viii

I. Figures index

II. Résumé

Dans plusieurs neurones du SNC l'activité somato-dendritique infraliminaire peut se propager passivement dans l'axone et augmenter transitoirement la transmission synaptique spontanée et évoquée par le potentiel d'action de manière dépendante du Ca 2+ . Les mécanismes sousjacents à ce type de plasticité synaptique, appelée facilitation "analogique" ou "analogo-digitale", restent largement inconnus pour la plupart des synapses centrales, principalement en raison de la difficulté à réaliser des enregistrements directs des petits boutons présynaptiques. Ici, nous utilisons de la photolyse de Ca 2+ et de l'imagerie aux niveau des terminaisons présynaptiques individuelles des interneurones de la couche moléculaire du cervelet (ICM), combinées à des enregistrements électrophysiologiques avec la technique du patch.

Nous décrivons un nouveau mécanisme de facilitation analogo-digitale qui est dépendant du Ca 2+ infraliminaire et dans lequel la fraction et la cinétique du pool de vésicules dites "prêtes à être libérées" (readily releasable pool ou RRP en anglais), est modulée par des modifications de la probabilité d'occupation des sites d'ancrage dans des contacts synaptiques individuels. Nos résultats ajoutent une nouvelle dimension à la compréhension de la manière dont l'activité infraliminaire module le flux d'information dans les circuits neuronaux xi

III. Abstract

In several neurons of the CNS, subthreshold somatodendritic activity can spread passively into the axon and transiently enhance spontaneous and spike-evoked synaptic transmission in a Ca 2+ -dependent and graded manner. Available evidence about the underlying mechanism of this type of synaptic plasticity, called "analog" or "analog to digital" facilitation (ADF), remains largely incomplete for the majority of central synapses, mainly due to the experimental inaccessibility to the small presynaptic boutons. Here we use both Ca 2+ photolysis and imaging at individual presynaptic terminals of the rat cerebellar molecular layer interneurons (MLIs), combined with whole-cell paired recordings from synaptically connected MLIs, to report a novel subthreshold Ca 2+ -dependent mechanism for ADF whereby the fraction and the kinetics of the pool of vesicles available for immediate release, the readily releasable pool (RRP), are modulated by changing the docking site occupancy probability in single synaptic contacts. Our results add a new dimension in the understanding of how subthreshold activity modulates information flow in neuronal circuits.

IV. Mots clés; Keywords:

Analog signaling, Analog-to-digital, Docking Sites, GABAergic interneurons, Readily Releasable Pool, Single synaptic contacts, Synaptic plasticity. A mí amada hija, por darme la oportunidad de experimentar una fuerza tan poderosa, y por alegrar mi mundo con su inocencia y belleza.

xv

VI. Acknowledgment

First of all, I would like to thank Federico, Alain and Isabel for accepting me into the lab. It's been amazing to work in Paris doing beautiful experiments. Thank you, Fede, for giving me the chance to explore and to think freely, and also for putting up with my stubbornness; I have learned a lot from you. Thank you, Isabel for your support and guidance. Thank you, Alain, for your support and advice; it's a pleasure to discuss results with you. A special thanks to Professor Juan Bacigalupo for your support, advice and trust over the years, none of these would have been possible without your help and mentorship; I have no words to express my gratitude.

I would like to thank to the members of the thesis committee: Dra. Brigitte Van Zundert, Dra. Ursula Wyneken, Dr. Stéphane Dieudonné, Dr. Pierre Vincent, Dra.

Magdalena Sanhueza, and Dra. Cecilia Vergara. I really appreciate your help, advice and support.

Thank you to all the past and present members of the Cell Physiology Lab, it is been a real pleasure to work in the lab.

Thank you to David and Celine for being so nice to me in spite of my poor English and French. Thank you to Philippe and Boris, it was a pleasure to share the working space with you. Thank you to Brandon for always smiling and your xvi willingness to help. Thank you to Michael for being such a nice guy, it was great to work next to you the last year. Thank you, Thibault, for helping me every time I had a question related to science or the French language. A super special thank you to Jorge, Gerardo, Taka, Camila, Lupe, Javier, Merouann, Bastian, Laura Castro, Laura Gomez, Enrico and Maria, you are totally awesome!!! I am very happy to have met you. Thanks to Patrick, Linda and Chantal for being so kind and helpful.

Thank you to all my Parisian-Chilean friends; it's been a real pleasure to share part of my life with you. A special thank you to "los terribles voisins" Vale and Enrique, for your friendship and support.

Thank you to my parents for your love and the enormous effort you have put into my education. Thanks to my big brother Israel for walking with me through life and for your unconditional love. Thanks to my little sisters Nathalie and Ambar for your love and support, you are amazing. Thanks to all my friends for the advices and amazing conversations.

Thanks to my mother-in-law Lita for your love and support. A super mega thank to my beloved wife for all your love, patience, kindness and bravery, you are wonderful. It is been amazing to share my life with you. Finally, I would like to thank to my baby Violeta, you are everything to me now, thank you for making me a better person.

1 Introduction

Subthreshold somatic depolarization enhances neurotransmitter release in cerebellar molecular layer interneurons and other central synapses

It is well known that the probability of neurotransmitter (NT) release depends on the recent history of synaptic activity, which can influence residual calcium (Ca 2+ )

and modulate the amount of NT released when an action potential (AP) arrives to the presynaptic terminal (short-term synaptic plasticity). Traditionally, APdependent transmission has been considered as the only fast way of communication between the somatodendritic compartment and the presynaptic terminals [START_REF] Shu | Modulation of intracortical synaptic potentials by presynaptic somatic membrane potential[END_REF]. However, a growing body of evidence suggests that the neuronal activity on which short-term synaptic plasticity depends, not only involves intracellular Ca 2+ changes as a result of the "all-or-nothing" (or "digital") nature of the AP, but also involves the subthreshold, AP-independent or "analog" synaptic activity that produces changes in membrane potential without reaching the AP threshold. On invertebrate preparations it was demonstrated long ago that the application of subthreshold presynaptic depolarization can increase the amount of NT released to the synaptic cleft [START_REF] Shimahara | Multiple interneuronal afferents to the giant cells in Aplysia[END_REF][START_REF] Nicholls | Modulation of transmission at an inhibitory synapse in the central system of the leech[END_REF]. In mammals, this feature was demonstrated more recently by recording synaptic activity in pairs of connected neurons [START_REF] Shu | Modulation of intracortical synaptic potentials by presynaptic somatic membrane potential[END_REF][START_REF] Alle | Combined analog and action potential coding in hippocampal mossy fibers[END_REF]. Direct recordings from hippocampal mossy fiber boutons in slices have revealed that subthreshold somatic depolarization (SSD) can passively spread hundreds of microns into the axon and modulate the process of NT release at the mossy fiber bouton-CA3 pyramidal neuron synapse [START_REF] Alle | Combined analog and action potential coding in hippocampal mossy fibers[END_REF]).

In the Calyx of Held (where direct pre-and postsynaptic recordings is achievable), it has been shown that subthreshold depolarization of the presynaptic terminal is capable of potentiating NT release by a mechanism that involves both, an increase in the basal Ca 2+ levels [START_REF] Awatramani | Modulation of transmitter release by presynaptic resting potential and background calcium levels[END_REF] and an increase in the APdependent Ca 2+ influx as a consequence of a change in the AP shape [START_REF] Hori | Mechanisms underlying short-term modulation of transmitter release by presynaptic depolarization[END_REF]. In cortical neurons, it has been proposed that changes in the AP shape may be mediating the potentiation induced by the application of SSD [START_REF] Shu | Modulation of intracortical synaptic potentials by presynaptic somatic membrane potential[END_REF]. However, no changes have been found in the axonal Ca 2+ levels (neither basal nor AP-evoked) during this so-called "analogue signaling" in hippocampal mossy fibers [START_REF] Scott | Analog modulation of mossy fiber transmission is uncoupled from changes in presynaptic Ca 2+[END_REF]. Given the discrepancies, it has been proposed that different forms of analog (or analog-to-digital 1 ) facilitation (ADF), that are Ca 2+dependent or Ca 2+ -independent, can co-exist in different synapses and with different contributions [START_REF] Scott | Analog modulation of mossy fiber transmission is uncoupled from changes in presynaptic Ca 2+[END_REF].

Little is known about the molecular mechanisms underlying this form of synaptic plasticity; however, important advances were recently made in this direction using cerebellar molecular layer interneurons (MLIs) as a model [START_REF] Bouhours | Somatic depolarization enhances GABA release in cerebellar interneurons via a calcium/protein kinase C pathway[END_REF].

In these cells, SSD pulses are electrotonically spread to the presynaptic terminals, where they produce an enhancement of NT release through a signaling pathway involving the activation of the P/Q type, voltage-dependent Ca 2+ channels (VDCC), followed by an increase in the basal Ca 2+ levels and a local activation of protein kinase C (PKC) [START_REF] Bouhours | Somatic depolarization enhances GABA release in cerebellar interneurons via a calcium/protein kinase C pathway[END_REF]; the target of PKC that mediates this effect is unknown. Additionally, for the same preparation it has been also proposed that the SSD-evoked Ca 2+ influx facilitates the AP-evoked Ca 2+ entry and enhances NT release [START_REF] Christie | Ca 2+ -dependent enhancement of release by subthreshold somatic depolarization[END_REF]. Interestingly, in the same study was reported that 4 out of 10 MLI-MLI recorded pairs showed a depression instead of a facilitation when the SSD pulse was preceded by the application of a high frequency train of action potentials. More recently, some controversy has arisen because the group of Christie did not see any SSD-mediated facilitation in MLIs in PKC knockout animals. Instead, they attributed the presence of analog signaling to a broadening of the AP due to the rapid inactivation of the Kv 3 type of potassium channels in the presynaptic terminals, which modifies the currents accounting for the AP waveform, directly enhancing the spike-evoked Ca 2+ influx independently of PKC [START_REF] Rowan | Rapid state-dependent alteration in Kv3 channel availability drives flexible synaptic signaling dependent on somatic subthreshold depolarization[END_REF]. Previous studies showed that both, the application of NMDA and the application of SSD in cerebellar MLIs can significantly increase the frequency of miniature GABAergic events [START_REF] Glitsch | Presynaptic effects of NMDA in cerebellar Purkinje cells and interneurons[END_REF], thus suggesting that physiological subthreshold activity can efficiently modulate the axonal function. Additionally, the application of SSD also produces an increase in the frequency of the so-called "preminis" [START_REF] Trigo | Presynaptic miniature GABAergic currents in developing interneurons[END_REF], which correspond to synaptic events that emerge from the autocrine activation of the presynaptic GABAergic receptors in the axon of MLIs. It was recently shown that these preminis can have an impact on the interneuron excitability, because the voltage changes that they produce are transmitted to the soma antidromically (de San

Martin JZ et al., 2015). Furthermore, since preminis are prominent during the formation of the molecular layer (P11-15), it is thought that they could guide the formation of the neuronal circuits in growing GABAergic neurons through an increase in the release probability of newly formed synaptic contacts by a positive feedback mechanism.

During the last decade, a growing body of evidence suggests that many of the neurons in the nervous system can express ionotropic receptors in the axonal terminals that can modify the membrane potential locally. For example, in several preparations in which the SSD-mediated facilitation effect has been studied, the presynaptic terminal expresses GABA A or glycine receptors [calyx of Held [START_REF] Turecek | Presynaptic glycine receptors enhance transmitter release at a mammalian central synapse[END_REF]; mossy fibers [START_REF] Ruiz | GABAA receptors at hippocampal mossy fibers[END_REF][START_REF] Alle | GABAergic spillover transmission onto hippocampal mossy fiber boutons[END_REF]; MLIs [START_REF] Pouzat | Somatic recording of GABAergic autoreceptor current in cerebellar stellate and basket cells[END_REF][START_REF] Trigo | Enhancement of GABA release through endogenous activation of axonal GABA(A) receptors in juvenile cerebellum[END_REF]]. In addition, unlike the somatodendritic compartment, where the activation of these receptors is inhibitory (or mixed inhibitory/excitatory), their activation in the axonal compartment is excitatory, leading to membrane depolarization, increase in the axonal Ca 2+ levels and increase in NT release [START_REF] Trigo | Axonal GABAA receptors[END_REF]. Consequently, it has been proposed that the activation of these presynaptic receptors and the SSD-mediated facilitation could share a common signaling pathway.

In summary, the analog-to-digital signaling, by which subthreshold synaptic activity reaches the presynaptic terminals and modules the AP-evoked process of NT release, has deep implications in the way the NS encodes and processes information. Indeed, in a "digital" synapse NT release follows the all-or-nothing nature of the AP, hence being a binary process that either does ("1") or does not ("0") happen, while in an analog synapse the dynamic range in which information can be transferred is very large [START_REF] Rama | Modulation of spike-evoked synaptic transmission: The role of presynaptic calcium and potassium channels[END_REF]. Thus, a hybrid type of synaptic transmission where subthreshold activity modulates the following APdependent release will largely increase the amount of information that can be transmitted in neural circuits. Furthermore, if we consider that many neurons in the mammalian brain possess axons with lengths of a few hundred microns, it became clear that the extent of SSD penetration can result in that a high proportion of presynaptic terminals are modulated; for example, the space time constant (distance at which the change in the membrane potential has experienced a decrease of 63%) in hippocampal neurons where ADF has been described is approximately 430 μm [START_REF] Alle | Combined analog and action potential coding in hippocampal mossy fibers[END_REF].

It would be very helpful, to determine the synaptic processes controlling NT release in central synapses, to describe the specific synaptic parameters that are being modulated during ADF, their differences between individual synaptic contacts of the same neuron, and from different neuronal types at different developmental stages.

