distinction between first and higher-order connections close to impossible. Therefore, the development of a trans-synaptic tracer that is restricted to mono-synaptic connected cells was a ground-breaking development in the field of circuit neuroscience. The method is based on a pseudotyped rabies virus derived from the vaccine strain of rabies virus, Street Alabama Dufferin (SAD) B19. Rabies virus is a single-stranded enveloped RNA-virus and a member of the Rhabdoviridae family.

The negative stranded RNA contains only five genes, encoding for the nucleoprotein (N), the phosphoprotein (P), the matrixprotein (M), the glycoprotein (G) and the polymerase (L). It was possible to reconstitute the SAD B19 strain from the individual cDNAs and an antisense genome construct. As a result the function of all gene products can be examined and modifications can be engineered in the virus genome (Conzelmann and Schnell, 1994). Furthermore, it permitted the introduction of foreign genes, rendering rabies virus useful as vector for transgene expression. The next important step was the development of a recombinant rabies virus strain, SAD∆G, in which the endogenous gene encoding the G protein was deleted [START_REF] Mebatsion | Matrix protein of rabies virus is responsible for the assembly and budding of bullet-shaped particles and interacts with the transmembrane spike glycoprotein G[END_REF]. This renders the virus incapable of crossing synapses (Etessami et al., 2000). The retrograde variant of this virus is generated by pseudotyping this recombinant virus with its own glycoprotein, thereby generating the single-step retrograde tracer SAD∆G -(SAD-G) (Wickersham et al., 2007a).

To generate the mono-trans-synaptic tracer, the virus was instead pseudotyped with an envelope protein (EnvA) derived from the subgroup A avian sarcoma and leukosis virus -(ASLV-A) (Wickersham et al., 2007b). This pseudotyping makes the virus infectious exclusively to cells expressing the TVA viral receptor, a protein found in birds but not in mammals.

Using Magnetic Resonance Imaging to Study Brain Networks

Magnetic resonance imaging (MRI) is a widely employed non-invasive technology for taking images of tissue using a strong magnetic field. MRI is being used for a range of different types of applications and can be used repeatedly on a patient since it does not involve a harmful procedure like the ionizing radiation used for X-ray computer tomography. While the underlying mechanisms of MRI are complex as their physical, biophysical mechanisms can just be correctly described using quantum mechanics, the overview here will cover mainly the practical aspects, which are required to understand and interpret the results.

Diffusion Tensor Imaging

Diffusion tensor MR imaging (DT-MRI) is an advanced non-invasive imaging technique to measure the large-scale structural connectivity of brains. DT-MRI is based on the diffusion of water in the brain tissue in multiple orientations. At every voxel the combination of several vectors, each corresponding to the diffusion in a different direction, is reconstructed into a diffusion ellipsoid. The directionality and anisotropy of the ellipsoid gives information about how structured the brain tissue is.

Cellular membranes, axonal bundles and organelles, all restrict the diffusion of the water in the tissue. Therefore the shape and the size of the diffusion ellipsoid reflects whether and how cells and fibers have a preferential orientation. This technique is therefore frequently used to look at tissue that has a parallel orientation like the axonal bundles of the white matter (WM) and in some cases for grey matter [START_REF] Basser | MR diffusion tensor spectroscopy and imaging[END_REF]. The diffusion tensor can be analyzed with respect to its mean diffusivity (MD) revealing how restricted the overall diffusion is due to cells in the tissue. The MD is calculated per voxel as the average of the vectors into three perpendicular directions. Another commonly used parameter is the diffusion anisotropy (or fractional anisotropy, FA), which reveals the directionality or in other words, how pointy (the maximal FA value is 1) or round the ellipse (the minimal FA value is 0) is shaped. The FA value of cerebro-spinal fluid is 0 since it can diffuse equally in all directions, whereas the values in parallel fiber bundles or the white matter (WM) are high. It is important to note that DT-MRI however deals inadequately with crossing fibers and therefore the FA is rarely used in the grey matter. For example, the FA value could approach 0 in a scenario where three fibers cross perpendicular to each other in one voxel, since now the ellipsoid would have no preferred orientation.

Images of single slices are usually acquired in 100 ms or less to eliminate the chance of a head motion, since even a tiny shift can lead to significant artifacts in the acquisition and reconstructed image [START_REF] Alexander | Diffusion tensor imaging of the brain[END_REF].

DT-MRI has often been used in to study the structural integrity in cognitive diseases, e.g. for Alzheimer's Disease where a reduced FA in the WM and an increased MD in the grey matter was found. This data has to be interpreted carefully, but usually additional methods can help to define the origin of changes in DT-MRI values. In Alzheimer's Disease patients it was found that the altered FA and MD values correspond to a degradation of myelin and axons in the WM and a neuronal loss in the grey matter, respectively [START_REF] Clerx | New MRI markers for Alzheimer's disease: a meta-analysis of diffusion tensor imaging and a comparison with medial temporal lobe measurements[END_REF].

Resting-state functional MRI

Activity in a brain region causes a locally and temporary increased blood flow [START_REF] Roy | On the Regulation of the Blood-supply of the Brain[END_REF] interactions. An extensive review about the merits and drawbacks of fMRI can be found in [START_REF] Logothetis | What we can do and what we cannot do with fMRI[END_REF] [START_REF] Logothetis | What we can do and what we cannot do with fMRI[END_REF]. To address shortcomings of this technique it is often complemented with other techniques, for example electroencephalography (EEG), which provides a high temporal resolution (less than a ms) but lacks the spatial resolution, or with methods that can provide conclusive evidence for a structural connection.

Fragile X Syndrome and Brain Wiring Defects

Fragile X Syndrome (FXS) is the most common form of inherited mental retardation passed along by the X-Chromosome affecting approximately 1/4000 males and 1/8000 females (Bassell and Warren, 2008;[START_REF] Garber | Fragile X syndrome[END_REF]. FXS is caused by a mutation silencing a single gene, Fmr1, encoding the Fragile X mental retardation protein (FMRP). FMRP is a regulatory protein controlling the expression and function of a large number of other proteins, which causes the myriad of effects found in its absence in FXS. In order to understand how the cellular defects lead to the behavioural symptoms that patients with FXS develop we also need a better understanding about changes on the intermediate level, the neuronal networks.

Cause of the Fragile X Syndrome

The primary cause of FXS is the absence of the gene product of the Fmr1 gene, the Fragile X mental retardation protein (FMRP) [START_REF] Pieretti | Absence of expression of the FMR-1 gene in fragile X syndrome[END_REF]. The cause of the silencing of the Fmr1 gene is an excessive expansion of the CGG trinucleotide repeat in the 5' untranslated region (UTR) of the gene (O' Donnell and Warren, 2002). The number of CGG repeats is normally ~6-54, 55-200 in premutation carriers and >200 in FXS patients [START_REF] Willemsen | CGG repeat in the FMR1 gene: size matters[END_REF]. In the case that more than 200 CGG repeats are accumulated, the CpG island in the 5'-untranslated region (UTR) becomes hypermethylated, which causes a suppressed transcription and therefore a lack of the FMRP protein [START_REF] Sutcliffe | DNA methylation represses FMR-1 transcription in fragile X syndrome[END_REF]. Trinoclueotide repeats can expand from one to the next generation and cause that the offspring of a premutation carrier develops FXS [START_REF] Loesch | Unstable mutations in the FMR1 gene and the phenotypes[END_REF][START_REF] O'donnell | A decade of molecular studies of fragile X syndrome[END_REF]. A recent study estimated that 1/151 females (or ~1 million women in the United States) and 1/468 males, (or ~320,000 men in the United States) are carrier of a FXS premutation [START_REF] Seltzer | Prevalence of CGG expansions of the FMR1 gene in a US population-based sample[END_REF].

Fragile X Phenotype

The most striking deficit observed in FXS patients is a mild to severe mental retardation. Their intelligence quotient (IQ) is typically in the range of 40-70 (Merenstein et al., 1996). In addition, FXS patients usually suffer from a number of neurological and behavioral alterations. The most frequent symptom, found in almost all male and around 30% of the female patients, is a pronounced attention deficit hyperactivity disorder (ADHD) [START_REF] Mclennan | Fragile x syndrome[END_REF]. Another common feature is hypersensitivity to sensory stimuli, in particularly of tactile, auditory and visual modalities [START_REF] Hagerman | The fragile X premutation: into the phenotypic fold[END_REF][START_REF] Miller | Electrodermal responses to sensory stimuli in individuals with fragile X syndrome: a preliminary report[END_REF]. Indeed, hypersensitivity to visual stimuli and visual avoidance is present in more than 90% of male patients [START_REF] Merenstein | Molecular-clinical correlations in males with an expanded FMR1 mutation[END_REF]. FXS patients often exhibit autistic features, with autism occurring in ~30% of the males with FXS and autism spectrum disorder (ASD) in an additional ~30% of males that did not meet the criteria for autism. In fact social anxiety is one of the most common problems of FXS patients [START_REF] Harris | Autism profiles of males with fragile X syndrome[END_REF][START_REF] Kaufmann | Autism spectrum disorder in fragile X syndrome: communication, social interaction, and specific behaviors[END_REF] [START_REF] Berry-Kravis | Epilepsy in fragile X syndrome[END_REF][START_REF] Sabaratnam | Epilepsy and EEG findings in 18 males with fragile X syndrome[END_REF]. Difficulties with their vision are also common in FXS patients (in ~25%), often caused by strabismus and refractive errors [START_REF] Hatton | Ocular status of boys with fragile X syndrome: a prospective study[END_REF]. Besides these pronounced neurological, cognitive and sensory problems, FXS often also leads to visible alterations in the peripheral tissues causing prolonged faces, macroorchidism, and hyperextensible joints [START_REF] Hagerman | The fragile X premutation: into the phenotypic fold[END_REF].

Fragile X Mental Retardation Protein (FMRP) Function

FMRP occurs in a number of different tissues but it is foremost expressed in neurons of the central nervous system (CNS) and in testes. FMRP operates as RNAbinding protein [START_REF] Darnell | Fragile X mental retardation protein targets G quartet mRNAs important for neuronal function[END_REF][START_REF] Siomi | Essential role for KH domains in RNA binding: impaired RNA binding by a mutation in the KH domain of FMR1 that causes fragile X syndrome[END_REF]. It targets a large variety of mRNA molecules, with up to as many as 800 mRNAs as binding partners (Brown et al., 2001). This equals ~ 4% percent of all mRNA transcripts that occur in the mammalian brain (Bassell and Warren, 2008). A recent study identified that hundreds of targets of FMRP are mRNAs encoding part of the pre-and postsynaptic proteome (Figure 1.5; [START_REF] Darnell | FMRP stalls ribosomal translocation on mRNAs linked to synaptic function and autism[END_REF]). This large number of interactions explains why a single-gene deficit leads to such a complex sequence of events and makes it difficult to assess the full extent of the consequences of this cognitive disorder. It could further explain why FXS is one of the few single gene mental retardation disorders, since the loss FMRP might affect so many other genes that compensatory mechanisms are insufficient to avoid strong cognitive defects. FMRP has a number of RNA binding motifs [START_REF] Darnell | Fragile X mental retardation protein targets G quartet mRNAs important for neuronal function[END_REF][START_REF] Siomi | Essential role for KH domains in RNA binding: impaired RNA binding by a mutation in the KH domain of FMR1 that causes fragile X syndrome[END_REF] (metadata recently reviewed in [START_REF] Suhl | Analysis of FMRP mRNA target datasets reveals highly associated mRNAs mediated by G-quadruplex structures formed via clustered WGGA sequences[END_REF]) through which it mainly represses the translation of mRNAs [START_REF] Li | The fragile X mental retardation protein inhibits translation via interacting with mRNA[END_REF] and only in the case of a few proteins increased translation levels where found in the absence of FMRP [START_REF] Zalfa | The fragile X syndrome protein FMRP associates with BC1 RNA and regulates the translation of specific mRNAs at synapses[END_REF]. It is thought that phosphorylated FMRP suppresses translation of target mRNAs and conversely dephosphorylated FMRP enhances translation [START_REF] Ceman | Phosphorylation influences the translation state of FMRP-associated polyribosomes[END_REF]. It has further been shown that FMRP can also reversibly stall polyribosomes, hindering the translation of the associated mRNAs [START_REF] Darnell | FMRP stalls ribosomal translocation on mRNAs linked to synaptic function and autism[END_REF].

In addition to its strong effects on the translational level, FMRP is also thought to have a role in the transport of mRNAs to the synapse. For example, it was shown that blocking the FMRP mediated transport to the synapse lead to altered spine morphology, resembling the changes found in the absence of FMRP [START_REF] Dictenberg | A direct role for FMRP in activity-dependent dendritic mRNA transport links filopodial-spine morphogenesis to fragile X syndrome[END_REF]. This identified a strong overlap with the postsynaptic proteome (>30% of FMRP targets)

and also, to a lesser extend, with the presynaptic proteom (13% of FMRP targets)

Adapted from [START_REF] Darnell | FMRP stalls ribosomal translocation on mRNAs linked to synaptic function and autism[END_REF]

Fragile X Mouse Model

A number of different approaches have been taken to get a better understanding of FXS and the role of FMRP in health and disease. The help of different genetically modified organisms, most importantly Drosophila [START_REF] Zhang | Drosophila fragile X-related gene regulates the MAP1B homolog Futsch to control synaptic structure and function[END_REF] and knock-out mice (1994) have brought a wealth of findings that are important for human patients.

In fact much of what we know today about the function of FMRP in vivo was originally discovered in studies using those two animal models.

Fmr1, which is the homologue gene in the mouse of the human FMR1 is 97% identical to the human gene at the amino acid level and to 95% identical at the nucleotide level in the coding region [START_REF] Ashley | Human and murine FMR-1: alternative splicing and translational initiation downstream of the CGG-repeat[END_REF]. Also the expression patterns of FMR1 and Fmr1 are similar in human and mouse tissues [START_REF] Verheij | Characterization of FMR1 proteins isolated from different tissues[END_REF]. The first Fmr1 knockout (Fmr1KO) mouse was generated by introducing a neomycin gene into exon 5 of the murine Fmr1 gene through homologous recombination in embryonic stem cells (1994). Many parallels to human FXS patients have been found in the Fmr1KO mouse model with biochemical and behavioral tests [START_REF] Bakker | Understanding fragile X syndrome: insights from animal models[END_REF][START_REF] Kooy | Of mice and the fragile X syndrome[END_REF]. For example, Fmr1KO mice also suffer from cognitive impairments, even though they are less severe than those described in FXS patients [START_REF] D'hooge | Mildly impaired water maze performance in male Fmr1 knockout mice[END_REF][START_REF] Dobkin | Fmr1 knockout mouse has a distinctive strain-specific learning impairment[END_REF][START_REF] Kooy | Transgenic mouse model for the fragile X syndrome[END_REF][START_REF] Van Dam | Spatial learning, contextual fear conditioning and conditioned emotional response in Fmr1 knockout mice[END_REF]1994). The knockout mice also display macroorchidism (1994), which is common in male FXS patients and various other important aspects of the disease, including sensory hypersensitivity and thin, immature dendritic spines. Craniofacial abnormalities have been reported in zebrafish [START_REF] Tucker | Contribution of mGluR and Fmr1 functional pathways to neurite morphogenesis, craniofacial development and fragile X syndrome[END_REF] they were not found in an MRI study of Fmr1KO mice [START_REF] Ellegood | Anatomical phenotyping in a mouse model of fragile X syndrome with magnetic resonance imaging[END_REF]. Although Fmr1 KO mice do not express FMRP, the Fmr1 promoter in those mice is intact and residual Fmr1 transcription was found in these mice. To resolve the uncertainty whether the residual transcript functions in some way the Fmr1 KO2 mouse line, a complete null knockout mouse, has been created by excision of the Fmr1 promoter and first exon [START_REF] Mientjes | The generation of a conditional Fmr1 knock out mouse model to study Fmrp function in vivo[END_REF]. These animal models have become vital not only for studying the cellular basis of deficits in FXS but also for testing promising drugs that can rescue individual or several of the cellular, behavioral or cognitive deficits.

Mechanisms of Fragile X Syndrome

Concurrent Theories

The currently dominant mechanistic hypothesis about FXS is known as 'mGluR theory' [START_REF] Bear | Therapeutic implications of the mGluR theory of fragile X mental retardation[END_REF][START_REF] Bear | The mGluR theory of fragile X mental retardation[END_REF]) (reviewed in [START_REF] Bhakar | The pathophysiology of fragile X (and what it teaches us about synapses)[END_REF]).

This theory proposes (a) that the core symptoms in FXS are caused by an overactivation of the metabotrobic glutamate receptor 5 (mGluR5) because (b) the principal role of FMRP for the synaptic function is to control the mGluR-dependent forms of long-term plasticity. A number of indications support this theory: (i) FMRP is increasingly synthesized in response to mGluR activation (Weiler et al., 1997) (ii) mGluR-dependent long-term depression (LTD) is elevated in Fmr1KO mice in the hippocampus and cerebellum [START_REF] Hou | Dynamic translational and proteasomal regulation of fragile X mental retardation protein controls mGluR-dependent long-term depression[END_REF][START_REF] Huber | Altered synaptic plasticity in a mouse model of fragile X mental retardation[END_REF][START_REF] Koekkoek | Deletion of FMR1 in Purkinje cells enhances parallel fiber LTD, enlarges spines, and attenuates cerebellar eyelid conditioning in Fragile X syndrome[END_REF] (iii) mGluR5 blockers, like MPEP showed correction of several symptoms in the KO mouse model (reviewed in [START_REF] Berry-Kravis | Targeted treatments for fragile X syndrome[END_REF]D'Hulst and Kooy, 2009)) and even reversal of already established FXS phenotypes [START_REF] Michalon | Chronic pharmacological mGlu5 inhibition corrects fragile X in adult mice[END_REF].

Today the theory is widely accepted, but despite all hopes the drug tests in humans showed much less effect than expected and the disease might be more complex than initially thought. Novartis stopped in April 2014 the drug trials done with the mGluR5 antagonist mavoglurant (AFQ056) as a consequence of negative results in the large international clinical trial in adult FXS patients (http://www.fraxa.org/novartis-discontinues-development-mavoglurant-afq056fragile-x-syndrome/). Drug trials of two large pharmaceutical companies are ongoing with GRN-529 (Pfizer) and RG7090 (Roche), which both also target mGluR5 receptors.

Besides the mGluR theory a second prominent theory has been developed to describe the disease status in Fragile X, the 'GABA theory of Fragile X Syndrome'.

GABA is the principal inhibitory neurotransmitter in adults. The GABA theory is based on the observation that expression levels of GABA receptor type A is reduced [START_REF] D'hulst | Decreased expression of the GABAA receptor in fragile X syndrome[END_REF] in animal models of FXS and a decreased GABAergic inhibition was found in the hippocampus [START_REF] Curia | Downregulation of tonic GABAergic inhibition in a mouse model of fragile X syndrome[END_REF] and amygdala [START_REF] Olmos-Serrano | Defective GABAergic neurotransmission and pharmacological rescue of neuronal hyperexcitability in the amygdala in a mouse model of fragile X syndrome[END_REF]). An impaired inhibition could contribute to epilepsy and other symptoms of Fragile X. To this end several groups proposed rescue approaches targeting GABA receptors to restore the excitation/inhibition balance with the hope to cure the ADHD and hyperexcitability phenotypes [START_REF] Braat | Insights into GABAAergic system deficits in fragile X syndrome lead to clinical trials[END_REF][START_REF] Heulens | Involvement and therapeutic potential of the GABAergic system in the fragile X syndrome[END_REF][START_REF] Feldmeyer | Excitatory neuronal connectivity in the barrel cortex[END_REF].

Again transforming the results from animal research into a successful human application have been difficult. Seaside Therapeutics announced in May 2013 the end of any clinical trials with their GABA-B agonist Arbaclofen (STX209) for FXS patients, although positive results were reported in a number of patients.

Apart from those two prominent receptor alterations in the absence of Fmr1 an increasing number of differences have been found with potential implications for the cellular and network function and which might contribute to certain behavioral phenotypes. Several groups reported a circuit hyperexcitability, or extended upstates in the activity of networks (Gibson et al., 2008;[START_REF] Gonçalves | Circuit level defects in the developing neocortex of Fragile X mice[END_REF]. Most likely there are several factors that contribute to this hyperexcitability, ranging from receptor and cellular mechanisms to changes in the network.

On the cellular level, besides the mGluR5 and GABA receptors, also a number of other alterations have been described that may contribute to the phenotype of the disease. Cellular excitability was shown to be altered and a broadening of action potentials was found [START_REF] Deng | FMRP regulates neurotransmitter release and synaptic information transmission by modulating action potential duration via BK channels[END_REF]. In the hippocampal CA3-CA1 synapse a recent study found an elevated release probability during repetitive activity (Wang et al., 2014). Synaptic plasticity was shown to be altered in a number of studies using different stimulating protocols, animal ages, brain areas. A reduced long-term potentiation was found in the anterior somatosensory cortex (at 8-10 weeks) [START_REF] Li | Reduced cortical synaptic plasticity and GluR1 expression associated with fragile X mental retardation protein deficiency[END_REF] in the primary olfactory cortex (only from 6 months on) [START_REF] Larson | Agedependent and selective impairment of long-term potentiation in the anterior piriform cortex of mice lacking the fragile X mental retardation protein[END_REF], in the anterior cingulated cortex and the lateral amygdala (at 6-8weeks) [START_REF] Zhao | Deficits in trace fear memory and long-term potentiation in a mouse model for fragile X syndrome[END_REF], the visual cortex layer (L)5 [START_REF] Wilson | Absence of metabotropic glutamate receptormediated plasticity in the neocortex of fragile X mice[END_REF], and the temporal cortex

(2-3 months) [START_REF] Hayashi | Inhibition of p21-activated kinase rescues symptoms of fragile X syndrome in mice[END_REF]. Apart from the deficits on the synaptic and cellular level it is also important to understand whether the circuitry is intact or altered in FXS to get a better understanding about the cause of the cognitive defects in FXS.

Neuroanatomy in Fragile X Syndrome

Defective brain wiring can have devastating neurological and psychiatric consequences and those defects are suspected to underlie several brain disorders including autism [START_REF] Geschwind | Autism spectrum disorders: developmental disconnection syndromes[END_REF][START_REF] Müller | Underconnected, but how? A survey of functional connectivity MRI studies in autism spectrum disorders[END_REF]. FXS is the leading single gene mutation causing ASD and several ASDs have already been associated with structural changes in the synaptic contacts and connectivity alterations (reviewed in [START_REF] Peça | Cellular and synaptic network defects in autism[END_REF]). Yet, the data about circuit alterations in FXS is sparse when we compare it to the numerous studies addressing basic functions of FMRP (recently reviewed in [START_REF] Darnell | The translation of translational control by FMRP: therapeutic targets for FXS[END_REF]). However, among the list of putative targets of FMRP there are a number of mRNAs known to regulate cytoskeletal structure (reviewed in [START_REF] Bagni | From mRNP trafficking to spine dysmorphogenesis: the roots of fragile X syndrome[END_REF]). Moreover, several studies already suggested that changes occur in the circuitry of FXS patients and animal models, which will be summarized in the subsequent sections.

Studies addressing the wiring of the brain in FXS have been conducted mainly on two levels, first top-down approaches -mostly implicating human patients -that looked at large scale structural (reviewed in [START_REF] Lightbody | Gene, brain, and behavior relationships in fragile X syndrome: evidence from neuroimaging studies[END_REF]) and functional network connectivity [START_REF] Hall | Identifying large-scale brain networks in fragile X syndrome[END_REF][START_REF] Van Der Molen | Resting-state EEG oscillatory dynamics in fragile X syndrome: abnormal functional connectivity and brain network organization[END_REF], and second using bottom-up approaches that examined local networks or single cells in the different animal models (Patel et al., 2014).

To date we have not found studies combining those different approaches or providing the link of small-to large-scale network alterations in FXS. Even though it would be of major importance to improve our understanding of this complex syndrome given that sensory information is processed on different scales in parallel.

Each individual brain area may act as a computational unit with a very defined function, but the real functionality required to process sensory information will only emerge, by arranging these individual processing units in the very specific manner our brains do. Therefore the wiring on the small scale is inseparably linked to the wiring on the large scale, both are equally relevant and just in conjunction they allow that the sensory information is faithfully processed in the brain network. Affected networks could be involved in visual, auditory, somatosensory sensation and motor control or more elusive functions as perception, learning and memory or motivation. Therefore in any neurologic disorder it is possible that altered networks may play a role either being the cause or consequence and depending on the type and number of networks affected this may be reflected in specific or global deficits. While it will become very difficult to untangle if network changes are the cause or a consequence it would be a good first step to identify which networks are mainly affected. Studies of behavior and performance are an excellent starting point to identify the candidate brain areas/pathways that should be further inspected. Also large-scale studies, performed on the whole brain, may provide good indications. Fragile X Syndrome is an intellectual disability and the range of neurological changes and behavioral effects in human patients and mouse models is wide, as described above. In the subsequent paragraphs large-and fine-scale studies of brain network alterations in Fragile X Syndrome in humans and model organisms will be discussed in more detail. Since those studies did not establish direct links between findings in the large and fine scale networks they will be treated in separate sections.

Large-Scale Neuroanatomy in Fragile X Syndrome

From studies in human FXS patients we know about a number of changes in the size of several brain structures (caudate nucleus, hippocampus, superior temporal gyrus, cerebellar vermis, amygdala). Furthermore, also changes in the activation of certain brain areas where found (insula, the caudate, hippocampus) when individuals with FXS perform cognitive tasks e.g. involving eye gaze processing (reviewed in [START_REF] Lightbody | Gene, brain, and behavior relationships in fragile X syndrome: evidence from neuroimaging studies[END_REF]).

Initial neuroanatomical studies in FXS patients focused on gross-scale volume measurements of the brain structures. The first brain abnormality found in human FXS patients was a reduced size of the cerebellar vermis (the structure that connects the right and left hemisphere of the cerebellum) by using magnetic resonance imaging (MRI) [START_REF] Reiss | Preliminary communication: neuroanatomical variations of the posterior fossa in men with the fragile X (Martin-Bell) syndrome[END_REF]. The same deficit was confirmed in later studies, looking during development and adulthood to the cerebellar vermis [START_REF] Hoeft | Morphometric spatial patterns differentiating boys with fragile X syndrome, typically developing boys, and developmentally delayed boys aged 1 to 3 years[END_REF]. It is thought that this bears relevance for the behavioural deficits in FXS patients since the cerebellum is important for visual-spatial processing, learning, executive function and language [START_REF] Stoodley | Functional topography in the human cerebellum: a meta-analysis of neuroimaging studies[END_REF] and the vermis is linked to the amygdala and hippocampus [START_REF] Hessl | The neuroanatomy and neuroendocrinology of fragile X syndrome[END_REF]). In fact it was possible to use the posterior vermis size to predict the visual-spatial performance and executive function in FXS patients [START_REF] Gothelf | Neuroanatomy of fragile X syndrome is associated with aberrant behavior and the fragile X mental retardation protein (FMRP)[END_REF].

The second unusual change that is consistently found in FXS patients is an enlarged caudate nucleus [START_REF] Hallahan | In vivo brain anatomy of adult males with Fragile X syndrome: an MRI study[END_REF]. The caudate nucleus is part of (the striatum and) the basal ganglia, a system which is responsible not only for voluntary movement but also several other non-motor functions like learning, memory, sleep, and social behavior and plays a major role in planning [START_REF] Grahn | The cognitive functions of the caudate nucleus[END_REF]. Several studies found an inverse correlation between the IQ and the caudate size in FXS patients [START_REF] Gothelf | Neuroanatomy of fragile X syndrome is associated with aberrant behavior and the fragile X mental retardation protein (FMRP)[END_REF][START_REF] Reiss | Neurodevelopmental effects of the FMR-1 full mutation in humans[END_REF] but the link remains to be understood.

A few studies also noticed a reduced volume of the amygdala in FXS patients at young and adolescent age [START_REF] Gothelf | Neuroanatomy of fragile X syndrome is associated with aberrant behavior and the fragile X mental retardation protein (FMRP)[END_REF][START_REF] Hazlett | Teasing apart the heterogeneity of autism: Same behavior, different brains in toddlers with fragile X syndrome and autism[END_REF][START_REF] Kates | Reliability and validity of MRI measurement of the amygdala and hippocampus in children with fragile X syndrome[END_REF]. The amygdala is major player in emotions and has therefore it also been carefully examined on the cellular level, which also found alterations in FXS models [START_REF] Zhao | Deficits in trace fear memory and long-term potentiation in a mouse model for fragile X syndrome[END_REF]. It is thought that changes in the amygdala and insula are related to the anxiety and emotional troubles found in FXS patients. It is unclear what the reasons are for regional changes of brain volume in FXS but it has been suggested that it could be linked to changes in the synaptic pruning and dendritic maturation during development with deficient or absent FMRP [START_REF] Lightbody | Gene, brain, and behavior relationships in fragile X syndrome: evidence from neuroimaging studies[END_REF].

The brain areas are not only affected individually but also the connections between them are changed in FXS. Changes were found in the white matter of FXS patients by using DT-MRI. A loss of axonal order or structure was found in frontostriatal pathways and in parietal sensory-motor tracts indicated by lower Fractional Anisotropy (FA) values in the DT-MRI [START_REF] Goraly | White matter tract alterations in fragile X syndrome: preliminary evidence from diffusion tensor imaging[END_REF][START_REF] Haas | Early white-matter abnormalities of the ventral frontostriatal pathway in fragile X syndrome[END_REF]. In a later study [START_REF] Hall | Identifying large-scale brain networks in fragile X syndrome[END_REF] looked at large-scale resting-state networks in children and adolescent (aged 10-23 years) FXS patients and identified region-specific alterations in brain structures [START_REF] Hall | Identifying large-scale brain networks in fragile X syndrome[END_REF]. The FXS patients showed a widespread reduction in their functional connectivity across multiple cognitive and affective brain networks. Five large-scale networks were identified that were significantly decreased in their functional connectivity: the salience, precuneus, left executive control, language, and visuospatial networks. The salience network is thought to play a role in recruiting brain regions for sensory information processing;

the precuneus is involved in self-referential processing, imagery, and memory and is suggested to be a core node or hub of the default mode network. Both, structural and functional abnormalities were found in the left insular cortex. The insula has also been implicated in autism spectrum disorders and is thought to be important for a variety of functions, including interoceptive awareness and emotions. The authors further suggested that their method of combining structural and functional in this study could also be used to establish an imaging biomarker for FXS [START_REF] Hall | Identifying large-scale brain networks in fragile X syndrome[END_REF].

A recent study used resting-state EEG to characterize functional brain connectivity and brain network organization in male FXS patients and healthy controls. A decrease in global functional connectivity was found for upper alpha (8)(9)(10)(11)(12)(13)(14)(15) Hz) and beta frequency bands (16)(17)(18)[START_REF]New technologies for imaging synaptic partners 1-7[END_REF][START_REF] Lundh | Spread of vesicular stomatitis virus along the visual pathways after retinal infection in the mouse[END_REF][START_REF] Van Den Pol | Enhanced cytomegalovirus infection of developing brain independent of the adaptive immune system[END_REF][START_REF] Van Den Pol | Viral strategies for studying the brain, including a replication-restricted self-amplifying delta-G vesicular stomatis virus that rapidly expresses transgenes in brain and can generate a multicolor golgi-like expression[END_REF][START_REF] Etessami | Spread and pathogenic characteristics of a G-deficient rabies virus recombinant: an in vitro and in vivo study[END_REF][START_REF] Wickersham | Retrograde neuronal tracing with a deletionmutant rabies virus[END_REF][START_REF] Chandran | Endosomal proteolysis of the Ebola virus glycoprotein is necessary for infection[END_REF][START_REF] Beier | Anterograde or retrograde transsynaptic labeling of CNS neurons with vesicular stomatitis virus vectors[END_REF][START_REF] Conzelmann | Rescue of synthetic genomic RNA analogs of rabies virus by plasmid-encoded proteins[END_REF][START_REF] Schnell | Infectious rabies viruses from cloned cDNA[END_REF][START_REF] Lawson | Recombinant vesicular stomatitis viruses from DNA[END_REF][START_REF] Whelan | Efficient recovery of infectious vesicular stomatitis virus entirely from cDNA clones[END_REF][START_REF] Mebatsion | A CXCR4/CD4 pseudotype rhabdovirus that selectively infects HIV-1 envelope protein-expressing cells[END_REF]. For theta oscillations (4-7 Hz), the authors saw an increased connectivity in long-range (fronto-posterior) and short-range (frontal-frontal and posterior-posterior) clusters (van der [START_REF] Van Der Molen | Resting-state EEG oscillatory dynamics in fragile X syndrome: abnormal functional connectivity and brain network organization[END_REF].

Over the last years the resolution of MRI studies has been constantly improving and it became therefore possible to also use this technique to study the KO mouse model. While earlier studies did not see large differences [START_REF] Kooy | Neuroanatomy of the fragile X knockout mouse brain studied using in vivo high resolution magnetic resonance imaging[END_REF] a more recent study using a 7.0 Tesla MRI scanner in developing mice (postnatal age P30) found that cerebellum and striatum both decreased in size. Significant volume decreases were also discovered in two regions in two deep cerebellar nuclei (nucleus interpositus and the fastigial nucleus). The authors saw a decrease in NeuN positive signal (and conversely a increase in GFAP positive cells) and concluded that the decreased size is due to a loss of neurons in the deep cerebellar nuclei. Here DT-MRI measurements did not reveal any significant changes in the white matter structures of the Fmr1KO mice compared to the WT mice [START_REF] Ellegood | Anatomical phenotyping in a mouse model of fragile X syndrome with magnetic resonance imaging[END_REF].

Fine-Scale Neuroanatomy in Fragile X Syndrome

When considering the structural deficits on the fine scale the literature can be grouped in the (i) cellular defects, mainly addressing the dendritic spine morphology and density and (ii) short range networks within the immediate proximity.

Neuronal Structure in Fragile X Syndrome

An altered spine phenotype -with increased density and a shift towards long, thin immature appearing spines -is often proclaimed as a major hallmark in FXS and in fact it is probably the most commonly described structural change, which has been described in both human patients [START_REF] Hinton | Analysis of neocortex in three males with the fragile X syndrome[END_REF] and animal models (reviewed in [START_REF] Bagni | From mRNP trafficking to spine dysmorphogenesis: the roots of fragile X syndrome[END_REF][START_REF] He | The trouble with spines in fragile X syndrome: density, maturity and plasticity[END_REF]). Over time however the picture has become increasingly complex and the initially thought striking phenotype is not found in all brain areas and actually, for unclear reasons, several studies could not confirm the previous findings. Figure 1.6 summarizes the complicating findings concerning the spine phenotype in mouse models of FXS. The prevalent opinion today is that Fmr1KO mice have an increased number of spines due to defects in dendritic pruning (Galvez et al., 2003;[START_REF] Pfeiffer | Fragile X mental retardation protein induces synapse loss through acute postsynaptic translational regulation[END_REF] but several studies reported the deficits occur just transiently [START_REF] Nimchinsky | Abnormal development of dendritic spines in FMR1 knock-out mice[END_REF].

Even though we know now that the spine phenotype is more complex than initially thought (e.g. see [START_REF] Wijetunge | Stimulated emission depletion (STED) microscopy reveals nanoscale defects in the developmental trajectory of dendritic spine morphogenesis in a mouse model of fragile X syndrome[END_REF]) it has been and still is often being used as a parameter to evaluate for example the efficacy of a rescue approach [START_REF] Dolan | Rescue of fragile X syndrome phenotypes in Fmr1 KO mice by the small-molecule PAK inhibitor FRAX486[END_REF][START_REF] Henderson | Reversal of disease-related pathologies in the fragile X mouse model by selective activation of GABAB receptors with arbaclofen[END_REF] and it is speculated to provide a possible link between several causes of ASDs [START_REF] Tang | Loss of mTOR-Dependent Macroautophagy Causes Autistic-like Synaptic Pruning Deficits[END_REF]. Curiously much less is known about the counterpart of the spines, the presynaptic boutons in FXS. This is remarkable given that a study using a mosaic model of Fmr1 KO mouse, found a reduced connectivity in the CA3-CA3 network, which was independent of the FMRP in the postsynaptic cell, but instead determined by the pre-or absence of FMRP in the presynaptic cell (Hanson and Madison, 2007).

A later study, also using the mosaic model, showed that FMRP in the presynaptic cell was responsible for the loss of connection strength onto inhibitory neurons [START_REF] Patel | A target cell-specific role for presynaptic Fmr1 in regulating glutamate release onto neocortical fast-spiking inhibitory neurons[END_REF]. Even though the pre-dominant opinion is that FMRP acts mostly on the postsynapse by regulating translation and pruning of spines there are indications that FMRP plays a role in the initial axonal guidance. FMRP expression has been observed in axons and axon growth cones (Antar et al., 2006), and growth cones from developing neurons of Fmr1KO mice exhibit a hyper-abundance of filopodia and a reduction in dynamicity in hippocampal cultures (Antar et al., 2006). In the zebrafish, a knockdown of Fmr1 was associated with abnormal axon branching [START_REF] Tucker | Contribution of mGluR and Fmr1 functional pathways to neurite morphogenesis, craniofacial development and fragile X syndrome[END_REF]; although it was later contested as an artifact by using a newly generated zebrafish null knock-out [START_REF] Broeder | Generation and characterization of FMR1 knockout zebrafish[END_REF]. In drosophila, FMRP is required for efficient activity-dependent pruning of axon branches in the Mushroom Body [START_REF] Tessier | Drosophila fragile X mental retardation protein developmentally regulates activity-dependent axon pruning[END_REF]. Immunoelectron microscopy revealed FMRP in axons and presynaptic terminals [START_REF] Akins | Systematic mapping of fragile X granules in the mouse brain reveals a potential role for presynaptic FMRP in sensorimotor functions[END_REF][START_REF] Christie | The FXG: a presynaptic fragile X granule expressed in a subset of developing brain circuits[END_REF]. In the analysis of mRNA translational profiles of Fmr1KO mice a large number of different presynaptic FMRP targets appeared were revealed [START_REF] Brown | Microarray identification of FMRP-associated brain mRNAs and altered mRNA translational profiles in fragile X syndrome[END_REF][START_REF] Darnell | Cytoplasmic RNA-binding proteins and the control of complex brain function[END_REF] and proteomic studies found that the levels of various presynaptic proteins was affected due to the loss of FMRP [START_REF] Klemmer | Proteomics, ultrastructure, and physiology of hippocampal synapses in a fragile X syndrome mouse model reveal presynaptic phenotype[END_REF][START_REF] Liao | Quantitative proteomic analysis of primary neurons reveals diverse changes in synaptic protein content in fmr1 knockout mice[END_REF]. In the drosophila a neuronal overexpression of the Fmr1 homologue, dfxr, resulted in fewer and larger synaptic boutons, while knocking the gene out caused enlarged synaptic terminals, which was mediated through a regulatory role in the microtubuledependent growth of synapses [START_REF] Zhang | Drosophila fragile X-related gene regulates the MAP1B homolog Futsch to control synaptic structure and function[END_REF].

Small-Scale Networks in Fragile X Syndrome

Our knowledge about the short-range connectivity in FXS is limited, derived from individual studies and restricted to a few brain areas at a defined age or during a narrow time window. Most of those studies examined the connectivity in the neocortex of Fmr1 KO mice.

Gibson et al. examined the within-layer excitation in L4 of the somatosensory cortex in Fmr1 KO mice and found that it is extensively reduced (~50%) to fast spiking (FS) inhibitory neurons and to a lesser extend also decreased to excitatory spiny stellate neurons as well as the between neighboring excitatory neurons (Gibson et al., 2008). This loss of connection to FS neurons was later attributed to the lack of FMRP in the presynaptic neuron, resulting in a reduced neurotransmitter release [START_REF] Patel | A target cell-specific role for presynaptic Fmr1 in regulating glutamate release onto neocortical fast-spiking inhibitory neurons[END_REF]. Bureau et al. also found a reduction in intralayer connectivity within the same barrel column in the Fmr1KO mice. Photostimulation of L4 neurons lead to a less intense response in L3 neurons but when examining the strength of individual connections from L4 to L3 the authors found them to be unaltered. This suggests that a reduced number of connections is the reason for the changes seen in the Fmr1 KO. Interestingly, filling of individual L4 cells and axon reconstruction showed similar axonal arborization in L2/3, speaking against a problem in axon growth. Furthermore the authors noted that the connectivity, which they found altered at 2 weeks of age was normal again at 4 weeks of age (Bureau et al., 2008). Opposed to the decrease in local connectivity in the previous studies, Teste-Silva et al. found an increased mid-range connectivity in Fmr1 KO mice. Here L5 pyramidal cells in the medial prefrontal cortex were examined in 25 -100 µm distance and an increased connectivity was found in between them in Fmr1KO mice [START_REF] Testa-Silva | Hyperconnectivity and slow synapses during early development of medial prefrontal cortex in a mouse model for mental retardation and autism[END_REF].

Interestingly the connectivity changes found in all of those studies were only transient and disappeared at a later point during development even though the synaptic strength changes remained (Bureau et al., 2008;Gibson et al., 2008;[START_REF] Testa-Silva | Hyperconnectivity and slow synapses during early development of medial prefrontal cortex in a mouse model for mental retardation and autism[END_REF].

A more recent study looking at the interconnectivity of L5A pyramidal cells, specifically the intratelencephalic type, using paired recordings surprisingly found a different effect due to an impairement in synaptic pruning. The L5A-L5A connectivity decreased between the third and fifth postnatal weeks in WT animals, which indicates a period of connection pruning, while it did not decrease in the Fmr1KO mice, leaving these neurons in a hyperconnected state at postnatal day P30.

The use of the mosaic mouse model showed that the pruning was dependent on the postsynaptic neuron (Patel et al., 2014).

Besides the neocortex it has also been inferred that the wiring in the amygdala is affected in Fmr1 KO mice. Recordings from principle neurons in the basolateral amygdala (BLA) showed a decreased number of spontaneous and miniature inhibitory postsynaptic currents (sIPSCs and mIPSCs), which is likely the result of a reduced input from surrounding inhibitory neurons [START_REF] Olmos-Serrano | Defective GABAergic neurotransmission and pharmacological rescue of neuronal hyperexcitability in the amygdala in a mouse model of fragile X syndrome[END_REF].

Taken together these data suggest that FMRP plays a role in the formation or maintenance of connections in cortical and subcortical areas, which could be exhibited by the axon guidance or the spine pruning, but most likely it has an important role regulating both.

Network States

The wiring and the excitation/inhibition balance as well as the synaptic strength and intrinsic properties of neurons, all contribute to the response a network exhibits to a given stimulus. Since so many of these parameters were found to be altered it is not surprising that also the response and the dynamics of the network are changed in FXS.

And a number of studies (Gibson et al., 2008;[START_REF] Gonçalves | Circuit level defects in the developing neocortex of Fragile X mice[END_REF][START_REF] Hays | Altered neocortical rhythmic activity states in Fmr1 KO mice are due to enhanced mGluR5 signaling and involve changes in excitatory circuitry[END_REF][START_REF] Ronesi | Disrupted Homer scaffolds mediate abnormal mGluR5 function in a mouse model of fragile X syndrome[END_REF] found a hyperexcitable cortical network in the Fmr1KO mice that reacted with a prolonged activated state upon stimulation, which could explain the epileptic features often found in FXS .

Aims of this Study

The aim of this work was to study the anatomical and functional connectivity of neuronal networks and develop novel tools for this purpose. At the beginning of this study rabies virus was available as a retrograde polysynaptic, as a retrograde mono-transsynaptic and as a single-cycle retrograde vector. For several research questions it would be beneficial, to use the same viral vector to infect neurons at the injection site -and hence mark those neurons and their projections anterogradelytogether with one of the retrograde variants to label the input. We therefore first set out to develop an anterograde variant of the rabies virus, which would allow us to also study the morphological features of the cells in the injection site together with the input after a single stereotactic injection.

We applied this technique in combination with other approaches to probe neuronal connectivity defects in Fragile X Syndrome (FXS). One of the morphological hallmarks of FXS is an alteration in the shape/number of spines, the major sites for synaptic input. In addition, FMRP regulates the RNAs of molecules that regulate the anatomical connectivity (cytoskeleton, axon and dendrite branching, spines) within cortical circuits. In fact an altered anatomical and functional connectivity has been proposed to play a role in both FXS and ASDs (Geschwind and Levitt, 2007), yet our understanding of the nature of these defects is limited due to the complex nature of these syndromes.

Profound knowledge of deficits in the neuronal circuits will be necessary to bridge the gap, between alterations on the molecular level/local circuitry, and the global changes in behavior and cognition. Correct wiring is critical for maintaining normal brain function and for the processing of sensory information. In particular, the long-range connections between different brain areas are crucial for a successful sequential processing and transmission of sensory information from lower to higher hierarchical areas. Since hypersensitivity to visual, auditory, tactile, and olfactory stimulation is a prominent feature of human FXS patients and Fmr1KO mice, we were interested in studying connectivity changes both to-and in between-the neocortical sensory areas. We hypothesized that long-range connections between brain areas might be altered in FXS given the deficits previously found in axonal outgrowth and spine morphologies. Yet, long-range wiring deficits underlying FXS have been largely unexplored as a potential mechanism underlying cognitive deficits.

Therefore we set out to quantify brain-wide connectivity features in the Fmr1KO mouse model and to correlate them with functional connectivity features. We have probed changes in neuronal connectivity, and the functional consequences of these changes on information processing in the neocortical circuits in Fmr1KO mouse model of Fragile X Syndrome. For this, we used a combination of cutting-edge approaches ranging from viral tracing, magnetic-resonance-imaging and optical imaging to novel anatomical quantification methods.

Results

Novel Tools to Unravel the Neuronal Wiring

Mono-trans-synaptic input mapping

Newly developed methods need a phase of thorough evaluation and often improvement to aim for the highest standards and ensure that the results are reproducible, can be fully trusted and biases are avoided by all means possible.

Mono-trans-synaptic tracing with a glycoprotein deleted variant of rabies virus has been a method offering unprecedented possibilities of mapping the input of a group of or individual cells. In the next section we described in a review article the results that have been gained with this method by several groups over the last couple of years until the date of publication as well as possibilities and limitations the method bears.

A more recent update on this matter can be found in our recent book chapter, in the Appendix.

Publication N.

INTRODUCTION

How does the brain process information, encode perception, and generate behavior? An answer to these questions requires a mechanistic understanding of the operation of the brain's neural circuits. The operation of these neural circuits is determined by their structure, the physiological properties of the neuronal connections, and the integrative properties of the neurons. Thus, an understanding of the brain's computations is inevitably linked to knowledge of the relationship between the structure of its neuronal circuits and their function (Lichtman and Denk, 2011;Rancz et al., 2011). Rabies virus (RABV) has outstanding properties as a retrograde tracer of synaptically connected neuronal populations (Kelly and Strick, 2000;Wickersham et al., 2007a;Ugolini, 2011). Recently, the development of a glycoprotein genedeleted ( G) RABV based method has enabled the tracing and functional investigation of monosynaptic connections of defined neurons (Wickersham et al., 2007b;Callaway, 2008;[START_REF] Marshel | Targeting single neuronal networks for gene expression and cell labeling in vivo[END_REF]. This methodology can be readily combined with the expression of a variety of genes, optical/electrophysiology methods, and behavioral assays to drive integrative studies of neural circuits (Callaway, 2008;Arenkiel and Ehlers, 2009;Osakada et al., 2011;[START_REF] Wickersham | New technologies for imaging synaptic partners[END_REF].

In this review we present an update on recent developments in the field of RABV G based trans-synaptic tracing technology. We explain the basic principles of this technology, and show how, in recent studies, it has been adapted to reveal specific aspects of neuronal circuit structure/function. We also discuss what we believe to be the major potential and shortcomings of recombinant RABV technology. Lastly, we provide an overview of how this approach complements other recent technological developments in the field of neural circuit analysis and the kind of questions that can be addressed using a combination of these approaches.

TRANS-SYNAPTIC TRACERS FOR THE STUDY OF NEURAL CIRCUIT CONNECTIVITY POLYSYNAPTIC TRACERS

Conventional anterograde and retrograde tracers have significantly advanced our knowledge of connectivity between different brain areas [START_REF] Cowan | The emergence of modern neuroanatomy and developmental neurobiology[END_REF]Luo et al., 2008;Lichtman and Denk, 2011). These tracers reveal which brain areas are connected to one another and the location and identity of the projections neurons. However, since these tracers do not cross synapses, they are unable to establish direct synaptic connectivity between neurons.

Polysynaptic tracers, on the other hand, have the ability to spread from one neuron to another, permitting the identification of networks of synaptically connected neurons. These polysynaptic tracers can be divided into two types: non-viral and viral tracers. Non-viral polysynaptic tracers are, for the most part, limited to certain types of plant lectin and bacterial toxins [START_REF] Cowan | The emergence of modern neuroanatomy and developmental neurobiology[END_REF][START_REF] Carter | Guide to Research Techniques in Neuroscience[END_REF]. These tracers have a number of advantages including their safety, ease of use and ability to be genetically encoded. However, they also suffer from one or more shortcomings (reviewed in references Callaway, 2008;Luo et al., 2008;Lichtman and Denk, 2011;[START_REF] Wickersham | New technologies for imaging synaptic partners[END_REF], importantly severe dilution of signal with each synaptic step.

These limitations can be overcome by the use of certain polysynaptic viral tracers, in particular those belonging to the classes of α-herpes viruses (herpes simplex virus type 1 and pseudorabies virus) and RABV (reviewed in Callaway, 2008;Ekstrand et al., 2008). In contrast to non-viral tracers, trans-synaptic viral labeling is amplified rather than diluted due to the replicative nature of viruses. This labeling can be revealed by the expression of suitable morphological markers such as native viral proteins or reporter molecules in the case of recombinant virus strains (reviewed in Ekstrand et al., 2008). Not all of the polysynaptic viral tracers spread exclusively at synaptic sites, thereby labeling neurons that are not necessarily connected by synapses. The CVS derived strains of RABV and the Bartha strain of pseudorabies virus (PRV), however, have been shown to spread in a synapsespecific manner (reviewed in references Callaway, 2008;Ekstrand et al., 2008;Luo et al., 2008;Ugolini, 2011). Furthermore, these two viruses spread in a defined (retrograde) direction, enhancing their value for neural circuit analysis. RABV has two main advantages over PRV, namely a significantly reduced cytotoxicity and the ability to be used in primates (Callaway, 2008). The relative merits of these polysynaptic viruses for neural circuit analysis have been discussed in detail elsewhere (Callaway, 2008;Ekstrand et al., 2008;Ugolini, 2011).

Altogether, these features make RABV a very useful virus for the study of neural circuit connectivity [START_REF] Ugolini | Specificity of rabies virus as a transneuronal tracer of motor networks: transfer from hypoglossal motoneurons to connected second-order and higher order central nervous system cell groups[END_REF]Kelly and Strick, 2000;Callaway, 2008;Ugolini, 2011). However, the fact that native RABV (like other trans-synaptic viruses) is a polysynaptic tracer causes potential ambiguity in the interpretation of how many synaptic steps have been crossed at any given time. For instance, such ambiguity would occur if RABV crosses synapses at different rates depending on their strength or simply because of differences in the RABV transport time along presynaptic axons that span several orders of magnitude in length (for discussion of synapse strength see references [START_REF] Ugolini | Specificity of rabies virus as a transneuronal tracer of motor networks: transfer from hypoglossal motoneurons to connected second-order and higher order central nervous system cell groups[END_REF]Ugolini, , 2011;;Callaway, 2008;[START_REF] Wickersham | New technologies for imaging synaptic partners[END_REF]. Furthermore, it is not possible to concomitantly use this method for both circuit tracing and the manipulation of first-order connections (see below), since higher-order connections will also be affected.

RABV AS A MONOSYNAPTIC TRACER

Revealing first-order connections

An elegant solution to this problem led to a technique, which has come to be known as monosynaptic trans-synaptic tracing (Wickersham et al., 2007b) or mono-trans-synaptic tracing for short [START_REF] Miyamichi | Cortical representations of olfactory input by trans-synaptic tracing[END_REF]. Mono-trans-synaptic tracing uses a pseudotyped, recombinant RABV to identify direct presynaptic inputs to a defined target cell population. These connections are "mono-synaptic" because trans-synaptic traversal is limited to one synaptic step between initial infected cells (i.e., the source cells) and their immediate presynaptic partners. The exquisite specificity of the RABV ensures that only synaptically connected neurons are labeled.

To implement this highly specific tracing system, Wickersham et al. (2007b) employed a deletion mutant RABV (RABV G) (Mebatsion et al., 1996a), deficient in the expression of the RABV envelope glycoprotein (RG) (Figures 1A,B). RG is essential for the assembly of infectious virus particles during the natural life cycle of RABV (Mebatsion et al., 1996a), as well as for mediating trans-synaptic crossing of the virus (Mebatsion et al., 1996a;Etessami et al., 2000) (Figure 1B). Infectious properties can be rescued by cultivating the virus in a trans-complementing cell line (pseudotyping; Figures 1C,D). The resulting virus, however, behaves as a single-cycle tracer and is trapped in the source cells. Exogenous expression of RG in the same source cell population is sufficient to rescue the trans-synaptic capabilities of the virus-but in a highly restricted manner, which is limited to first order connections (Wickersham et al., 2007b) (Figure 1E).

In the original description of the method, pseudotyping was employed as a means of limiting the initial infection to a defined source cell population. To achieve this, Wickersham et al. (2007b) exploited the envelope glycoprotein (EnvA) of the avian sarcoma and leukosis virus, whose cognate receptor (TVA) has no homologue in mammalian cells (Bates et al., 1993;Young et al., 1993;Federspiel et al., 1994). Ectopic TVA expression is required to permit infection by RABV G(EnvA) and needs to occur in the same cells expressing RG, to correctly demarcate the source cell population (Figure 1E). In order to visualize the resulting transsynaptically labeled neurons, RABV was modified to express a fluorescent protein (eGFP) and source cells are distinguished by the addition of a second fluorescent marker (Wickersham et al., 2007b) (Figure 1B). Pseudotyping can also be achieved with a variety of viral glycoproteins provided that the glycoprotein is suitably engineered to contain the cytoplasmic domain of the native RABV glycoprotein (Mebatsion et al., 1995;[START_REF] Conzelmann | Nonsegmented negative-strand RNA viruses: genetics and manipulation of viral genomes[END_REF]Choi et al., 2010;[START_REF] Choi | Monosynaptic inputs to ErbB4expressing inhibitory neurons in mouse primary somatosensory cortex[END_REF]. In such cases, the source cell population is defined by the resulting tropism of the pseudotyped virus.

Features of RABV vectors

RABV is, in many ways, the prototypic neurotropic virus since its life cycle is entirely adapted to survival and spread in the nervous system. RABV derived vectors thus possess a number of features, which endow them with excellent properties for a viral tracer. These include efficient long-range transport in neurons, low cytotoxicity, and high-level gene expression (Mebatsion et al., 1996b;Wickersham et al., 2007a;[START_REF] Dietzschold | Concepts in the pathogenesis of rabies[END_REF]. Wild-type RABV has a broad host species range that includes almost all terrestrial mammals, bats, and birds [START_REF] Gough | Rabies antibodies in sera of wild birds[END_REF][START_REF] Nel | Lyssaviruses[END_REF], and this likely extends to all RABV derived vectors making them suitable for use in a range of animal model systems. The RABV genome [START_REF] Conzelmann | Molecular cloning and complete nucleotide sequence of the attenuated rabies virus SAD B19[END_REF]) is small, modular, and readily accepts foreign genes without the issues of instability inherent to other RNA viruses (Mebatsion et al., 1996b;[START_REF] Conzelmann | Nonsegmented negative-strand RNA viruses: genetics and manipulation of viral genomes[END_REF]. Moreover, RABV is confined to the cytoplasm throughout its life cycle [START_REF] Albertini | Chapter 1 -Rabies virus transcription and replication[END_REF]. This precludes integration into the host genome and the subsequent disruption or inappropriate transcriptional activation of host genes, which is reported to occur for other vectors such as lentivirus [START_REF] Sakuma | Lentiviral vectors: basic to translational[END_REF]. The highly stable and tightly bound ribonucleoprotein core [START_REF] Albertini | Chapter 1 -Rabies virus transcription and replication[END_REF] also prevents recombination between the RNA genome and cellular RNAs.

Transgene expression from a RABV vector is integrally tied to viral genome expression and is thus constitutively ON. RABV transcription starts at the 3 end (upstream of the N gene; Figure 1A) and proceeds sequentially throughout the genome. Attenuation at gene boundaries generates a gradient of transcripts with respect to gene order (reviewed in references [START_REF] Conzelmann | Molecular cloning and complete nucleotide sequence of the attenuated rabies virus SAD B19[END_REF][START_REF] Albertini | Chapter 1 -Rabies virus transcription and replication[END_REF], resulting in a finely tuned ratio of gene products in the native virus (reviewed in references [START_REF] Conzelmann | Nonsegmented negative-strand RNA viruses: genetics and manipulation of viral genomes[END_REF][START_REF] Albertini | Chapter 1 -Rabies virus transcription and replication[END_REF]. This feature can be manipulated to some extent to influence the level of expression of a transgene. In addition, "engineered" gene border transcription signals (Finke et al., 2000) or regulatory elements from other RNA viruses can also be deployed to modify transgene expression levels [START_REF] Marschalek | Attenuation of rabies virus replication and virulence by picornavirus internal ribosome entry site elements[END_REF]. RABV G vectors expressing a single transgene have typically made use of the endogenous transcription signals, which normally flank the glycoprotein gene (Wickersham et al., 2007b;Osakada et al., 2011). In addition, it is possible to insert additional cistrons as long as each is flanked by suitable transcription start/stop signals [START_REF] Conzelmann | Nonsegmented negative-strand RNA viruses: genetics and manipulation of viral genomes[END_REF]Osakada et al., 2011). This principle was used to express twoindependent genes from a RABV G vector (Osakada et al., 2011). In this case 3.6 kb of exogenous sequence was effectively integrated into the RABV G viral genome and subsequently packaged into infectious particles. Unlike other vectors, the most important factor limiting "cloning capacity" is probably not the total insert size, but instead the number of additional transcription initiation start sites introduced with the exogenous genes.

The RABV genome never encompasses a DNA form and this poses particular constraints on RABV derived vectors. Certain genetic tools such as lox P sites, tet-regulatory sequences or cellspecific promoters simply do not function with a negative-sense single-stranded RNA [(-)ssRNA] virus since a double stranded DNA binding site or substrate is required. Unfortunately, this precludes the use of a number of genetic tools that are normally employed for cell-specific or inducible expression of the viraldriven transgene (see Figure 4). The (-)ssRNA RABV genome also presents practical difficulties for the production of recombinant viral particles. Although improved methods for RABV G rescue have recently been described (Wickersham et al., 2010;Osakada et al., 2011;[START_REF] Ghanem | Significantly improved rescue of rabies virus from cDNA plasmids[END_REF], reverse genetics approaches, required for the recovery of infectious RABV G from cloned DNA, remain several orders of magnitude less efficient than for positive strand RNA or DNA viruses (Schnell et al., 1994;[START_REF] Ghanem | Significantly improved rescue of rabies virus from cDNA plasmids[END_REF]. This being said, it is worth noting that once a new recombinant form of RABV G has been recovered from DNA, it is straightforward to amplify the virus using an RG-expressing cell line (Osakada et al., 2011).

STRATEGIES FOR TRACING MONOSYNAPTIC CONNECTIONS TO SPECIFIC BRAIN REGIONS, NEURON TYPES, OR INDIVIDUAL NEURONS TARGETING SPECIFIC BRAIN REGIONS

A specific brain region can simply be targeted via stereotaxic injection of CNS competent viral vectors expressing TVA/RG, followed several days later by injection into the same region of the RABV G(EnvA). For example, mono-trans-synaptic tracing was crucial for determining synaptic connections between oxytocinexpressing neurons of the hypothalamic accessory magnocellular nucleus and neurons of the central amygdala (Knobloch et al., 2012).