Possible role of protein kinase C in the subthreshold somatic depolarization-mediated facilitation of neurotransmitter release

Protein kinase C is a family of kinases that are implicated in controlling the Because post-tetanic potentiation as well other types of short-term synaptic plasticity (including depression) are elicited by different temporal patterns of presynaptic activity, it has been hypothesized that they may contribute to the 2. The short-term synaptic plasticity is often separated into three categories according to their temporal scales: facilitation (which lasts for tens of milliseconds), augmentation (lasts for seconds) and potentiation (also called post-tetanic potentiation), that lasts for minutes. It is thought that all depend in one way or another on the residual presynaptic Ca In a recent study in the calyx of Held [START_REF] Chu | Calcium-dependent PKC isoforms have specialized roles in short-term synaptic plasticity[END_REF] it was demonstrated that, depending on the developmental stage of the animal, the activation of different conventional PKC isoforms is capable of mediating post-tetanic potentiation either by an increase in pNR or through an increase in the RRP; however, the PKC substrate that could be mediating these processes is unknown. Interestingly, MUNC18-1 presents multiple sites of phosphorylation by PKC [START_REF] Barclay | Phosphorylation of Munc18 by protein kinase C regulates the kinetics of exocytosis[END_REF][START_REF] Fujita | Phosphorylation of Munc-18/n-Sec1/ rbSec1 by protein kinase C: its implication in regulating the interaction of Munc-18/n-Sec1/rbSec1 with syntaxin[END_REF]; phosphorylation of these sites has been previously implicated in vesicle replenishment after intense stimulation [START_REF] Nili | Munc18-1 phosphorylation by protein kinase C potentiates vesicle pool replenishment in bovine chromaffin cells[END_REF] and in the control of RRP size in GABAergic and glutamatergic synapses [START_REF] Toonen | Munc18-1 expression levels control synapse recovery by regulating readily releasable pool size[END_REF]. Additionally, it is known that MUNC18-1 is rapidly phosphorylated by PKC under depolarizing conditions [START_REF] Craig | Physiological regulation of Munc18/nSec1 phosphorylation on serine-313[END_REF][START_REF] De Vries | Dynamics of munc18-1 phosphorylation/dephosphorylation in rat brain nerve terminals[END_REF]. All these observations make the MUNC18-1 protein the most likely candidate to mediate the effects of PKC activation during subthreshold somatic depolarizations. to propose that the differences observed between individual synaptic contacts could emerge as a consequence of differences in the number of docking sites (NDS), which would increase in direct proportion with the synaptic size. They also suggested that the application of depolarizing subthreshold pulses could significantly modify the docking site occupancy, which would have a direct impact on the synaptic performance during high-frequency stimulation (40 Hz). In this way, variations in the synaptic size might account for the differences that were reported in both presynaptic (release probability) and postsynaptic parameters such as the peak amplitude and the kinetics of the postsynaptic current (PSC). This neuronal property could be particularly important in synapses with a marked saturation of postsynaptic receptors such as in GABAergic synapses between MLIs (~70%), since in these synapses an increase in the amount of released NT would not generate a substantial change in the amplitude of the PSCs, unless both an increase in the amount of NT released as well as an increase in the number of postsynaptic receptors occurs in a coordinated way. Thus, in the long-term, a fixed synaptic size would markedly decrease the efficiency of the transmission process. The figure illustrates the active zone, the docking sites, the docked vesicles (RRP), the recycling vesicle pool (which fill the empty docking sites after release) and the postsynaptic receptors of a single synaptic contact. This central synapse, unlike the typical models of synaptic transmission (the calyx of Held, the amphibian neuromuscular junction, or the squid giant synapse), has a single active zone associated with a single postsynaptic density. In this model of Pulido et al., (2015), NT release is described according to the following assumptions: a synapse contains "N" independent docking sites, each of which has a probability () of being occupied by a vesicle. At the same time, a docked vesicle has a probability (p) of fusing with the plasma membrane and releasing its content to the synaptic cleft. After the exocytosis, each empty docking site will be refilled according to a replacement constant rate "R".

Synapse

Docking site

Synaptic vesicle

Active zone

Postsynaptic receptors

The importance of studying the mechanisms of modulation of synaptic transmission at the level of single synaptic contacts

One of the ultimate goals of neuroscience is the detailed understanding of the processes by which humans and other animals with a nervous system are capable of sensing and processing the environmental information in order to perform an action that allows an adequate adaptation to a particular situation. Virtually all the evidence accumulated to this day support the idea that all this amazing capacity of the nervous system depends on the ability to generate and propagate electrical signals between its essential neuronal components during the process of synaptic transmission.

The importance of the electrical activity for the physiology of the nervous system was highlighted more than two centuries ago by the Italian physicist and anatomist Luigi Galvani [START_REF] Piccolino | Animal electricity and the birth of electrophysiology: the legacy of Luigi Galvani[END_REF], and since then the research has mainly focused on understanding the cellular and molecular bases by which this electrical activity allows the transmission of information in a fast and efficient way between excitable cells 3 , and how this activity relates to animal behavior. Consistent with this idea, most of the information that we have today about the physiological aspects of synaptic transmission has been obtained by electrophysiological approaches. Although this approximation has a series of remarkable advantages 3. Those cells that present some type of electrical signaling as a result of a differential gene expression pattern that allows them to express ion channels through which they are able to generate and propagate an electrical signal: neurons, muscle cells, glia, endocrine pancreatic cells, endocrine pituitary cells, adrenal medulla cells, gametes and some endothelial cells.

(e.g., high temporal resolution of the synaptic current), electrophysiological recordings have proved insufficient to study the functional properties of synaptic transmission at the level of individual synaptic contacts. This is mainly because most electrophysiological recordings usually reflect the averaged electrical activity of many different synapses that are activated in a determined time interval. This is very relevant if we consider that mammalian central synapses differ widely in morphological and functional aspects, even within the same family of synapses, where the pre-and postsynaptic cells belong to the same neuronal type [START_REF] Branco | The probability of neurotransmitter release: variability and feedback control at single synapses[END_REF].

On the other hand, the experimental preparations that have been commonly used for the study of synaptic physiology correspond to "special" types of synapses that do not represent the vast majority of the central synapses. These are: the amphibian neuromuscular junction, the squid giant synapse, and the mammalian Calyx of Held. These synapses have been very useful as models; however, they markedly differ in several important aspects with the great majority of the central synapses;

for example, central synapses are usually smaller (up to orders of magnitude) and possess well-defined synaptic contacts that are morphologically and functionally differentiable from each other [START_REF] Schikorski | Quantitative ultrastructural analysis of hippocampal excitatory synapses[END_REF]Stevens, 1997, Nusser et al., 1997).

As mentioned before, one important parameter of the synapse is the size, especially if we consider that a decisive and often limiting aspect in the study of central synapses is the relationship between the amplitude of the response evoked by an AP and the quantal response. Unlike the amphibian neuromuscular junction, the squid giant synapse or the Calyx of Held, where the quantal responses produce only modest fluctuations in the AP-evoked response, in the central synapses the quantal and AP-evoked responses can be similar in magnitude, hence the influence of a single quantum in the AP-evoked response becomes much more significant.

Although the probability of a single quantum to be released (p) does not differ markedly between central synapses and the traditional models of synaptic transmission, these last synapses have a quite distinctive feature that makes them very special: they present a very low probability of failures in the transmission process, thus ensuring that NT is released every time that an AP arrives to the presynaptic terminal. In the amphibian neuromuscular junction and the squid giant synapse, a low failure rate confers to the animal the ability to reliably move away from potential life-threatening dangers, while the Calyx of Held confers the ability to efficiently locate the source of a sound. These exceptional features are a direct consequence of the high number of actives zones (hundreds) in the presynaptic terminal, which allow them to have both a high probability of an AP generating NT release (synaptic reliability), and also a high average postsynaptic response (synaptic efficacy). Contrary to this, a typical central synapse in the brain has a single active zone in the presynaptic terminal and presents a high rate of failures, which translates directly into a low synaptic reliability and efficacy in the process of synaptic transmission. Although this may be seen as a disadvantage, in evolutionary terms this feature plays a fundamental role: it allows the neurotransmitter release process to be modulated within a wide dynamic range, which is extremely important for the phenomenon of synaptic plasticity and in consequence for the processes of memory and learning [START_REF] Kandel | Principles of neural science[END_REF]. A comparison between the sizes of the traditional synaptic models and a typical central synapse is presented in Fig. 12.

Considering the arguments presented so far, it should be evident that studying synaptic transmission and its mechanisms of modulation, as for example the SSDmediated facilitation in more representative models of synaptic transmission and at the level of individual synapses is crucial. Nevertheless, it is also clear that the experimental interrogation of a single and small synapse that is immersed in the complexity of brain tissue is an extremely difficult task. It is absolutely necessary to overcome this barrier in order to understand the details of the mechanisms that could be governing the different aspects of synaptic physiology and that may account for the differences that have been observed among different synapses, for example, the differences in Ca 2+ dependency during the SSD-mediated facilitation.

Approaches for the study of single synaptic contacts in brain slices

Given the extreme difficulty of studying NT release at the level of single synaptic contacts, the problem has usually been solved by the use of cellular cultures. In these preparations the accessibility to the small and well-defined cellular structures makes relatively easy to induce the exocytosis of NT by direct electrical stimulation of the presynaptic terminals, to visualize the release events by performing imaging experiments and to perform recordings of synaptic current from single synaptic contacts [START_REF] Liu | Properties of synaptic transmission at single hippocampal synaptic boutons[END_REF][START_REF] Ryan | Optical detection of a quantal presynaptic membrane turnover[END_REF][START_REF] Forti | Loose-patch recordings of single quanta at individual hippocampal synapses[END_REF]. However, the relevance of these results is uncertain, since it is highly probable that the synaptic function is altered in this type of preparations. A more physiological approach, although more difficult, is to search for pairs of synaptically connected neurons in brain slices, since in some cases a single synaptic contact is established between them (Gulyás et al., 1993, Kondo andMarty, 1998).

One approach that has been extensively used is the technique called "minimal synaptic stimulation of single synapses", where extracellular electrical stimulation attempts to trigger release from a single presynaptic fiber, for example, in CA3 to CA1 connections in the hippocampus [START_REF] Stevens | Facilitation and depression at single central synapses[END_REF]. However, with this methodology it becomes very difficult to control of the extracellular stimulation well enough to ensure that a single presynaptic fiber is being stimulated.

The exocytosis-inducing effect of the α-latrotoxin has previously been used to study synaptic transmission between cerebellar MLIs at single synaptic contacts [START_REF] Auger | Heterogeneity of functional synaptic parameters among single release sites[END_REF]. This toxin, when applied in low doses, is able to stochastically activate one synaptic site in periods of about one minute, resulting in a burst of quantal signals arising from a single contact. This method has been used

to study the number, the unitary conductance and the opening probability of the postsynaptic channels in GABAergic synapses [START_REF] Auger | Heterogeneity of functional synaptic parameters among single release sites[END_REF] and to study the replenishment of vesicle and the degree of desensitization of AMPA receptors in glutamatergic synapses [START_REF] Crowley | Fast vesicle replenishment and rapid recovery from desensitization at a single synaptic release site[END_REF]. However, the main disadvantage of this approach is that it does not allow identifying the sites that are activated and is difficult to control the duration and intensity of the presynaptic stimulus.

On the other hand, at the level of individual glutamatergic synapses the so-called "quantal optical analysis" method has been used to study synaptic vesicular release; the method relies in visualizing the Ca 2+ rises produced by the opening of NMDA receptors on the postsynaptic dendritic spines [START_REF] Yuste | Mechanisms of calcium influx into hippocampal spines: heterogeneity among spines, coincidence detection by NMDA receptors, and optical quantal analysis[END_REF][START_REF] Oertner | Facilitation at single synapses probed with optical quantal analysis[END_REF]. This approach has provided convincing information in favor of multivesicular release in synapses where previous results obtained by using the minimal synaptic stimulation approach had been interpreted in favor of the onesite-one-vesicle hypothesis 4 . These Ca 2+ imaging experiments, while providing relatively high spatial resolution of synaptic events, do not reliably reflect the current through the postsynaptic receptors, and lack the temporal resolution required to analyze in detail the effects of receptor activation (as can be obtained by measuring the synaptic currents).

The ideal approach to study synapses at the level of individual synaptic contacts with high spatial and temporal resolution should be capable of detecting individual synaptic release events.

A method that allows studying single synaptic contacts between cerebellar MLIs without the disadvantages of the previous methodologies was recently developed.

It combines the photorelease of Ca 2+ from a photosensitive "cage" in the presynaptic terminal and the simultaneous measurement of postsynaptic currents by dual pre-and postsynaptic whole-cell recordings. This methodology has already been used successfully to estimate the size of the RRP at individual synaptic contacts in the same preparation used in the present study [START_REF] Trigo | Readily releasable pool of synaptic vesicles measured at single synaptic contacts[END_REF].

4. Hypothesis that propose that although a synaptic contact can contain several docked vesicles in the active zones, only one of them fuses with the plasma membrane and releases its content as consequence of the arrival of an action potential to the terminal.

Thesis project proposal

The discovery of the dual nature of the rapid communication between the soma and the presynaptic terminals (through the generation of APs and by analog-to-digital signaling) has profound implications in our understanding of the mechanisms by which the brain processes information: the analog signaling phenomenon can significantly modify the synaptic output that determines the functioning of cellular circuits, either through an increase in the amount of information transmitted by a particular neuron or by modulating the synaptic activity in groups of neurons subjected to fluctuations of membrane potential, such as those produced by oscillatory activity [START_REF] Alle | Analog signalling in mammalian cortical axons[END_REF].