TARGETING A DEFINED NEURONAL CELL POPULATION

While the aforementioned approach maps the connectivity of multiple neuron types within the respective brain structure, it may be desirable to target a defined neuronal cell population. In the next paragraphs we will highlight the variety of approaches that have been developed to achieve neuron type specific monotrans-synaptic tracing.

One way to achieve this is by taking advantage of an everincreasing list of mice expressing cre-recombinase in specific neuronal cell types (e.g., reference [START_REF] Gong | A gene expression atlas of the central nervous system based on bacterial artificial chromosomes[END_REF]. TVA/RG is then selectively expressed in these cell types using a cre-dependent viral vector (Wall et al., 2010;Watabe-Uchida et al., 2012) (Figure 2A). This strategy was applied to map reciprocal synaptic connections between PKC-δ -and PKC-δ + interneurons in the central amygdala [START_REF] Haubensak | Genetic dissection of an amygdala microcircuit that gates conditioned fear[END_REF], and to perform brain-wide labeling of neurons providing direct input to dopaminergic neurons of the midbrain (Watabe- Uchida et al., 2012).

Alternatively, neuron types can be targeted based on their expression of cell surface receptors that recognize specific ligands. For example, a specific class of interneurons in the neocortex expresses the receptor ErbB4 that binds the soluble ligand neuregulin β1. A recent study took advantage of this interaction to target ErbB4-positive neurons by pseudotyping RABV G with an avian virus envelope protein (EnvB) that binds to a neuregulin β1-EnvB receptor (TVB) bridge protein [START_REF] Choi | Monosynaptic inputs to ErbB4expressing inhibitory neurons in mouse primary somatosensory cortex[END_REF]. A lentivirus was pseudotyped in the same way to permit RG expression in the same source cell population [START_REF] Choi | Monosynaptic inputs to ErbB4expressing inhibitory neurons in mouse primary somatosensory cortex[END_REF]. Such approaches may also be of interest for studies of schizophrenia, which have been linked to a dysfunction in neuregulin signaling [START_REF] Mei | Neuregulin 1 in neural development, synaptic plasticity and schizophrenia[END_REF]. This approach could also provide a means for targeting RABV G infection to particular cell populations in situations where genetic-based targeting approaches are not available, for example in primate systems [START_REF] Choi | Monosynaptic inputs to ErbB4expressing inhibitory neurons in mouse primary somatosensory cortex[END_REF].

An alternative way to target a very specific source cell population is the use of a helper virus capable of propagating exclusively in dividing cells. Certain retroviruses, such as the Moloney murine leukemia virus (MMLV), are restricted in this manner and have classically been used to mark newly born neurons in the dentate gyrus of the hippocampus (reviewed in [START_REF] Van Praag | Functional neurogenesis in the adult hippocampus[END_REF][START_REF] Ming | Adult neurogenesis in the mammalian central nervous system[END_REF]. Stereotaxic injection of the MMLV expressing TVA/RG/fluorescent marker, followed by subsequent injection of RABV G(EnvA) was thus used as a means of following the functional integration of newly born granule cells into existing neuronal circuits (Vivar et al., 2012).

All of the aforementioned examples necessitate two sequential stereotaxic injections into the same brain region [e.g., virus expressing TVA/RG, followed by RABV G(EnvA)]. One can bypass the first step of this procedure by using RABV G(EnvA) together with a bi-transgenic mouse line, in which TVA/RG is induced in a brain region-or cell-type specific manner under the control of a tet-regulatable promoter [START_REF] Weible | Transgenic targeting of recombinant rabies virus reveals monosynaptic connectivity of specific neurons[END_REF]. This approach is suited to tracing long-range connections between nuclei that can be discretely targeted by cell-specific tetracycline transactivator expression. However, carefully designed controls are required to ensure that RG expression only occurs in the targeted source cell population and not in directly presynaptic neurons, as this would confound the interpretation of the tracing results (first-order vs. higher-order connectivity). Mono-trans-synaptic tracing from defined source cell populations can also be achieved using a simpler targeting strategy which eliminates the need for the EnvA-TVA system, and instead takes advantage of the inherent qualities of certain retrogradelytransported viruses. Recent studies utilized this approach, by co-injecting RABV G pseudotyped with its native G together with an RG-expressing retrogradely transported helper virus [START_REF] Stepien | Monosynaptic rabies virus reveals premotor network organization and synaptic specificity of cholinergic partition cells[END_REF]Yonehara et al., 2011). The source cell population was thus defined by their specific projection characteristics, rather than by their molecular identity. Injections were made into known target regions for the axons of certain neurons, thereby permitting the infection of axon terminals. With this approach, mono-trans-synaptic tracing enabled the identification of direct synaptic inputs to motor neurons targeting discrete muscle groups [START_REF] Stepien | Monosynaptic rabies virus reveals premotor network organization and synaptic specificity of cholinergic partition cells[END_REF], and to ON direction-selective retinal ganglion cells (Yonehara et al., 2011).

Monitoring network activity

STRATEGIES TO TARGET INDIVIDUAL NEURONS

The aforementioned strategies (sections "Targeting specific brain regions" and "Targeting a defined neuronal cell population") may not permit the unambiguous identification of the presynaptic partners of a given neuron, because more than one source cell is typically targeted. Furthermore, in situations where a presynaptic partner of a source cell also expresses RG, an additional trans-synaptic crossing step may occur, resulting in the labeling of higher-order connections. One strategy to reduce this ambiguity is exemplified by Arenkiel et al. (2011). In this study the authors developed an elegant strategy to allow mono-trans-synaptic tracing of a very sparse and defined neuronal population. They transfected neuronal progenitor cells in the subventricular zone of the lateral ventricles in early postnatal mice with a targeting construct expressing TVA/RG. Adult-born granule cells derived from these progenitors migrate to the olfactory bulb and establish functional connections [START_REF] Panzanelli | Early synapse formation in developing interneurons of the adult olfactory bulb[END_REF]. However, only a small number of migrated, functionally integrated daughter cells retained the ability to express TVA/RG, by virtue of rare integration events leading to stable expression of this targeting construct (Arenkiel et al., 2011). These source cells were then infected with RABV G(EnvA) by stereotaxic injection into the olfactory bulb. The authors were thus able to trace microcircuits derived from sparsely distributed individual source granule cells.

The ability to target sparsely-distributed source cells may also be important for studies of long-range connections, where the topography or convergence of inputs needs to be examined and therefore the source cell number needs to be strictly limited. To achieve this, a complex strategy involving a conditional bi-transgenic mouse line and an AAV vector expressing TVA/RG under the control of a tetracycline-dependent promoter was employed [START_REF] Miyamichi | Cortical representations of olfactory input by trans-synaptic tracing[END_REF]. RABV G(EnvA) was subsequently injected into the same region as the AAV helper virus 14 days later. These tools not only permit tracing in a very sparse neuronal population, but also precise temporal control over TVA/RG expression.

Although mono-trans-synaptic tracing has most frequently been applied to populations of source cells, it also has great power for determining inputs into a single neuron in vitro and in vivo (Figure 2B). To target a single neuron, the plasmids encoding the tracing components (TVA/RG) and a fluorescent marker are first introduced together into this neuron. Two approaches have thus far been employed to achieve this in vivo-two-photon-guided electroporation and whole cell recording [START_REF] Marshel | Targeting single neuronal networks for gene expression and cell labeling in vivo[END_REF]Rancz et al., 2011). Such in vivo approaches are painstaking, but permit the unambiguous identification of neurons presynaptic to a defined individual neuron. Furthermore, when combined with for instance whole-cell recordings and sensory stimulation, information regarding the anatomical receptive field of this neuron can be linked with its synaptic receptive field (Rancz et al., 2011).

RECENT ADVANCES IN NEURONAL CIRCUIT ANALYSIS EMPLOYING THE RABV G APPROACH MAPPING OF LONG-RANGE CONNECTIONS

One of the great advantages of RABV G mediated monotrans-synaptic tracing over other assays for synaptic connections [e.g., electron microscopy (EM) and paired electrophysiological recordings] is the ability to identify long-range synaptic connections. For example, RABV G has been used to map direct long-range connections between mitral cells in the olfactory bulb and their postsynaptic targets in basal forebrain regions [START_REF] Miyamichi | Cortical representations of olfactory input by trans-synaptic tracing[END_REF]. Such connections had previously been inferred from the presence of axons (e.g., reference [START_REF] Scott | The organization of projections from the olfactory bulb to the piriform cortex and olfactory tubercle in the rat[END_REF], but direct synaptic connectivity had not been proven. Similarly, Knobloch et al. (2012) employed the RABV G technology as part of a complex toolkit to elucidate novel connections between oxytocin expressing neurons in the hypothalamus and neurons of the central amygdala. In this case, synaptic connections were strongly suggested by a variety of supporting evidence, obtained from optogenetic-based mapping and electron microscopy data. However, mono-trans-synaptic tracing was essential for confirming the presence of a functional synapse.

RABV G based tracing approaches have also been exploited to map inputs into specific dopaminergic populations of the midbrain-in this case, on the scale of the whole brain (Watabe- Uchida et al., 2012). The authors were able to correct or refine previous findings obtained with conventional tracing or optogenetic approaches. By targeting genetically defined source cell populations they identified more specific subsets of presynaptically connected neurons. In addition, the authors showed that striatal neurons do in fact provide direct input to dopaminergic neurons of the ventral tegmental area or substantia nigra (Watabe-Uchida et al., 2012). Lastly, the exquisite labeling of the neuronal morphology afforded by RABV-driven eGFP expression aided the identification of the type of presynaptic neurons.

FUNCTIONAL INTEGRATION OF POSTNATAL-BORN NEURONS INTO A NEURAL CIRCUIT

In addition to the validation of known connections, or the discovery of novel connections, a recent study examined the plasticity-induced remodeling of neural circuits (Arenkiel et al., 2011). In this study, the authors traced the connections formed by postnatal-born, newly migrated, differentiated granule cells following their integration into established circuits of the olfactory bulb. The authors further showed that odor stimulation resulted in increased input onto these neurons, as well as remodeling of their dendritic morphology. This study demonstrates the immense power of RABV G based monosynaptic tracing for both screening of neural circuit changes as well as for fine morphological analysis.

A different study (Vivar et al., 2012) describes the timedependent incorporation of adult-born granule cells of the dentate gyrus into existing neuronal circuits. To do this, the authors performed mono-trans-synaptic tracing at various time points after the initial labeling of adult-born daughter cells. They were then able to demonstrate quantitative changes in the type and number of the presynaptic neurons over time. In particular, they showed a transient period of input coming from mature granule cells, and that intra-hippocampal and cortical inputs increased with time. They also identified a novel "back-projection" from CA3 pyramidal cells. This study demonstrates the utility of RABV G based mono-trans-synaptic tracing for the identification of novel connections, as well as for studying the remodeling of circuits.

THREE-DIMENSIONAL TOPOGRAPHY OF PRESYNAPTIC NEURONS

With the use of RABV G based trans-synaptic tracing, [START_REF] Stepien | Monosynaptic rabies virus reveals premotor network organization and synaptic specificity of cholinergic partition cells[END_REF] were able to demonstrate that distinct populations of premotor neurons project to functionally-defined motor neuron pools, and that these populations extend over a large three-dimensional space along the spinal cord. In a related study the authors showed that premotor interneurons controlling antagonistic extensor-flexor muscles are segregated from one another along the medial lateral axis of the dorsal spinal cord (Tripodi et al., 2011). In an unrelated study [START_REF] Miyamichi | Cortical representations of olfactory input by trans-synaptic tracing[END_REF] examined the topography of inputs from olfactory bulb into different olfactory cortex regions. In doing so, the authors were able to correlate topography of post/presynaptic neurons and divergence/convergence of connections with the presumed functionality of each type of olfactory processing center. These studies demonstrate that the topography of connections can yield additional information about the functionality of circuits.

NOVEL RABV G VARIANTS AND THEIR UTILITY FOR NEURONAL CIRCUIT ANALYSIS

A range of RABV G variants have recently been described. These variants include vectors expressing the trans-acting factors required for conditional expression of other transgenes (e.g., Cre, FLP recombinase; Figure 2C), a genetically encoded calcium sensor (Osakada et al., 2011;Kiritani et al., 2012) (Figure 2D) and molecules for the activation 51 or silencing of circuits (Osakada et al., 2011) [e.g., allatostatin receptor, channelrhodopsin-2 (ChR2); Figure 2E]. Variants expressing additional fluorescent markers to permit a greater array of tracing possibilities are also described (Wickersham et al., 2007b;Osakada et al., 2011). These aforementioned tools can be combined with a variety of mouse models, for example those expressing Cre-or FLP-dependent reporters or conditional mutations permitting loss-or gain-of-function studies in defined neuronal circuits. A RABV G vector expressing the light-activatable ChR2 has recently been used for the analysis of defined spino-cortical and cortico-striatal circuits (Apicella et al., 2012;Kiritani et al., 2012).

PERSPECTIVES FOR THE ANALYSIS OF NEURAL CIRCUIT STRUCTURE/FUNCTION

To date, our understanding of the wiring diagrams of neuronal circuits and the spatiotemporal dynamics of their electrical signals is rather limited. Critical steps toward this goal are the identification of the inputs of the circuit's diverse neuron types, and an understanding of their interaction within the circuit. The last years have seen numerous innovations in methods that rapidly advance the analysis of neural circuits. These include improvements in optical methods for stimulation and imaging, genetics, and computational methods. In the next paragraphs we will discuss how the RABV G technology in concert with these methods can help shed light on some of the outstanding questions in this field.

TRACING STRUCTURE-FROM THE NANOSCALE TO THE MESOSCOPIC SCALE

RABV G has outstanding properties for the labeling of the fine-scale neuronal morphology, due to its high-level expression of fluorescent proteins (Wickersham et al., 2007b). This feature is advantageous for visualization approaches where further signal amplification is not feasible. Such approaches include fluorescence imaging of entire mouse brains following a chemical clearing procedure (Dodt et al., 2007;[START_REF] Hama | Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain[END_REF] or using serial two-photon tomography (STP tomography) (Ragan et al., 2012). Similarly, the high-intensity expression of morphological markers permits the dynamic observation of processes such as spine stability/turnover in living animals (for a recent review see [START_REF] Holtmaat | Experience-dependent structural synaptic plasticity in the mammalian brain[END_REF], and likely satisfies the requirements for super-resolution imaging at nanoscale resolution (for review, see references [START_REF] Huang | Super-resolution fluorescence microscopy[END_REF][START_REF] Sigrist | Optical super-resolution microscopy in neurobiology[END_REF]. Furthermore, RABV mediated expression of horseradish peroxidase may be a way to enhance the intensity of labeling required for ultrastructural techniques such as serial block-face scanning electron microscopy [START_REF] Denk | Serial block-face scanning electron microscopy to reconstruct threedimensional tissue nanostructure[END_REF][START_REF] Briggman | Wiring specificity in the direction-selectivity circuit of the retina[END_REF][START_REF] Li | In vivo time-lapse imaging and serial section electron microscopy reveal developmental synaptic rearrangements[END_REF].

RABV G pseudotyped with RG can be used as a retrograde tracer (Figure 1D) to infer connectivity between brain regions by labeling cells projecting to a specific target region (Wickersham et al., 2007a;Apicella et al., 2012;Kiritani et al., 2012). In combination with large-scale fluorescence imaging techniques (Dodt et al., 2007;[START_REF] Hama | Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain[END_REF]Ragan et al., 2012) this approach allows systematic brain-wide mapping of neuronal circuits at the mesoscopic scale (Bohland et al., 2009) (see also Mouse Brain Architecture Project).

Thus, the RABV G technology has the potential to aid visualization of neuronal morphology over spatial dimensions ranging from the nanoscale (electron microscopy/super-resolution light microscopy) to the entire mouse brain (light-sheet microscopy/STP tomography).

CLASSIFYING NEURONAL CELL TYPES

One of the intractable problems with neuronal circuit analysis is the ability to define the neuronal cell types present within such a circuit. Cellular identity is based on an evermore refined list of criteria such as electrical properties, patterns of gene expression and morphological features [START_REF] Interneuron | Petilla terminology: nomenclature of features of GABAergic interneurons of the cerebral cortex[END_REF]Arenkiel and Ehlers, 2009). Ideally one would also link cell identity to the expression of a fluorescent marker to facilitate analysis of the properties of these cells within a circuit (Arenkiel and Ehlers, 2009;Lichtman and Denk, 2011;[START_REF] Steinmeyer | High-throughput singlecell manipulation in brain tissue[END_REF]. For certain cell types this has been achieved with high specificity in transgenic mice [START_REF] Gong | A gene expression atlas of the central nervous system based on bacterial artificial chromosomes[END_REF] (reviewed in reference Lichtman and Denk, 2011). Pseudotyping of RABV G permits the targeting of specific cellular populations, for example projection neurons or those expressing a certain surface protein (Mebatsion et al., 1997;[START_REF] Choi | Monosynaptic inputs to ErbB4expressing inhibitory neurons in mouse primary somatosensory cortex[END_REF]. This enables the identification/classification of a neuron based on its morphology, projections, and in the case of mono-transsynaptic tracing their connectivity properties. In the latter case, the morphological labeling of the presynaptic neurons aids their identification as well. RABV G technology has been used, for example, to characterize neurons in the primate visual cortex [START_REF] Nassi | Specialized circuits from primary visual cortex to V2 and area MT[END_REF]Nhan and Callaway, 2012) rodent neocortex (Larsen, 2008;[START_REF] Marshel | Targeting single neuronal networks for gene expression and cell labeling in vivo[END_REF]Rancz et al., 2011;Apicella et al., 2012;Kiritani et al., 2012) and rodent midbrain together with their presynaptic partners (Watabe-Uchida et al., 2012).

REVEALING SYNAPTIC CONNECTIVITY

The construction of neural circuit connectomes requires comprehensive knowledge of the underlying synaptic connectivity. We are still lacking quantitative measurements regarding the numbers and sub-cellular organization of the different inputs received by a neuron. EM, paired electrophysiological recording, and optical simulation approaches have done much to advance our knowledge of synaptic connections between neurons. However, these approaches are either time-intensive (EM, paired recordings), currently limited to microcircuits (EM), or can only demonstrate the existence of synaptic connections, but not their precise sub-cellular location (paired recordings, optical stimulation methods).

The elegance and power of the RABV G mono-transsynaptic tracing has now been amply demonstrated (as discussed above). Specifically, this technology provides information about the type, localization, and number of presynaptic neurons forming synaptic connections with particular target cell types. As such, it can be used for the validation of known neuronal synaptic connections (e.g., [START_REF] Stepien | Monosynaptic rabies virus reveals premotor network organization and synaptic specificity of cholinergic partition cells[END_REF], but may also reveal hitherto unknown connections or a re-organization of wiring diagrams under physiological and pathophysiological conditions (Figure 3A). For example, the question of whether memories are stored in the connections formed by neurons [START_REF] Seung | Reading the book of memory: sparse sampling versus dense mapping of connectomes[END_REF] could be tested using this approach. Similarly, the integration of adult-born neurons (Arenkiel and Ehlers, 2009) into functional neuronal circuits can be examined (Figure 3B). A range of targeting approaches is now available (see above) to permit experimental design to be tailored to meet the requirements of a particular study. Lastly, in combination with techniques labeling synaptic contacts (GRASP/BLINC), mono-trans-synaptic tracing may be used to identify the sub-cellular location of synaptic inputs [START_REF] Kim | mGRASP enables mapping mammalian synaptic connectivity with light microscopy[END_REF][START_REF] Wickersham | New technologies for imaging synaptic partners[END_REF].

PROBING SYNAPTIC PROPERTIES AND SYNAPTIC INTEGRATION

One important parameter defining neuronal circuits is the physiological properties of specific anatomical connections (i.e., release probability, strength, short-term dynamics etc.). So far, the most precise method to achieve this is the use of simultaneous electrophysiological recordings between synaptically connected neurons (e.g., reference [START_REF] Frick | Monosynaptic connections between pairs of L5A pyramidal neurons in columns of juvenile rat somatosensory cortex[END_REF]. Specific labeling of pre-and post-synaptic neurons with mono-trans-synaptic tracing greatly enhances the sampling efficiency of synaptic connections for a given postsynaptic neuron (Wickersham et al., 2007b) (Figure 3C). In addition, the integration of synaptic inputs from a number of presynaptic neurons within an individual neuron can be studied. As an alternative approach to simultaneous electrophysiological recordings, presynaptic partners could be stimulated optically following RABV G driven expression of light-activated ion channels or heterologous receptor systems (Osakada et al., 2011) (Figures 2E and3C). We can learn more about plasticity in the adult brain by studying the integration of adult-born neurons into existing neuronal networks. Similar approaches might be used to study circuit integration following stem cell therapy. (C) The analysis of the physiological properties of synaptic connections between defined neuron types (in addition to the anatomical wiring diagram) is greatly aided by this technique. Furthermore, the spatial and temporal integration of signals from a larger number of presynaptic neurons can be examined in postsynaptic neurons. (D) The population activity of a defined neuronal circuit can also be measured during active sensory stimulation or behavior and subsequently correlated with the anatomical input network of a defined starter population.

(E) The receptive field properties of individual neurons can be combined with the analysis of the anatomical wiring diagram of their presynaptic network.

COMBINING PHYSIOLOGY WITH ARCHITECTURE

Information processing in neural circuits depends on the spatiotemporal dynamics of the electrical signals within its neurons.

It is therefore desirable to measure or manipulate the activity of these neurons during a particular behavioral task and to link the cellular activity with the circuit's structure (Figures 2D,E, and3D). Electrophysiology is the most direct method to measure electrical activity, and provides a very high signal-to-noise ratio. Recently, it has become feasible to combine direct electrophysiological measurements of individual neurons in vivo with RABV G mono-trans-synaptic tracing (Rancz et al., 2011) (Figures 2B and3E).

In other instances it is advantageous to use imaging approaches to monitor the cellular activity of identified populations of neurons during a specific task (Figure 3D). The advent of genetically-encoded indicators for calcium ions, membrane potential, and neurotransmitters (for a recent review, see reference Looger and Griesbeck, 2012) makes it possible to express such sensors of cellular activity using RABV G technology (Osakada et al., 2011) (Figure 2D). For instance, the cellular resolution afforded by two-photon microscopy imaging of changes in calcium ions provide a proxy for action potentials in neuronal populations in vivo (recently reviewed in reference [START_REF] Grewe | High-speed in vivo calcium imaging reveals neuronal network activity with near-millisecond precision[END_REF]. Although measurements of changes in membrane potential provide a more direct readout of electrical activity, the lack of suitable genetically encoded voltage sensors has, so far, hampered in vivo measurements of neuronal activity with cellular resolution. A new class of voltage sensors may permit this notion to become a reality [START_REF] Kralj | Optical recording of action potentials in mammalian neurons using a microbial rhodopsin[END_REF][START_REF] Looger | Genetically encoded neural activity indicators[END_REF]. These sensors can be readily incorporated into the RABV G expression system for combined functional neural circuit studies. Further developments in the form of new variants (see Figure 4) are likely to increase the range of applications for which the unique properties of RABV G vectors can be exploited for imaging of neuronal circuits in the intact animal.

Finally, gain-or loss-of-function experiments can also be implemented employing the RABV G technology (Osakada et al., 2011). There is now a large selection of available lightactivated ion channels and heterologous receptors to control neuronal excitability (for reviews see references Arenkiel and Ehlers, 2009;Fortin et al., 2011;[START_REF] Yizhar | Optogenetics in neural systems[END_REF]. For example, specific firing patterns can be triggered or neurons be silenced in targeted neuronal circuits (as defined by the RABV G transfection strategy) within freely moving mice. This optical control of neuronal activity can then be integrated with measurements of the compatible readout, such as behavior, fluorescence measurements, electrical recordings, or fMRI signals (reviewed in reference [START_REF] Yizhar | Optogenetics in neural systems[END_REF], and finally linked to the structural circuit information.

SHORTCOMINGS OF THIS APPROACH AND ALTERNATIVE MONOSYNAPTIC VIRAL TRACERS UNDERSAMPLING OF PRESYNAPTIC NEURONS

Although immensely powerful, RABV G based tracing is, like any technology, affected by particular shortcomings. Certainly the most perplexing issue is the undersampling of labeled presynaptic neurons. This has been most clearly demonstrated by targeting single pyramidal neurons in the primary visual cortex in vivo [START_REF] Marshel | Targeting single neuronal networks for gene expression and cell labeling in vivo[END_REF]Rancz et al., 2011). Mono-trans-synaptic tracing from individual layer 2/3 pyramidal neurons resulted in the labeling of fewer than 100 presynaptic neurons [START_REF] Marshel | Targeting single neuronal networks for gene expression and cell labeling in vivo[END_REF], while up to ∼700 presynaptic neurons were labeled by targeting individual layer 5 pyramidal neurons (Rancz et al., 2011). Furthermore, mono-trans-synaptic tracing from a single starter cell in layer 5 of the S2 somatosensory cortex resulted in the tracing of 249 presynaptic cells [START_REF] Miyamichi | Cortical representations of olfactory input by trans-synaptic tracing[END_REF]. These differences may be partially due to cell type and/or other factors such as plasmid delivery or regulation of transgene expression. Nonetheless, the number of labeled presynaptic neurons was substantially lower than that expected from calculations based on dendritic spine numbers and the average number of synapses formed between connected pairs of neocortical pyramidal neurons (e.g., references [START_REF] Larkman | Dendritic morphology of pyramidal neurones of the visual cortex of the rat: III. Spine distributions[END_REF][START_REF] Defelipe | The pyramidal neuron of the cerebral cortex: morphological and chemical characteristics of the synaptic inputs[END_REF][START_REF] Markram | Physiology and anatomy of synaptic connections between thick tufted pyramidal neurones in the developing rat neocortex[END_REF][START_REF] Feldmeyer | Synaptic connections between layer 4 spiny neurone-layer 2/3 pyramidal cell pairs in juvenile rat barrel cortex: physiology and anatomy of interlaminar signalling within a cortical column[END_REF]. One possible explanation for this undersampling is the level of expression of RG which may fail to meet the stoichiometry required for the efficient assembly and trans-synaptic crossing of RABV particles in vivo [START_REF] Marshel | Targeting single neuronal networks for gene expression and cell labeling in vivo[END_REF]. It remains to be shown whether certain properties of the synaptic contacts (strength, activity, or molecular signature of presynaptic neuron) could influence the efficacy of synaptic crossing. A recently developed single-cell targeting technique using a recombinant polysynaptic RABV (Nguyen et al., 2012) should help shed some light on this issue, as should a more detailed analysis of the molecular characteristics of the synapses that are efficiently crossed.

NEUROINVASIVENESS

At least one study has pointed to a limited time window for experimental infection by RABV G following intramuscular injection. [START_REF] Stepien | Monosynaptic rabies virus reveals premotor network organization and synaptic specificity of cholinergic partition cells[END_REF] reported that motor neurons are refractory to infection by the peripheral route after approximately postnatal day 10. This limitation most likely stems from the fact that the RABV G mono-trans-synaptic tracing approach employs a recombinant virus derived from the Street Alabama Dufferin (SAD) B19 strain of RABV. This strain is highly attenuated for infection by the peripheral route and is only neuroinvasive following peripheral injection in young mice [START_REF] Finke | Replication strategies of rabies virus[END_REF][START_REF] Rasalingam | Comparative pathogenesis of the SAD-L16 strain of rabies virus and a mutant modifying the dynein light chain binding site of the rabies virus phosphoprotein in young mice[END_REF][START_REF] Dietzschold | Concepts in the pathogenesis of rabies[END_REF]. This may perhaps lead to the misconception that RABV G technology can only be applied over a limited postnatal time window. For direct injections into the CNS, however, there is no such impediment. Efficient tracing has been observed in a variety of brain areas in both rats and mice of 1-6 months of age [START_REF] Haubensak | Genetic dissection of an amygdala microcircuit that gates conditioned fear[END_REF]Arenkiel et al., 2011;Rancz et al., 2011;Knobloch et al., 2012;Watabe-Uchida et al., 2012).

TIME WINDOW FOR TRACING

Features that make RABV extremely efficient both as pathogen and as viral tracer are also associated with its reduced cytotoxicity [START_REF] Dietzschold | Concepts in the pathogenesis of rabies[END_REF][START_REF] Lafon | Evasive strategies in rabies virus infection[END_REF][START_REF] Rieder | Interferon in rabies virus infection[END_REF]Ugolini, 2011). Deleting RG is suggested to further decrease its cytotoxicity (Wickersham et al., 2010). Nevertheless, there is a finite time window for RABV G mediated tracing experiments. RABV G infected cortico-thalamic neurons maintain normal electrophysiological parameters up to 12 days following infection (Wickersham et al., 2007b). However, morphological defects or cell death were observed from 14-16 days post-infection (Wickersham et al., 2007b;Arenkiel et al., 2011). Osakada et al. (2011) suggest a 5-11 days (post-infection) time window for viable physiological experiments, which would also take into account any increased toxicity related to the nature of the virally expressed protein. Altogether, these data suggest a sufficiently broad time-window for RABV G based tracing experiments. These findings may be contrasted with the significant perturbations in neuronal physiology that were reported to occur as early as 72 h post-infection with a GCaMP2-expressing PRV strain derived from the highly attenuated PRV-Bartha [START_REF] Granstedt | Fluorescence-based monitoring of in vivo neural activity using a circuit-tracing pseudorabies virus[END_REF].

ALTERNATIVE VIRUS-BASED MONO-TRANS-SYNAPTIC TRACING TECHNIQUES

In spite of the many advantages of RABV G, this technology is currently restricted to mapping input to a neuronal population. An alternative approach, permitting anterograde trans-synaptic tracing from a defined source cell population was recently described [START_REF] Lo | A Cre-dependent, anterograde transsynaptic viral tracer for mapping output pathways of genetically marked neurons[END_REF]. This approach employs a modified, thymidine kinase-deleted herpes simplex virus 1 (HSV-1) derived from strain 129 and capable of propagating in a selective manner in cre-expressing starter cells. This virus was tested in a variety of cre-expressing transgenic mouse lines and resulted in reliable anterograde trans-synaptic mapping of defined neuronal circuits. Notably, this system can be readily combined with the large number of existing cre-/BAC-cre transgenic mouse lines and appears to be specific with respect to trans-synaptic transmission. Unfortunately, this approach is limited to the extent that it is not monosynaptic. In addition, it suffers from the cytotoxicity inherent to HSV-1 viruses and may also have the disadvantage of infecting axon terminals at the site of injection [START_REF] Lo | A Cre-dependent, anterograde transsynaptic viral tracer for mapping output pathways of genetically marked neurons[END_REF]. A cell-targetable, anterograde monosynaptic tracer derived from a glycoprotein-deleted vesicular stomatitis virus has recently been described (Beier et al., 2011). However, it is uncertain whether these findings can be reproduced in vivo (see Beier et al., 2012).

CONCLUDING REMARKS

Recent developments in the field of RABV G technology convincingly establish the importance and power of this tool for neural circuit mapping. Combined with other imaging, physiological, and behavioral approaches, it promises to open new avenues of research permitting a greater understanding of the mammalian connectome and its function. RABV G is applicable to a range of experimental paradigms and permits not only the study of circuit structure, but also the ability to manipulate or monitor its function.

RABV G is invaluable as a viral tracer. In addition, it has considerable potential as a neuronal expression vector. In contrast to classical polysynaptic viral tracers, the current technology permits a greater degree of control over initially infected source cells, by virtue of pseudotyping technology and other genetic targeting approaches. RABV G technology allows a comprehensive reconstruction of the input map of defined neurons as well as physiological monitoring of defined neuronal populations. This tool shows great promise for improving our understanding of neuronal circuit structure/function and its plastic modification during physiological processes or in disease. Future strategies for neuronal circuit analysis will involve a close alliance of genetics, circuit tracing, light-or heterologous receptor-mediated control, high-resolution imaging, and electrophysiology, and RABV G will be an essential part of this tool box.

GLOSSARY

Anterograde tracing: Tracing of projection neurons by transport from the cell bodies to the axon terminals.

Connectome: Here referred to as a complete map of all synaptic connections of the brain or a neural circuit.

Mono-trans-synaptic tracing or monosynaptic transsynaptic tracing:

The process of tracing connections between source cells and first-order synaptically connected partners.

Neurotropic virus: Virus preferentially infecting neurons.

Polysynaptic tracer: This term is often used quite loosely to describe tracers that spread from one neuron to another (transneuronal tracer), but not always exclusively between synaptically connected cells. In contrast to mono-trans-synaptic tracers, tracing can occur between chains of connected neurons.

Pseudotyping: The process of replacing the native surface glycoprotein of a virus with that of another virus to alter viral tropism.

Retrograde tracing: Tracing of projection neurons by transport from the axon terminals to the cell bodies.

Reverse genetics: In the context of virology the term reverse genetics implies the reconstitution of live infectious virus particles using cDNAs, transfected into heterologous cells, as starting material.

Source cells: Initially infected postsynaptic neuron/s that allows retrograde RABV G spread upon RG transcomplementation, thereby revealing directly presynaptic partners.

Trans-neuronal tracer: A tracer that passes from one neuron to another, for which synapse specificity has not been adequately identified.

Trans-synaptic tracer: A tracer that spreads exclusively between neurons that are connected by synaptic contacts.

Tropism: Preferential or specific infection of, and replication within, a certain cell-type.

Introduction

The reconstruction of neuronal circuits is central to many questions in neuroscience. Indeed, knowledge of the finescale morphology of neurons provides not only insight into the identity and function of individual neurons, but also into the function of neural circuits [START_REF] Douglas | Neuronal circuits of the neocortex[END_REF]Lichtman and Denk 2011;Oberlaender et al. 2012a;Parekh and Ascoli 2013;[START_REF] Svoboda | The past, present, and future of single neuron reconstruction[END_REF]. Successful neuronal reconstruction depends on a number of key parameters: (1) Neurons must be labeled in a way that permits visualization of all neuronal structures (dendrites, spines, axons, boutons). ( 2) The full extent of neuronal processes should be efficiently labeled. In particular, this applies to the axons, which extend over large brain volumes [START_REF] Oberlaender | Three-dimensional axon morphologies of individual layer 5 neurons indicate cell type-specific intracortical pathways for whisker motion and touch[END_REF]). ( 3) Labeling would ideally permit visualization by high-resolution light microscopy approaches (such as confocal, two-photon, or super-resolution microscopy). In effect, this means efficient expression of a volume-filling fluorescent marker and a high signal-tonoise ratio for the labeled structure. ( 4) The ideal labeling method would not only be suited to bulk labeling of populations of neurons, but importantly also provide intense labeling of sparse populations of neurons, or even single neurons. This is, because to date the most successful reconstructions of complete neuronal morphology require sparse or single-cell labeling since the axons of bulk labeled neurons become indistinguishable in the densely packed neuropil unless resolved with electron microscopy (da Costa and Martin 2013; Helmstaedter 2013).

Viral vectors fulfill many of these criteria due to their self-amplifying properties (ensuring a high-level expression of volume-filling markers) (Callaway 2008;van den Pol et al. 2009). In particular, genetically modified rabies virus (RABV) is well suited to this approach due to its highly neurotropic nature, rapid, high-level expression of encoded proteins and relatively low cytotoxicity (Wickersham et al. 2007a;Ginger et al. 2013b). The glycoprotein gene-deleted RABV variant, (RABV DG) is an especially useful tool that permits the manipulation of the tropism of the virus through pseudotyping approaches (Mebatsion et al. 1997;Ginger et al. 2013b). This principle has previously been exploited for both retrograde labeling of neurons (Wickersham et al. 2007a;[START_REF] Larsen | Retrograde tracing with recombinant rabies virus reveals correlations between projection targets and dendritic architecture in layer 5 of mouse barrel cortex[END_REF]Nhan and Callaway 2012) and for labeling inputs into a specific cell population (i.e., mono-trans-synaptic tracing) (Wickersham et al. 2007b;Choi et al. 2010).

Here, we employ a RABV DG-based method that allows direct transduction of cell bodies, permitting the tracing and complete 3D reconstruction of dendritic and axonal arbors of sparsely labeled neurons. This method combines the advantages of an anterograde tracer with the brilliant morphological labeling previously described for recombinant RABV DG (Wickersham et al. 2007a). This vector fulfills all of the aforementioned criteria for neuronal labeling. Low cytotoxicity, fast and strong expression and intense labeling of even the most distant processes set it apart from 'classical' viral and non-viral anterograde neuroanatomical tracing approaches. Moreover, the ability to infect a range of cell types over a large age window makes this vector a versatile tool for a large number of experimental situations.

Results

To render RABV DG capable of cell body infection, we pseudotyped it with a chimeric envelope protein containing the N-terminal domain of the vesicular stomatitis virus glycoprotein (VSV-G). VSV-G binds to highly ubiquitous receptors [START_REF] Finkelshtein | LDL receptor and its family members serve as the cellular receptors for vesicular stomatitis virus[END_REF], thus conferring the ability to transduce a wide range of cell types, a property that has previously been exploited for the production of VSV-G pseudotyped viral vectors such as retro-and lentiviruses [START_REF] Burns | Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells[END_REF]. We replaced the membrane anchor and C-terminal cytoplasmic sequence of the authentic VSV-G with that of the RABV-G protein ( RtmC ) to support selective incorporation of the protein into the RABV DG envelope (Fig. 1a). We have generated vectors expressing either a membrane-targeted form of tdTomato labeling the neuronal membrane for subsequent surface reconstruction (Fig. 1c, lower panels; Fig. 3c), or cytoplasmic fluorescent proteins (eGFP and mCherry) (all other figures). The resulting vector [RABV DG (VSV G RtmC )] consistently transduced cells at the site of injection (Figs. 1,2,3,4,5,6).

Important criteria determining the suitability of a viral vector for neuronal tracing are the time course for expression, the intensity of expression, and the morphological detail provided by the labeling. Thus, we compared RABV DG (VSV G RtmC ) with two commonly used anterograde viral tracers, namely lentivirus, and adeno-associated virus (AAV). We quantified fluorescence intensity and the signal-to-noise ratio for eGFP expressing neurons following injection of these vectors into the hippocampal dentate gyrus (DG) region (Fig. 1b). Fluorescence intensity and signal-to-noise ratio measured at the cell bodies of transduced neurons were significantly higher for RABV DG compared to both other vectors (one-way Anova followed by Tukey multiple-comparison; p \ 0.001). Importantly, these superior labeling qualities were achieved (with no sign of cytotoxicity) at much shorter time scales of infection (6 days post infection for RABV DG (VSV G RtmC ) versus 12 and 22 days for lentivirus and AAV, respectively). Moreover, dendrites, spines, axons, boutons, as well as filopodia were readily distinguished within RABV DG (VSV G RtmC ) infected neurons, aiding their visualization and reconstruction (Fig. 1c). Notably, the labeled neurons presented here are DG granule cells, neurons that are difficult to infect using the native RABV (reviewed in Ohara et al. 2009).

We noted that the transduction with RABV DG (VSV-G RtmC ) occurred in a purely anterograde manner (i.e., infection of cell bodies and subsequent labeling of axons and dendrites). To exclude the possibility that RABV DG (VSV-G RtmC ) is transported in a retrograde or trans-synaptic manner, we performed injections into the whisker-Brain Struct Funct related barrel cortex (BC) and visually inspected regions known to connect to this structure. While neurons were infected locally at the site of injection, no labeled cell bodies were found in any of the following regions: thalamic ventral posterior medial (VPm) division and posterior medial (POm) division, other areas of the primary somatosensory cortex (S1), secondary somatosensory cortex (S2), primary motor cortex (M1), and contralateral S1 (not shown). In fact, we saw no infected cell outside the local injection site in S1. This property of RABV DG (VSV-G RtmC ) permits its combined use with a retrogradely transducing RABV DG variant (Wickersham et al. 2007a) to unambiguously trace projections both to-, and from-, a given region. An example for this type of experiment is shown for the labeling of reciprocal projections between the VPm division of mouse thalamus and the primary somatosensory barrel cortex (S1-BC; Fig. 1d). Importantly, we found a complete absence of retrograde infection in all injections using RABV DG (VSV-G RtmC ).

In the aforementioned examples, we demonstrate the utility of this vector for the transduction of populations of neurons. However, as stated previously, reconstruction of the complete structure of a neuron, including its complex and wide-reaching axonal arbors, requires methods for sparse-or single-neuron labeling. This is often achieved by intracellular filling with biocytin (or its analogs), as most viral-based tracers are completely unsuited to this task. RABV, however, is somewhat unique due to its ability to amplify sufficiently from a single infectious particle (reviewed in Callaway 2008) to confer robust, high intensity labeling to all morphological aspects of an infected cell. Although this property is known from the wild-type CVS strain of RABV (Callaway 2008), it has not been demonstrated for glycoprotein-deleted pseudotyped variants of the SAD B19 strain. To examine the ability of our anterograde RABV DG vector to confer the sparse labeling necessary for single neuron reconstruction, we injected 5-10 viral particles into the thalamic POm division of a 16-week-old mouse. Within the investigated thalamic volume of *400 lm 9 400 lm 9 400 lm, only seven cells were labeled (Fig. 2a), including three excitatory projecting neurons, one inhibitory interneuron and three astrocytes (Fig. 2a,b). However, all cells were intensely labeled throughout the extent of their processes. Using high-resolution, large-scale confocal microscopy and automated image processing routines [START_REF] Oberlaender | Transmitted light brightfield mosaic microscopy for three-dimensional tracing of single neuron morphology[END_REF], we reconstructed the somata, dendrites and axons of the four RABV-infected neurons within 12 consecutive 50-lm-thick brain sections (Fig. 2). The high labeling quality in terms of fluorescent intensity, signal-tonoise ratio, and homogeneity across cells (Fig. 2c) including their entire axonal arbor (Fig. 2d) enabled the automated tracing of all morphological fine-structures within the imaged volume. To demonstrate this, we reconstructed the complete 3D morphology of all dendrites, the complete axon of the interneuron, and the initial parts of the axons from the three projecting neurons. Furthermore, we traced and reconstructed one long-range axon. In this case, the neuron projected to the nucleus reticularis (RT) and continued to traverse the white matter tract before entering the vibrissal cortex (S1). This type of axon trajectory has been reported previously for POm neurons [START_REF] Deschenes | The organization of corticothalamic projections: reciprocity versus parity[END_REF]. The axon showed no obvious decrease in labeling quality with distance from the soma, allowing identification of individual boutons within thalamus and cortex (Fig. 2d). These results clearly demonstrate the ability of our anterograde RABV DG tracer to amplify its genome sufficiently, following infection of a cell by a single particle, to enable tracing of long-ranging axons. As a result of this analysis, we found that the initial axonal segments of the traced neurons were less unidirectional than those described using other methods [START_REF] Deschenes | The organization of corticothalamic projections: reciprocity versus parity[END_REF], possibly indicating that POm projects more diversely to regions other than S1.

To better characterize the efficacy/versatility of our vector, we performed injections into a variety of brain areas and over a range of ages. RABV DG (VSV G RtmC ) transduced cells in all mouse brain areas tested, i.e., somatosensory cortex, thalamus, hippocampal dentate gyrus, substantia nigra and cerebellum (Figs. 1c,d,2b,3,4). RABV DG (VSV-G RtmC ) was capable of transducing excitatory and inhibitory neurons, as well as glial cells, as illustrated in Figs. 2, 4 and 6. For example, in the cerebellum, we found that the virus efficiently transduced inhibitory stellate cells, basket cells and Purkinje cells, as well as the excitatory granule cells. Notably, this vector mediated efficient and intense cell labeling in mice of all ages tested (1-15 months), permitting its use for the neuroanatomical study of neurodegenerative disorders (AD) or aging, as demonstrated in Fig. 5. To investigate its tropism in the neocortex in greater detail, we performed injections into layer 5 of S1-BC (Fig. 6), where the composition of cell types has been previously well characterized (i.e., *70 % neurons vs. *30 % glial cells [START_REF] Tsai | Correlations of neuronal and microvascular densities in murine cortex revealed by direct counting and colocalization of nuclei and vessels[END_REF][START_REF] Meyer | Inhibitory interneurons in a cortical column form hot zones of inhibition in layers 2 and 5A[END_REF]). We found that *71 % of the labeled cells in layer 5 were neurons (and thus 29 % were glial cells). Interestingly, only 0.4 % of infected neurons were inhibitory interneurons in contrast to the *80 % excitatory versus *20 % inhibitory neurons previously reported for layer 5 of the barrel cortex [START_REF] Meyer | Inhibitory interneurons in a cortical column form hot zones of inhibition in layers 2 and 5A[END_REF]. This suggests a strong bias for the transduction of excitatory neurons, as previously reported for VSV-G pseudotyped lentiviral vectors [START_REF] Nathanson | Preferential labeling of inhibitory and excitatory cortical neurons by endogenous tropism of adeno-associated virus and lentivirus vectors[END_REF]). To further characterize the types of infected glial cells, we used markers for astrocytes [S100b, [START_REF] Zuo | Fluorescent proteins expressed in mouse transgenic lines mark subsets of glia, neurons, macrophages, and dendritic cells for vital examination[END_REF]] and microglia [Iba1, [START_REF] Schafer | Microglia sculpt postnatal neural circuits in an activity and complement-dependent manner[END_REF]]. We found that *15 % of infected cells fell into the former category (Fig. 6g-i), while virtually no microglia were labeled (data not shown). We would like to point out, however, that glial cells form a non-homogeneous group of differing origins and several markers coexist in the various types (Cahoy et al. 2008). Our finding is therefore only the first step in characterizing the infection of various glial cell types by RABV DG (VSV G RtmC ). Nonetheless, our data provide evidence for the ability of pseudotyped RABV DG variants to infect glial cells and thus extends the range of cell types that may be amenable to manipulation with RABV DG.

Discussion

Here, we demonstrate the utility of RABV DG (VSV-G RtmC ) for neuroanatomical studies involving not only bulk populations of neurons, but also sparse or individual neurons. Our vector is exclusively anterograde, permits rapid high intensity labeling, without cytotoxic side effects and can be used over a sufficiently extended time window to permit physiological experiments. In addition, it can transduce a range of cell types/brain areas in both young and aged animals. These qualities are unequaled by any other type of viral tracer, and in addition render it useful for the study of aging or neurodegenerative disorders (Fig. 5) where age/toxicity might be a factor limiting labeling success.

We show that RABV DG (VSV-G RtmC ) is able to amplify sufficiently from single-particle infection of a host cell to confer the high intensity labeling necessary for automated detection of morphological features (without prior amplification of the signal) (Fig. 2). Most other viral vectors such as lentivirus and AAV do not provide sufficient labeling intensity for reconstruction upon sparse Brain Struct Funct infection. Indeed, only Sindbis virus [START_REF] Ghosh | Sensory maps in the olfactory cortex defined by long-range viral tracing of single neurons[END_REF] has been reported to amplify sufficiently from a single particle to allow the visualization of long-ranging axonal structures from individual neurons in 3-week-old animals. However, extreme cytotoxicity and reduced efficacy in adult animals [START_REF] Chen | Imaging high-resolution structure of GFP-expressing neurons in neocortex in vivo[END_REF] hamper the practical use of Sindbis virus for the purpose of quantitative morphological tracing.

Our vector may be regarded as a viable alternative to classical single cell labeling approaches, such as those based on biocytin delivery via patch pipettes. The latter are limited by low success rates for recovering complete axonal morphologies [e.g., *60 % (Oberlaender et al. 2012b)] and require histological post-processing to stain the biocytinlabeled structures. Moreover, reduced penetrability of axon bundles such as the white matter limits the success of tracing long-range axons using biocytin-labeling (where post-processing with immunological agents is required). RABV DG (VSV-G RtmC ), on the other hand, is not affected by any of these issues (Fig. 2). In addition to the aforementioned qualities, the large size of RABV DG (VSV-G RtmC ) particles limits diffusion, allowing very targeted infection of a spatially restricted brain volume. We propose that co-injection of RABV DG (VSV-G RtmC ) variants expressing different fluorescent markers, together with large-scale reconstruction of single-cell morphology, could aid the classification of neuron types within a specific brain region or nucleus.

Recently, a glycoprotein-deleted form of another closely related rhabdovirus, the vesicular-stomatitis-virus (rVSV), has also been used as a single-cycle (i.e., non-trans-synaptic) anterograde tracer (van den Pol et al. 2009). Despite strong morphological labeling following fluorescent marker expression, the use of this virus is limited due to severe, fastonset cytotoxic effects that cause shut-down of the host cell transcription and nuclear export [START_REF] Faul | Interferon response and viral evasion by members of the family rhabdoviridae[END_REF]. This cytotoxicity strongly restricts the time window for many anatomical/physiological studies to *1 day post infection, even for attenuated variants of rVSV DG (Beier et al. 2011).

The presumed amphotropic qualities of the chimeric VSV-G envelope protein likely enable transduction of a wider range of species and cell types than those presented here. Of note, a similar VSV-G pseudotyped RABV DG vector has recently been described (Gomme et al. 2010). This vector was also shown to be anterograde (Wickersham et al. 2013), although it may have a slightly different tropism due to differences in the transmembrane domain of the glycoprotein-packaging construct. Our findings, together with the latter study, suggest novel applications for RABV DG in addition to its use as a retrograde (Wickersham et al. 2007a) or mono-trans-synaptic tracer (Wickersham et al. 2007b). For example, RABV DG (VSV-G RtmC ) may be used to define a spatially confined starter cell population for mono-trans-synaptic tracing. It may also be employed as a tool to manipulate/monitor neural circuit activity following the expression of, for example, calcium/ voltage indicators or photo-activatable channels (Osakada et al. 2011). In addition, it can be readily combined with retrogradely transducing RABV variants. Unlike other previously reported combinations of anterograde and retrograde agents, the present approach enables the exploitation of two vectors with the same diffusion characteristics, high quality of labeling and short time course for expression as shown in Fig. 1d. Lastly, this tool together with other technologies, e.g., permitting dendritic or synaptic protein profiling (Ginger et al. 2013a;[START_REF] Micheva | Single-synapse analysis of a diverse synapse population: proteomic imaging methods and markers[END_REF] or gross-scale reconstruction approaches (as described here), could greatly aid the classification of cell-type identity. In conclusion, the combination of different RABV variants with optical, physiological and computational approaches, offers a wide range of possibilities for the investigation of the structure-function relationship of neuronal circuits.

Methods

Engineering of the hybrid glycoprotein

The chimeric VSV/SAD G (VSV G RtmC ) cDNA was constructed to encode the ectodomain (aa 1-454) of VSV Indiana G (kindly provided by Dr. John K. Rose) fused to the entire transmembrane and cytoplasmic domain (amino acids 450-524) (Mebatsion et al. 1995) of RABV SAD G. This construct differs from the packaging construct employed by Wickersham et al. (2013), which contained the surface-and trans-membrane domain of the VSV glycoprotein and cytoplasmic domain of the RABV glycoprotein.

Virus production

The production of G-gene deficient RABV SAD DG-eGFP and SAD DG-mCherry was described previously (Wickersham et al. 2007a). SAD DG myr-TdTom was constructed in the same way to encode a protein in which two Zurich, Switzerland). This labeling also revealed anatomical abnormalities like tortuous dendrites, which have previously been described as an effect of aging in humans [right panel 2 [START_REF] Tsamis | Properties of CA3 dendritic excrescences in Alzheimer's disease[END_REF]]. b Similar to the dendrites/spines, the axons of granule cells (mossy fibers) and their boutons in the CA3 area of the hippocampus were strongly labeled (upper and middle panel). Automated surface reconstruction (Imaris) of an isolated mossy fiber bouton showing its fine morphological details (lower panel). Scale bars 5 lm Brain Struct Funct tandem copies of a myristoylation signal are fused to tdTomato for membrane targeting [START_REF] Trichas | Use of the viral 2A peptide for bicistronic expression in transgenic mice[END_REF].

Stocks of VSV G RtmC -pseudotyped rabies viruses [hereafter referred to as RABV DG XFP (VSV G RtmC )] were prepared essentially as described in (Rancz et al. 2011), with the exception that BSR T7/5 cells [START_REF] Buchholz | Generation of bovine respiratory syncytial virus (BRSV) from cDNA: BRSV NS2 is not essential for virus replication in tissue culture, and the human RSV leader region acts as a functional BRSV genome promoter[END_REF]

Stereotaxic injections

All experimental procedures were performed in accordance with French law and the European Directive covering the use of experimental animals (2010/63/EU) and approved by the Ethics Committee of Bordeaux (approval # 5012024-A). Stereotaxic injections were performed as previously described [START_REF] Cetin | Stereotaxic gene delivery in the rodent brain[END_REF]) in C57Bl/6 J mice (aged 1-15 months) or in 15-month-old APP/PS1 mice [a mouse model for Alzheimer's disease [START_REF] Reiserer | Impaired spatial learning in the APPSwe ? PSEN1DeltaE9 bigenic mouse model of Alzheimer's disease[END_REF]]. In brief, viral vectors were injected into the brains of isoflurane anesthetized and head-fixed mice using a 10-ll glass syringe fitted with a 35-gauge needle or a pulled glass pipette. Volume and speed of the injections were controlled using a WPI Ultra Micro Pump. The stereotaxic coordinates were as follows: ( 1 7) substantia nigra: A/P -3.00 mm, L 1.56 mm, D/V 4.1 mm. A/P and L coordinates are given with respect to the bregma, D/V coordinates with respect to the brain surface. Injection volumes were 200-600 nl for RABV DG XFP (VSV G RtmC ), 400 nl for AAV (diluted 1/50) and 600 nl for lentivirus. Sparse labeling was achieved by diluting anterograde rabies virus to titers of *2 9 10 4 infectious particles/ml and injection of 250 nl, which resulted in injection of 5-10 infectious particles.

Mouse perfusion and brain sectioning

Mice were deeply anesthetized with a lethal dose of sodium pentobarbital and then transcardially perfused with 30 ml normal Ringer's solution (135 mM NaCl, 5.4 mM KCl, 1 mM CaCl 2 , 1.8 mM MgCl 2 , 5 mM HEPES, pH 7.4) followed by 100 ml of a 4 % PFA solution (prepared in 19 phosphate buffered saline (PBS, pH 7.4). Fixed brains were dissected and postfixed in 4 % PFA solution for either 24 h, or 6 h in the case where immunohistology was performed. Free-floating slices (50 lm) were cut using a vibratome (Leica).

Immunohistological determination of the cellular tropism of RABVDG(VSV-G RtmC ) Cell types were identified using antibodies against NeuN (dilution: 1:500; Millipore, clone A60, MAB377) to mark neurons, the 67 kDa isoform of glutamate decarboxylase GAD67 (1:1,500 dilution; Millipore, clone 1G10.2, MAB5406) to label GABAergic neurons [START_REF] Meyer | Inhibitory interneurons in a cortical column form hot zones of inhibition in layers 2 and 5A[END_REF], S100b (1:1,500; Sigma S2532) to mark astrocytes [START_REF] Zuo | Fluorescent proteins expressed in mouse transgenic lines mark subsets of glia, neurons, macrophages, and dendritic cells for vital examination[END_REF], or against Iba1 (1:500;Wako Cat. #019-19741) to mark microglia [START_REF] Schafer | Microglia sculpt postnatal neural circuits in an activity and complement-dependent manner[END_REF]. NeuN and S100b were detected using Alexa488-conjugated goat antimouse H ? L (Life technologies), and GAD67 was detected using Alexa647-conjugated goat anti-mouse (subtype IgG2A) (Life technologies).

Immunohistochemical protocols were adapted from [START_REF] Meyer | Inhibitory interneurons in a cortical column form hot zones of inhibition in layers 2 and 5A[END_REF]. In brief, free-floating slices were blocked with MOM blocking reagent (Vector Labs) (1 h) in the presence of 0.5 % triton X-100, then for 30 min in 3 % BSA, 4 % NGS, 0.5 % triton in 19 PBS. Slices were incubated 40 h with the primary antibody in 3 % BSA, 2 % NGS in 19 PBS at 4 °C, washed two times with 0.1 M PB then two times with 0.1 M PB ? 1 % NGS. Slices were then incubated 2 h at RT with the secondary antibody (1:500) in the presence of 0.3 % triton X-100, washed five times with 0.1 M PB, counter-stained with TO-PRO-3 to label nuclei (1:5,000; Life Technologies), and mounted in prolong gold mounting media. The number of RABV DG (VSV G RtmC ) transduced cells expressing cell-type specific markers was quantified by manually counting the immunohistochemically stained cells in confocal images stacks.

High-resolution microscopy, tracing and quantification

For reconstruction of thalamic neurons, images were acquired using a prototype confocal laser scanning system (based on LAS AF SP5, Leica Microsystems), equipped with a glycerol immersion objective (HCX PL APO 63x, 1.2 N.A.), a tandem scanning system (Resonance Scanner), spectral detectors with hybrid technology (GaAsP photocathode), and mosaic scanning software [Matrix Screener (beta-version), provided by Frank Sieckmann, Leica Microsystems]. Mosaic image stacks of volumes up to 2 mm 9 2 mm 9 0.05 mm (in thalamus) and 0.6 9 0.6 9 0.05 mm (in cortex) were acquired at a resolution of 0.094 lm 9 0.094 lm 9 0.5 lm per voxel (2.59 digital zoom, 89 line average, 8 kHz scanning speed, *20 9 20 and *6 9 6 fields of view in thalamus and cortex, respectively) for each of 12 consecutive 50-lm-thick brain sections. 3D reconstructions were performed using previously described automated tracing algorithms [START_REF] Oberlaender | Transmitted light brightfield mosaic microscopy for three-dimensional tracing of single neuron morphology[END_REF]). Automated tracings were proof-edited [START_REF] Dercksen | Interactive visualization: a key prerequisite for reconstruction Brain Struct Funct 123 of anatomically realistic neural networks[END_REF]) and semi-automatically aligned Brain Struct Funct across brain sections [START_REF] Dercksen | Automatic Alignment of Stacks of Filament Data[END_REF]) using Amira Visualization Software (Visage Imaging).

All remaining images were acquired using either a commercial confocal microscope (Leica SP5) or a commercial spinning disk system (Leica SP2). Hippocampal mossy fiber boutons were reconstructed from image stacks using the Imaris surface tool (Bitplane, Zurich, Switzerland). To compare the fluorescence intensities and signalto-noise ratios of RABV DG (VSV G RtmC ), lentivirus-(LV), and adeno-associated virus (AAV) infected brain sections were imaged with identical microscopy settings. Image stacks were acquired at 16 bit-depth at a resolution of 141.47 nm 9 141.47 nm 9 125.89 nm per voxel (639 magnification, 1.79 digital zoom, 1024 9 1024 pixel per image, 39 line average, 700 Hz scanning speed). Cellular somata were detected automatically in these 3D image stacks using Imaris, and the maximum fluorescence intensity of the somata was quantified as arbitrary unit (0-65536 levels of grey) from 16 bit images. Background levels were calculated for each image stack as the average mean intensity value of several larger distributed areas devoid of any cellular processes (i.e., signal). For illustration purposes, the intensity levels of all three images in Fig. 1b were enhanced to the same extent. Cell counts for immunological cell-type characterizations were performed manually with the use of Amira Visualization Software (Visage Imaging) on confocal image stacks.

Fluorescence intensity comparison

Fluorescence intensities were compared using brains of mice injected at 4 months of age with either lentivirus (MND-eGFP-WPRE, a kind gift of Dr. N. Abrous), AAV 2/9 CAG eGFP-WPRE (Penn Vector Core), or RABV DG-eGFP(VSV G RtmC ). Injected animals were killed at 6 days post infection (RABV DG), 12 days post infection (lentivirus), or 22 days post infection (AAV). eGFP-labeled cells were imaged and analyzed as described above.

Statistics

Significance was evaluated using one-way ANOVA followed by a post hoc Tukey test for multiple comparisons using GraphPad Prism 6 software (San Diego, CA). *** p \ 0.001. Data are represented as mean ± SEM.

Introduction 45

Fragile X syndrome (FXS) is the most prevalent inherited mental retardation and most common single gene cause of autism spectrum disorder (ASD) in humans, occurring with a prevalence of 1/4000 in male and 1/8000 in female children 1 .