Although today we know that this type of signaling is capable of modulating NT release in both excitatory and inhibitory synapses, we still have much to discover about the mechanisms by which this modulatory effect is set; for example, it is still unknown whether the Ca 2+ -dependency of this phenomenon is a particular feature of some experimental preparations or whether it is a general mechanism in central The evidence presented so far suggests that the analog signaling phenomenon in MLIs of the cerebellum could act through a mechanism that involves a subthreshold Ca 2+ -dependent modification of the dynamics of the occupation of the docking sites; this mechanism may depend on the activation of PKC and the downstream phosphorylation of some of the proteins of the release machinery, like MUNC18-1. This would generate changes in the amount of NT that is released both asynchronously as well as evoked by a suprathreshold increase in Ca 2+ levels, which in turn would generate changes in the reliability and/or efficacy of the synaptic transmission.

In this thesis work, we studied the relationship between the ADF phenomenon and the synaptic parameters accounting for NT release (like peak amplitude, charge and synaptic latency of postsynaptic currents). Additionally, we studied how possible changes on these parameters are associated with the occupancy probability of docking sites, and the size of the RRP at single synaptic contacts of cerebellar MLIs. In order to do this, we used a methodology that combines optical and electrophysiological techniques (see methods) that provides the temporal and spatial resolution required to perform direct measurements of the synaptic activity at the single synaptic level.

Hypothesis

The subthreshold Ca 2+ rises reached during analog-to-digital facilitation in cerebellar MLIs can effectively modulate GABA release by a mechanism that involves an increase in the occupancy probability of docking sites, which translates directly into an increase in the RRP size and an enhancement of the synaptic reliability/efficacy in single synaptic contacts.

General objective

To explore the effects of the direct manipulation of presynaptic subthreshold Ca 2+ levels on the synaptic parameters controling the features of the postsynaptic response, and their possible differences between individual GABAergic MLI-MLI synapses of the rat cerebellum.

Specific objectives

1. To study the nature of the RRP at the single synaptic level and its possible modulation by subthreshold Ca 2+ rises.

2. To directly determine the number of docking sites (NDS), their occupancy probability () and its putative modulation by subthreshold Ca 2+ changes.

3. To explore the effects of modulating subthreshold Ca 2+ influx on the features of PSC by the application of presynaptic SSD in pairs of synaptically connected MLIs.

6 Methods

General methods used in this work

In this thesis work we studied quantal release in single synaptic contacts between MLIs of the rat cerebellar cortex. Individual synaptic connections were interrogated by measuring quantal events evoked postsynaptically by presynaptic Ca 2+ uncaging with a localized laser spot and after establishing a recording from a synaptically connected pair of MLIs. Direct control of Ca 2+ levels in the sub-and

Preparation of cerebellar slices

Sagittal cerebellar slices from young (11-17 days old) Sprague-Dawley rats were obtained according to the established procedures used in the Brain Physiology Lab [START_REF] Llano | Synaptic-and agonist induced excitatory currents of Purkinje cells in rat cerebellar slices[END_REF]. Rats were killed by direct decapitation in accordance with institutional guidelines for ethical procedures. The cerebellar vermis was carefully dissected (see Figure 6-2) and placed in ice-cold bicarbonate-buffered solution (BBS; see composition below) that was constantly gassed with a mixture of 5%

(vol/vol) CO 2 and 95% O 2 .

The meninges that were still attached to the surface of the vermis were carefully removed by using tweezers under a stereomicroscope in order to facilitate the slicing procedure. 200 μm thick slices were cut with a vibroslicer (VT1200S;

Leica) in ice-cold BBS and then placed in an incubating chamber at 34°C for 45 min, to recover from the slicing damage. Thereafter, they were kept at room temperature until use. The slices were transferred to the recording chamber and used during a period of up to 8 h after the decapitation procedure. Synaptic connectivity was tested by delivering short depolarizing voltage steps of 1 ms to 0 mV to the presynaptic cell. Immediately after the connectivity test was successful (as ascertained by the presence of postsynaptic currents), TTX (0.2 µM final concentration) was included in the bath solution throughout the entire experiment in order to minimize contamination from AP dependent neurotransmitter release. Recordings from both pre-and postsynaptic cells were acquired at a sampling rate of 50 kHz and low-pass filtered at 2.9 KHz. Most data were obtained from cells located in the proximal 1/3 part of the molecular layer (basket cells). However, interneurons located in the outer 2/3 of the molecular layer (stellate cells) were also included.

Pure electrophysiological recordings in pairs of molecular layer interneurons

Molecular layer interneurons were selected following morphological criteria and The number of repetitions that can be performed under these experimental conditions is limited. The minimum and maximal number of repetitions obtained in this work was 10 and 46, respectively. This restriction is not a consequence of photodamage produced by the 405 nm laser because it does not happen when other cage compounds like glutamate or GABA are photolised in the extracellular milieu (Trigo et al., 2009b) using the exact same technique. It may be caused by oxidizing byproducts of photolysis (nitrosoacetophenone; Kaplan and Ellis-Davies, 1988), but the exact mechanism of this phenomenon remains to be explored. The traces correspond to a group of PSCs evoked by the photorelease of Ca 2+ at different laser energies. Laser timing is indicated by the black dashed lines. First and second pulses were separated by 30 ms; the grey dashed lines correspond to the end of the 5 ms interval after triggering the laser. Any postsynaptic event that has a first latency within this range was considered as part of the RRP. From the figure it is possible to observe that there was no second response for laser energies of 0.4 µJ (last 2 traces in green), thus we concluded that at this energy the RRP was fully released by the first laser pulse.

Determination of the readily releasable pool at a single synaptic contacts

The onset kinetics of all the detected events was analyzed for a time window of 100 ms after the laser pulse and histograms of synaptic latency were constructed.

Single exponential fit of the histograms were used to determine the event distribution and to set a criteria to estimate whether a fusion event belonged to the RRP. A time corresponding to 4 time constant (4 =10 ms) in the histogram of all events (in the 100 ms time window), at which 98% of the events have occurred, was defined to associate an event with the laser pulse. Considering this limit, normal and cumulative histograms of the first latencies were constructed to determine the superior limit to associate a first event with the laser pulse. where the differences (in terms of the synchronicity of fusion events) between physiological (AP-evoked) and laser-evoked responses can be observed. The optimal situation for a precise estimation of the RRP by using laser stimulation is when a high level of desynchronization of fusion events is achieved; unfortunately, this was not commonly observed. Nevertheless, the typical postsynaptic response to laser stimulation, which generates a partial level of desynchronization of the fusion events, provides valuable information that can be used to extract the synaptic parameters controlling NT release (Figure 6-5b) and also to estimate the number and the dynamics of individual fusion events in the presynaptic terminal.

Counting single fusion events and estimating docking site occupancy probability

The counting of individual fusion events was done in a first instance by detecting the number of inflexions in the PSC. Nevertheless, because typically the laserevoked Ca 2+ transients can only produce a partial desynchronization of the fusion process, this inspection is expected to fail in detecting some closely spaced fusion events; hence, this approach results in an underestimation of the number of vesicles in the RRP because it does not consider the differences observed in the amplitude of individual peaks. To overcome this limitation, we used the amplitude of single fusion events to correct our first estimation by adding quantal information. Single quantal responses are typically seen during the laser stimulation repetitions in any particular experiment either as a single and isolated event (for example at low laser energies, as in the first sweeps in Figure 6-4) or as the first inflexion that has the smallest size in the composed PSC. Figure 6-6 shows and example where the detected number of inflexions in the rising phase of the PSC does not account for the differences in the peak amplitude: there are some responses with the same number of inflexions but with obvious differences in the peak amplitude, for example, the first and last PSC (both present a single inflexion in the PSC, however, the last response is several times larger than the first one, thus they hardly reflect the same number of fusion events).

We used both the number of inflexion and the quantal amplitude to estimate the number of fusion events for each laser-evoked PSC. This approach gives a better estimate of the number of released quanta in the time period where more synchronicity occurs, which usually corresponds to the first detected valley, and where the lower level of saturation takes place.

By using these criteria, we estimate the average value of the RRP (RRP avg ), the number of docking sites in the presynaptic terminals (NDS), which correspond to the maximal observed value for the RRP across repetitions [START_REF] Trigo | Readily releasable pool of synaptic vesicles measured at single synaptic contacts[END_REF] and the docking site occupancy probability (). These parameters were calculated for each presynaptic terminal tested in this work, and according to the following equations.

1) RRPavg = ∑ RRPn n 1 n
; where "n" corresponds to the number of sweeps per experiment.

2)  = RRPavg NDS ; where NDS corresponds to the number of docking sites The image describes the counting methodology used in this work, and corresponds to a selected group of traces (from a total number of 38 repetitions). The detected inflexions in the curve are denoted by the sky-blue lines. The number of estimated quantum is denoted by the red asterisk. It is possible to observe that traces with the same number of inflexions can have very different peak amplitudes, thus the number of inflexion is not a good indicator of the number of fused vesicles (compare first and last traces). Hence, we used both the number of inflexion and the quantal amplitude to estimate the number of vesicles in the RRP for each repetition. When comparing the latencies of the postsynaptic events, an increase in both the mean value and the CV was observed in the laser-evoked responses (see Figure 7-1b, bottom histogram). This can be interpreted in two ways: late events may correspond to some vesicles of the RRP that were not released by the Ca 2+ levels reached after the arrival of an AP or they may correspond to replenishment of the docking sites from which release occurred (latency: 1.65 ± 0. For a given presynaptic terminal and across all the synapses tested here, the number of vesicles in the RRP varied from sweep to sweep but exhibited a fixed maximal value, which we interpreted as the number of docking sites in a particular presynaptic active zone [START_REF] Trigo | Readily releasable pool of synaptic vesicles measured at single synaptic contacts[END_REF]. For example, the synaptic terminal analyzed in Figure 7 In order to accurately categorize a fusion event as belonging to the RRP, and to set a criteria for a precise and reliable estimation of this important parameter, we determined the onset kinetics of all the detected events in a time window of 100 ms after the laser pulse (t=0 ms) for a total number of 31 individual synapses. Figure 7-2 shows the latency histograms at low (upper) and high (bottom) time resolutions both for all latencies (left) and for the first event latencies (first latencies, right).

From the exponential fit of the histogram of all latencies in Figure 7-2a (upper panel, left), it is expected that for a t=4 =4*(2.5) ms=10 ms, 98% of the events had already occurred. For this work we have hence considered as belonging to the RRP all the laser-evoked events in a time window between the laser pulse (t=0 ms) and 10 ms (Figure 7-2a, bottom). Additionally, from the first latencies histograms it is possible to observe that for this time window (0-10 ms), almost the totality of the events occur within the first 5 ms (Figure 7-2b, bottom), hence, we have considered only belonging to the laser-evoked RRP the events having latencies in a time window from 0 to 10 ms and with first latency values below than or equal to 5 ms. All together the results presented in this section show our ability to release and measure the RRP at the single synaptic level. influx that does not depend on subthreshold Ca 2+ rises or PKC activation (Rowan and Christie, 2017), but it depends on the inactivation of a K + conductance.

In the present work we took advantage of the methodology described so far, where one can effectively control and measure vesicle release without the complexity introduced by the coupling between the somatic and the axonal compartments. We tested the hypothesis that the subthreshold Ca 2+ increases (like the ones that are reached during ADF in MLIs) can effectively modulate GABA release by a mechanism that involves an increase in the occupancy probability of docking sites in single active zones, which translates directly into an increase in the RRP size.

In order to test this idea, we performed Ca 2+ uncaging experiments in single synapses, where a direct and reliable control of both sub-and suprathreshold Ca 2+ levels can be produced (see methods for further details). In the first set of experiments, we applied two different and interleaved laser stimulation protocols.

The control protocol consisted of a single suprathreshold laser pulse that released the RRP; the test protocol consisted of the same suprathreshold pulse preceded by a low intensity train of 5 subthreshold laser pulses at 10 Hz (200 ms delay) that generates an increase in the basal Ca 2+ level (see later section 7.4: Ca 2+ imaging at single synaptic contacts). For each experiment, the subthreshold laser energy was set to reach the maximal intensity that does not produce a postsynaptic response, while for the suprathreshold RRP-releasing laser pulse the energy was set to reach the minimal value that does not produce a postsynaptic response when a second pulse of the same energy was applied 30 ms later (see Methods for further details).

Figure 7-3 shows the experimental approach where the laser protocols and the PSCs are illustrated in black and red, for control and test conditions, respectively.

A clear alignment between the PSCs and the laser onset is observed (Figure 7-3a).

Additionally, it is possible to observe that the frequency of miniature GABAergic events before and after the suprathreshold laser pulse was relatively low (see Figure 7-3a, and the summary histograms of events for 21 individual synaptic contacts in the panels of Figure 7-3b).

The subthreshold laser energies were set to avoid the trigger of the vesicle fusion process, nevertheless there is a possibility that the elevations of Ca 2+ levels during the application of the train can increase the probability of neurotransmitter release, enhancing the frequency of miniature GABAergic events and modifying in consequence the number of available docked vesicles in the RRP previous to its experimental interrogation by the suprathreshold pulse. The results presented in the histograms (middle and bottom) of Figure 7-3b show that there was no significant change in the frequency of GABAergic miniature events between control and test conditions during the time window between the start of the train and the start of the suprathreshold pulse (-600 to 0 ms). This confirms our ability to control the Ca 2+ dependent triggering of the vesicle fusion process. In order to obtain a precise and reliable measurement of the amount of GABA released in control and test conditions (and considering the presence of postsynaptic GABA A receptor saturation in the laser-evoked postsynaptic response), we decided to measure the following synaptic parameters of the PSC to have a better idea of the timing and amount of NT released by the suprathreshold laser pulse; peak amplitude, synaptic charge, rate of failures, first and all latencies (in the burst of multivesicular release) and time to peak. The results are presented in Table 2 and Table 3 and illustrated in Figure 7-4 and Figure 7-5.

The results show that when the relation between control and test condition for individual experiments is considered, a small but significant increase in both peak amplitude and synaptic charge (including failures) arises. This indicates that the amount of GABA released after applying the conditioning protocol is significantly larger than the interleaved control amplitudes without the train (peak amplitude ratio (test/control)=1.11 ± 0.04, p=0.0088; synaptic charge ratio (test/control)=1.18 ± 0.06, p=0.0061, Figure 7-4a and Figure 7-4c, respectively). Additionally, we evaluated whether the effect of subthreshold Ca 2+ rises can modify the dispersion of these parameters in single synapses. To do this we calculated the CV of the peak amplitude and synaptic charge for each condition in single experiments. The results show a significant decrease in the CV for synaptic charge but not for peak amplitude (see Table 2), indicating that the application of the conditioning protocol can also modify the degree of variation in the amount of GABA released (which accounts for the total charge crossing the postsynaptic membrane) (peak amplitude CV: 0.58 ± 0.06 vs. 0.51 ± 0.06, p=0.0576; synaptic charge CV: 0.65 ± 0.06 vs.

0.57 ± 0.06, p=0.0495 for control and test conditions, respectively).

The fact that the effect on synaptic charge was larger than the effect on peak amplitude (ratio test/control= 1.11 ± 0.04 vs. 1.18 ± 0.06, for peak amplitude and synaptic charge, respectively) is in agreement with what is expected if we consider that the integral of the curve (charge=current * time) is much more informative than the peak amplitude of PSCs, especially for synapses displaying postsynaptic receptor saturation. Thus, the synaptic charge is a better indicator of the amount of neurotransmitter that is released in a particular condition, as had been already

shown by [START_REF] Pulido | Vesicular release statistics and unitary postsynaptic current at single GABAergic synapses[END_REF]. The results shown so far for both peak amplitude and synaptic charge show that the amount of neurotransmitter released after applying the conditioning protocol is larger than that obtained in control conditions, thus indicating that subthreshold Ca 2+ rises can increase the number of docked vesicles that constitute the RRP in single presynaptic terminals. Next and regarding the analysis of the rate of failures in both conditions, if the hypothesis proposed in this work is correct, one would expect to obtain a lower number of failures in test condition as a direct consequence of an increased docking site occupancy probability, which will translate directly into an enhanced number of vesicles composing the RRP.

First, the results show a considerable number of failures in both conditions (See Table 2). This indicates that, under our experimental conditions, the presynaptic terminal goes through phases where there are no docked vesicles in the active zone to be released (meaning RRP=0). Our results further suggest that the failures that are often observed when the system is interrogated by using other methods, like electrophysiological paired recordings, can be a consequence of a combination of factors, like the presence of empty docking sites and failures in reaching the suprathreshold Ca 2+ levels required for triggering the fusion of a docked vesicle, like for example as a consequence of AP propagation failures.

Concerning the differences in the rate of failures in control and test conditions, the results show a not significant decrease (p=0.0654) in the number of failures after applying the conditioning train (control failure rate: 0.20 ± 0.04 vs. 0.16 ± 0.03, for control and test conditions, respectively, see Figure 7-4e). This result suggests that the increased levels of subthreshold Ca 2+ obtained by the application of the conditioning train of subthreshold laser pulses do not modify the average rate of failures between control and test conditions.

Next and regarding the possible differences in the release kinetics of the RRP, the results showed no significant difference in the averaged latency of all the events in the burst of multivesicular release after the application of the conditioning train (synaptic latency: 2.53 ± 0.18 ms vs. 2.55 ± 0.17 ms, p=0.4298 for control and test condition, respectively; ratio (test/control)=1.04 ± 0.05, p=0.1997; Figure 7-5a-I and Figure 5a-III). This result suggests that the conditioning treatment does not modify the average kinetics of the pool of fusion events that account for the RRP.

Nevertheless, a significant increase in the CV was observed in the test condition Figure 7-5a-II), probably as a consequence of the enhanced number of vesicles that account for the effect in the peak amplitude and synaptic charge, which possibly introduce a source of variability in the timing of fusion events, increasing in that way the heterogeneity of synaptic latencies (CV: 0.61 ± 0.04ms vs. 0.76 ± 0.05, p=0.0072, for control and test condition, respectively; n=21).

Although the averaged latency of all the events in the multivesicular burst of RRP release did not change with the conditioning treatment, a significant decrease in both the average latency and CV was observed for the first events in the burst of multivesicular release (synaptic latency: 1.81 ± 0. Considering that the results so far show an increase in the number of vesicles in the RRP and a decrease in the first latencies of PSCs as a consequence of subthreshold Ca 2+ rises, we next evaluated whether this increment in the number of docked vesicles can modify the timing at which the peak amplitude is reached with respect to the first latency, because a larger number of vesicles being released in a semiasynchronous manner should in theory take longer to reach the maximal concentration of GABA that is detected by the postsynaptic receptors. For this purpose, we measured the time to peak for each experiment by making the subtraction between the time at which peak amplitude was reached (peak time) and the first latency. The results of Table 3 7-5c, panels I, II and III).This suggests that while there is a larger amount of GABA being released in test condition, the release seems to occur in a more synchronized way, probably as a consequence of an increment in the probability of a docked vesicle to undergo exocytosis (as can be suggested by the decrease in the first latency), thus the time at which the peak of synaptic current is reached does not change.

Additionally, and for all of these temporal parameters the results show no significant correlation between the strength of the effect and the values obtained in control condition (Fig. 5 b-IV, c-IV; p value from Pearson (R) calculator: p=0.0649, p=0.7173, and p=0.2189, for all latencies, first latencies and time to peak, respectively).

Time dependency of the effects of subthreshold calcium rises on synaptic transmission

Considering that the previously described effect of SSD on synaptic transmission has been categorized as a facilitation type of synaptic plasticity (lasting for dozens to hundreds of ms), we decided to explore the time dependence of the effect of subthreshold Ca 2+ rises on the synaptic parameters that we have described so far. In order to do this, we increased the delay between the end of the subthreshold train and the RRP-releasing suprathreshold pulse from 200 ms to 2 s. The results obtained for this second pool of experiments are presented in Tables 4 and5, and illustrated in Figure 7-6, Figure 7-7 and Figure 78.

The results show that when the suprathreshold laser pulse was applied a long time (2 s) after the end of the subthreshold train, there was no change in the amount of GABA released (measured using peak amplitude and synaptic charge values) and in the failure rate (peak amplitude ratio (test/control)=0.93 ± 0.04, p=0.0684; synaptic charge ratio (test/control)=0.95 ± 0.05, p=0.1599; rate of failures: 0.14 ± 0.05 vs. 0.20 ± 0.06, p=0.1249; n=9; see Table 4 and Figure 7-6). Nevertheless, the observed tendency towards a depressing effect of the conditioning train applied 2 s before the suprathreshold laser pulse is probably due to an enhanced frequency of miniature events prior to RRP interrogation with the suprathreshold laser pulse. To correlation is significant for both the peak amplitude and synaptic charge analysis, suggesting that the strength of the effect (depression for this case) might depends on the location/or the size of the synapse (p value from Pearson (R) calculator: p=0.0321 and p=0.0346, for peak amplitude and synaptic charge, Figure 7-6b and These results additionally suggest that the mechanism(s) that account for the effect in the kinetic parameters that were observed in the previous set of experiments are vesicle-dependent, acting in a relatively short time scale and not lasting beyond the release of the "newly" docked vesicle(s). So once the vesicle(s) is released, the effect on the first latency is gone. All these results suggest that the facilitating effect of subthreshold Ca 2+ rises on synaptic transmission acts in the same time scale that the previously reported subthreshold somatic depolarization-induced facilitation, in the milliseconds range. Our results further suggest that the net effect on synaptic transmission (facilitation/depression) will depend on both the time at which the RRP is interrogated and the size or location of the synapse with respect to the postsynaptic neuron. We also evaluated whether the application of the conditioning protocol can modify the suprathreshold Ca 2+ transient that accounts for the release of the RRP. The results from Figure 7-9-c shows that the fluorescent signal evoked by the conditioning protocol did not return to the baseline before applying the suprathreshold pulse, thus indicating a slight enhancement in the level of basal Ca 2+ previous to the interrogation of the RRP (left inset). Regarding the kinetics of the suprathreshold Ca 2+ transients, the results showed no statistical differences between the time constants  1 and  2 and the amplitude A 1 and A 2 values from the double exponential fit that describe the recovery phase (A 1 = 0.4887 ± 0.0847,

 1 =0
.0579 s ± 0.0091, A 2 = 0.1867 ± 0.0402,  2 =0.4895 s ± 0.0268 and A 1 =0.2607 ± 0.0329,  1 = 0.1225 s ± 0.07005, A 2 = 0.4203 ± 0.1362,  2 =0.5829 s ± 0.3029, for control and test condition, respectively; n=3, Figure 7-9-d). However, an average increase in the recovery phase can be seen in the figure, probably as a consequence of a summation effect between the sub-and the suprathreshold Ca 2+ transients. ] is inferior to 170 nM (subthreshold level, no NT release). (c) Fluorescence (F/F0) signal of Ca 2+ transients for control (black) and test (red) conditions. In test condition, a train of 5 subthreshold laser pulses (range=0.0085-0.0245uJ) was applied 600 ms before the high energy pulse stimulation (range=01-04 uJ). (d) Amplitudes (A1, A2) and decay time constants () obtained from the double exponential fit of the suprathreshold Ca 2+ transient.

Docking site occupancy probability accounts for the subthreshold calcium-dependent modulation of GABA release in single synaptic contacts

For any given experiment the RRP varies from trial to trial but exhibits a fixed maximal value (RRP max ) that most likely represents the number of docking sites in the presynaptic terminal [START_REF] Trigo | Readily releasable pool of synaptic vesicles measured at single synaptic contacts[END_REF][START_REF] Pulido | Vesicular release statistics and unitary postsynaptic current at single GABAergic synapses[END_REF].

With the intention to perform a detailed analysis of the mechanism(s) by which the subthreshold Ca 2+ rises modulates GABA release in MLIs, we performed vesicular release statistics by counting individual fusion events in control and test condition for both 200 ms and the 2 second delay. By doing this it is possible to obtain a reliable estimation of the RRP and the docking site occupancy probability in single presynaptic terminals.

We counted individual fusion events by both counting the number of inflexions in the PSC of each trace and by comparing their amplitudes with the value obtained for the release of a single quantum. This method allowed us to obtain a better estimation of the number of vesicles in the RRP to allow detecting possible differences between control and test condition (see methods for further details). The distribution of docking sites among all the synaptic terminals evaluated in this section was: 3.3%, 16.7%, 50%, 26.67% and 3.3% for 1,2,3,4 and 5 docking sites, respectively. Table 6 shows the summary of distribution of the number of docking sites for the two pools of experiments presented in this section. The results for the number of docking sites, RRP avg and docking sites occupancy probability are Interestingly, for the 200 ms and 2 s delays it is possible to observe that there was a slight negative correlation between the strength of the facilitating effect on both the RRP avg and the docking site occupancy probability and their initial values in control conditions (only significant for the docking site occupancy probability in (facilitation/depression) to the same conditioning stimulus, depending on their initial state in terms of RRP size and docking site occupancy probability. We hypothesized that if both mechanisms coexist, one way to dissect them is to consider that a signaling pathway requires one single step to trigger the cascade (an increase in Ca 2+ levels in this case), and then the cascade will continue even if the Ca 2+ returned to its basal level.

We decided to use a stimulation protocol where the presynaptic spike is triggered at 3 different times from the end of the SSD: a) 0 ms (no delay), b) 200 ms and c) 2 s. The three delays (test condition) were applied in an interleaved manner within the same experiment and each of them has its own control where no SSD was applied. The results are illustrated Figure 7-13 and presented in Table 9, Table 10 and Table 11 for peak amplitude of both PSC and autoreceptor current "ARC", (which corresponds to the current obtained by activation of the presynaptic GABA A receptors in the axon, see introduction) and synaptic latency, respectively.

A scheme of the protocol used is presented in Figure 7-13a. Although an average increase in PSC amplitude can be seen after the application of the SSD at no delay condition, the effect is larger and only significant at 200 ms delay. This result suggests that there is a summation process that requires time, probably as a consequence of a signaling pathway or another relatively slow mechanism(s) that is contributing to the net effect. In addition, and as it was expected from our previous results by using Ca 2+ uncaging, no significant effect was observed for a 2 s delay.

Discussion

In this study we found that local subthreshold Ca 2+ rises at single presynaptic terminals of young rat cerebellar MLIs enhances GABA release by a mechanism that is likely to involve an increase in the occupancy probability of docking sites, which directly translates into an increase in the number of vesicles available for immediate release under high Ca 2+ conditions (RRP). We also show that subthreshold Ca 2+ rises can decrease the latency of the first fusion events, thus enhancing the temporal precision at which released GABA is first detected by the postsynaptic receptors. The strength of these effects appears to depend on both the initial number of docking sites and their initial occupancy level: when the delay between the conditioning and the suprathreshold stimuli was 2 seconds, there was an enhancement of the asynchronous release that had decreased the initial occupancy level of docking sites previous to its interrogation by the suprathreshold pulse, hence weakening the facilitating effect or instead, even producing a depression in some synapses. Furthermore, synapses exhibiting larger presynaptic terminals (in terms of number of docking sites) tend to produce depression rather than facilitation. This occurs probably as a consequence of an elevated sensitivity to subthreshold Ca 2+ rises, which causes a decrease in the occupancy level of docking sites as well. Our results suggest that subthreshold Ca 2+ rises resulting from physiological processes like analog-to-digital signaling or periods of high suprathreshold activity leading to high levels of residual Ca 2+ can efficiently change the occupancy of docking sites at single synaptic contacts and thereby modulate the synaptic output in a synapse-specific manner. [START_REF] Thanawala | Presynaptic calcium influx controls neurotransmitter release in part by regulating the effective size of the readily releasable pool[END_REF]. This is relevant if we consider that the distance between docking sites and the high Ca 2+ microdomains (determined by individual or cluster of voltage-dependent Ca 2+ channels) can limit the percentage of docked vesicles that fuse with the plasma membrane. Here we have generated a high and uniform Ca 2+ transient; hence we propose that our estimate of the RRP includes all the vesicles in the docked state in the active zone (independent of the distance to the VSCCs), although it is highly likely that this value also includes the contribution of vesicles that can be in an intermediate state,

close to reaching the releasable-competent state of docking (priming state), and that perhaps can account for the average increase in the latency of all the events when comparing AP-evoked vs. laser-evoked responses (Fig 7 -1). These delayed vesicles cannot be considered as a consequence of a full replenishment from the reserve pool, because it takes hundreds of milliseconds to seconds for this process to occur (Takeshi [START_REF] Sakaba | Two Ca( 2+ )-dependent steps controlling synaptic vesicle fusion and replenishment at the cerebellar basket cell terminal[END_REF]. Instead, we think that they could reflect a semicompetent state of docking in the steps of the synaptic vesicle cycle.