Symptoms include learning and memory deficits, a high prevalence of autistic behavior, seizures, hypersensitivity to sensory stimuli and alterations/perturbations in 50 the processing of sensory information 2 . The Fmr1 knock-out (KO) mouse model 3 exhibits several neuropathologic features that are similar to those in human FXS patients, which suffer a loss of function of the FMR1 gene 4 . While many studies focus on the misregulation of individual neuronal receptors or compartments our knowledge about the network organization is sparse. Today we only have some indications about 55 small-scale network changes and we do not know how they are involved in the disease, or how they contribute to the cognitive and perceptional changes in FXS. At the cellular level, an altered anatomical connectivity has been proposed to play a role in both FXS and ASD 5 . Both dendritic spine morphology and axon outgrowth 6 are thought to be altered in Fmr1 KO mice. Therefore altered brain circuits are believed 60 to play an important role in FXS. Several reports indicated changes in the neocortical short-range connectivity in Fmr1 KO mice but often those appeared during a narrow time window and disappeared in adolescence 7-10 . This poses the question of whether wiring changes actually play a role in the adulthood in FXS? Here, a profound knowledge of deficits in the neuronal circuit wiring would be necessary to bridge the 65 gap between alterations found on the molecular/micro-circuit level and behavioral/intellectual deficits.

Correct structural wiring is critical for maintaining normal brain function, like the processing of sensory information. Long-range connections between different brain areas are crucial for the faithful sequential transfer of sensory information in a 70 feedforward and feedback manner. Short-ranging connections need to be intact to integrate and process the information step-wise in each area. We hypothesized that both, the connectivity within and in between brain areas might be altered in FXS given the deficits, which have been described previously in axonal outgrowth and spine morphologies. Yet, long-range wiring deficits underlying FXS have been 75

largely unexplored as a potential mechanism underlying cognitive deficits. Therefore we set out to quantify brain-wide and local structural connectivity features in the Fmr1 KO mouse model and correlate them to the functional connectivity.

Here we used diffusion tensor imaging (DTI) to measure structural integrity of the white matter in the Fmr1 KO mice and found a reduced organization or number of 80 axons within the fiber bundles of the corpus callosum below several cortical areas.

Retrograde labeling from one adjacent and therefore presumably affected region, the primary visual cortex, revealed that in fact the number of long-ranging inputs to this area is reduced. Overall we find a shift towards more local and less long ranging input. Using resting state functional MRI (rs-fMRI) we found that in fact the 85 structural changes correspond to a functional de-synchronization/decoupling of several brain areas, which are crucial for the processing of sensory, visual and auditory information.

Results

90

Altered diffusion tensor fractional anisotropy

We hypothesized that an altered network organization/connectivity in the neocortex could lead to the deficits that have been described in the processing of sensory information in FXS and ASD. Therefore we measured the large-scale anatomical connectivity of adult mice of the Fmr1 KO FXS model 3 ultra-high-field 95 MRI (11.7 T). We used diffusion tensor imaging (DTI) to measure structural connectivity parameters. In particular we measured the fractional anisotropy (FA) values, which allow an exploration of the organization and integrity of the structures in parallel oriented tissue. We found that the white matter is significantly affected in several parts of the brain (Fig. 1). In particular, the FA values were decreased in the 100 corpus callosum below the visual cortex, auditory cortex and parts of the somatosensory cortex. The FA values of the corpus callosum were collectively measured in several horizontally planes from -0.2mm -2.7mm and separately from -3.2mm to -3.7mm from the bregma, with an interplane distance of 0.5mm (WT: n=12; Fmr1KO: n=8). The FA values were significantly reduced in Fmr1 KO mice at -1.2mm (p=0.0274) -2.2mm (p=0.0178) and at -3.2mm (in both hemispheres; right: p=0.0423; left p=0.0314); unpaired t-test.

105

Local-and long-ranging structural input connectivity in Fmr1 KO mice 105

Our results show that the organization of the white matter is structurally altered, and that the primary visual cortex (amongst others) may be strongly affected by these changes. To test whether this white matter defect is caused by a reorganization of anatomical connections, we quantified projections into the primary visual cortex. In particular, we investigated alterations in the local connectivity of the 110 visual cortex versus long-ranging projections into this region. To address these questions, we employed a retrograde rabies virus (SAD∆G-eGFP(SAD G)) injected into the primary visual cortex (V1). To control the precise location of the injection site and the diffusion of the virus in the tissue we co-injected a local/anterograde variant of the virus SAD∆G-mCherry(VSV Ctm ) 11 . The average diffusion of the virus in 115 the tissue is ~200µm with a maximal distance of locally infected cells at ~500µm (Table 1) distance calculated from the center of mass of the infected cells. All labeled cells were introduced as fiducal markers into a 3D average brain model (MRI generated; 16µm resolution) of the C57/Bl6 mouse 12 , to compute the Euclidian distance in the whole-brain from the injection site (Fig. 2). The average distance of 120 input to the brain region is at ~1mm distance. A summary of the most important values calculated from the tracer injections can be found in Table 1. We found a reduced average distance of presynaptic cells in the Fmr1 KO mice. Conversely the contribution of presynaptic cells from within the visual cortex is increased in the Fmr1 KO mice. 125

Table 1. Quantification of the labeled cells

The center of mass of the anterogradely labeled cells determines the center of the injection site. The distance of the anterogradely labeled cells gives an indication about the maximum diffusion of rabies virus in the tissue. The distribution of the retrogradely labeled cells is indicated for cells in the Cortex, the Visual Cortex and the Primary Visual Cortex.

Key: Total: all retrogradely labeled cells; Cortex: all retrogradely labeled cells in the cortex; Vi (Visual Cortex): all retrogradely labeled cells in the primary and secondary visual cortex V1 (primary visual cortex): all retrogradely labeled cells in the primary and secondary

Functional connectivity in the neocortex of Fmr1 KO mice

To evaluate the relevance of the anatomical deficits we proceeded by 130 assessing whether the structural alterations in Fmr1 KO mice would correspond/correlate to an altered functional connectivity (Fig. 3). Resting state functional MRI (rs-fMRI) measurements were performed in head-restrained Fmr1 KO (n=7) and wild-type (n=10) mice under light isofluorane aneasthesia. We performed rs-fMRI experiments to measure the functional connectivity of somatosensory, 135 auditory, visual, and motor cortex and the dorsal and ventral hippocampus, as well as the subcortical areas (Fig. 3, Supplementary Fig. S1). Resting state fMRI measurements revealed a reduced functional connectivity between several brain areas in the Fmr1 KO mice (n=7) compared to age-matched wild type mice (n=10). The functional decoupling affected largely connections from hippocampus to neocortical areas and the intracortical connectivity. The auditory cortex is less linked to the dorsal and ventral hippocampus, to the visual cortex and to the retrosplenial cortex. The connectivity between visual cortex and the motor and somatosensory cortices is also deficient. Overall all measured cortical areas appear to be strongly affected. None of the examined connections showed a statistically significant increased functional connectivity. The pre-dominant pattern is a functional decoupling of numerous brain areas. Key: Dorsal hippocampus (DH), ventral hippocampus (VH), auditory cortex (Au), motor cortex (Mo), primary somatosensory cortex (S1), primary visual cortex (V1), retrosplenial cortex (RS), Piriform Cortex (Pi), Amygdala (Amy), Pretectal Area (PtA), Caudate Putamen (CPu), lateral geniculate nucleus (DLG), globus pallidus (LGP), parafascicular nucleus (PF), ventral posterolateral nucleus and ventral posteromedial nucleus (VPN). Right hemisphere (R), left hemisphere (L).

140

Discussion

We found converging connectivity changes in Fmr1 KO mice, structurally on a whole-brain and single-cell-resolution level and a corresponding functional 145 decoupling of several brain areas, in particular in the neocortex. Alterations in the way the brain is wired provide a reasonable explanation for deficits in the sensory information processing and could also be linked to learning deficits either by being cause or being effect.

In particular we found a reduced fractional anisotropy in several parts of the 150 corpus callosum, which could indicate that either the number of axons running through these areas is reduced or that they are running less in parallel, e.g. they could be ascending more into the cortex. Since these measurements allow more than one interpretation we decided to inject viral tracers that allowed us to quantify the number of connections. The retrograde tracer allowed us to determine more specifically how 155 these changes could be caused. When examining the connections arriving to the visual cortex we found a reduced number of neuronal input from long-ranging connections to V1 in the Fmr1 KO mice. Conversely we found an increased number of local connections (<1mm of distance). This shift could lead to an imbalance in the cognitive processing of sensory information, by underrepresentation of external 160 information in the cortex and overrepresentation of ongoing activity in the own brain area. We hypothesized that this overrepresentation of the local information and a reduction of input from other regions would cause the region to be more isolated and functionally decoupled from other brain areas. We tested this idea using resting state functional MRI and found that this decoupling in fact occurs between V1 and several 165 other brain areas. Interestingly a reduced functional connectivity appears to be a widespread phenomenon, which we found between several cortical and subcortical areas. We conclude that connectivity changes are an important feature in FXS, which using a WPI Ultra Micro Pump. The coordinates for the injections were (i) anterior/posterior (A/P) 3.0 mm, lateral (L) 0.94 mm, dorso/ventral (D/V) 0.5 mm for the primary visual cortex (V1). A/P and L coordinates are given with respect to the 205 bregma, D/V coordinates with respect to the brain surface. Viral injections were performed with 200nl viral solution consisting of 1/10 th anterograde rabies virus (VSV tm ) mCherry together with 9/10th retrograde (RG) eGFP rabies virus.

Anterograde RABV was utilized to verify injection coordinates and to normalize the amount of injected viral solution. Subsequent comparisons between animals were 210 done normalizing the number of retrogradely infected cells to the number of locally (anterogradely) labeled cells in the same animal. Ratios of the retrogradely labeled cells in different areas were calculated in relation to the total number of retrogradely labeled cells in the same animal.

215

Mouse brain slice preparation

Mice were perfused and brains sectioned as described previously 11 . Briefly, mice were administered a lethal dose of sodium pentobarbital and then trans-cardially perfused with 30ml normal Ringer's solution followed by 100ml of a 4% PFA solution in 1 X phosphate buffered saline (PBS). The mouse brains were dissected and postfixated in 220 4% PFA solution and slices were cut using a vibratome (Leica). For whole-forebrain sectioning brains where emerged in 10% Gelatine, post-fixed 2h in 4% PFA, sectioned in 50µm slices and mounted using Prolong Gold Antifade Reagent.

Immunohistology was used for signal amplification for the axon quantification using dsRed antibody (polyclonal Rabbit) and Alexa 594 goat anti-rabbit. 225

Fluorescence microscopy and analysis

Images of the entire forebrain where acquired using a scanning mosaic widefield fluorescence acquisition system (Nanozoomer, Hamamatsu) equipped with a 20X 0.75 NA objective. Images were acquired by scanning each section at multiple (~5-6) 230 z-positions with 8µm step size. Analysis of retrogradely and locally labeled cells was performed blind to the genotype by placing manually fiducal markers on the cell position. Images where segmented using the mouse brain atlas ('The Mouse Brain in Stereotaxic Coordinates', Paxinos and Franklin) and fiducal points where transformed into a 3D average brain atlas, using Vaa3D software 13 , to compute the Euclidian 235 distance of each marker in the whole-brain in xyz directions from the injection site.

The precise coordinates of each injection site was confirmed by calculating the center of mass of infected cells by the local tracer SAD∆G-mCherry(VSV-G).

MR imaging 240

MRI was performed on mice from the same line of mice in adult male mice (9-12 weeks old). Breeding of the Fmr1 KO mice 3 Diffusion of water was imaged as described previously 14,15 . In short, 22 axial slices 260 covering the whole brain were acquired with a four-shot SE-EPI protocol. B0 shift compensation, navigator echoes and an automatic correction algorithm to limit the occurrence of ghosts and artefacts were implemented. Encoding b-factors of 0 s/mm 2 (b0 images; 5×) and 1000 s/mm 2 were used and diffusion-sensitizing gradients were

Functional connectivity measurements 270

The rsfMRI datasets were processed as described previously (Zerbi et al., J Neuoscience 2014 online); briefly, the data were first realigned using a least squares method and rigid-body transformation with Statistical Parametric Mapping (SPM) mouse toolbox (SPM5, University College London, 16 ).

The mean SE-EPI images of each mouse were then used to generate and normalize 275 the data into a study-specific template through linear affine and non-linear diffeomorphic transformation (ANTs. V1.9, http://picsl.upenn.edu/ANTS/). On the template, seventeen areas were selected in left and right hemisphere and backtransformed in each subject space using the inverse of the affine and diffeomorphic transformations. Brain regions where segmented based on based on a MRI atlas 12 , and 280 includes: dorsal hippocampus (DH), ventral hippocampus (VH), auditory cortex (Au), primary motor cortex (M1), somatosensory cortex (So), primary visual cortex (V1), retrosplenial cortex (RS), Piriform Cortex (Pi), Amygdala (Amy), Pretectal Area (PtA), Caudate Putamen (CPu), lateral geniculate nucleus (DLG), globus pallidus (LGP), parafascicular nucleus (PF), ventral posterolateral nucleus and ventral 285 posteromedial nucleus (VPL, VPM). In-plane spatial smoothing (0.4×0.4mm) and temporal high-pass filtering (cut-off at 0.01Hz) were applied to compensate for small across-mouse misregistration and temporal low frequency noise using the FEAT tool of FSL (FSL 5.0, 17 ). Functional connectivity (FC) between ROIs was calculated from the BOLD time series after movement regression using total correlation analyses 290 implemented in FSLNets (FSLNets, V0.3, www.fmrib.ox.ac.uk/fsl). Pearson's correlation values were Fisher transformed to Z-scores for group comparisons and statistical analysis.

Diffusion tensor MRI parameters estimation 295

The calculation of the two commonly used DT-MRI parameters, mean diffusivity (MD) and fractional anisotropy (FA), was performed following a protocol as described previously 15 . Briefly, the diffusion images were first realigned with SPM mouse toolbox, to compensate for small movement artefacts; thereafter, the datasets were spatially normalized to a study-specific template through linear affine and non-300 linear diffeomorphic transformation using ANTs. Following these pre-processing

General Discussion and Future Perspectives

During this work we developed a new anterograde viral tracer, based on the glycoprotein-deleted variant of rabies virus, and tested its potential for studying neuronal circuits. We found that the rapid high-level expression of transgenes, and limited cytotoxicity is advantageous to delineate the neuronal morphology and to reconstruct neuromorphological features. Importantly the intense labeling is independent of the age of the animal, which provides an advantage over several other viral tracers that normally depend on cellular promoters [START_REF] Gray | Optimizing promoters for recombinant adeno-associated virus-mediated gene expression in the peripheral and central nervous system using self-complementary vectors[END_REF][START_REF] Hong | Functional analysis of various promoters in lentiviral vectors at different stages of in vitro differentiation of mouse embryonic stem cells[END_REF]. We tested the new anterograde tracer for bulk and sparse labeling. Diluting the virus and injecting only a few viral particles (<10) allowed us to sparsely label neurons in vivo, still achieving intense labeling and therefore facilitating subsequent reconstructions. Stereotactically co-injecting the anterograde variant together with the retrograde (not trans-synaptic) glycoprotein-deleted rabies virus allows us to study a brain region of interest in a combined fashion. We can thus precisely define the center of the injection site (as the center of mass of anterogradely labeled cells), meanwhile having the input of this region labeled together with the axonal projections coming from it, in a different color.

We took advantage of this approach and combined it in a quantitative way, together with non-invasive whole-brain measurements of structural and functional connectivity. We employed these techniques to study connectivity features in the Fmr1 KO mouse model. We found alterations of both local-and long-range connections when comparing the Fmr1 KO mice to WT littermates.

In particular, using DTI we found a reduced FA in the corpus callosum adjacent to several cortical areas. Those deficits in the WM integrity might be caused by a reduction in the number of axons in these areas, less in parallel-organized axons or a reduction in the myelination of the axons. Each of these interpretations could have strong implications for the function of long-ranging connections. Myelination provides an important mechanism for the signal transmission [START_REF] Hildebrand | Myelinated nerve fibres in the CNS[END_REF] and recent evidence suggests that along individual axons the thickness of myelin can vary substantially [START_REF] Tomassy | Distinct profiles of myelin distribution along single axons of pyramidal neurons in the neocortex[END_REF]. It would be possible that the regulation of myelination is disturbed in FXS, e.g. by changes in the oligodendrocytes, the type of glial cells that are responsible for the myelination in the CNS. In fact, evidence suggests that -unlike earlier reports proposed -FMRP is not only expressed in oligodendrocyte progenitor cells and immature oligodendrocytes [START_REF] Pacey | Developmental expression of FMRP in the astrocyte lineage: implications for fragile X syndrome[END_REF][START_REF] Wang | Developmentally-programmed FMRP expression in oligodendrocytes: a potential role of FMRP in regulating translation in oligodendroglia progenitors[END_REF], but also in mature oligodendrocytes [START_REF] Giampetruzzi | FMRP and myelin protein expression in oligodendrocytes[END_REF]. Furthermore, also the mRNA of Fmr1 was found in the corpus callosum of adult mice [START_REF] Hinds | Tissue specific expression of FMR-1 provides evidence for a functional role in fragile X syndrome[END_REF]. Furthermore in vitro studies showed that FMRP can act as a translational repressor of myelin basic protein (MBP), which is a crucial protein for the formation of myelin in the CNS (reviewed in [START_REF] Boggs | Myelin basic protein: a multifunctional protein[END_REF]). This however would suggest that a lack of FMRP in the oligodendrocytes would lead to an increase of MBP. An increased myelination would restrict the water diffusion, and should lead to increased rather than decreased FA values. More recent evidence from in vivo studies showed that the MBP and other myelin proteins are similar in the Fmr1 KO mouse compared to wild type mice. Furthermore this study found that the FMRP concentrations in the oligodendrocytes in vivo are likely too small to act as translational repressor on MBP or other proteins, and that in comparison, the level of FMRP present in cortical neurons nearby the oligodendrocytes is much higher [START_REF] Giampetruzzi | FMRP and myelin protein expression in oligodendrocytes[END_REF].

We therefore consider that the reason for the reduction in FA reported here is most likely due to a mechanism altering neuronal features, rather than acting through the oligodendrocytes. To investigate the two possible neuronal mechanisms that might play a role in the reduced FA, we would need to quantify the number of axons, and also examine how they run in the tissue (parallel or dispersed). A decreased number of axons would, in the worst-case scenario, mean that the number of cells sending projections to the adjacent cortical areas is reduced. We therefore decided to quantify the number and the origin of cells projecting to one of the presumably affected cortical regions, the primary visual cortex (V1). When quantifying their average distance to the injection site we found less long-distance connections and that more of the input is coming from the immediate proximity (<1mm of distance). Overall, we see a hyper-connectivity phenotype for local neocortical circuits, but a hypoconnectivity for long-ranging ones. In the literature, a local change in connectivity has been described in several publications (Bureau et al., 2008;Patel et al., 2014;[START_REF] Testa-Silva | Hyperconnectivity and slow synapses during early development of medial prefrontal cortex in a mouse model for mental retardation and autism[END_REF], but very often this change was only seen transiently (Bureau et al., 2008;[START_REF] Testa-Silva | Hyperconnectivity and slow synapses during early development of medial prefrontal cortex in a mouse model for mental retardation and autism[END_REF]. Whereas in other studies it was suggested that a change is due to a deficit in the pruning of synapses, which occurs between the 2 nd and the 5 th postnatal week in mice (Patel et al., 2014). These somewhat contradictory results did not leave an indication how the connectivity of small-networks would be manifested in adult Fmr1 KO mice. Here we found that both, the short-and longrange wiring is altered and conclude that the connectivity features might play an important role in sensory and cognitive deficits of adults with FXS.

The overall large number of neurons labeled in the short-range distance (>90% in both genotypes) might appear surprising at first. However, a recent study performed in macaque monkeys quantified the input into several visual areas as fraction of labeled neurons (FLN). The authors found that the FLN from subcortical areas makes only 1.3% of the total neurons labeled and that the long-ranging corticocortical connectivity is also low with 3% FLN. Local intrinsic connectivity on the other hand is extremely high with 80% FLN, while neighboring areas comprise 15% cumulative FLN [START_REF] Markov | Weight consistency specifies regularities of macaque cortical networks[END_REF]. The authors concluded that the "high investment in local projections highlights the importance of local processing". This is a reasonable conclusion, considering the dominance of intrinsic connectivity over exterior input. Whereas the additional prominent connection strength from neighboring areas is another proof of the wiring efficacy of the brain, which is often described as the concept of small-world networks [START_REF] Bullmore | Complex brain networks: graph theoretical analysis of structural and functional systems[END_REF][START_REF] Watts | Collective dynamics of "small-world" networks[END_REF]. This implies that functionally related brain areas should be kept in close physical proximity to reduce the wiring costs for the organism.

We used rs-fMRI to further assess, in the Fmr1KO mouse model, whether the structural alterations in the KO mice would lead to an altered functional processing in the brain. We found that the resting-state functional connectivity was significantly reduced between numerous brain areas. Which led us to the conclusion that (i) the alteration in the connectivity is causing, or at least involved in functionally relevant changes in the neuronal network; (ii) that this phenomenon is not specific for V1 but rather widespread in the brain of Fmr1 KO mice. We therefore propose that FXS in characterized by a strong component of connectivity deficits, which should be further explored to understand their role in the cognitive deficits.

Deficits of Connectivity in FXS as a Potential Biomarker

Hypersensitivity to visual, auditory, tactile, and olfactory stimulation is a feature that is common to both human FXS patients and Fmr1 KO mice. We were therefore interested in studying connectivity changes to-and in between-the neocortical sensory areas. We examined which anatomical alterations occur in brain wiring and neuronal connectivity in FXS and what functional role they play in the brain. We found connectivity changes in Fmr1 KO mice, structurally on a large-and fine-scale level, and a corresponding functional decoupling of several brain areas, in particular in the neocortex. Changes in the way the brain is wired provide a reasonable explanation for deficits in the sensory information processing and could also be linked to learning deficits, either by being the cause or being the effect of such change. We propose that the non-invasive measurements of brain connectivity -demonstrated here in the mouse -could be repeated in human patients. Our study thus points to suitable biomarkers that could be applied for testing the efficacy of therapeutic interventions. This could facilitate and improve the outcome of clinical trials, by testing their capability of reversing brain wiring deficits in FXS.

The recent discontinuation of several large-scale drug trials emphasizes the need for a better mechanistic understanding of FXS. Those drugs did not bring the anticipated advantageous effect on patients e.g. Arbaclofen (Seaside Therapeutics/Roche) or Mavoglurant/mGluR5 (AFQ056) (Novartis), even though they act on particularly well-described molecular targets (mGluR5 and GABAR).

Alterations in the anatomical wiring provide a mechanism that could explain (i) the drastic effects found on the processing of sensory information in the neuronal circuits and (ii) the complications in rescue strategies aiming at curing molecular phenotypes.

We believe that our work will not only strengthen our understanding of the pathologic deficits of FXS (and ASDs) but also that it will provide a framework for testing the effectiveness of novel therapeutic agents for their capability to reverse changes in the anatomical and functional wiring in FXS mice and in the future in human patients.

Importantly our study was specifically performed only in adult mice (9)(10)(11)(12) weeks postnatal). Previous pre-clinical studies and tests have often been performed in cell cultures or young animals (e.g. starting chronic treatment in mice at 4-5 weeks [START_REF] Michalon | Chronic pharmacological mGlu5 inhibition corrects fragile X in adult mice[END_REF]). It is thus important to consider additional tests, both in vivo and ex vivo which evaluate intermediate measures like the capability of potential therapeutics to reverse wiring deficits. Performing those trials in adult animals can provide better indications for clinical efficacy in adult FXS patients since neuronal wiring is much more plastic in the young animals or infants, where the brain is still undergoing strong rewiring and pruning effects. We suggest that our approach has the potential to become an analysis pipeline to test the new drug candidates. Such drug candidates can be identified at an increasing speed in large screenings for bioactive small molecules with a desirable effect [START_REF] Dominguez | Integrated phenotypic and activitybased profiling links Ces3 to obesity and diabetes[END_REF]. One potential compound for FXS is the recently identified small molecule (FRAX486) that acts by inhibiting a protein modulating the actin cytoskeleton dynamics [START_REF] Dolan | Rescue of fragile X syndrome phenotypes in Fmr1 KO mice by the small-molecule PAK inhibitor FRAX486[END_REF].

The component has been used in adult mice and provided positive effects on several behavior deficits. It would therefore be interesting to test whether this drug would be efficient to reverse the connectivity features found here. To test this we could examine the same parameters of structural and functional connectivity in the adult mice with and without treatment.

A last point that should be considered is the complexity of FXS and the variability of associated phenotypes. The cause of this variability is not entirely clear but it likely depends on several other genetic or environmental factors. Therefore it seems reasonable that probably not all FXS patients are equally affected by changes in mGluR and GABA activity or connectivity features. Some patients develop strong epilepsy, which could be caused by an excitation/inhibition imbalance and might be corrected by targeting GABA receptors. Depending on the neurologic deficits in each patient might be treated for the combination of the most severe problems he/she experiences. All in all, rather than removing promising drugs from the market because they do not provide the benefit in a sufficiently large number of patients it would be better to develop a personalized medicine for FXS patients. Here large-scale wiring and functional connectivity have the advantage that they can be measured in the patient and predictions could be made how beneficial certain treatments might be.

Alternative Tracers

The most important aspects and limitations concerning mono-trans-synaptic tracing and anterograde viral tracing have been discussed in the Publications N.1 and N.2 respectively.

In spite of their numerous advantages, rabies virus derived vectors -even glycoprotein deleted forms -are still mostly limited by eventual cytotoxic side effects. This cytotoxicity is in part due to certain aspects of rabies virus biology.

However the high level production of virally encoded genes, including transgenes, which is an advantage for tracing, may also contribute to this cytotoxicity.

Reducing the toxicity of RABV to permit long-term infection might therefore be achieved by reducing the expression levels of the rabies virus polymerase, which can for example be achieved by changing the position of the polymerase (L) gene of the rabies virus (Finke et al., 2000) to a different position further downstream.

Another interesting target for manipulating the virus to reduce neurotoxicity could be the matrixprotein (M). This is an essential protein for the assembly of the virus, but at the same time it has a crucial regulatory role for the balance of virus transcription and replication. While a reduced M expression causes an increased transcription and reduced replication (Finke and Conzelmann, 2003;Finke et al., 2003) an increased M expression might reduce the transcription and therefore decrease the translation and make the infection less exhaustive for the cell metabolism.

In some cases, like long-term studies, alternative approaches might be favorable. While there are different options for anterograde viral vectors that exhibit very low toxicity, most notable AAVs, the options are more limited for retrograde vectors. To date most of them are either not single-cycle vectors, and cross further trans-synaptic steps to connected neurons, or are toxic to the cell or both. The most promising candidates as alternative retrograde vectors are (i) a retrograde serotype of the AAV [START_REF] Towne | Efficient transduction of non-human primate motor neurons after intramuscular delivery of recombinant AAV serotype 6[END_REF] (ii) lentivirus pseudotyped with the rabies virus glycoprotein [START_REF] Kato | Efficient gene transfer via retrograde transport in rodent and primate brains using a human immunodeficiency virus type 1-based vector pseudotyped with rabies virus glycoprotein[END_REF] and (iii) a Canine Adeno-Virus (CAV). Despite enormous efforts, the first two still exhibit a low retrograde efficacy and often lead to only a small number of retrogradely labeled cells or are mixed with somatically infected cells and further they were shown to work mainly for specific projections [START_REF] Aschauer | Analysis of transduction efficiency, tropism and axonal transport of AAV serotypes 1, 2, 5, 6, 8 and 9 in the mouse brain[END_REF][START_REF] Carpentier | Enhanced pseudotyping efficiency of HIV-1 lentiviral vectors by a rabies/vesicular stomatitis virus chimeric envelope glycoprotein[END_REF][START_REF] Kato | Improved transduction efficiency of a lentiviral vector for neuron-specific retrograde gene transfer by optimizing the junction of fusion envelope glycoprotein[END_REF][START_REF] Nelson | A circuit for motor cortical modulation of auditory cortical activity[END_REF].

Meanwhile, CAV has been improved over the last years and it is now possible to reach high titers and efficient retrograde tracing. To date there is only a limited number of studies that applied this technique this far to questions about the neural circuitry, even though the potential is promising. It has been shown that CAV is specific for neurons and does not infect glial cells [START_REF] Soudais | Preferential transduction of neurons by canine adenovirus vectors and their efficient retrograde transport in vivo[END_REF] has a large cloning capacity 27-kb and leads to a long-lasting transfection shown up to 1 year, but the expression levels seem to somewhat decrease over time [START_REF] Soudais | Long-term in vivo transduction of neurons throughout the rat CNS using novel helper-dependent CAV-2 vectors[END_REF].

Notably CAV serotype 2 was recently combined with designer drug application technology (DREADD) [START_REF] Boender | Combined use of the canine adenovirus-2 and DREADDtechnology to activate specific neural pathways in vivo[END_REF], which might be of great interest to explore the effects of neuromodulators and alter them in long-term experiments. There is however some concern about pathway specific tropism [START_REF] Senn | Long-range connectivity defines behavioral specificity of amygdala neurons[END_REF] and the retrograde tracing properties (mono-versus polysynaptic) have not been evaluated extensively.

All of the viral vectors mentioned in chapter 3 are under constant development, to reduce toxicity, increase expression levels, increase packaging size, achieve cell type specificity and it is therefore difficult to specify a single best technique, each having its own merits and drawbacks, which need to be taken into consideration. Therefore the viral vector should be chosen carefully for a specific purpose, to find the best type for each experiment, considering all advantages and disadvantages.

Future Perspectives

Based on our findings we suggest the following long-term experimental plan:

(1) In order to verify the local hyperconnectivity, we have begun to quantify the local axonal arbors (i.e. the output) of bulk-infected neurons in the visual cortex. To quantify the axonal arbors of neurons we recently developed a novel anterograde RABV ∆G based method, conferring intense labeling of neuronal structures [START_REF] Haberl | An anterograde rabies virus vector for high-resolution large-scale reconstruction of 3D neuron morphology[END_REF]. We are now using this approach with algorithms delineating the axonal arborization of the infected neurons [START_REF] Broser | Automated axon length quantification for populations of labelled neurons[END_REF]. We are therefore combining anterograde RABV injections in V1 with confocal scanning microscopy in the same area over several layers, and the white matter. Automated tracing and quantification methods are applied to measure changes in the number and localization of axons.