One of the results of the present work confirms the previous observation that the RRP exhibits a high degree of fluctuation [START_REF] Trigo | Readily releasable pool of synaptic vesicles measured at single synaptic contacts[END_REF]. We interpreted this as reflecting spontaneous variations in the occupancy of docking sites. An alternative explanation could be that these fluctuations may reflect an incomplete recovery from the previous laser stimulation, nevertheless, this is highly unlikely considering that the inter-sweep interval for all the uncaging experiments was 1 minute, which is long enough to allow the full recovery from the previous laser stimulation. cooperativity of the release process; instead, a Ca 2+ -dependent increase of the RRP also contributes in one-third to the net effect [START_REF] Thanawala | Presynaptic calcium influx controls neurotransmitter release in part by regulating the effective size of the readily releasable pool[END_REF].

Hence, our results suggest that the Ca 2+ dependent modulation of the RRP size could be a general mechanism for modulation of the strength of central synapses.

The average increase in peak amplitude and synaptic charge observed in this work was relatively small (ratio test/control= 1.11 ± 0.04 vs. 1.18 ± 0.06, for peak amplitude and synaptic charge, respectively) in comparison with previous data from our laboratory showing an increase of around 42% when using a subthreshold somatic depolarization as the conditioning stimuli [START_REF] Bouhours | Somatic depolarization enhances GABA release in cerebellar interneurons via a calcium/protein kinase C pathway[END_REF]. We interpret that this difference might be due to the specific characteristics of the two protocols used. Bouhours et al, 2011 utilized a 10 s subthreshold somatic depolarization that generates a subthreshold Ca 2+ rise that extends for several seconds, and which is likely to set the initial occupancy state of docking site to a higher level than what we have achieved here by the application of the train of conditioning pulses (occupancy levels change from 0.53 to 0.58; see Fig. [START_REF] Auger | Heterogeneity of functional synaptic parameters among single release sites[END_REF][START_REF] Auger | Multivesicular release at single functional synaptic sites in cerebellar stellate and basket cells[END_REF][START_REF] Awatramani | Modulation of transmitter release by presynaptic resting potential and background calcium levels[END_REF][START_REF] Barclay | Phosphorylation of Munc18 by protein kinase C regulates the kinetics of exocytosis[END_REF][START_REF] Bouhours | Somatic depolarization enhances GABA release in cerebellar interneurons via a calcium/protein kinase C pathway[END_REF].

Additionally, because Bouhours et al., did not use a RRP-releasing suprathreshold stimuli (they used an AP), it is also highly probable that the effect on the peak amplitude resulted from changes in both release probability and RRP size; thus the net facilitating effect observed by Bouhours et al., would mainly reflect the release of a larger fraction of the RRP rather than an increase in the RRP.

From the results of Fig. 7-4, it is possible to observe that the effect on synaptic charge was larger than the effect on peak amplitude. This is in agreement with previous studies indicating that synaptic charge is a much better indicator of neurotransmitter release [START_REF] Pulido | Vesicular release statistics and unitary postsynaptic current at single GABAergic synapses[END_REF], because while peak amplitude reflects the maximum level of charges crossing the plasma membrane in a particular time, synaptic charge includes the contribution of individual fusion events occurring after the peak current level.

(ii) Synaptic latency: For the calyx of Held, it has been recently proposed that the large differences in strength and properties of short-term plasticity observed across glutamatergic synapses can be explained assuming that a fraction of vesicles in the active zone are in a "superprimed" state that confers them an enhanced release probability (Taschenberger, Woehlerb, and Neher, 2016). Furthermore, in the same study was suggested that the enhanced strength after post-tetanic potentiation or by the activation of the phospholipase-C-diacylglycerol (PLC-DAG) pathway result from an increase in the fraction of vesicles in the superprimed state. This effect is probably associated to the activation of the protein MUNC-13 which is an essential component of the release machinery that can control both forms of synaptic potentiation trough a regulation of the energy barrier for the fusion of synaptic vesicles [START_REF] Schotten | Additive effects on the energy barrier for synaptic vesicle fusion cause supralinear effects on the vesicle fusion rate[END_REF]. In this thesis work we show that subthreshold Ca 2+ rises can speed up GABA release and increase its temporal precision by decreasing both the average first latency values and its coefficient of variation CV (Fig. 7-5), thus suggesting that the same mechanism that appears to modulate the primed state of the RRP vesicles in the calyx of Held can be acting in GABAergic MLIs of the cerebellum. Furthermore, the fact that only the first latencies were affected by the conditioning stimuli suggests that a small fraction of the vesicles in the RRP adopt the superprimed state (in addition to the increase in the RRP size). Another possibility to explain our results is what has been suggested for the ribbon synapse, where the existence of an inverse relationship between the size of the RRP and the latency of the first fusion events has been proposed to mediate the fast vesicular release (Wittig and Parsons, 2008). It is possible that the same relationship could be valid for MLI synapses, in which case an increase in the RRP size could potentially increase the probability of any vesicle to undergo exocytosis, producing a decrease in the first latency values. Furthermore, it is also possible that this last effect and the superpriming effect could coexist within the same synapse.

(iii) RRP and docking site occupancy: In addition to the macroscopic analysis of PSC peak amplitude, charge and latency, we performed quantal analysis of the RRP at the level of single synaptic contacts. With this "microscopic" approach we measured the number of vesicles composing the RRP and the occupancy probability of docking sites before and after the conditioning treatment. Our results

show that presynaptic terminals of MLIs possess a small number of docking sites, ranging from 1 to 5, with a modal value of 3. The average number of vesicles composing the RRP was 1.70 and the average docking site occupancy probability was 0.53, which is slightly lower than previously reported values (1.9 and 0. Marty, 2017. Assuming that this is true, the most likely explanation for the depression/facilitation observed here is that synapses expressing larger initial values of docking site occupancy are more sensitive to the effect of the conditioning treatment. This can be understood if we consider that an elevated number of vesicles available for immediate release will increase the probability that any of them to fuse with the plasma membrane and undergo exocytosis, hence these synapses tend to release (asynchronously) part of the RRP previous to its interrogation with a high Ca 2+ pulse (this assumes that asynchronous and evoked release share the same vesicle pool). This means that while the conditioning stimuli increases the RRP it tends to be balanced by the accompanying increase in asynchronous release. Such a process will have a higher impact on the synapses exhibiting a high level of docking site occupancy or RRP size.

If this last suggestion is correct, it is logical to think that the net balancing effect will depend on the time window between the end of the conditioning stimuli and the suprathreshold pulse; in other words, the net effect will depend on how long is the interval before testing the RRP: the longer that interval, the higher the level of asynchronous release and the higher the probability of obtaining a small facilitating effect or even a depression. In fact, this is exactly what we observed here in the experiments where the delay between the end of the conditioning train and the test pulse was increased from 200 ms to 2 seconds. In this case, instead of facilitation a small depression (although not significant) was evidenced when looking at both macroscopic (peak amplitude and charge) and microscopic (RRP and docking site occupancy probability) parameters. All together, these results suggest that variations in docking site occupancy account for differences in short-term plasticity [START_REF] Pulido | Vesicular release statistics and unitary postsynaptic current at single GABAergic synapses[END_REF]. Furthermore, they highlight the importance of the docking sites for synaptic plasticity. uncaging (200 ms of delay). Furthermore, it appeared that there was a depression with 2 second delay (not statistically significant). These results confirm our previous findings and validate our main approach, where we supposed that the photorelease of Ca 2+ can mimic the subthreshold Ca 2+ levels reached during analog-to-digital facilitation. Nevertheless, the interpretation of these last results must be taken with caution because of the high free [Ca 2+ ] used in this set of experiments.

Possible mechanisms for the subthreshold calcium dependent modulation of the RRP and its relation to the analog-to-digital facilitation phenomenon in MLIs

Previous From these experiments it is possible to observe that the Ca 2+ levels reached during the conditioning treatment did not return to the basal level before interrogating the RRP, therefore it could be argued that the facilitating effect seen in our experiments was the result of the enhanced Ca 2+ during the suprathreshold laser pulse, which in turn would generate the fusion of a larger faction of the RRP. This is unlikely to be the case because, as mentioned before, the intensity of the suprathreshold laser pulse in control condition was set to release the entire RRP.

Overall our results present the first evidence that subthreshold Ca 2+ rises can modulate the RRP and its kinetics in presynaptic terminals of MLIs. The remaining obvious question concerns the molecular mechanism(s) underlying this effect. As

- 1 . 13 Figure 1 - 2 . 19 Figure 6 - 1 . 28 Figure 6 - 2 . 30 Figure 6 - 3 . 35 Figure 6 - 4 . 38 Figure 6 - 5 . 42 Figure 6 - 6 . 45 Figure 7 - 1 . 52 Figure 7 - 2 . 54 Figure 7 - 3 . 58 Figure 7 - 4 . 61 Figure 7 - 5 . 62 Figure 7 - 6 . 71 ixFigure 7 - 7 . 72 Figure 7 - 8 . 73 Figure 7 - 9 . 77 Figure 7 - 10 . 80 Figure 7 - 11 . 84 Figure 7 - 12 . 85 Figure 7 - 13 .

 1131219612862306335643865426645715272547358746175627671777278737977710807118471285713 Figure 1-1. Vesicular release model for a single synaptic contact between cerebellar MLIs. .......................................................................................................13 Figure 1-2. Traditional synapses used as synaptic transmission models and a typical central synapse. ............................................................................................19 Figure 6-1. Main components of the electrophysiological setup for simultaneous dual whole-cell patch-clamp recordings and photolysis experiments. ....................28 Figure 6-2. Dorsal and caudal view of the rat brain. ...............................................30 Figure 6-3. Fluorescence image and synaptic site localization of a synaptically connected pair of MLIs. ...........................................................................................35 Figure 6-4. RRP release by Ca 2+ uncaging from the cage compound DM-nitrophen (DMNP)....................................................................................................................38 Figure 6-5. Comparison between the optimal situation where a high level of desynchronization of single fusion events is obtained vs. the typical postsynaptic response evoked by presynaptic Ca 2+ uncaging at single synaptic contacts. ..........42 Figure 6-6. Quantal analysis and estimation of the RRP for each sweep. ...............45 Figure 7-1. Direct control of GABA release at single synaptic contacts by photorelease of caged Ca 2+ . ...............................................................................................52 Figure 7-2. Latency histograms of the laser-evoked PSCs. .....................................54 Figure 7-3. Experimental approach to evaluate the effect of subthreshold Ca 2+ transients on asynchronous and evoked synaptic transmission at single synaptic contacts. ....................................................................................................................58 Figure 7-4. Peak amplitude, synaptic charge and rate of failures for control and test conditions. ................................................................................................................61 Figure 7-5. All latencies, first latencies and time to peak for control and test conditions. ................................................................................................................62 Figure 7-6. Peak amplitude, synaptic charge and rate of failure for control and test conditions for 2 second delay. .................................................................................71

  : subthreshold somatic depolarization 4. ADF: Analog-to-digital facilitation 5. MLI: Molecular layer interneuron 6. VDCC: Voltage-dependent calcium channels 7. PKC: Protein kinase C 8. NMDA: N-methyl-D-aspartate 9. DAG: Diacylglycerol 10. RRP: Readily releasable pool 11. pNR: Probability of neurotransmitter release 12. PSC: Postsynaptic current 13. p: Probability of a single quantum to be released 14. IS: intracellular solution xiii 15. OGB-1: Oregon Green BAPTA-1 16. DMNP: DM-Nitrophen xiv A mi amada esposa, quien con su amor y coraje me recuerda día a día el real sentido del éxito.

  function of others proteins by the phosphorylation of their hydroxyl group of Ser/Thr amino acid residues. The conventional isoforms of PKC (PKCα, PKCβI/βII, and PKCγ) are expressed in several tissues (including the brain) and require the binding of the lipid second messenger diacylglycerol (DAG) and Ca 2+ for their full activation (Hirokazu Hirai, 2017). The effects of the activation of the conventional isoforms of PKC by DAG (through its C 1 binding domain) or directly by Ca 2+ (through its C 2 domain) have been implicated in many studies with the induction of synaptic enhancement/augmentation (Francis et al., 2002, Majewski and Iannazzo, 1998, Stevens and Sullivan, 1998). There is also evidence that PKC plays a critical role in the phenomenon of post-tetanic potentiation 2 , which results from a high-frequency or "tetanic" stimulation of 30-50 or 100 Hz (commonly, 50 Hz for 5 seconds). (Alle et al., 2001, Beierlein et al., 2007, Brager et al., 2003; Fioravante et al., 2011; Korogod et al., 2007; Lee et al., 2008; Wierda et al., 2007).

  phenomenon of temporal filtering by facilitating or impeding the transmission of different patterns of activity(Fortune and Gary, 2000). Additionally, post-tetanic potentiation is thought to correspond to one of the mechanisms that significantly contribute to the formation of short-term memory, synaptic filtering and information processing in the nervous system(Abbott and Regehr, 2004; Klug et al., 2012; Silva et al., 1996). Many forms of regulation of neurotransmitter release are mediated through an increase in the size of the readily releasable pool (RRP) (fraction of vesicles in the active zone available for immediate release upon strong stimulation) and/or by an increase in the probability of NT release (pNR) (Pan and Zucker, 2009, Regehr et al., 2009, Zucker and Regehr, 2002).

From a functional

  perspective, knowing which of the two mechanisms (changes in pNR or the RRP size) is governing the properties of NT release in different synapses is very important, since they have very different outcomes in response to the application of high frequency stimulation trains[START_REF] Pan | A general model of synaptic transmission and short-term plasticity[END_REF][START_REF] Thanawala | Presynaptic calcium influx controls neurotransmitter release in part by regulating the effective size of the readily releasable pool[END_REF]: an increase in RRP proportionally increases the amplitude of the synaptic responses (an effect similar to increasing the number of postsynaptic receptors), while an increase in pNR has also the effect of increasing the use-dependent depression of the response. In this way under tetanic stimulation, the total amount of NT released is doubled if the RRP is doubled, while it essentially remains unchanged if pNR doubles[START_REF] Chu | Calcium-dependent PKC isoforms have specialized roles in short-term synaptic plasticity[END_REF].Proteins of the release machinery (SNAP25, MUNC18-1, GAP-43, and synaptotagmin-1) and some voltage-dependent ion channels (K + and Ca 2+ ) have been identified as putative presynaptic substrates of PKC, whose phosphorylation could be involved in the phenomenon of synaptic plasticity. However, with the notable exception of MUNC-18, evidence for a role of the rest of the proteins in the PKC-mediated potentiation is either incomplete or has been ruled out[START_REF] Parfitt | Phorbol esters enhance synaptic transmission by a presynaptic, calcium-dependent mechanism in rat hippocampus[END_REF][START_REF] Finley | SNAP-25 Ser187 does not mediate phorbol ester enhancement of hippocampal synaptic transmission[END_REF][START_REF] Hulo | A point mutant of GAP-43 induces enhanced short-term and long-term hippocampal plasticity[END_REF][START_REF] Nagy | Different effects on fast exocytosis induced by synaptotagmin 1 and 2 isoforms and abundance but not by phosphorylation[END_REF]. MUNC18-1 is an essential component of the vesicle fusion complex and is indispensable in the process of NT release[START_REF] Verhage | Synaptic assembly of the brain in the absence of neurotransmitter secretion[END_REF][START_REF] Zilly | Munc18-bound syntaxin readily forms SNARE complexes with synaptobrevin in native plasma membranes[END_REF].

  -specific differences in the number of docking sites and their occupancy probability as key parameters in the subthreshold somatic depolarization-mediated synaptic facilitation Central mammalian synapses differ substantially among each other in several key parameters like quantal size (postsynaptic response to the release of NT from a single vesicle), pNR and short-term synaptic plasticity. One of the most basic parameters within the set of synaptic heterogeneities is the size. It is known that differences in synaptic size can reach up to an order of magnitude, and that the dimensions of the presynaptic active zones and the postsynaptic densities, as well as the number of synaptic vesicles and the number of postsynaptic receptors, vary in direct proportion with the synaptic size[START_REF] Harris | Dendritic spines of rat cerebellar Purkinje cells: serial electron microscopy with reference to their biophysical characteristics[END_REF], Schikorski and Stevens, 1997, Nusser et al., 1997). Recently, it was demonstrated in the Calyx of Held (by local electrophysiological recordings in the presynaptic terminal, Sheng et al., 2012) and in hippocampal glutamatergic synapses (by optical quantal analysis, Holderith et al., 2012; see later section 1.5: Approaches for the study of single synaptic contacts in brain slices) that the number of Ca 2+ channels and the corresponding pNR increases linearly with the size of the active zone.In a recent publication using MLIs of the cerebellum as a model, the differences between presynaptic parameters (such as the number of docking sites and release probability) and postsynaptic parameters (such as the amplitude and kinetics of the synaptic currents) were explored at the level of single synaptic contacts. This was carried out using an approach that allows studying individual contacts by simultaneously recording the pre-and postsynaptic activity in pairs of connected neurons in slices of rat cerebellum[START_REF] Pulido | Vesicular release statistics and unitary postsynaptic current at single GABAergic synapses[END_REF], based on the fact that a significant percentage of the MLI-MLI connections consist of single synaptic contact with a single active zone and a single postsynaptic density[START_REF] Nusser | Differences in synaptic GABA(A) receptor number underlie variation in GABA mini amplitude[END_REF][START_REF] Kondo | Synaptic currents at individual connections among stellate cells in rat cerebellar slices[END_REF]. Pulido et al. (2015) used a simple binomial model (briefly explained in Figure 1-1)

Figure 1 - 1 .

 11 Figure 1-1. Vesicular release model for a single synaptic contact between cerebellar MLIs.The figure illustrates the active zone, the docking sites, the docked vesicles (RRP), the recycling vesicle pool (which fill the empty docking sites after release) and the postsynaptic receptors of a single synaptic contact. This central synapse, unlike the typical models of synaptic transmission (the calyx of Held, the amphibian neuromuscular junction, or the squid giant synapse), has a single active zone associated with a single postsynaptic density. In this model of Pulido et al., (2015), NT release is described according to the following assumptions: a synapse contains "N" independent docking sites, each of which has a probability () of being occupied by a vesicle. At the same time, a docked vesicle has a probability (p) of fusing with the plasma membrane and releasing its content to the synaptic cleft. After the exocytosis, each empty docking site will be refilled according to a replacement constant rate "R".

Figure 1 - 2 .

 12 Figure 1-2. Traditional synapses used as synaptic transmission models and a typical central synapse. (a) Image of the calyx of Held and its large presynaptic terminal in the mammalian brain stem. The calyx was filled with Lucifer Yellow. The presynaptic terminal and the postsynaptic cell body are presented in pseudocolors, yellow and blue, respectively (adapted from Meinrenken, Borst, and Sakmann 2003). (b) Giant synapses in the squid nervous system. The squid brain possesses "firstorder" giant neurons (left) whose activation excites second-order giant neurons (middle) that make connections with the stellate ganglia at the squid mantle. The giant axons of several third-order giant neurons (~1 mm in diameter; right) radiate from the ganglia to the mantle muscles and cause contraction (Animal Physiology, Fourth edition, box extension 12.3 Giant Axons). (c) Electron micrograph of a cross-section through the nerve terminal of the frog neuromuscular junction where the actives zones are indicated by the white arrows (adapted from S. O. Rizzoli and W. J. Betz, 2005). (d) Electron micrograph of a central synapse in the brain (image by Linnaea Ostroff and adapted from Cell Biology by the Numbers by Ron Milo, Rob Phillips).

  synapses. On the other hand, we do not know what are the parameters associated with the vesicular cycle that could be modified during SSD-mediated facilitation and if they exhibit or not variations among individual synaptic contacts within the same neuron.In cerebellar MLIs, a SSD-dependent increase in both asynchronous release (during SSD) and AP-evoked release (after SSD) has been reported(Glitsch and Marty, 2009;[START_REF] Trigo | Readily releasable pool of synaptic vesicles measured at single synaptic contacts[END_REF][START_REF] Bouhours | Somatic depolarization enhances GABA release in cerebellar interneurons via a calcium/protein kinase C pathway[END_REF][START_REF] Christie | Ca 2+ -dependent enhancement of release by subthreshold somatic depolarization[END_REF]. It has also been suggested that the strength of the effect on AP-evoked NT release could depend on the availability of fusion-competent vesicles to release their content upon the arrival of an AP[START_REF] Christie | Ca 2+ -dependent enhancement of release by subthreshold somatic depolarization[END_REF].

  suprathreshold range was made by varying the laser intensity. The presynaptic cell was recorded with an intracellular solution (IS) containing a high millimolar concentration of the Ca 2+ cage DM-nitrophen and the fluorescent dye Alexa 594 to allow identification. In some experiments, the Ca 2+ indicator Oregon Green BAPTA-1 (OGB-1) was included in the IS to monitor the laser-evoked Ca 2+ changes. The postsynaptic cell was recorded using a KCl-based IS to obtain large quantal sizes. Alexa 488 was included to distinguish the postsynaptic MLI from the presynaptic MLI. An additional set of experiments were performed using purely electrophysiological interrogation of synaptic contacts in pairs of connected MLIs, where subthreshold somatic depolarizing steps were delivered to modulate synaptic transmission. Figure6-1 shows the principal components of the setup for simultaneous dual patch-clamp recording and photolysis experiments.

Figure 6 - 1 .

 61 Figure 6-1. Main components of the electrophysiological setup for simultaneous dual whole-cell patch-clamp recordings and photolysis experiments.

Figure 6 - 2 . 1 5 MgCl 2 , 4 . 2

 6215242 Figure 6-2. Dorsal and caudal view of the rat brain. (a) Illustration of the dorsal aspect of the brain. Red asterisks denote the structures relevant for this work: the cerebellum and the cerebellar vermis (https://instruct.uwo.ca/anatomy/530/ratpix.pdf). (b) Picture showing the caudal view of the head of a rat where the vermis has been removed.

6 . 4

 64 photosensitive Ca 2+ chelator DM-nitrophen (Kaplan and Ellis-Davies, 1988; see

Figure 6 - 3 shows

 63 and example for the reconstruction of a pair of connected MLIs and the localization of the interrogated synaptic contact.

Figure 6 - 3 . 5

 635 Figure 6-3. Fluorescence image and synaptic site localization of a synaptically connected pair of MLIs. The image corresponds to the composite of the individual z-projections of the stacks taken at 594 nm and 488 nm of excitation. Cells are presented with pseudocolors: presynaptic in red, postsynaptic in green. The arrow indicates the site chosen for uncaging.

Figure 6 - 4 .

 64 Figure 6-4. RRP release by Ca 2+ uncaging from the cage compound DM-nitrophen (DMNP).The traces correspond to a group of PSCs evoked by the photorelease of Ca 2+ at different laser energies. Laser timing is indicated by the black dashed lines. First and second pulses were separated by 30 ms; the grey dashed lines correspond to the end of the 5 ms interval after triggering the laser. Any postsynaptic event that has a first latency within this range was considered as part of the RRP. From the figure it is possible to observe that there was no second response for laser energies of 0.4 µJ (last 2 traces in green), thus we concluded that at this energy the RRP was fully released by the first laser pulse.

6 . 6

 66 Calcium imagingFor Ca2+ imaging experiments, 200 µM of the high-affinity indicator (K d =170 nM) Oregon Green BAPTA-1 (OGB-1) (Invitrogen) was added to the presynaptic IS (see composition above) to monitor changes of Ca 2+ upon photolysis of DMNP.The axonal compartment of the recorded cell was identified by using Alexa 594 and the selected varicosity for uncaging was imaged after letting the dye diffuse for at least 10 min after break-in. A subregion of the CCD comprising sixteen 4 × 4 binned pixels, giving a resolution of 1 μm, was imaged in 2.11 ms exposure at 259.7 Hz. Fluorescence was corrected for background, determined in pixels at the periphery of the subregion. The traces shown in this work are averaged responses from individual varicosities in which averaging was done from at least 5 repetitions. The experiments were performed in the same experimental set-up as detailed for the uncaging work (see above). Imaging data was analyzed with IGOR Pro (Wavemetrics) using a routine written in the laboratory by Brandon M. Stell and Jorge Ramirez. The fluorescence signals are reported as F/F 0 corresponding to changes in background subtracted fluorescence with respect to the values before stimulation (F: F-F 0 ), normalized to the pre-stimulus values (F 0 ). For the analysis of the kinetics of the axonal Ca 2+ signal, exponential functions were fitted to the recovery phases of the responses.

  A time limit corresponding to t = 5 ms (at which almost 100 % of the first events have occurred) was defined as the superior limit to associate a first event with the laser pulse. Hence, in this work any event (or series of events) was considered as part of the RRP if it had latency values up to 10 ms and first latency values were below than or equal to 5 ms. Peak amplitude, synaptic charge (from 0-150 ms), rate of failures, first and all latencies and time to peak values were calculated for all the PSCs responses associated with the release of the RRP. These synaptic parameters were calculated for each of the individual and interleaved PSCs in control and test conditions. The comparison between conditions was made considering the average values per experiment. The statistical results are given as mean ± SEM; n is the number of independent experiments.As it was evidenced in Figure6-4, the postsynaptic response to local presynaptic Ca2+ uncaging typically results in a multiphasic pattern of PSCs where several inflexions points can be detected in the curve. These inflexions represent the semiasynchronous release of the NT from several synaptic vesicles in the presynaptic terminal, thus allowing a direct estimation of the number of vesicles composing the RRP. However, the precision in the counting of individual fusion events depends on the level of desynchronization achieved. Figure 6-5 shows examples of PSCs

Figure 6 - 5 .

 65 Figure6-5. Comparison between the optimal situation where a high level of desynchronization of single fusion events is obtained vs. the typical postsynaptic response evoked by presynaptic Ca 2+ uncaging at single synaptic contacts. (a) The photolysis of DMNP triggers the asynchronous release of 4 vesicles (green traces). A magnified view is presented in the right panel. The black asterisks represent individual fusion events. An example of the physiological AP-evoked PSC (purple traces), where a high level of synchronization is observed, was included for reference. (b) Laser-evoked postsynaptic response where a semi-asynchronous release of several synaptic vesicles is observed. In this case a progressive decrease in the amplitude of individual peaks is evidenced and presumably reflects the saturation of postsynaptic receptors. In this case the asterisks represent single inflexions of the curve, not necessarily single fusion events.