From this data we will establish an axonal density plot ranging from layer 1-6 and the white matter in Fmr1 KO and in WT mice. This approach should provide an additional quantitative measure of the local connectivity of neocortical circuits of Fmr1 KO mice and of the number of outgoing connections.

(2) We are currently measuring the spine density of excitatory neurons in the target area, in order to evaluate how changes in the local-and long-ranging connectivity could influence the density. We therefore started reconstructing sparsely labeled pyramidal neurons in layer 3 of adult Fmr1 KO and WT mice. We are using 3D reconstruction software (Imaris, Bitplane) to quantify the number of spines per dendritic segment. We will then test whether the spine density changes occur in the second generation of the Fmr1 KO mouse model. We will determine whether the spine density correlates with the calculated sum of changes of local pre-synaptic cells and long ranging pre-synaptic cells as well as the local axonal density.

(3) In the next step we will further define the local contribution of excitatory / inhibitory input to the observed changes by quantifying the number of retrogradely labeled GABAergic interneurons in the intra-areal fraction of labeled neurons. We will therefore use immunohistochemistry to stain GABAergic neurons and quantify the number of RABV labeled inhibitory neurons in both conditions. This will allow us to determine whether the local change of presynaptic neurons implies a change in the ratio of excitatory vs. inhibitory input.

(4) Once these experiments regarding the characterization of the connectivity phenotype in Fmr1KO mice are completed, this knowledge will provide a baseline for

Introduction

Recent years have seen a surge of interest in mapping the functional architecture of the brain. Magnetic resonance imaging approaches have greatly contributed to the overall map of distinct brain regions and the functional (non-directional) interaction of these regions (e.g.( 1)). According to the Allen Brain Atlas (http://www.alleninstitute.org/) there are more than 700 brain structures and we are only at the beginning of our quest to understand the precise synaptic organization within and between these neural circuits. Entire maps of the synaptic connectivity of these neural circuits would enable the possibility to decipher their role in defined behavioral tasks. This endeavor is evidenced by large-scale initiatives such as the Human Brain Project, as well as the plethora of articles describing connectivity data at a nano-, meso-and macroscopic level (2)(3)(4). The evolution of sophisticated chemical and optical approaches (e.g. CLARITY, two-photon tomography, array tomography (5-7) -permitting the visualization of functional circuits on a brain-wide scale -are an essential part of this initiative.

However, the characterization of the anatomical wiring diagram on such a scale also requires tools for marking and identifying interconnected neurons. Traditionally this has been performed using two main tracing approaches, based on either chemical/biochemical tracers, or on neurotropic viruses. Whilst each method has a number of strengths and limitations, the crucial advantages of several neurotropic viruses are their highly synapse-specific inter-neuronal transfer and self-amplifying nature, thereby enabling the visualization of fine-detailed morphological features of strictly synaptically connected neurons (e.g. (8)(9)(10)). In addition to the ability to visualize neuronal circuits, the capacity to control and monitor their activity is also essential for understanding their function. This latter aspect is almost exclusively within the domain of neurotropic viruses, due to their amenability to genetic manipulation.

Most studies of neuronal circuit organization have exploited two main classes of virus -the alpha herpes viruses and rhabdoviruses (11,12). The focus of this chapter is the use of recombinant rhadoviruses for the dissection of the structure and function of neuronal circuits. Alternative approaches employing, for example alpha herpes viruses or chemical/biochemical tracers are reviewed elsewhere (e.g. (11,(13)(14)(15)(16)(17)).

Rhabdoviruses for the Analysis of Neuronal circuits

Rabies and vesicular stomatitis viruses

Certain rhabdoviruses, such as rabies virus (RABV) and the vesicular stomatitis virus (Indiana strain; VSV) are ideally suited to studies of neuronal circuit organization due to their ability to infect the nervous system (18). To the best of our knowledge, however, the only rhabdoviruses that have thus far been used for circuit analysis are and the closely related lyssavirus, rabies virus. Conceivably, other lyssaviruses might also be useful for studies in the nervous system due to their infectious properties. Rabies virus (RABV) has a long history of use as a transneuronal tracer and is thus, in many ways, the prototypic neurotropic tracer. Through the pioneering work of Strick, Ugolini and colleagues, much is known about the specific features of this tracer, in particular it's exclusively retrograde, trans-synaptic spread and the high level expression of its gene products (reviewed in (9,11)). The native RABV is a poly-synaptic trans-synaptic tracer, meaning that after it has crossed synapses of the first-order neurons it will continue to do so, thereby infecting second-and then higher-order neurons (Fig. a,b). Although this property has certain advantages for addressing particular experimental questions (for example the flow of information from the CNS to the periphery) it is also a limitation, not in the least because of the convergent nature of many circuits (11,[START_REF]New technologies for imaging synaptic partners 1-7[END_REF]. Indeed, over the course of the trans-synaptic crossing and infection it becomes impossible to distinguish first-order from second-or high-order connected neurons.

Vesicular stomatitis virus (VSV) is a relative new-comer to the field of neuronal circuit analysis and it is uncertain whether wild-type forms are trans-synaptic in nature, or if they spread by diffusion [START_REF] Lundh | Spread of vesicular stomatitis virus along the visual pathways after retinal infection in the mouse[END_REF][START_REF] Van Den Pol | Enhanced cytomegalovirus infection of developing brain independent of the adaptive immune system[END_REF] reviewed in [START_REF] Van Den Pol | Viral strategies for studying the brain, including a replication-restricted self-amplifying delta-G vesicular stomatis virus that rapidly expresses transgenes in brain and can generate a multicolor golgi-like expression[END_REF]. Recent studies, using recombinant forms of VSV have indicated that they are also trans-synaptic. Glycoprotein-deleted forms of both VSV and RABV have been described [START_REF] Van Den Pol | Viral strategies for studying the brain, including a replication-restricted self-amplifying delta-G vesicular stomatis virus that rapidly expresses transgenes in brain and can generate a multicolor golgi-like expression[END_REF][START_REF] Etessami | Spread and pathogenic characteristics of a G-deficient rabies virus recombinant: an in vitro and in vivo study[END_REF][START_REF] Wickersham | Retrograde neuronal tracing with a deletionmutant rabies virus[END_REF][START_REF] Chandran | Endosomal proteolysis of the Ebola virus glycoprotein is necessary for infection[END_REF][START_REF] Beier | Anterograde or retrograde transsynaptic labeling of CNS neurons with vesicular stomatitis virus vectors[END_REF], demonstrating not only the important role of the envelope glycoprotein in trans-synaptic spread, but also the diversity of applications feasible with these glycoproteindeleted vectors. We will therefore dedicate our discussion mostly to the potential of recombinant glycoprotein-deleted forms of both RABV and VSV for studying the physical wiring diagram of defined circuits, and for monitoring and manipulating their activity.

Glycoprotein-deleted rhabdoviruses

To overcome the limitations of 'classical' poly-synaptic viral tracers, a novel strategy was developed that restricted tracing to first order or 'mono-synaptic' synapses (8,11). This strategy, which has come to be known as monosynaptic trans-synaptic tracing (or mono-trans-synaptic tracing, for short) was a consequence of several critical developments/innovations from the field of virus biology:

(1) The ability to rescue infectious rhabdovirus particles entirely from transfected DNA and thus manipulate the rhabdovirus genome [START_REF] Conzelmann | Rescue of synthetic genomic RNA analogs of rabies virus by plasmid-encoded proteins[END_REF][START_REF] Schnell | Infectious rabies viruses from cloned cDNA[END_REF][START_REF] Lawson | Recombinant vesicular stomatitis viruses from DNA[END_REF][START_REF] Whelan | Efficient recovery of infectious vesicular stomatitis virus entirely from cDNA clones[END_REF].

Schematic representation of different tracing possibilities A) A retrograde poly-synaptic tracer moves from the dendrite of the post-synaptic neuron to the axon of the pre-synaptic, whereas an anterograde tracer (B) moves from the axon of a presynaptic neuron to the dendrites of a post-synaptic neuron. In both cases tracing is not limited to a single order synapse -the tracer continues to spread to the next neuron in a chain of synaptically-connected neurons. C) A mono-synaptic retrograde tracer, on the other hand, is only capable of crossing first order synapses from a postsynaptic neuron (orange) to those immediately presynaptic to it (green). D) Photomicrograph of a network of neurons traced in this manner. The single source cell (red/yellow) is connected to the presynaptic neurons in green. Panel C adapted from (9).

Using World Scientific's Review Volume MS Word Template 121 (2) The finding that the infectious properties of glycoprotein-deleted forms of rhabdovirus (Rbd∆G) could be rescued by either transcomplementation with their own glycoprotein or hybrid glycoproteins from other viral species (8,[START_REF] Etessami | Spread and pathogenic characteristics of a G-deficient rabies virus recombinant: an in vitro and in vivo study[END_REF][START_REF] Chandran | Endosomal proteolysis of the Ebola virus glycoprotein is necessary for infection[END_REF][START_REF] Mebatsion | A CXCR4/CD4 pseudotype rhabdovirus that selectively infects HIV-1 envelope protein-expressing cells[END_REF][START_REF] Mebatsion | Specific infection of CD4+ target cells by recombinant rabies virus pseudotypes carrying the HIV-1 envelope spike protein[END_REF][START_REF] Mebatsion | Mokola virus glycoprotein and chimeric proteins can replace rabies virus glycoprotein in the rescue of infectious defective rabies virus particles[END_REF][START_REF] Schnell | Construction of a novel virus that targets HIV-1-infected cells and controls HIV-1 infection[END_REF].

(3) The demonstration that Rbd∆G is incapable of trans neuronal spreading (8,[START_REF] Van Den Pol | Viral strategies for studying the brain, including a replication-restricted self-amplifying delta-G vesicular stomatis virus that rapidly expresses transgenes in brain and can generate a multicolor golgi-like expression[END_REF][START_REF] Etessami | Spread and pathogenic characteristics of a G-deficient rabies virus recombinant: an in vitro and in vivo study[END_REF][START_REF] Wickersham | Retrograde neuronal tracing with a deletionmutant rabies virus[END_REF][START_REF] Beier | Anterograde or retrograde transsynaptic labeling of CNS neurons with vesicular stomatitis virus vectors[END_REF]. ( 4) The development of the EnvA/TVA targeting system [START_REF] Young | Isolation of a chicken gene that confers susceptibility to infection by subgroup A avian leukosis and sarcoma viruses[END_REF][START_REF] Federspiel | A system for tissue-specific gene targeting: transgenic mice susceptible to subgroup A avian leukosis virus-based retroviral vectors[END_REF][START_REF] Bates | A receptor for subgroup A Rous sarcoma virus is related to the low density lipoprotein receptor[END_REF], which has also been used in cancer biology [START_REF] Fisher | Development of a flexible and specific gene delivery system for production of murine tumor models[END_REF] and which permits targeting approaches which circumvent the natural tropism of a virus. Note that an avian virus receptor for subgroup B and its related glycoprotein (EnvB/TVB) has also been used in some studies [START_REF]Monosynaptic inputs to ErbB4-expressing inhibitory neurons in mouse primary somatosensory cortex[END_REF][START_REF] Choi | Selective viral vector transduction of ErbB4 expressing cortical interneurons in vivo with a viral receptor-ligand bridge protein[END_REF][START_REF] Osakada | Design and generation of recombinant rabies virus vectors[END_REF].

As a result of further innovations, namely the introduction of a fluorescent marker into the viral genome [START_REF] Wickersham | Retrograde neuronal tracing with a deletionmutant rabies virus[END_REF]and approaches for transcomplementing the missing glycoprotein, the prototypic monosynaptic trans-synaptic tracing approach was developed (8). Since this approach was first developed for RABV, and has now been very well characterized using this virus, we will devote a larger part of our discussion to this work.

3 Mono-trans-synaptic tracing using RABV ∆G Essentially this approach involves the production of a pseudotyped form of RABV∆G employing a hybrid glycoprotein containing sequences pertaining to the extracellular domain of a bird virus glycoprotein (avian sarcoma and leukosis virus EnvA glycoprotein). The resulting pseudotyped vector, RABV ∆G XFP(EnvA) (where XFP is any fluorescence marker) is incapable of infecting mammalian cells (8). Ectopic expression of the receptor for the EnvA glycoprotein (TVA) together with a trans-complementing rabies virus glycoprotein (RG), however, is sufficient to permit infection of a limited population of neurons. Since these neurons (or source cells) express RG, the production of infectious RABV ∆G particles in these cells permits transneuronal crossing, driving subsequent labeling of both the source cells and those immediately pre-synaptic to the source cell population (Fig. c,d).

Because of the constraints of the aforementioned tracing approach, the only neurons infected are the source cells and their first order presynaptic partners (source cells are distinguished by the expression of an additional fluorescent marker). The major consequence is of this development is the loss of ambiguity with the analysis of transneuronal tracing experiments. It is not only possible to discern the identity of the source cell population, but it also, for the most part, eliminates the problem of convergent connectivity patterns.

To understand the importance of this development, it is important to understand that the unequivocal synaptic pairing of neurons can only otherwise be demonstrated by paired electrophysiological recordings or by electron microscopy (EM).

Light-mediated activation of the presynaptic neuron, for example by the expression of a light-sensitive ion channel such as channel rhodopsin (ChR2) in the presynaptic cell, can be used to infer connectivity. However other methods such as light activation, paired with electrophysiological recordings are needed to confirm this finding. While EM and paired electrophysiological recordings remain the gold standard for proving connectivity, they are unsuited to brain-wide circuit discovery or global circuit mapping approaches. The development of this mono-synaptically restricted tracing approach was this a major boon for field of circuit analysis.

Innovative targeting strategies (see below) permit the experimenter to determine, with more or less specificity, the identity of the target cell population. This permits not only the extraction of detailed information about the cell-type composition of certain circuits, but in certain cases, the elucidation of entirely novel connectivity patterns (e.g. [START_REF] Vivar | Monosynaptic inputs to new neurons in the dentate gyrus[END_REF][START_REF] Deshpande | Retrograde monosynaptic tracing reveals the temporal evolution of inputs onto new neurons in the adult dentate gyrus and olfactory bulb[END_REF][START_REF] Takatoh | New modules are added to vibrissal premotor circuitry with the emergence of exploratory whisking[END_REF]). A second major advantage is the ability to both map and manipulate the circuit under investigation. Thirdly, although this point remains the issue of some debate, this strategy also removes the aspect of ambiguity, which might theoretically arise, if certain circuits would be traced with more efficiency than others (see below). Lastly, the deletion of the glycoprotein has the effect of enhancing the survival of infected neurons, Using World Scientific's Review Volume MS Word Template 123 reducing cytotoxicity [START_REF] Wickersham | Production of glycoprotein-deleted rabies viruses for monosynaptic tracing and high-level gene expression in neurons[END_REF][START_REF] Ohara | Rabies virus vector transgene expression level and cytotoxicity improvement induced by deletion of glycoprotein gene[END_REF], and perhaps improving expression of the transgene [START_REF] Ohara | Rabies virus vector transgene expression level and cytotoxicity improvement induced by deletion of glycoprotein gene[END_REF].

Different strategies for mono-trans-synaptic tracing

As mentioned previously, the great advantage of the mono-trans-synaptic tracing approach is the ability to determine the identity of the source or starter cell population. In effective terms, this means devising a means of delivering the machinery required for mono-trans-synaptic tracing (i.e. TVA, RG and additional fluorescent marker) and limiting the expression of these factors to a more-or-less defined population of neurons. Various approaches have been developed to achieve this and may be roughly grouped into three main strategies -the use of accessory viruses, DNA transfection approaches or the use of transgenic animals. For a more detailed discussion of this issue, the reader is referred to a recent review (9,[START_REF] Luo | Genetic dissection of neural circuits[END_REF][START_REF] Arenkiel | Molecular genetics and imaging technologies for circuit-based neuroanatomy[END_REF][START_REF] Packer | Targeting neurons and photons for optogenetics[END_REF].

The use of accessory viruses to determine the source cell population.

The wide range of expression vectors used in modern neuroscience have, in most cases, provided ample possibilities for addressing the problem of targeting the sources cell population. These vectors can be adapted, for example by the use of a cell-type specific promoter, to target initial infection and thus mono-trans-synaptic tracing to a particular neuron type [START_REF] Liu | Tracing inputs to inhibitory or excitatory neurons of mouse and cat visual cortex with a targeted rabies virus[END_REF]. More frequently, the use of a Cre-restricted expression vector is used, in conjunction with a transgenic or knock-in mouse expressing Cre in a particular neuron type (e.g. [START_REF] Kohara | Cell typespecific genetic and optogenetic tools reveal hippocampal CA2 circuits[END_REF][START_REF] Watabe-Uchida | Whole-brain mapping of direct inputs to midbrain dopamine neurons[END_REF][START_REF] Wall | Differential innervation of direct-and indirectpathway striatal projection neurons[END_REF][START_REF] Wall | Monosynaptic circuit tracing in vivo through Cre-dependent targeting and complementation of modified rabies virus[END_REF][START_REF] Miyamichi | Dissecting local circuits: parvalbumin interneurons underlie broad feedback control of olfactory bulb output[END_REF][START_REF] Betley | Parallel, redundant circuit organization for homeostatic control of feeding behavior[END_REF]. In other cases, a more complex strategy involving a conditional bi-transgenic mouse line and an adeno associated virus (AAV) vector expressing TVA/RG under the control of a tetracycline-dependent promoter has been employed [START_REF] Miyamichi | Cortical representations of olfactory input by trans-synaptic tracing[END_REF][START_REF] Fu | A cortical circuit for gain control by behavioral state[END_REF]. It has also been possible to take advantage of the inherent properties of certain certain vectors. For example retrovirus-mediated expression limits, with certain caveats (see [START_REF] Deshpande | Retrograde monosynaptic tracing reveals the temporal evolution of inputs onto new neurons in the adult dentate gyrus and olfactory bulb[END_REF]), the expression of TVA, RG and fluorescent marker encoding transgenes to proliferating neurons, a characteristic of early progenitors or neuroblasts. This approach permits the tracing of de novo circuits formed as adult-born neurons integrate into an existing network (as described in [START_REF] Vivar | Monosynaptic inputs to new neurons in the dentate gyrus[END_REF][START_REF] Deshpande | Retrograde monosynaptic tracing reveals the temporal evolution of inputs onto new neurons in the adult dentate gyrus and olfactory bulb[END_REF][START_REF] Li | Molecular layer perforant path-associated cells contribute to feed-forward inhibition in the adult dentate gyrus[END_REF]). AAV serotype 6 (AAV6) and herpes simplex virus (HSV), on the other hand, have the quality of being retrogradely transported. Approaches in which RGexpressing AAV6 and HSV vectors are coinjected with an alternatively pseudotyped form of RABV ∆G, which also has exclusively retrograde properties (see below) have permitted mono-trans-synaptic tracing from source cell population defined by their 'projection/connectivity signature' rather than by their molecular identity (60-62) [START_REF] Yonehara | Spatially asymmetric reorganization of inhibition establishes a motion-sensitive circuit[END_REF][START_REF] Yonehara | Motion detection: neuronal circuit meets theory[END_REF]. In this case the EnvA/TVA system is unnecessary because spatial targetting is used to limit infection to a specific population of projection neurons. Lastly, accessory vectors have been repackaged using novel pseudotyping strategies to limit their tropism to particular cellular populations (e.g. as described in [START_REF]Monosynaptic inputs to ErbB4-expressing inhibitory neurons in mouse primary somatosensory cortex[END_REF][START_REF] Choi | Selective viral vector transduction of ErbB4 expressing cortical interneurons in vivo with a viral receptor-ligand bridge protein[END_REF]).

Transfection approaches to delineate the source cell population.

In contrast to the aforementioned approaches permitting the 'bulk' targeting of populations of neurons, single cell transfection techniques can be used to identify neurons providing inputs into an individual neuron. To achieve this, expression plasmids encoding the tracing components are introduced using either two-photon-guided electroporation or whole cell patch-clamp mediated transfection [START_REF]Targeting Single Neuronal Networks for Gene Expression and Cell Labeling In[END_REF][START_REF]Transfection via whole-cell recording in vivo: bridging single-cell physiology[END_REF]. Although painstaking, and difficult to achieve, such approaches enable the unambiguous identification of neurons presynaptic to a defined individual neuron. In combination with instance whole-cell recordings they provide unparalleled information about both the physiology and the connectivity matrix of a particular neuron.

In addition to the targeting of single neurons, in vivo transfection approaches as also highly suited to the targeting of specific populations of neurons, through, for example, the use of in utero-or early postnatal electroporation techniques. This approach has been used to target, as source cells, the neuronal progenitors of either olfactory bulb granule cells [START_REF] Arenkiel | Activityinduced remodeling of olfactory bulb microcircuits revealed by monosynaptic tracing[END_REF], or layer 2/3 pyramidal neurons in the neocortex [START_REF] Mori | Rabies virus glycoprotein variants display different patterns in rabies monosynaptic tracing[END_REF]. Further specificity was achieved by spatial and temporal separation of the initial electroporation event and subsequent RABV ∆G(EnvA) infection.

The use of transgenic mouse for targeting of a source cell population.

A more generic option for source cell targeting is the use of a transgenic mouse line expressing TVA and RG, or TVA alone in a conditional manner. Both proteins can be placed under the control of a tetracyclineregulatable promoter (69) limiting their expression to a selccted cell populations by the choice of an appropriate tetracycline trans-activator driver line (tTA). This has enabled, for example, mono-trans-synaptic tracing from a defined population of neurons of the medial entorhinal cortex [START_REF] Rowland | Transgenically targeted rabies virus demonstrates a major monosynaptic projection from hippocampal area CA2 to medial entorhinal layer II neurons[END_REF]. Similarly, a floxed-STOP mouse line delivering either TVA, RG and fluorescent marker or TVA and RG, both in a Cre-dependent manner has been used to permit targeted tracing in combination with specific Cre driver lines [START_REF] Takatoh | New modules are added to vibrissal premotor circuitry with the emergence of exploratory whisking[END_REF][START_REF] Li | Molecular layer perforant path-associated cells contribute to feed-forward inhibition in the adult dentate gyrus[END_REF]. The advantage of these approaches is that there is no need for two surgical procedures -RABV ∆G (EnvA) can be injected in a single operation. However, this approach is best suited to the analysis of long-range connections rather than local connections. Lastly, mice expressing TVA only in a Cre dependent manner [START_REF] Sun | Cell-Type-Specific Circuit Connectivity of Hippocampal CA1 Revealed through Cre-Dependent Rabies Tracing[END_REF] or through the use of a highly restricted promoter have also been described [START_REF] Deshpande | Retrograde monosynaptic tracing reveals the temporal evolution of inputs onto new neurons in the adult dentate gyrus and olfactory bulb[END_REF]. In these two cases RG was delivered separately through the use of accessory virus. This measure was designed to limit false positives through lower levels of ectopic TVA expression.

Recent advances in mono-trans-synaptic tracing technology

Although the advances in mono-trans-synaptic tracing technology have recently been summarized in review form (9), we feel that the flurry of new work implicating this technology and arising over the last year merits an updated assessment of the current 'state of the art'.

Mono-trans-synaptic tracing may be used to address a multitude of questions, as well as providing a means of confirming findings from other mapping approaches or for the discovery of novel connectivity patterns. Here we highlight the most interesting aspects of recent studies.

3.2.1.Dissecting the relationship between structure and function

The distribution of inputs can be used to extract information about the underlying functionality of a circuit. For example, there are two major outputs in the striatum and mono-trans-synaptic tracing from these two cell populations identified an asymmetry of inputs from cortical and subcortical brain areas respectively [START_REF] Wall | Differential innervation of direct-and indirectpathway striatal projection neurons[END_REF]. There are also examples in which RABV∆G and mono-trans-synaptic tracing have been used to describe neuronal subpopulations within a well-defined brain nucleus based on their projection diagram (see [START_REF] Betley | Parallel, redundant circuit organization for homeostatic control of feeding behavior[END_REF][START_REF] Cruz-Martín | A dedicated circuit links directionselective retinal ganglion cells to the primary visual cortex[END_REF]). In the latter, the authors described a novel di-synaptic circuit transmitting sensory information from the retina to the visual cortex via an intermediate structure in the thalamus. Interestingly, this circuit was spatially segregated -in both the neocortex and thalamus -from a parallel circuit bringing different visual information from the retina to the visual cortex.

In addition, it is also possible to incorporate genetically encoded sensors into the RABV ∆G genome to monitor circuit modulation (for example see [START_REF] Yonehara | Motion detection: neuronal circuit meets theory[END_REF]). Elaborate toolboxes -encompassing mono-trans-synaptic tracing, retrograde tracing, classical tracers, optogenetics, pharmacology and behaviour studies -can now be put to use, as exemplified by Lammel et al (73). Importantly, these tools (e.g. RABV ∆G-mediated expression of ChR2) can even be used to mediate a behavioural response at the neuronal population level [START_REF] Lammel | Input-specific control of reward and aversion in the ventral tegmental area[END_REF].

The use of mono-trans-synaptic tracing as an aid to circuit discovery.

Betley et al [START_REF] Betley | Parallel, redundant circuit organization for homeostatic control of feeding behavior[END_REF] demonstrate the utility of the mono-trans-synaptic approach for aiding circuit discovery. Another remarkable possibility with RABV ∆G is to identify the inputs of a specific neuron type. Indeed, the brain is made of a large number of structures, each composed of a vast amount of neuron types. For example, the cortex contain more than ten interneuron subtypes [START_REF] Markram | Interneurons of the neocortical inhibitory system[END_REF] and nine excitatory neuron subtypes [START_REF] Oberlaender | Cell type-specific three-dimensional structure of thalamocortical circuits in a column of rat vibrissal cortex[END_REF]. Using transgenic mice, with or without additional vectors, it is possible to target a specific cell type for primary infection, such as parvalbumin positive interneurons or mitral cells in the olfactory bulb [START_REF] Miyamichi | Dissecting local circuits: parvalbumin interneurons underlie broad feedback control of olfactory bulb output[END_REF] or vasoactive-intestinal peptide positive interneurons of the primary visual cortex [START_REF] Fu | A cortical circuit for gain control by behavioral state[END_REF]. Such approaches have also been used to target monotrans-synaptic tracing to a specific layers of the entorhinal cortex, an important structure for spatial learning paradigms [START_REF] Rowland | Transgenically targeted rabies virus demonstrates a major monosynaptic projection from hippocampal area CA2 to medial entorhinal layer II neurons[END_REF].

The mono-trans-synaptic tracing technology has also been applied successfully to the identification of spinal cord circuits. For example, the segregation and topography of premotor circuits associated with specific muscle targets has been shown [START_REF]Monosynaptic Rabies Virus Reveals Premotor Network Organization and Synaptic Specificity of Cholinergic Partition[END_REF][START_REF] Tripodi | Motor antagonism exposed by spatial segregation and timing of neurogenesis[END_REF][START_REF] Pivetta | Motor-circuit communication matrix from spinal cord to brainstem neurons revealed by developmental origin[END_REF].

Analysis of circuit remodelling

One approach, for which this technique is ideally suited, is the analysis of circuit remodelling, following, for example activity or normal developmental programmes. Arenkiel et al [START_REF] Arenkiel | Activityinduced remodeling of olfactory bulb microcircuits revealed by monosynaptic tracing[END_REF] showed that odour stimulation resulted in enhanced tracing, resulting from an increased input onto olfactory bulb newborn neurons of odour-stimulated mice, as well as remodeling of their dendritic morphology. Novel connections from long range-inhibitory neurons were observed with adult-born neurons of the olfactory bulb [START_REF] Deshpande | Retrograde monosynaptic tracing reveals the temporal evolution of inputs onto new neurons in the adult dentate gyrus and olfactory bulb[END_REF]. Interestingly, this study also suggested that migrating neuroblasts form transient connections with neurons of the rostral migrating stream and subependymal zone as they migrate to the OB. The integration of adult neurons into hippocampal circuits have also been described [START_REF] Vivar | Monosynaptic inputs to new neurons in the dentate gyrus[END_REF][START_REF] Deshpande | Retrograde monosynaptic tracing reveals the temporal evolution of inputs onto new neurons in the adult dentate gyrus and olfactory bulb[END_REF][START_REF] Li | Molecular layer perforant path-associated cells contribute to feed-forward inhibition in the adult dentate gyrus[END_REF] albeit over different time-scales. Athough common patterns immerge, a number of novel connectivity diagrams were reported such as a transient input from CA3 [START_REF] Vivar | Monosynaptic inputs to new neurons in the dentate gyrus[END_REF], novel inputs from a region adjoining the ventral tegmental area [START_REF] Li | Molecular layer perforant path-associated cells contribute to feed-forward inhibition in the adult dentate gyrus[END_REF] and early connection with neurons in the subgranular zone of the dentate gyrus [START_REF] Deshpande | Retrograde monosynaptic tracing reveals the temporal evolution of inputs onto new neurons in the adult dentate gyrus and olfactory bulb[END_REF]. Takatoh et al (44) used a modified mono-trans-synaptic tracing approach to map the premotor circuits involved in the control of coordinated whisking by premotor neurons. They analyzed the premotor neuron distribution at two distinct developmental stages, before and after the onset of whisking (i.e. postnatal day [11][12][13][14], and they were able to identify a developmental addition of projecting neurons in the premotor circuitry, which could explain the emergence of whisking. These brainstem premotor neurons also received input from the motor cortex, an area that has long been implicated in the control of whisking behaviour. Thus this study not only extended our knowledge of the circuits involved in the control of sensory information gathering, but also demonstrated age-dependent circuit remodeling using the mono-transsynaptic tracing technique.