Figure 6 - 6 .

 66 Figure 6-6. Quantal analysis and estimation of the RRP for each sweep.The image describes the counting methodology used in this work, and corresponds to a selected group of traces (from a total number of 38 repetitions). The detected inflexions in the curve are denoted by the sky-blue lines. The number of estimated quantum is denoted by the red asterisk. It is possible to observe that traces with the same number of inflexions can have very different peak amplitudes, thus the number of inflexion is not a good indicator of the number of fused vesicles (compare first and last traces). Hence, we used both the number of inflexion and the quantal amplitude to estimate the number of vesicles in the RRP for each repetition.

7 Results 7 . 1

 771 photolysis with electrophysiological whole-cell voltage-clamp paired recordings

  -1 has 4 docking sites and displays RRP values ranging from 2 to 4 vesicles, with a modal value of 3 (see histogram of Figure 7-1c). A schematic representation of this presynaptic terminal is illustrated in Figure 7-1e.Because MLI-MLI synapses display postsynaptic receptor saturation following multivesicular release[START_REF] Kondo | Synaptic currents at individual connections among stellate cells in rat cerebellar slices[END_REF][START_REF] Auger | Multivesicular release at single functional synaptic sites in cerebellar stellate and basket cells[END_REF][START_REF] Pulido | Vesicular release statistics and unitary postsynaptic current at single GABAergic synapses[END_REF], the relation between GABA release and the PSC can be complex. As illustrated in Figure7-1d, the laser-evoked PSC exhibits sublinear amplitude summation when PSC peak amplitudes are plotted against the number of vesicles in the RRP. For the representative experiment of Figure7-1, the release of two or three vesicles, corresponding to 50% and 75% of the maximal value of the RRP that was observed (and which reflects the number of docking sites), generates a receptor occupancy level of 70% and 94%, respectively. These results suggest that postsynaptic receptor saturation should be taken into consideration when using maximal amplitudes of PSCs as a reliable indicator of the RRP size in terms of number of vesicles.

Figure 7 - 1 .

 71 Figure 7-1. Direct control of GABA release at single synaptic contacts by photo-release of caged Ca 2+ . (a) Schematic representation of the experimental approach. The pre-and postsynaptic cells were loaded with the fluorescent dyes Alexa 594 and Alexa 488, respectively, to allow neuron identification and discrimination between pre-and postsynaptic neurites. The presynaptic cell also contains the photosensitive Ca 2+ cage DMNP. Presynaptic Ca 2+ uncaging was achieved by delivering a brief (100-200 µs) and spatially confined laser pulse (~5 μm of diameter; 405 nm λ) in one of several synaptic contacts. (b) AP-(gray, left) and laser-evoked (blue, right) PSCs. Laser-evoked single fusion events are indicated by the red asterisks. PSC peak amplitude and latency histograms are shown for both stimuli. (c) Histogram of the number of vesicles in the RRP. (d) Relationship between number of vesicles in the RRP and PSC peak amplitude. (e) Schematic representation of the presynaptic terminal. The synaptic vesicles bind to the docking sites (black) previous to the exocytosis. The RRP is represented by the number of docked vesicles, which varies from trial to trial. Postsynaptic receptors are represented in purple.

Figure 7 - 2 . 7 . 2

 7272 Figure 7-2. Latency histograms of the laser-evoked PSCs. Histograms of PSC latencies for 31 synaptic contacts displayed at intermediate (Top; 3-ms bins; Freq, frequency) and high (bottom; 0.4-ms bins) time resolutions, for (a) all entries, and (b) first events. The triggering time of the laser pulse correspond to t=0 ms. The laser energy range was: 0.1-0.4 μJ. The intervals were fitted with a single exponential function (red curves superimposed on the histograms; upper panels) with decay constants of 2.5 ms and 1.63 ms, corresponding to all and first latencies, respectively. The cumulative percent of events is superimposed on each bar histogram (dashed curves).

Figure 7 - 3 .

 73 Figure 7-3. Experimental approach to evaluate the effect of subthreshold Ca 2+ transients on asynchronous and evoked synaptic transmission at single synaptic contacts. (a) Representative experiment illustrating the experimental approach. The control protocol consisted of a single suprathreshold laser pulse that releases the RRP; the test protocol consisted of the same suprathreshold pulse preceded by a low intensity train of five subthreshold laser pulses at 1 Hz (200 ms delay); TTX (0.2 µM) was included in the bath solution to avoid contamination from AP-dependent release. A low frequency of miniature events and a clear alignment of PSCs with the laser were observed. (b) Histograms of laser-evoked PSCs at low (Top; 100 ms bins; Freq, frequency) and intermediate (middle and bottom; 10ms bins) time resolution for the time window comprehended between the start of the subthreshold train and the start of the suprathreshold laser pulse (-600 ms to 0 ms; n=21). Middle panel also shows the averaged PSC traces for control (black) and test (red). The cumulative distribution of asynchronous (miniature) events is presented in the bottom panel; KS test, Kolmogorov-Smirnov test.

Figure 7 - 4 .

 74 Figure 7-4. Peak amplitude, synaptic charge and rate of failures for control and test conditions. (a) Amplitude ratio between test and control conditions. (b) Difference between test and control peak amplitudes as a function of the initial peak amplitude in control. (c) Charge ratio between test and control conditions. (d) Difference between test and control current charges as a function of the initial charge in control. (e) Summary data for the rate of failures in both conditions. Mean values ± SEM, one tail T-Test for two dependent means, and Pearson correlation coefficient P value; ns P > 0.05, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. n=21.

Figure 7 - 5 .

 75 Figure 7-5. All latencies, first latencies and time to peak for control and test conditions. Summary data for mean values, coefficient of variation, ratio (test/control) and relationship between the "strength" of the effect and the initial value in control condition (I, II, III and IV, respectively) for (a) all latencies, (b) first latency, and (c) time to peak. Mean values ± SEM, one tail T-Test for two dependent means, and Pearson correlation coefficient P value; ns P > 0.05, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. n= 21.

Figure 7 -

 7 Figure 7-6d, respectively).

Figure 7 - 6 .Figure 7 - 7 .

 7677 Figure 7-6. Peak amplitude, synaptic charge and rate of failure for control and test conditions for 2 second delay. Summary data for ratios (test/control) and the relationship between the strength of the effect and the initial value in control condition: (a) and (b) peak amplitude; (c) and (d) synaptic charge. (e) Summary data for the rate of failures in both conditions. Mean values ± SEM, one tail T-Test for two dependent means and Pearson correlation coefficient P value; ns P > 0.05, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. n=9.

Figure 7 - 8 .

 78 Figure 7-8. All latencies, first latencies and time to peak for control and test conditions for 2 second delay. Summary data for mean values, coefficient of variation, ratio (test/control) and relationship between the strength of the effect and the initial value in control condition (I, II, III and IV, respectively) for (a) all latencies, (b) first latency, and (c) time to peak. Mean values ± SEM, one tail T-Test for two dependent means and Pearson correlation coefficient P value; ns P > 0.05, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. n=9.

7 . 4

 74 Calcium imaging at single synaptic contactsIt is well known that during normal synaptic release, the magnitude of synaptic strength, which depends on the number of vesicles that fuse with the plasma membrane, relies on the local and short-lived Ca 2+ increase in the surroundings of the docking sites, near open Ca 2+ channels[START_REF] Regehr | Short-term presynaptic plasticity[END_REF]. These Ca 2+ rises (10-30 µM, Meinrenken, Borst, and Sakmann 2003) occurs in small and localized sites in the presynaptic terminal called "microdomains" (tens of nanometers in length) and exhibit both rapid rise and decay kinetic (tens of microseconds; see Smith & Augustine, 1988; Augustine & Neher, 1992). During the type of artificial laserevoked synaptic transmission performed in this work, the vesicle fusion process relies on the "global" and long-lasting Ca 2+ increase of the entire volume of the presynaptic terminal, which is high enough to induce the fusion of all the docked and release-competent vesicles.We evaluated the features of the sub-and suprathreshold laser-evoked Ca 2+ rises in single presynaptic terminals by using the kinetic properties of the high-affinity Ca 2+ indicator OGB-1, in a pool of experiments where the indicator was included in the presynaptic IS. Using the saturation properties of OGB-1, we estimated the Ca 2+ level reached during the conditioning test protocol in single varicosities. To this end, a train of 50 subthreshold laser pulses at 10 Hz (see Figure7-9-b) was applied. The fluorescence changes recorded with such protocol indicate that the K d of OGB-1 (170 nM) is reached at the 7 th pulse. This result demonstrates our ability to effectively control the basal Ca 2+ levels in single presynaptic terminals of MLIs. Additionally, they indicate that when applying the train of 5 laser pulses utilized in the previous set of experiments (see above), the global Ca 2+ concentration in the terminal stays in the nanomolar range).

Figure 7 - 9 .

 79 Figure 7-9. Modulation of Ca 2+ levels in the sub-and suprathreshold range. (a) Epifluorescence image of a representative MLI filled via patch pipette with DMNP, Alexa 594 and OGB-1. The inset shows a zoom of the varicosity where DMNP was photolized. (b) Fluorescence records (F/F0) of Ca 2+ transients triggered by the application of a train of 50 low-energy laser pulses. The yellow line corresponds to the fitting of the data with a Hill equation (yellow line); the x-half value corresponds to pulse # 7. This result indicates that at pulse # 7 the [Ca 2+] is inferior to 170 nM (subthreshold level, no NT release). (c) Fluorescence (F/F0) signal of Ca 2+ transients for control (black) and test (red) conditions. In test condition, a train of 5 subthreshold laser pulses (range=0.0085-0.0245uJ) was applied 600 ms before the high energy pulse stimulation (range=01-04 uJ). (d) Amplitudes (A1, A2) and decay time constants () obtained from the double exponential fit of the suprathreshold Ca 2+ transient.

Figure 7 - 10 .

 710 Figure 7-10. Representative experiment showing the approach for quantal analysis of the RRP in single synaptic contacts. (a) Example of the PSCs obtained from the application of the control and test protocols (black and red, respectively). The estimated number of vesicles is indicated by the asterisks. (b) Superimposition of all the traces obtained for control and test condition (19 repetitions each). The dashed grey lines corresponds to the laser onset (t=0 ms). A clear enhancement in the peak amplitude of PSCs is observed for the test condition. Right panel shows an amplified view where the effect on the first synaptic latency is also evidenced. (c) Schematic representation of the synaptic terminal interrogated during the experiment. (d) RRP histogram for both conditions (e) Summary data for the average value of the RRP and (f) the docking sites occupancy probability for control and test condition.

=

  RRP avg /RRP max presented in Tables7 and 8for 200 ms and 2 second delays, respectively, and illustrated in Figure7-11.When comparing both RRP avg and docking site occupancy probability between control and test conditions in 200 ms delay, a clear and significant increase in both parameters was observed (RRP avg : 1.70 ± 0.18 vs. 1.86 ± 0.16, p=0.0148; docking site occupancy: 0.53 ± 0.04 vs. 0.58 ± 0.03, p=0.0106, for control and test condition, respectively; Figure7-11a and Figure7-11b). These results suggest that subthreshold Ca 2+ rises can control the strength of synaptic transmission by modulating the dynamics of docking site occupancy in single synapses. For the 2 second delay pool, as expected from the previous analysis of peak amplitude and synaptic charge, the results show a not significant average decrease in the value of the RRP avg and docking site occupancy probability (RRP avg : 1.83 ± 0.27 vs. 1.74 ± 0.27, p=0.0845; docking site occupancy: 0.6 ± 0.05 vs. 0.56 ± 0.04, p=0.0586, for control and test condition, respectively).

Figure 7 - 11 .Figure 7 - 12 .

 711712 Figure 7-11. Quantal analysis of RRP release in control and test conditions for 200 ms and 2 s delay. (a) Summary data for RRPavg. (b) Summary data for . (c) Relation between the strength of the effect on the RRPavg and the initial value in control condition. (d) Relation between the strength of the effect on the docking site occupancy and the initial value in control condition. (e) Summary data for RRPavg for 2 second delay. (f) Summary data for docking site occupancy probability for 2 second delay. (g) Relation between the strength of the effect on the RRPavg and the initial value in control condition for 2 second delay. (h) Relation between the strength of the effect on the docking site occupancy and the initial value in control condition for 2 second delay. Mean values ± SEM, one tail T-Test for two dependent means; ns P > 0.05, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001

Figure 7 -

 7 Figure 7-13b shows a representative experiment (for 200 ms delay) where the test

Figure 7 - 13 .

 713 Figure 7-13. Presynaptic subthreshold somatic depolarization enhances AP-evoked release in MLIs. (a) Schematic representation of the protocols. In control condition the presynaptic MLI was clamped at -75 mV and a 0.1 ms pulse to 10 mV that triggers an unclamped action potential was applied. In test condition the holding potential was held at -50 mV for 2 seconds previous to the AP, which was triggered 200 ms (n=8), 0 ms (no delay, n=7) or 2 s (n=8) later. The order was randomized across experiments. (b) Presynaptic and postsynaptic traces from a representative experiment. Control condition is shown in black and test condition is shown in purple and red for both ARC and PSC, respectively.(c), (d) and (e) Summary plots for the ratios of PSC peak amplitude, ARC peak amplitude and synaptic latency, respectively, for the 3 different tested delays. One tail T-Test for two dependent means was used for the analysis of ratios while Mann-Whitney U Test was used to compare the ratios between different delays; ns P > 0.05, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.