Confirming the wiring diagram

In addition to the aforementioned uses of this technology, RABV ∆Gmediated mono-trans-synaptic tracing may be used to confirm connectivity information obtained from other approaches. For example, the identity of the presynaptic layer 5 cell type providing input into layer 2/3 pyramidal neurons of the somatosensory neocortex [START_REF] Mori | Rabies virus glycoprotein variants display different patterns in rabies monosynaptic tracing[END_REF] has been previously described using electrophysiological and optogenetic methods (reviewed in [START_REF] Feldmeyer | Excitatory neuronal connectivity in the barrel cortex[END_REF]). In addition, mono-trans-synaptic tracing has been used to confirm novel connectivity patterns discovered using other methods (see [START_REF] Kohara | Cell typespecific genetic and optogenetic tools reveal hippocampal CA2 circuits[END_REF][START_REF] Yonehara | Spatially asymmetric reorganization of inhibition establishes a motion-sensitive circuit[END_REF][START_REF] Knobloch | Evoked axonal oxytocin release in the central amygdala attenuates fear response[END_REF][START_REF]Genetic dissection of an amygdala microcircuit that gates conditioned[END_REF]. Farrow et al. (79) used mono-transsynaptic tracing to confirm the identity of an inhibitory neuron providing presynaptic input to a defined class of ganglion cells. The identity of the presynaptic neuron was inferred by prior pharmacological experiments and by knowledge of the anatomical landscape of the retina. Undoubtedly, the intense morphological labeling conferred by the high level expression of RABV ∆G encoded fluorescence marker was an important aspect permitting the reconstruction of long-range axons from these neurons which extend over 1mm in the retina.

Other Tracing Strategies Using RABV ∆G

Using World Scientific's Review Volume MS Word Template 129

RABV ∆G as a single cycle retrograde tracer

In addition to its use for mono-trans-synaptic tracing, RABV ∆G, pseudotyped with its native glycoprotein, or that of a closely related RABV strain (RABV ∆G RG), acts as a classical retrograde tracer [START_REF] Etessami | Spread and pathogenic characteristics of a G-deficient rabies virus recombinant: an in vitro and in vivo study[END_REF][START_REF] Wickersham | Retrograde neuronal tracing with a deletionmutant rabies virus[END_REF][START_REF] Ohara | Rabies virus vector transgene expression level and cytotoxicity improvement induced by deletion of glycoprotein gene[END_REF]. RABV ∆G (RG) infects axon terminals [START_REF] Wickersham | Retrograde neuronal tracing with a deletionmutant rabies virus[END_REF] and is retrogradely transported to the cell body and dendrites of target neurons in the same manner as wild-type replication competent forms. However in the absence of trans-complementation, it remains confined to the initially infected neuron -thus acting as a 'single cycle' tracer [START_REF]Chapter 9 -Rabies Virus as a Research Tool and Viral Vaccine Vector[END_REF]. This enables the identification and also the concurrent modulation, monitoring and/or manipulation of neurons based on their projection characteristics. This approach has been used to label specific projection neurons of the somatosensory cortex of rodents and visual cortex of monkeys and cats [START_REF] Nhan | Morphology of superior colliculus-and middle temporal area-projecting neurons in primate primary visual cortex[END_REF][START_REF] Larsen | Retrograde tracing with recombinant rabies virus reveals correlations between projection targets and dendritic architecture in layer 5 of mouse barrel cortex[END_REF][START_REF] Connolly | Parallel feedback pathways in visual cortex of cats revealed through a modified rabies virus[END_REF]. Concurrent injection of RABV ∆G variants expressing different fluorophores has been used to map the inputs from lateral entorhinal cortex to olfactory bulb and anterior piriform cortex in order to understand how connectivity shapes odour discrimination [START_REF] Chapuis | Lateral entorhinal modulation of piriform cortical activity and fine odor discrimination[END_REF]. Retrogradely-targeted RABV∆G has also been used to express ChR2 to study the post-synaptic elements of circuits involving defined projection neurons [START_REF] Apicella | Laminarly orthogonal excitation of fastspiking and low-threshold-spiking interneurons in mouse motor cortex[END_REF][START_REF] Kiritani | Hierarchical connectivity and connection-specific dynamics in the corticospinal-corticostriatal microcircuit in mouse motor cortex[END_REF][START_REF] Kress | Convergent cortical innervation of striatal projection neurons[END_REF]. RABV ∆G (RG) expressing Cre can be used to created conditional loss-or gain-of-function in defined projection neurons. Using such an approach, for example, Dölen et al [START_REF] Dölen | Social reward requires coordinated activity of nucleus accumbens oxytocin and serotonin[END_REF] ablated oxytocin receptor function in a particular population of neurons projecting to the nucleus accumbens. Retrograde tracing approaches have also been used to confirm results obtained using mono-trans-synaptic tracing approaches (e.g. [START_REF] Takatoh | New modules are added to vibrissal premotor circuitry with the emergence of exploratory whisking[END_REF][START_REF] Wall | Differential innervation of direct-and indirectpathway striatal projection neurons[END_REF][START_REF] Miyamichi | Dissecting local circuits: parvalbumin interneurons underlie broad feedback control of olfactory bulb output[END_REF]. However care is needed interpreting such findings due to the lack of precise knowledge regarding the identity of the neurons receiving these inputs. Finally, a novel kind of retrograde tracing approach was recently described in which target neurons expressing TVA were infected with RABV ∆ G (EnvA) via their axon projections [START_REF] Huang | Convergence of pontine and proprioceptive streams onto multimodal cerebellar granule cells[END_REF]. While lacking the specificity of infection mediated via the axon terminals, it permits the un-ambiguous discrimination of TVAexpressing neurons based on their projection characteristics.

RABV ∆G as a single cycle antergrade tracer

The ability to pseudotype RABV ∆ G using an alternative envelope protein permits the development of new vectors with altered tropism (e.g. [START_REF] Mebatsion | A CXCR4/CD4 pseudotype rhabdovirus that selectively infects HIV-1 envelope protein-expressing cells[END_REF][START_REF] Mebatsion | Specific infection of CD4+ target cells by recombinant rabies virus pseudotypes carrying the HIV-1 envelope spike protein[END_REF]). To extend this observation, we pseudotyped RABV ∆G with a chimeric glycoprotein containing the ectodomain of the VSV glycoprotein (VSV-G) and transmembrane-and cytoplasmic domain of RG (10). In doing so, we wished to target infection to neuronal cell bodies rather than their axons, permitting infection of neurons located at the site of infection. Indeed, we found that RBAV ∆G pseudotyped in this manner can act as an anterograde tracer rather than a retrograde tracer. Comparable findings were reported using a similar vector (pseudotyped with a chimeric glycoprotein containing the ecto-and transmembrane domains of VSVG and cytoplasmic domain of RG [START_REF] Gomme | Characterization of a single-cycle rabies virus-based vaccine vector[END_REF][START_REF] Wickersham | Axonal and subcellular labelling using modified rabies viral vectors[END_REF]. These vectors have a range of advantages, namely rapid, Golgi-like labeling of neuronal structures at the site of injection and compatibility with retrograde RABV ∆G forms, permitting reciprocal tracing of projection both to-and from a region (10,[START_REF] Wickersham | Axonal and subcellular labelling using modified rabies viral vectors[END_REF]. Importantly, these vectors are suitable for use in aged animals, making them amenable to use in aging or neurodegeneration studies (10). This feature is not possible with most neurotropic vectors. Lastly, we were able to demonstrate that RABV ∆G can amplify from a single particle, conferring sufficient labeling to permit automated single neuron reconstruction approaches (10). The axon labeling achieved using this vector was superior to that achieved using intracellular introduction of biocytin -a method that has traditionally been use as a gold standard for single neuron reconstruction (16). This vector has a strong tropism for excitatory neurons but also permits the transduction of astrocytes (10) extending the range of cell types that can be transduced using RABV ∆G.

RABV as a Poly-synaptic Tracer

This subject has been the topic of numerous reviews ((13-15, 92)). We will therefore briefly highlight a number of interesting and innovative studies using this approach. Ohara et al [START_REF] Ohara | Dual transneuronal tracing in the rat entorhinalhippocampal circuit by intracerebral injection of recombinant rabies virus vectors[END_REF] developed a recombinant form of the attenuated RABV strain, the HEP-flury strain, modified to express the envelope glycoprotein derived from the CVS strain, as well as a number of fluorescent markers. These recombinant virus variants can be injected into different brain structures to examine convergent circuits in two colors [START_REF] Ohara | Dual transneuronal tracing in the rat entorhinalhippocampal circuit by intracerebral injection of recombinant rabies virus vectors[END_REF][START_REF] Ohara | Untangling neural networks with dual retrograde transsynaptic viral infection[END_REF][START_REF] Ohara | Organization of multisynaptic inputs to the dorsal and ventral dentate gyrus: retrograde trans-synaptic tracing with rabies virus vector in the rat[END_REF]. To overcome the difficulties in resolving first-order from higher-order connections in a polysynaptic tracing experiment, wild-type RABV was co-injected with cholera-toxin, a classical retrograde tracer [START_REF] Suzuki | Organization of cerebral projections to identified cerebellar zones in the posterior cerebellum of the rat[END_REF]. A complementary approach was previously suggested by López et al (97), who injected conventional biochemical tracers together with RABV to label inputs to-and from the thalamus. Finally, a recently developed single-cell targeting technique using a recombinant poly-synaptic RABV ( 98) may point the way to new strategies for the targeting of individual neurons.

Vesicular Stomatitis Virus as a Tracer of Neuronal Circuits

Although lesser known in neuroscience research, vesicular stomatitis virus (VSV) is also eminently suited to certain neuronal tracing applications. Possessing a broader tropism than RABV, it is also capable of infecting neuronal cells. Recombinant forms, suitable to genetic manipulation, permit the construction of a variety of vectors. Although capable of spreading in the CNS [START_REF] Lundh | Spread of vesicular stomatitis virus along the visual pathways after retinal infection in the mouse[END_REF][START_REF] Van Den Pol | Vesicular stomatitis virus with the rabies virus glycoprotein directs retrograde transsynaptic transport among neurons in vivo[END_REF], the specific trans-synaptic nature of its spread has only recently been confirmed using recombinant forms of the vector [START_REF] Beier | Anterograde or retrograde transsynaptic labeling of CNS neurons with vesicular stomatitis virus vectors[END_REF]100,101). Similar to RABV, glycoprotein deleted forms of VSV (VSV∆G) have been developed [START_REF] Van Den Pol | Viral strategies for studying the brain, including a replication-restricted self-amplifying delta-G vesicular stomatis virus that rapidly expresses transgenes in brain and can generate a multicolor golgi-like expression[END_REF][START_REF] Chandran | Endosomal proteolysis of the Ebola virus glycoprotein is necessary for infection[END_REF][START_REF] Beier | Anterograde or retrograde transsynaptic labeling of CNS neurons with vesicular stomatitis virus vectors[END_REF][START_REF] Schnell | Construction of a novel virus that targets HIV-1-infected cells and controls HIV-1 infection[END_REF]. VSV readily accepts foreign transgenes and is also amenable to pseudotyping approaches (e.g. [START_REF] Van Den Pol | Viral strategies for studying the brain, including a replication-restricted self-amplifying delta-G vesicular stomatis virus that rapidly expresses transgenes in brain and can generate a multicolor golgi-like expression[END_REF][START_REF] Beier | Anterograde or retrograde transsynaptic labeling of CNS neurons with vesicular stomatitis virus vectors[END_REF][START_REF] Schnell | Construction of a novel virus that targets HIV-1-infected cells and controls HIV-1 infection[END_REF]101,102). VSV ∆G encoding eGFP and pseudotyped with its own glycoprotein was capable of infecting a broad range of cells in the CNS and resulted in confined focal infection, suggesting that like RABV, the glycoprotein gene is also responsible to trans-synaptic spreading. Recent studies confirm this finding and suggest that the nature of the glycoprotein can determine the direction of spreading of the recombinant virus [START_REF] Beier | Anterograde or retrograde transsynaptic labeling of CNS neurons with vesicular stomatitis virus vectors[END_REF]100). Interestingly, this VSV can be engineered to spread in both an anterograde and retrograde direction permitting analysis of both inputs to and outputs from a target region (100). VSV ∆G can also be also be used to trace mono-synaptically connected circuits, however thus far this has only been demonstrated for the retrograde direction in vivo. The use of VSV ∆G vectors pseudotyped with the avian sarcoma and leukosis virus (EnvA) glycoprotein permit specific targeting of a defined starter cell population, as established for RABV ∆G. Traced neurons exhibit fine morphological labeling, as has been demonstrated for RABV. The advantage of this system over RABV ∆G-mediated tracing/expression system is the extreme rapidity of transgene expression (within ~1h of infection [START_REF] Van Den Pol | Viral strategies for studying the brain, including a replication-restricted self-amplifying delta-G vesicular stomatis virus that rapidly expresses transgenes in brain and can generate a multicolor golgi-like expression[END_REF]100)). This vector is thus well suited to experiments in vitro systems such as cultured tissue where a rapid result is needed at a particular development time-point. In may thus also be useful in acute slices. The disadvantage, however, is the rapid toxicity. Calcium imaging experiments (calcium dynamics are used as a surrogate measure for electrical activity) showed that neurons infected with VSV ∆G were indistinguishable from WT neurons 24 post inoculation in brain slices prepared from infected mice [START_REF] Van Den Pol | Viral strategies for studying the brain, including a replication-restricted self-amplifying delta-G vesicular stomatis virus that rapidly expresses transgenes in brain and can generate a multicolor golgi-like expression[END_REF]. Similar findings were reported by Beier et al (100), however by 2 days post infection, electrophysiological properties of infected neurons had become aberrant, thus demonstrating a very narrow time-window for physiological experiments (100).

A novel circuit tracing approach has also been demonstrated in which the specificity of tracing is limited at the level of the pre-synaptic neuron (101) VSV ∆G has been engineered to express a chimeric glycoprotein containing extracellular sequences derived from the EnvA glycoprotein. This restricted tracing to presynaptic neurons expressing TVA (in this case engineered by Cre dependent expression using a ChAT-Cre, conditional TVA bitransgenic mouse). This vector was then pseudotyped with the rabies virus glycoprotein to permit axon infection and longrange retrograde transport of the pseudotyped vector. Following injection into the lateral geniculate nucleus (a nucleus essential for processing visual information from the retina (and which receives inputs from a wide range of retinal ganglion cells, RGC), trans-synaptic tracing was limited to starburst amacrine cells (SACs) immediate presynaptic to RGCs. Using this technique, Beier et al, described novel elements of the RGC-SAC circuitry. They also extended their description of the specificity of trans-synaptic tracing using VSV. In spite of the extensive overlap of neuronal processes, there was no infection of neurons with closely apposed structures. However, this approach may be limited to Using World Scientific's Review Volume MS Word Template 133 certain types of synapses, because no tracing was observed via dendrodendritic synapses.

Caveats

Although this is an extremely powerful approach, a number of caveats remain. Firstly, the long-standing principles about the specificity of tracing using the RABV (13,[START_REF] Ugolini | Use of rabies virus as a transneuronal tracer of neuronal connections: implications for the understanding of rabies pathogenesis[END_REF]103) are derived mostly from studies using native (non-recombinant) forms of the virus, mostly derived from the Challenge Virus Standard (CVS). As noted (13) it is possible that other strains differ in their trans-neuronal tracing qualities. To date, almost all mono-trans-synaptic tracing studies have been performed with glycoprotein-deleted recombinant RABV derived from the highly attenuated Street Alabama Dufferin-derived strain, SAD B19. The transneuronal tracing qualities of this virus have now been evaluated in numerous brains regions and the following features immerge. Glycoprotein deleted SAD B19-based mono-trans-synaptic tracing is, in all but one example (see below), exclusively retrograde. Tracing occurs in an entirely synapse-specific manner [START_REF]Targeting Single Neuronal Networks for Gene Expression and Cell Labeling In[END_REF][START_REF] Arenkiel | Activityinduced remodeling of olfactory bulb microcircuits revealed by monosynaptic tracing[END_REF]. A number of synapses of various modalities are crossed, including certain 'non-canonical' synapses such as dendro-dendritic synapse (but see also below). There is no evidence of the infection of 'fibers of passage' (e.g. ( 43)) although certain studies [START_REF] Takatoh | New modules are added to vibrissal premotor circuitry with the emergence of exploratory whisking[END_REF][START_REF] Arenkiel | Activityinduced remodeling of olfactory bulb microcircuits revealed by monosynaptic tracing[END_REF] suggest that glial cells may be infected. The significance of the later observation has not fully been investigated. In our own work in the neocortex and hippocampus, we find no evidence of the infection of glial cells using EnvA-pseudotyped RABV ∆G of SAD B19 origin, but each brain area may be different. Finally, it is an oft-cited 'fact' that peripheral innoculation leads to transduction of motor neuron endplates, but not sensory neurons (15,[START_REF] Ugolini | Use of rabies virus as a transneuronal tracer of neuronal connections: implications for the understanding of rabies pathogenesis[END_REF]. Recent studies show that this is not necessarily true for either SAD-B19 derived vectors or wildtype CVS-strains [START_REF] Takatoh | New modules are added to vibrissal premotor circuitry with the emergence of exploratory whisking[END_REF]104,105). Recent experiments using vectors derived from glycoprotein-deleted forms of the HEP-Flury strain of show that it can also function as an effective trans-neuronal tracer when transcomplemented with exogenously expressed glycoprotein [START_REF] Mori | Rabies virus glycoprotein variants display different patterns in rabies monosynaptic tracing[END_REF]. The specificity of tracing with this novel vector has not be characterized in detail and it is difficult to extrapolate these characteristics from other studies using this strain (e.g. [START_REF] Ohara | Dual transneuronal tracing in the rat entorhinalhippocampal circuit by intracerebral injection of recombinant rabies virus vectors[END_REF][START_REF] Ohara | Organization of multisynaptic inputs to the dorsal and ventral dentate gyrus: retrograde trans-synaptic tracing with rabies virus vector in the rat[END_REF]) because in the latter case the vector has been engineered to express a CVSderived glycoprotein.

In spite of its enormous potential, mono-transynaptic tracing, like any other experimental approach, also has its limitations. Factors relating to the nature of the rabies negative-sense single-stranded RNA genome preclude the use of certain genetic tools possible with other neurotropic viruses (reviewed in (9,11). Undersampling of presynaptic populations [START_REF] Miyamichi | Dissecting local circuits: parvalbumin interneurons underlie broad feedback control of olfactory bulb output[END_REF][START_REF]Targeting Single Neuronal Networks for Gene Expression and Cell Labeling In[END_REF] and the restricted time window (less than postnatal day 11) for use of the SAD B19-derived forms for tracing from the peripheral nervous system ( 60) are all significant factors to consider in the design of a tracing experiment. Most perturbing is the observation that this approach sometimes fails to reveal known synaptic connections. For example Wall et al (53) reported a dramatic under-labelling of striatalinputs from substantia nigra pars compacta. This nucleus represents an important source of dopaminergic input to the striatum -a finding that was confirmed by injection of a non-trans-synaptic retrograde RABV∆G tracer (see also below). Wall and colleagues (53) speculate that the unusual structure of this synapse, which features a large extracellular space, may impede trans-synaptic tracing. Deshpande et al (43) observed a similar perplexing lack of tracing of between adult born neurons of the OB and mitral cells. This may reflect an aspect of their atypical dendrodendritic synapses synapse or may represent some feature inherent to this type of new-born neurons, that the synapse lacks an essential molecule for trans-synaptic crossing. The molecules required for trans-synaptic crossing have not entirely been defined, although certain candidate molecules have been proposed (namely nicotinic acetylcholine receptor, neuronal cell adhesion molecule, low affinity nerve growth factor receptor ( 106)). It is thus possible that certain molecules may absent or present in limiting quantities in certain synapses. Another potential limitation, is that RABV ∆G particles cross synapses in a stochastic manner and that neurons with multiple inputs onto a source cell have a greater chance of being labeled in a given time window than those with sparse inputs [START_REF] Takatoh | New modules are added to vibrissal premotor circuitry with the emergence of exploratory whisking[END_REF]. Another unanswered question is whether the probability of trans-synaptic crossing could linked to synaptic strength. This question is difficult to test experimentally, but evidence in its favour Using World Scientific's Review Volume MS Word Template 135 comes from the finding that trans-synaptic tracing was more favourable in animals trained on a running wheel that sedentary animals. Although one study has shown that the activity of a synapse does not play a role in the efficiency of crossing [START_REF] Arenkiel | Activityinduced remodeling of olfactory bulb microcircuits revealed by monosynaptic tracing[END_REF], this may also strongly depend on the cell type involved. Lastly, rabies virus has been considered to be a gold standard for trans-neuronal tracing because of its obligate retrograde mode of passage. However, recent finding suggest that in certain cases this may not be the case. Zampieri et al.(105) showed that not only can RABV ∆G infect peripheral sensory neurons, but it is also capable, in certain cases, of anterograde transport. In so doing, they were able to dissect circuits involving sensory neurons, motor neurons and certain inhibitory neurons of the peripheral nervous. Reassuringly, perhaps, they demonstrated that anterograde transport is limited to the peripheral nervous system and does not occur in the CNS. The authors speculate that this may be due to different cytoskeletal dynamics in sensory neurons (c.f. CNS neurons). It is important to note, however, that this was demonstrated in P4 mice and is yet to be demonstrated in mature animals. VSV on the other hand has a limited history as a trans-synaptic tracer. Thus more studies are required to unequivocally establish its properties. It is important to note that although capable of crossing synapses in both the anterograde and retrograde direction, mono-transsynaptic tracing has not yet been demonstrated in vivo. Lastly, it will be important to demonstrate whether attenuated forms of VSV such as those possessing the M15R mutation (107) can be used for physiological studies in vivo.

Perspectives

Recent developments in the field of RABV ∆G technology convincingly establish the importance and power of this tool for neural circuit mapping. Combined with other imaging, physiological, and behavioral approaches, it promises to open new avenues of research permitting a greater understanding of the mammalian connectome and its function. RABV ∆G is applicable to a range of experimental paradigms and permits not only the study of circuit structure, but also the ability to manipulate or monitor its function. VSV ∆G is a relatively new tracer and its capabilities remain to be tested. It is likely that certain features of this vector namely the rapidity of replication and potential for anterograde mono-trans-synaptic tracing in vivo and ease of accepting alternative glycoproteins will lead to its preferred use in certain experimental questions.

A number of new targeting vectors and RABV ∆G variants have greatly extended the capabilities of the RABV ∆G toolbox. Many of these innovations may also be possible with VSV. The development of RABV variant expressing calcium sensors [START_REF] Yonehara | Motion detection: neuronal circuit meets theory[END_REF]108), glutamate sensors (64), ChR2 [START_REF] Lammel | Input-specific control of reward and aversion in the ventral tegmental area[END_REF], subcellular markers [START_REF] Wickersham | Axonal and subcellular labelling using modified rabies viral vectors[END_REF] and Cre recombinase [START_REF] Dölen | Social reward requires coordinated activity of nucleus accumbens oxytocin and serotonin[END_REF]108) extends the spectrum of approaches possible using this technology. In addition, the demonstration, for the first time that mono-trans-synaptic tracing may be targeted to non-rodent species greatly enhances the potential of this approach [START_REF] Liu | Tracing inputs to inhibitory or excitatory neurons of mouse and cat visual cortex with a targeted rabies virus[END_REF]. RABV and VSV have been shown to have a very broad host range, including all mammalian species, birds and even insects (reviewed in (18)). Thus given the appropriate targeting strategy, experiments in other model systems ranging from perhaps drosophila to primates may soon be possible. The concomitant use of optical approaches (109,110) will also greatly increase the potential of this technology for high-throughput screening approaches, making these vectors suitable for the global understanding of circuit level defects in models of pathology. Taken together these tools shows great promise for improving our understanding of neuronal circuit organisation/function as well as its plastic modification during physiological processes or in disease.

:

  Traçage Virale, Syndrome du X Fragile, Connectivité Titel: Studie der neuronaler Konnektivität des Gehirns der Maus im normalen Zustand und im Fragilen X-Syndrom Zusammenfassung: Das Ziel dieser Arbeit war die Untersuchung der anatomischen und funktionellen Konnektivität neuronaler Netze und die Entwicklung neuer Werkzeuge für diesen Zweck. Da der letztgenannte Aspekt ein wichtiger Schwerpunkt der aktuellen neurowissenschaftlichen Forschung ist haben wir zuerst einen neuartigen viralen Tracer entwickelt der die neuronale Rekonstruktion erleichtert. Des weiteren haben wir dieses Virus zusammen mit anderen Techniken angewendet, um Änderungen der neuronalen Konnektivität im Fragilen-X-Syndrom zu untersuchen. Im ersten Teil erörterten wir die Vorteile und Nachteile einer Schwellentechnik, die es mit Hilfe eines neuen viralen Vektors in einzigartiger Weise erlaubt, die Eingänge in bestimmte Hirnregion zu bestimmen. Im zweiten Teil haben wir, ausgehend von diesem viralen Vektor, eine neue Variante entwickelt, um die Sichtbarmachung und Rekonstruktion von morphologischen Eigenschaften von Neuronen zu erleichtern. Wir zeigten die Stärke dieser anterograde Variante des rekombinanten Tollwut-Virus für die Rekonstruktion aller wichtigen morphologischen Merkmale von Neuronen: Dendriten, Spines, lange Axone durch das Gehirn und axonale Boutons. Im dritten Teil untersuchten wir Veränderungen in der Verdrahtung von Hirnstrukturen im Fragilen X Syndrom (FXS). FXS ist die häufigste vererbte geistige Behinderung und häufigste genetische Form des Autismus. FXS führt zu Lern-(z.B. visuell). Eine der bedeutendsten Hypothesen in dem Gebiet des Autismus geht von einer lokalen hyper-Konnektivität aber einer hypo-Konnektivität für weit enfernte Verbindungen aus. Um diese Hypothese in einem FXS-Mausmodell zu testen verwendeten wir Magnetresonanztomographie, um das gesamte Gehirn zu scannen und die anatomischen und funktionellen Konnektivität zu messen. Dies erlaubte uns, in mehreren Bereichen, Konnektivitätsänderungen zu identifizieren. Diese Änderungen konnten wir anschließend unter Verwendung von viralen Tracern weiter untersuchen.

  fonctionnelle des réseaux neuronaux et le développement des nouveaux outils à cet effet. Car le dernier aspect est une préoccupation majeure de la neuroscience actuelle, nous avons développé d'abord un nouveau traceur virale permettant la reconstruction neuronale. Nous avons ensuite appliqué cet et d'autres techniques pour sonder les défauts de connectivité neuronale. 15 Ici, nous avons étudié la connectivité neuronale du cerveau de la souris normale, et dans une forme héréditaire d'un retard mental, le syndrome de l'X fragile. Comment est-ce que l'information sensorielle est traitée dans le 20 cerveau, la perception codé, et le comportement généré? Une réponse à ces questions nécessite une compréhension des mécanismes du fonctionnement des circuits neural du cerveau. Le fonctionnement de ces circuits neuronaux est déterminé par leur structure, les propriétés physiologiques des connexions neuronales, et les propriétés d'intégration des neurones. Ainsi, la 25 compréhension des calculs du cerveau est inévitablement liée à la connaissance de la relation entre la structure de ses circuits neuronaux et de leur fonction. Le virus de la rage a des propriétés remarquables comme un traceur rétrograde de populations neuronales reliées par des synapses. Récemment, le développement d'une variante supprimé du géné de 30 glycoprotéine basée sur le virus de la rage a permis le dépistage et l'exploration fonctionnelle de connexions de neurones monosynaptiques définies. Cette méthodologie peut être facilement combiné avec l'expression Studying Neuronal Connectivity in the Mouse Brain in Normal Condition and Fragile X Syndrome L'étude de la connectivité neuronale dans le cerveau de la souris dans des conditions normales et le syndrome de l'X fragile Thèse présenté par Matthias Georg Haberl 2 d'une variété de gènes, optogénétique, électrophysiologie, les tests comportementaux et de conduire des études d'intégration d'information dans les circuits neuronaux.

  Dans la deuxième partie, nous avons développé, au départ de ce vecteur viral, une nouvelle variante pour faciliter le traçage et la reconstruction 20 des caractéristiques morphologiques de neurones. Ici, nous avons prouvé l'utilité de cette nouvelle variante antérograde du virus de la rage. On a démontré l'utilité de cet vecteur pour des nouvelles approches neuroanatomiques impliquant une infection neuronales sois en gros ou clairsemées dans une région du cerveaux. Cette technologie exploite les 25 caractéristiques avantageux et uniques des vecteurs du virus de la rage. Notamment un haut niveau d'expression de transgènes qui se développe autonome et rapide, combiné avec une cytotoxicité assez limitée. Notre vecteur permet la longue portée et à petite échelle traçage sans ambiguïté de l'ensemble arbre de axonale des neurones individuels dans le cerveau. Nous 30 avons montré la force de cette variante antérograde du virus de la rage pour Studying Neuronal Connectivity in the Mouse Brain in Normal Condition and Fragile X Syndrome L'étude de la connectivité neuronale dans le cerveau de la souris dans des conditions normales et le syndrome de l'X fragile Thèse présenté par Matthias Georg Haberl 3 la reconstruction de calcul de toutes les caractéristiques morphologiques clés de neurones: les dendrites, épines, les axones longs envergure dans tous les terminaux du cerveau et les boutons. Notamment, ce niveau de l'étiquetage peut être réalisé après l'infection avec une seule particule virale. Le vecteur est efficace dans les souris sur une gamme d'âges (jusqu'à 14 mois) et par 5 conséquent, il peut être utilisé pour aider les études de maladies neurodégénératives ou le vieillissement du cerveaux. Le vecteur infecte de nombreux types différentes de cellules neuronaux et des cellules gliales dans toutes les régions du cerveau testés. Enfin, ce type de virus peut également être facilement combiné avec différentes variantes du virus de la rage, comme 10 le type rétrograde. Ensemble avec d'autres technologies modernes, cet outil offre de nouvelles possibilités pour l'enquête de l'anatomie et de la physiologie des circuits neuronaux. Dans la troisième partie, nous avons examiné les modifications dans la 15 connectivité des structures cérébrales dans le syndrome du X fragile. Le syndrome du X fragile est le retard mental héréditaire la plus fréquente. C'est également la cause la plus courante et la plus bien décrit d'un seule gène d'un trouble du spectre autistique (TSA) chez l'homme. Le syndrome du X fragile se produit avec une prévalence de 1/4000 à 1/8000 mâle et femelle 20 dans enfants. Les symptômes comprennent apprentissage et de mémoire déficits, une prévalence élevée de comportement autistique, des convulsions, hypersensibilité aux stimuli sensoriels et modifications / perturbations dans le traitement des informations sensorielle. Le modèle de souris knock-out FMR1 (Fmr1 KO) présente plusieurs caractéristiques neuropathologiques qui sont 25 semblables à ceux des patients FXS humains, qui souffrent d'une perte de fonction du gène FMR1. Alors que de nombreuses études se concentrent sur la dérégulation des récepteurs neuronaux ou compartiments individuels nos connaissances 30 sur l'organisation du réseau est clairsemée. Aujourd'hui, nous ne avons que Studying Neuronal Connectivity in the Mouse Brain in Normal Condition and Fragile X Syndrome L'étude de la connectivité neuronale dans le cerveau de la souris dans des conditions normales et le syndrome de l'X fragile Thèse présenté par Matthias Georg Haberl 4 quelques indications sur les changements de réseau à petite échelle et nous ne savons pas comment ils sont impliqués dans la maladie, ou comment ils contribuent aux changements cognitifs et de perception dans FXS. Au niveau cellulaire, une connectivité anatomique modifié a été proposé de jouer un rôle à la fois dans le syndrome du X fragile et TSA. Les deux, morphologie des 5 épinés dendritique et la croissance axone sont pensés de être altéré chez les souris KO FMR1. Par conséquent, une altération des circuits du cerveau est censée de jouer un rôle important dans FXS. Plusieurs rapports ont indiqué des changements dans la connectivité à courte portée du néocortex chez les souris KO Fmr1 mais souvent ceux qui sont apparus au cours d'une étroite 10 fenêtre de temps et a disparu dans adolescence. Cela pose la question de savoir si les changements de câblage jouent effectivement un rôle dans l'âge adulte dans FXS? Ici, une connaissance approfondie des déficits dans le câblage du circuit neuronal serait nécessaire pour combler le fossé entre les altérations trouvés au niveau moléculaire / micro-circuits et les déficits 15 comportementaux / intellectuelle. Un câblage structurel adéquat est crucial pour le maintien de la fonction cérébrale normale, comme le traitement de l'information sensorielle. Connexions à longue portée entre différentes zones du cerveau sont cruciales pour le transfert séquentiel fidèle de l'information sensorielle de manière 20 prédictive et de rétroaction. Connexions allant courts doivent être intacts à intégrer et traiter les informations étape par étape dans chaque zone. Ici, nous émettons l'hypothèse que les deux, la connectivité entre l'intérieur et dans les zones du cerveau peuvent être modifiés en raison des déficits FXS, qui ont été décrites précédemment dans la colonne vertébrale et des 25 morphologies excroissance axonale. Pourtant, à long terme sous-jacents FXS câblage déficits ont été largement inexploré en tant que mécanisme potentiel déficits cognitifs sous-jacents. Une des hypothèses éminents dans le domaine de l'autisme suppose une phénotype de hyper-connectivité locale mais de hypo-connectivité pour les connexions longue portée. Pour tester cette 30 hypothèse dans un modèle de souris du syndrome du X fragile nous avons Studying Neuronal Connectivity in the Mouse Brain in Normal Condition and Fragile X Syndrome L'étude de la connectivité neuronale dans le cerveau de la souris dans des conditions normales et le syndrome de l'X fragile Thèse présenté par Matthias Georg Haberl 5 utilisé différentes techniques de l'imagerie. Conséquent, nous plaçons pour quantifier cerveau et au niveau local caractéristiques structurelles de connectivité dans le modèle de la souris Fmr1 KO et les corréler à la connectivité fonctionnelle. Ici nous avons utilisé l'imagerie résonance magnétique, pour balayer la 5 totalité du cerveau et de mesurer la connectivité anatomique et fonctionnelle. Cela nous a permis d'identifier des altérations de connectivité dans plusieurs domaines. On a calculé le tenseur de diffusion (DTI) pour mesurer l'intégrité structurelle de la matière blanche chez les souris KO Fmr1 et trouvé une organisation réduite ou nombre d'axones dans les faisceaux de fibres du 10 corps calleux ci-dessous plusieurs aires corticales. Après nous avons utilisé des traceurs viraux pour explorer un de ceux domaines plus détaillé. On a utilisé le virus de la rage rétrograde pour quantifier le nombre de neurones projetant vers ces zones. Nous avons trouvé une connectivité d'entrée modifié pour le cortex visuel primaire, ce qui pourrait contribuer au traitement visuel 15 altéré de l'information. La marquage rétrograde a révélé que le nombre d'entrées de longue allant à cette zone est réduite. Globalement, nous trouvons une évolution vers plus d'entrée locale et moins long allant. Utilisation de l'état-repos IRM fonctionnelle (IRMf) nous avons constaté que, en fait, les changements structurels correspondent à une fonction de 20 synchronisation / découplage de plusieurs zones du cerveau, qui sont cruciaux pour le traitement de l'information sensorielle, visuelle et auditive. Les changements dans la façon dont le cerveau est câblé fournir une explication raisonnable pour les déficits dans le traitement de l'information 25 sensorielle et pourrait également être liée à des déficits d'apprentissage, soit en étant la cause ou l'effet étant d'un tel changement. Nous proposons que les mesures non invasives de la connectivité du cerveau -démontrés ici à la souris -pourraient être répétées chez des patients humains. Notre étude souligne ainsi biomarqueurs appropriés qui pourraient être appliquées pour 30 tester l'efficacité des interventions thérapeutiques. Cela peut faciliter et Studying Neuronal Connectivity in the Mouse Brain in Normal Condition and Fragile X Syndrome L'étude de la connectivité neuronale dans le cerveau de la souris dans des conditions normales et le syndrome de l'X fragile Thèse présenté par Matthias Georg Haberl 6 améliorer les résultats des essais cliniques, en testant leur capacité d'inverser les déficits de câblage du cerveau chez le syndrome du X fragile. L'arrêt récent de plusieurs essais de médicaments à grande échelle souligne la nécessité d'une meilleure compréhension des mécanismes de FXS. Ces médicaments ne ont pas apporté l'effet avantageux prévu par 5 exemple sur les patients Arbaclofen (Seaside Therapeutics / Roche) ou Mavoglurant / mGluR5 (AFQ056) (Novartis), même se ils agissent sur les cibles moléculaires particulièrement bien décrits (mGluR5 et GABAR). Modifications dans le câblage anatomique fournissent un mécanisme qui pourrait expliquer (i) les effets drastiques trouvés sur le traitement de 10 l'information sensorielle dans les circuits neuronaux et (ii) les complications dans les stratégies de sauvetage visant à guérir phénotypes moléculaires. Nous croyons que notre travail ne sera pas seulement renforcer notre compréhension des déficits pathologiques du syndrome du X fragile (et TSA) mais aussi qu'il fournira un cadre pour tester l'efficacité de nouveaux agents 15 thérapeutiques pour leur capacité à inverser les changements dans le câblage anatomique et fonctionnelle dans souris FXS et à l'avenir chez les patients humains. Important de notre étude a été spécifiquement réalisée uniquement chez les souris adultes (9-12 semaines postnatal). Études et des essais pré-20 cliniques antérieures ont souvent été réalisées dans des cultures cellulaires ou de jeunes animaux (par exemple le début du traitement chronique chez la souris à 4-5 semaines). Il est donc important de tenir compte des tests supplémentaires, à la fois in vivo et ex vivo qui évalue les mesures intermédiaires telles que la capacité d'agents thérapeutiques potentiels pour 25 inverser les déficits de câblage. Effectuer ces essais chez les animaux adultes peut fournir de meilleures indications pour l'efficacité clinique chez les patients adultes FXS depuis câblage neuronal est beaucoup plus plastique dans les jeunes animaux ou des nourrissons, où le cerveau est toujours en cours effets de recâblage et élagage fortes. Nous suggérons que notre 30 approche a le potentiel pour devenir un pipeline d'analyse pour tester les Studying Neuronal Connectivity in the Mouse Brain in Normal Condition and Fragile X Syndrome L'étude de la connectivité neuronale dans le cerveau de la souris dans des conditions normales et le syndrome de l'X fragile Thèse présenté par Matthias Georg Haberl 7 nouveaux médicaments candidats. Ces candidats médicaments peuvent être identifiés à une vitesse de plus en plus grandes projections pour les petites molécules bioactives avec un effet souhaitable. Un composé potentiel de FXS est la petite molécule récemment identifié (FRAX486) qui agit en inhibant une protéine modulant la dynamique du cytosquelette d'actine. Le composant a 5 été utilisé chez des souris adultes et muni des effets positifs sur plusieurs déficits comportementaux. Il serait donc intéressant de tester si ce médicament serait efficace pour inverser les fonctions de connectivité trouvés ici. Pour tester cela, nous pourrions examiner les mêmes paramètres de connectivité structurelle et fonctionnelle chez les souris adultes avec et sans 10 traitement. Un dernier point qui doit être considéré est la complexité de FXS et la variabilité des phénotypes associés. La cause de cette variabilité ne est pas tout à fait clair, mais il dépend probablement de plusieurs autres facteurs génétiques ou environnementaux. Il semble donc raisonnable que 15 probablement pas tous les patients FXS sont également affectés par des changements de mGluR et l'activité de GABA ou des fonctions de connectivité. Certains patients développent une forte épilepsie, qui pourrait être causée par une excitation / inhibition déséquilibre et pourrait être corrigée en ciblant des récepteurs GABA. Selon les déficits neurologiques chez 20 chaque patient peut être traité pour la combinaison des problèmes les plus graves, il / elle rencontrera. Dans l'ensemble, plutôt que de supprimer médicaments prometteurs du marché parce qu'ils ne fournissent pas l'avantage dans un assez grand nombre de patients, il serait préférable de développer une médecine personnalisée pour les patients FXS. Voici le 25 câblage à grande échelle et la connectivité fonctionnelle ont l'avantage qu'ils peuvent être mesurés dans le patient et les prévisions pourraient être faites sur la manière bénéfique certains traitements pourraient être.

Figure 1 . 1 Figure 1 . 2

 1112 Figure 1.1 The organization of a follum of the cerebellar cortex.This drawing, made by Ramón y Cajal in 1894 for a conference at the Medical Sciences Academy of Catalonia, places all neural cells and afferent fibres in a three-dimensional perspective. In this parasagittal section, dots represent the parallel fibres. Figure from[START_REF] Sotelo | Viewing the brain through the master hand of Ramón y Cajal[END_REF]

Figure 1 . 3 Chapter 2 . 2 .

 1322 Figure 1.3 Canonical neocortical circuits Quantitative map of anatomical connections between the major excitatory and inhibitory neuron types in area 17 of the cat, including the X-type and Y-type afferents to area 17 from the dorsal LGN. Each arrow is labeled with a numberindicating the proportion of all the synapses that are formed between excitatory neurons. Figure from[START_REF] Binzegger | A quantitative map of the circuit of cat primary visual cortex[END_REF]

. 4 B

 4 ). Commonly used retrograde tracers are chemical tracers like horseradish peroxidase, cholera-toxin subunit B, or fluoro-gold. Neurotropic viruses like rabies virus or pseudorabies virus (mainly the Bartha strain) are often used to trace multiple steps trans-synaptically as described below. In our hands the use of viral tracers in general has the advantage that it can provide an intense, complete labeling of the cells, which facilitates the subsequent counting and localization of projecting cells, which might not always be achieved by chemical tracers. Chemical retrograde tracers also do not reveal whether axons branching in the region of interest make synaptic contacts onto particular cells.

Figure 1 . 4

 14 Figure 1.4 Scheme of different tracer types. A) Anterograde tracers label cells and projections from the cell body. B) Retrograde tracer enter the cells at the axons and label those projecting cells. C)

Figure 1

 1 Figure 1.5 Comparison of FMRP target mRNAs with the curated dataset of mouse brain synaptic proteins (from the Genes to Cognition database, G2Cdb).

Figure 1 . 6

 16 Figure 1.6 The spine phenotype in the Fragile X knock-out mouse model Regional distributions of reported spine defects in the brain of Fmr1 KO mice. Data on dendritic spines in Fmr1 KO mice are displayed on a sagittal view of the mouse brain. Data from the adult brain, developing brain (P20), and in vitro results from dissociated neurons in culture (hippocampus only) are shown in the top, middle and bottom rows, respectively. Colored stripes indicate the existence of published studies reporting different results on either spine density or maturity. The relative thickness of the colored stripes reflects approximately the relative number of studies supporting one finding or another. Studies that used imaging in living neurons (in vivo or in acute/organotypic brain slices) are indicated by arrowheads next to the stripes. Note that results of studies are much more consistent for the spine immaturity phenotype than for the spine density phenotype. Abbreviations used in the figures: BF: barrel field; OB: olfactory bulb; S1: somatosensory cortex; V1: visual cortex. Figure adapted from[START_REF] He | The trouble with spines in fragile X syndrome: density, maturity and plasticity[END_REF].

FIGURE 1 |

 1 FIGURE 1 | RABV-the trans-synaptic tracing toolbox. (A)The RABV genome encodes five proteins including the envelope glycoprotein (G). (B) Deletion of G ( G) is sufficient to prevent trans-synaptic spreading and results in a single-cycle vector. A gene of interest can then be inserted in the place of the deleted G. This gene of interest can encode a fluorescent protein such as GFP, permitting the visualization of virally traced neurons. It can also permit the expression of an almost unlimited choice of genetically encoded tools for the manipulation/visualization of neuronal circuits, e.g., biosensors, synapse markers, activators/repressors of neuronal activity. (C) RABV G can be pseudotyped, either with its native glycoprotein or an engineered surface protein (e.g., EnvA). Pseudotyping restores the normal infection capabilities or enables cell-type selective infection, respectively. (D) RABV G coated with its native glycoprotein is taken up by axon terminals and can be used to trace neurons in a retrograde manner. (E) EnvA pseudotyped RABV G [RABV G(EnvA)] only infects neurons expressing the TVA receptor. Trans-complementation with RG enables RABV G to cross one synaptic step to infect the presynaptic partners of a defined neuron or neuronal population. The virus is then trapped in these presynaptic partner cells and cannot spread further due to the deficiency of RG in these presynaptic cells, hence limiting the strategy to a mono-trans-synaptic event. Initially infected source cells are identified by the expression of an additional fluorescent marker.

FIGURE 2 |Frontiers 5 FIGURE 2 |

 252 FIGURE 2 | Strategies to reveal the secrets of defined neural circuits. (A,B) There are several strategies to target the rabies infection to individual neurons or specific cell types. (A) Genetic strategies such as the Cre-lox

FIGURE 3 |Frontiers 9 FIGURE 3 |

 393 Photostimulation of single cellsElectrode for electroporation and recording

Fig. 1

 1 Fig. 1 Novel rabies virus variant for anterograde tracing of neuronal morphology. a RABV DG expressing a fluorescent protein (XFP: eGFP, mCherry or myr-TdTom) was pseudotyped with a chimeric surface protein containing the transmembrane and cytoplasmic domain of the native RABV glycoprotein (RtmC) and surface domain of the G protein of VSV Indiana virus. b Comparison of fluorescence intensity and signal-to-noise ratio of cells infected with RABV DG (VSV G RtmC) (RABV), VSV G-pseudotyped lentivirus (LV) and adeno-associated virus (AAV). Data are mean ± SEM ***p \ 0.001 (one-way ANOVA test). c RABV DG(VSV G RtmC ) infection of the hippocampal dentate gyrus (DG) region resulted in intense labeling of

Fig. 2

 2 Fig. 2 3D reconstruction of thalamic neurons sparsely labeled with RABV DG (VSV-G RtmC ). a Three excitatory neurons, one inhibitory interneuron (soma/dendrites: red, axon: blue) and three astrocytes (green) were reconstructed within the imaged volume in thalamus and cortex. b Cells were reconstructed from 12 consecutive 50-lm-thick brain sections (long dashes of the anterior-posterior axis). Maximum projection images of three sections containing the neuronal somata are shown (red within the anterior-posterior axis). c Left zoom of panel b. Right Semi-automated reconstructions of the neuronal skeletons are superimposed. In case of the interneuron in panel 3, only the soma and axonal arbor are shown. Please note: Reconstructed branches that are

Fig. 3

 3 Fig. 3 Efficiency of transduction of targeted brain areas. RABV DG XFP (VSV-G RtmC ) transduced cells in all brain structures examined. Confocal microscope images of fluorescently labeled neurons following stereotaxic injection into a substantia nigra, b anterior amygdaloid, dorsal region, and c layers 5 and 6 of the somatosensory cortex. Scale bars left panels 1 mm, right panels 50 lm

Fig. 4

 4 Fig. 4 Transduction in the cerebellum. A multiple-step injection at different depths was applied to investigate the transduction efficacy of RABV DG (VSV-G RtmC ) in the cerebellar cortex. a Injection scheme, black bar illustrates the virus injections into different depths along the needle track. b Injection overview of RABV DG (VSV-G RtmC ) infected cells (red) overlaid with calbindin-(CB?) positive cells (green). b, c Transduced cells were found in the molecular layer (ML), the Purkinje cell layer (PCL) and at high abundance in the granule cell layer (GCL). c In the molecular layer both of the

Fig. 5

 5 Fig. 5 Labeling and surface reconstruction in aged animals. RABV DG-mCherry(VSV-G RtmC ) infection of hippocampal dentate gyrus neurons in a 15-month-old APP/PS1 mouse-a mouse model for Alzheimer's disease-enables the fine-detailed reconstruction of dendrites and spines (a), as well as of axonal boutons (b). a Strong labeling facilitated automated reconstruction of the dendritic tree (left and middle panel; scale: 15 lm) and of dendritic spines (right panels 1 and 1*; scale 5 lm) of a dentate gyrus cell using Imaris (Bitplane,

Fig. 6

 6 Fig. 6 Tropism of RABV DG (VSV-G RtmC ) in CNS. Determination of cell-type identity of RABV DG (VSV-G RtmC ) infected mCherry expressing cells in layer 5 of the barrel cortex (representative images in a, d and g) in 50-lm-thick sections using immunohistochemistry against NeuN (total neuron marker; b), GAD67 (marker of inhibitory interneurons; e) and S-100b (marker of astrocytes, h). Overlay of mCherry and cell-specific marker (c, f and i). Images are either max

  ) Thalamic ventral posteromedial (VPm) nucleus: anterior/ posterior (A/P) -1.70 mm, lateral (L) 1.60 mm, dorso/ ventral (D/V) 3.20 mm; (2) thalamic posteromedial (POm) nucleus: A/P -1.80 mm, L 1.25 mm, D/V 2.75 mm; (3) hippocampal dentate gyrus (DG): A/P -1.90 mm, L 1.20 mm, D/V 1.90 mm; (4) layer 5 (L5) of primary somatosensory barrel cortex (BC): A/P -0.94 mm, L 3.00 mm, D/V 0.80 mm; (5) cerebellum: A/P -5.68 mm, L 0.68 mm, D/V 1.70-0 mm (6) amygdala: A/P -0.82 mm, L 2.45 mm, D/V 4.75 mm; (

Fig 1 .

 1 Fig 1. Reduced structural integrity of the corpus callosum in Fmr1KO mice Diffusion tensor imaging (DTI) in the Fmr1 KO mice shows a reduced fractional anisotropy in the white matter, indicating a reduction in the number of axons, or less organized fibers.

Fig. 2 .

 2 Fig. 2. Input to the primary visual cortex of Fmr1 KO mice (a) Example image displaying the injection site. (b) Overview of the 3D model with all retrogradely labeled cells in green. (c) Recoloring of the retrogradely labeled cells into, primary visual cortex, secondary visual cortex other cortical areas and the thalamus. (d) Distance of the retrogradely labeled cells to the injection site in the Fmr1 KO mice and the WT mice. The relative number of cells (in % of total), plotted as cumulative and relative frequency per distance to the injection site, indicates a strong increase in the cells with less than 1mm distance.

Fig 3 .

 3 Fig 3. Functional decoupling of brain areas in Fmr1KO mice Upper panel: Difference of the Z-scores of rs-fMRI measurements calculated as average Z-score of Fmr1 KO mice minus average Z-score of WT mice. Lower panel: Statistically significant differences in the functional connectivity matrix are color coded for the p-value.

  in a C57BL/6 background and wild-type littermates was continued in Nijmegen (Netherlands) in the same conditions. MRI measurements were performed with an 11.7 T BioSpec Avance III small animal MR system (Bruker BioSpin, Ettlingen, Germany) equipped with an actively shielded 245 gradient set of 600 mT/m and operated by Paravision 5.1 software. We used a circular polarized volume resonator for signal transmission and an actively decoupled mouse brain quadrature surface coil for signal reception (Bruker BioSpin). During the MR experiments, low-dose isoflurane was used (3.5% for induction and ~1.5% for maintenance), slightly adjusted throughout the experiment to maintain a fast and 250 stable breathing frequency (>130 bpm). The mice were placed in a stereotactic device in order to immobilize the head. Body temperature was measured with a rectal thermometer and maintained at 37° C by a heated air flow device. After standard adjustments and shimming, rsfMRI datasets were acquired using a single shot spin echo sequence combined with echo-planar imaging (SE-EPI) 255 sequence. Six hundred repetitions with a repetition time (TR) of 1.8s and echo time (TE) of 16.9ms were recorded for a total acquisition time of 18 minutes. Other imaging parameters: field of view = 25 × 25 mm; image matrix = 96 × 96; spatial resolution = 260 × 260 × 500 µm; number of slices = 9.

  applied along 30 non-collinear directions in three-dimensional space. Other imaging 265 parameters: TR = 7.55 s; TE = 20ms; field of view = 20 × 20 mm; image matrix = 128 × 128; spatial resolution = 156 × 156 × 500 µm; total acquisition time = 18 min.

  1

	Revealing the secrets of neuronal circuits
	with recombinant rabies virus technology
	Melanie Ginger*, Matthias Haberl*,
	Karl-Klaus Conzelmann, Martin K. Schwarz and Andreas Frick
	*Equal contributions
	Frontiers in Neural Circuits, Published online January 24, 2013

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Acknowledgments

Summary

Acknowledgments

ACKNOWLEDGMENTS

We are grateful to N. Rebola, M. Helmstaedter, S. Silva, and members of our teams for their helpful comments. The following funding sources are acknowledged: INSERM, Max-Planck-Society, Region of Aquitaine, Erasmus-Mundus/ENC Network and DFG SFB 870.

Acknowledgments We thank Drs. Nuno Da Costa and Kevan Martin for fruitful discussion, Drs. Kamill Balint and Botond Roska for their kind and helpful advice on handling RABV, Drs. Aude Panatier and Axel Nimmerjahn for their advice about glial cells, Dr. Panatier for providing the S100ß antibody and Dr. Nora Abrous for providing the lentivirus. Imaging data was acquired using equipment of the Bordeaux Imaging Center (BIC) and the Max Planck Florida Institute. The AAV2/9 eGFP vector was obtained from the Penn Vector Core using materials made available by Dr. Hongkui Zeng, Ph.D. of the Allen Institute for Brain Science. This project has been funded with support from the European Commission (European Erasmus Mundus programme; M.G.H.). This publication reflects the views only of the authors, and the Commission cannot be held responsible for any use, which may be made of the information contained therein. This study was also supported by INSERM (A.F.), the PDBEB Programm CNC Coimbra (S.V.S.), LabEx BRAIN (M.G.H., A.F., M.G., C.M.), Conseil de la Region d'Aquitaine (A.F., M.G.), ANR (10-MALZ-0009; C.M.), DFG (SFB 870; K.K.C. and A.G.), the Bernstein Center for Computational Neuroscience in Tuebingen (funded by the German Federal Ministry of Education and Research (BMBF; FKZ: 01GQ1002)) (M.O.), Max Planck Institute for Biological Cybernetics, Tuebingen (M.O.), Max Planck Florida Institute for Neuroscience, Jupiter (J.M.G, M.O.), and the Werner Reichardt Center for Integrative Neuroscience, Tuebingen (M.O.).

Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.

List of Abbreviations

AAV

Adeno-Associated Virus

Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Anterograde Rabies Virus for 3D Reconstruction of Neuronal

Morphology

Much of our current knowledge about the circuits of the brain comes from studies labeling the output of a brain region, anterogradely, either in bulk or sparsely followed by a projection-quantification or a precise reconstruction of individual axons, respectively. In particular sparse labeling has always been difficult to achieve and cumbersome, which could be facilitated by genetic strategies. The next section describes the generation of a new viral vector that facilitates the reconstruction of the neuronal structure. The experimental methods and materials used as well as the results are thoroughly described in the publication included in the subsequent section.

The computational reconstruction and figure preparation shown in Figure 2 has been performed by Jason Guest and Marcel Oberlaender. The author of the thesis was involved in the design, the performing and analysis of all experiments, partially or fully, and produced the here presented novel viral vectors.

Publication N. 2

An anterograde rabies virus vector for high-resolution large-scale

Connectivity Features in the Fmr1KO mouse

The following manuscript describes experiments that were performed to probe defects in the structural and functional connectivity in a mouse model of Fragile X Syndrome.

This project was partially performed as EuroBioImaging project in the BioImaging 5

Abstract

Structural and functional wiring of the brain provides the fundamental basis that 30 determines its capabilities and limitations. Fragile X syndrome, a frequent form of inherited mental retardation and cause of autism in humans, leads to learning and memory deficits, a high prevalence of autistic behavior, seizures, hypersensitivity to sensory stimuli and alterations in the processing of sensory information. Here, we probed alterations in the functional and structural connectivity, both locally and in the 35 long-range, of neocortical circuits in the Fmr1 KO mouse model. To measure these various connectivity aspects, we combined ultra-high-field in vivo diffusion tensor MRI, resting-state functional MRI and viral tracing. Our results show a hyperconnectivity phenotype for local neocortical circuits, but a hypo-connectivity for long-ranging ones. This anatomical data is matched by a functional decoupling of 40 various neocortical regions from one another. We therefore identify Fragile X disease as a "connectopathy", which could explain altered sensory information processing in this disorder, and the complications of rescue strategies acting on molecular targets. should be further assessed. The altered communication of the sensory cortical areas might play an important role in the overall processing of sensory information in FXS 170 and contribute largely to the cognitive and behavioral defects in the disease.

We consider that combining non-invasive measurements with a fine-scale dissection of circuit structure is an important step for a better understanding of the neuropathology. We expect that this work will pave the way in the future in two directions. First we hope it will lead to future studies in human FXS patients to 175 determine the role that connectivity changes play in the human disease. And secondly we think this study should provide a useful framework/baseline, to test the effects of therapeutic agents for their ability to reverse wiring deficits in Fmr1 KO. The prospect of those two directions would offer a mode for a fast MRI based-assessment of rescue approaches and facilitate the transition of strong candidate drugs to human 180 patients, providing new objective criteria to evaluate their success.

Materials and Methods

Adult (9-12 weeks old) male Fmr1 KO2 mice 3 in a C57BL/6 background and male 185 wild-type littermates were used in all experiments. Genotypes were determined by a PCR analysis of DNA extracted from tail samples. All experiments and analysis were performed with the experimenter being blind to the genotype.

Virus production 190

RABV SAD ∆G-eGFP (RG) was produced as described previously 11 . VSV G RtmCpseudotyped SAD ∆G-mCherry was produced as described previously using BSR T7/5 cells 11 .

Stereotaxic injections 195

All stereotactic injections and subsequent experiments were performed according to French law and the European Directive covering the use of experimental animals (2010/63/EU) and approved by the Ethics Committee of Bordeaux (approval #5012024-A). Stereotaxic injections were performed in Fmr1 KO and WT mice at 10-12 weeks of age. The stereotactic injections of viral vectors were performed in 200 isofluorane anesthetized and head-fixed mice using a 10µl glass syringe fitted with a 34-gauge needle or a pulled glass pipette. Injection volume and speed were controlled steps, the diffusion tensor was estimated for every voxel using the PATCH algorithm 18 .

Regions of interest in several white matter (WM) and grey matter (GM) areas were drawn on the template image based on an anatomical atlas ('The mouse brain in 305 stereotaxic coordinates', Keith B.J. Franklin, George Paxinos, 1997) and the resulting FA and MD values were measured for further statistical analyses.

Supplementary Material 310

Supplementary Fig S1. Functional Connectivity Matrix of WT and Fmr1KO mice

Resting state fMRI measurements were performed in Fmr1KO mice (n=7) and age-matched wild type mice (n=10). The intracortical functional connectivity is very strong in the wildtype animals whereas it is less in the Fmr1 KO mice. Key: Dorsal hippocampus (DH), ventral hippocampus (VH), auditory cortex (Au), motor cortex (Mo), primary somatosensory cortex (S1), primary visual cortex (V1), retrosplenial cortex (RS), Piriform Cortex (Pi), Amygdala (Amy), Pretectal Area (PtA), Caudate Putamen (CPu), lateral geniculate nucleus (DLG), globus pallidus (LGP), parafascicular nucleus (PF), ventral posterolateral nucleus and ventral posteromedial nucleus (VPN). Right (R) and left hemisphere (L).

testing the effect of different drugs on correcting alterations of the cortical wiring in FXS. Since we are correlating fine-structural measurements with non-invasive functional and structural magnetic-resonance imaging (MRI) we hope that the MRI measurements can be performed similarly on human patients. Since they can be performed repeatedly and provide another objective criteria to evaluate the efficacy of therapeutic agents. The ability to completely map all synaptic connections (i.e. the connectome) of the brain would provide neuroscientists the possibility to test predictive hypotheses about the brain organization/behavioral capability, compare variations between two brains and find common denominators between species. Yet, the techniques to take on such a challenging task in the mammalian brain (with ~10 11 -10 15 synapses depending on the species) are just evolving. Large research and funding efforts are underway devoted to the unravelling of the synaptic connectivity of individual neurons, specific neuron types, and of neural circuits to link their function to the underlying structure. Modern methodological approaches from a variety of disciplines are applied to tackle the immense complexity of the task. Amongst those approaches, recombinant neurotropic viruses of the rhabdoviridae family (in particular rabies virus and vesicular stomatitis virus) have become important tools. Advances in molecular biology, mouse genetics and in virology have increased the repertoire of tools available for defining the initial targets for infection as well as permitting improved tracing of synaptically connected neurons. At the same time, new constructs enhance our ability to manipulate circuits and to monitor their activity. Recombinant neurotropic viruses based strategies, together with optogenetic, electrophysiological, imaging methods and behavioural tasks enable previously unimaginable experiments. In this chapter, we will summarize the most recent advances in the use of recombinant rhabdoviruses for understanding the function and structure of neuronal circuitry.