8. 2

 2 Modulation of the RRP and docking site occupancy probability by subthreshold calcium levels and its impact on the features of the postsynaptic response (i) PSC peak amplitude and synaptic charge: In this work we tested the hypothesis that subthreshold Ca 2+ rises, in analogy to what has been reported during the phenomenon of analog-to-digital facilitation in MLIs, can effectively modulate GABA release by a mechanism that involves an increase in the occupancy probability of docking sites, which translates directly into an increase in the size of the RRP. We found that the application of a conditioning protocol consisting of a train of five low intensity laser pulses (10 Hz) produces an average increase in the peak amplitude and synaptic charge of the PSC. This was also accompanied by a decrease in the latency of the first fusion events (Fig7-4). These results are in agreement with what had been previously showed in the calyx of Held, where an increase in the Ca 2+ influx into the presynaptic terminal caused by changes in the external Ca 2+ concentration produces and increase in the size of the excitatory postsynaptic current (EPSC) that cannot be explained merely by the Ca 2+

8. 3

 3 Differences in synaptic output (facilitation/depression) depend on both the number and occupancy of docking sites and the timing at which the effect is evaluated (i) Uncaging experiments: Different synapses from the same neuron can express different forms of synaptic plasticity (Abbott and Regehr, 2004; Trommershauser et al., 2003; Reyes et al., 1998; Markram et al., 1998). It has been recently proposed that differences in the number of docking sites can account for differences in the properties of short-term synaptic plasticity in MLIs (Pulido et al., 2015; Pulido and Marty, 2017). Here we show that the strength of the facilitation effect evoked by subthreshold Ca 2+ rises seems to exhibit an inverse relationship with both, the initial value of PSC peak amplitude and/or the synaptic charge ("macroscopic" view) and the initial value of RRP and docking site occupancy probability ("microscopic" view): synapses with larger values of peak amplitude and charge are less facilitated than those with lower values. Furthermore, many of them exhibited depression instead of facilitation. The same tendency was observed when looking at the averaged values of RRP and the initial docking site occupancy state: synapses exhibiting values of RRP larger than 2 vesicles and/or docking site occupancy probabilities larger than 0.5 tend to exhibit depression instead of facilitation (Fig.7-4, Fig. 7-5 and 7-11). This can be understood if we consider that synaptic facilitation and depression are the consequence of an increased and decreased docking sites occupancy level, as recently discussed by Pulido and

(

  ii) Time dependency of the effect of subthreshold somatic depolarization in purely electrophysiological experiments: With the intention of testing our findings in a more physiological context, we performed electrophysiological experiments attempting to mimic the laser-evoked subthreshold Ca 2+ rises of our uncaging experiments by applying subthreshold somatic depolarizations in pairs of synaptically connected neurons, according to what has been described before for MLIs (Bouhours et al., 2011; Christie et al., 2011). Our results show that under these experimental conditions the application of a 2 second subthreshold somatic depolarization to the presynaptic neuron can produce a facilitation of the PSC depending on the delay between the sub-and the suprathreshold stimuli: we only observed a significant effect when the delay between the end of the subthreshold depolarization and the AP was equivalent to that used in experiments with Ca 2+

  mentioned in the introduction, the protein MUNC-18 is the most likely candidate to mediate the facilitating effect on GABA release. MUNC18-1 is an essential component of the release machinery[START_REF] Verhage | Synaptic assembly of the brain in the absence of neurotransmitter secretion[END_REF][START_REF] Zilly | Munc18-bound syntaxin readily forms SNARE complexes with synaptobrevin in native plasma membranes[END_REF]) that is phosphorylated by PKC under depolarization[START_REF] Craig | Physiological regulation of Munc18/nSec1 phosphorylation on serine-313[END_REF][START_REF] De Vries | Dynamics of munc18-1 phosphorylation/dephosphorylation in rat brain nerve terminals[END_REF] and has been implicated in the replenishment of vesicles under intense stimulation and in the control of the RRP in GABA and glutamatergic synapses[START_REF] Nili | Munc18-1 phosphorylation by protein kinase C potentiates vesicle pool replenishment in bovine chromaffin cells[END_REF][START_REF] Toonen | Munc18-1 expression levels control synapse recovery by regulating readily releasable pool size[END_REF]. On the other hand, the MUNC-13 protein (not related with MUNC-18), which is a component of the cytoskeletal matrix in the active zone and essential for all forms of vesicular release has been implicated in the superpriming phenomenon. As mentioned above, superpriming reflects the enhancement in the probability of NT release that is mediated by a decrease in the energy barrier for the fusion process to occur[START_REF] Lee | Superpriming of synaptic vesicles after their recruitment to the readily releasable pool[END_REF] Taschenberger et al., 2016). Like MUNC-18, MUNC-13 also presents C 1 and C 2 domains, possibly conferring to the protein modulation by Ca 2+ ions, PKC and DAG. Additionally, it has been shown that two different and essential signaling pathways generating synaptic potentiation can exhibit an interplay between each other: one that depends on PKC activation by DAG or Ca 2+ followed by phosphorylation of MUNC-18 (see introduction) and another that depends on the direct activation of MUNC-13 by DAG[START_REF] Wierda | interdependence of PKC-dependent and PKC-independent pathways for presynaptic plasticity[END_REF]. This interplay occurs because DAG potentiates the effects of residual Ca 2+ on synaptic transmission[START_REF] Lou | Allosteric modulation of the presynaptic Ca 2+ sensor for vesicle fusion[END_REF][START_REF] Wu | Protein kinase c increases the apparent affinity of the release machinery to Ca 2+ by enhancing the release machinery down-stream of the Ca 2+ sensor[END_REF], and residual Ca 2+ can also stimulate the production of DAG[START_REF] Micheva | Regulation of presynaptic phosphatidylinositol 4,5-biphosphate by neuronal activity[END_REF]). Here we propose that the subthreshold Ca 2+ influx during analog-to-digital facilitation activates these two signaling pathways, which in turn produce an increase in the probability of docking site occupancy, enhancing the amount of GABA released by increasing the RRP size and producing a superpriming of the vesicles after their recruitment. The most straightforward way to probe the implication of these two signaling pathways acting on MUNC-18 and MUNC-13, respectively, would be to perform uncaging experiments in single synaptic contacts of genetically modified animals in which the sensitivity of MUNC-18 to PKC and the sensitivity of MUNC-13 to DAG is abolished (knock-out of any of these proteins results in the total arrest of spontaneous and evoked neurotransmitter release). In this way, we could test our hypothesis and even determine the relative contribution of these two pathways in single synaptic contacts. Such experiments also have the potential of providing important information to determine whether there are differences among individual presynaptic terminals in the same cell.Another option would be to disrupt or alter each signaling pathway by using specific inhibitors of PKC and PLC, such as the PKC inhibitor Go694 and/or the PLC inhibitor U73122.

  KCl, 1.3 NaH 2 PO 4 , 26 NaHCO 3 , 25 glucose, 5 Na-Pyruvate, 2 CaCl 2 , and 1 MgCl 2 , with an osmolality of 300 mOsm/kg, and pH 7.4 when gassed with 5% (vol/vol) CO 2 and 95 O 2 .

The composition of the BBS for cutting, storing slices and recording was (in mM): 115 NaCl, 2.5

  14 ms vs. 2.56 ± 0.21 ms, p=0.0008; ratio (laser/AP)=1.69 ± 0.16, p=0.0002; and CV: 0.28 ± 0.04 vs. 0.57 ± 0.04, p=0.000004, for AP and laser-evoked PSCs, respectively). The latency and peak amplitude values for the AP-evoked and the laser-evoked PSCs analyzed in this work are presented in Table1(which can be found on the appendix).

  II and III). These results suggest that presynaptic subthreshold Ca 2+ rises can modulate the temporal precision of GABA at the time it is first detected by GABA A receptor in the postsynaptic compartment, by speeding-up GABA release and decreasing the CV of first latencies.

	10 ms vs. 1.64 ± 0.11 ms,
	p=0.0003; ratio (test/control)=0.91 ± 0.02, p=0.0005; CV: 0.46 ± 0.03 vs. 0.36 ±
	0.03, p=0.0027, for control and test condition, respectively; Figure 7-5b, panels I,

8.1 Approaches for release and measurement of the readily releasable pool in single synaptic contacts (

  i) RRP release: In this work we used an approach to study small synaptic contacts that relies on the local photorelease of Ca 2+ in single axonal presynaptic varicosities of MLIs in slices. The spatial restriction of the Ca 2+ signals that are produced by this method has been previously demonstrated to be sufficient to PSCs with both their amplitude (local peaks) and the quantal size for each synapse.as the number of vesicles that are bound to a few docking sites[START_REF] Alle | Combined analog and action potential coding in hippocampal mossy fibers[END_REF][START_REF] Alle | Analog signalling in mammalian cortical axons[END_REF][START_REF] Alle | GABAergic spillover transmission onto hippocampal mossy fiber boutons[END_REF][START_REF] Alle | PTP and LTP at a hippocampal mossy fiber-interneuron synapse[END_REF][START_REF] Auger | Quantal currents at single-site central synapses[END_REF] in the active zone[START_REF] Neher | Multiple roles of calcium ions in the regulation of neurotransmitter release[END_REF][START_REF] Ruiz | Active zones and the readily releasable pool of synaptic vesicles at the neuromuscular junction of the mouse[END_REF][START_REF] Sätzler | Three-dimensional reconstruction of a calyx of Held and its postsynaptic principal neuron in the medial nucleus of the trapezoid body[END_REF][START_REF] Nagwaney | Macromolecular connections of active zone material to docked synaptic vesicles and presynaptic membrane at neuromuscular junctions of mouse[END_REF]. However, recent findings suggest that not all but several (or many) of the vesicles in the docked state can contribute to the RRP, and that undocked vesicles can also be part of the RRP by rapid recruitment to activated ready-torelease docking sites[START_REF] Kaeser | The readily releasable pool of synaptic vesicles[END_REF]). In the same study it is also highlighted that estimates of RRP from Ca 2+ photolysis, hypertonic sucrose application or prolonged presynaptic voltage steps often provide larger values than the ones based on evoking RRP release by AP trains (which as mentioned before, is thought to represent a physiological situation). The main difference between these techniques is that the Ca 2+ rises evoked by the APs are both spatially an

	2015; see Figure 7-1). This strongly suggests that the synaptic signals presented
	here originate from single synaptic contacts. Our approach presents the advantage
	that photorelease permits an accurate and flexible control of Ca 2+ levels which can
	trigger vesicular release with submillisecond precision from unambiguously
	identified synaptic contacts. This allows the measurement of kinetic parameters
	without the inherent error of having unknown delays due to the propagation of the
	AP (Kondo and Marty, 1998). Additionally, our approach can generate a high
	enough level of desynchronization of individual fusion events to perform quantal
	analysis of the RRP. The main disadvantage of our approach is that is does not
	allow a high number of repetitions, probably as a consequence of oxidizing
	avoid triggering the unspecific release of neurotransmitter from neighboring byproducts from the photolysis reaction (Kaplan and Ellis-Davies, 1988),
	synaptic terminals (Trigo et al., 2012). Additionally, it has been previously precluding a more efficient statistical analysis. temporally constrained, creating what has been defined as Ca 2+ micro or
	demonstrated that in the postsynaptic side GABAergic synapses are sparse and (ii) RRP measurement: Here we used two approaches with different resolutions to nanodomains, while Ca 2+ rises produced by uncaging generate prolonged and
	compact, thus ensuring that GABA release from a single terminal reaches a single estimate RRP from single synaptic contacts; both of them rely on the features of unconstrained signals
	postsynaptic density (Nusser, Cull-Candy and Farrant, 1997). The main features of PSCs as a proxy of presynaptic GABA release. The first one consisted in using the
	the results presented here are in agreement with the hallmarks that have been values of peak amplitude, synaptic charge and latency as indicators of the amount
	previously used to identify synaptic release originating from single synaptic and timing of GABA release. The second approach consisted in combining the
	contacts: low variability of peak amplitude and occlusion between events that counting of individual inflexions in the multiphasic rising phase of laser-evoked
	occur closely in time (Auger and Marty, 2000; Trigo et al., 2012; Pulido et al.,

  studies have proposed that subthreshold somatic depolarizations can modulate vesicular release in MLIs in a Ca 2+ dependent way[START_REF] Bouhours | Somatic depolarization enhances GABA release in cerebellar interneurons via a calcium/protein kinase C pathway[END_REF][START_REF] Christie | Ca 2+ -dependent enhancement of release by subthreshold somatic depolarization[END_REF]. From those studies it is clear that the application of subthreshold somatic depolarizations generates an influx of Ca 2+ into the presynaptic terminal through voltage-dependent Ca 2+ channels. However, different mechanisms were proposed. Bouhours et al., 2011 have suggested that the amount of Ca 2+ entry associated to one action potential is not affected, while Christie et al., 2011 have suggested that subthreshold somatic depolarization facilitates AP-evoked Ca 2+ influx. Furthermore, more recently was proposed that subthreshold somatic depolarization modulates AP-evoked Ca 2+ influx by increasing the width of the AP waveform[START_REF] Rowan | Rapid state-dependent alteration in Kv3 channel availability drives flexible synaptic signaling dependent on somatic subthreshold depolarization[END_REF]. From a functional point of view, the net effect of subthreshold somatic depolarization is always the same: it increases in the amount of GABA that is released by the arrival of an AP.

	by a single high energy laser pulse would exhibit a slow recovery phase that is well
	described by a double exponential function, like previously reported for juvenile
	MLIs (basket cells) in the mouse (Collin et al., 2015), nevertheless, there was no
	significant change in the kinetic parameters from the double exponential fit
	between control and test condition. More experiments are needed to confirm this
	Nevertheless, knowing the presynaptic parameters affected during this type of because the observation relies in only three experiments.
	facilitation, it is fundamental to understand the processes governing synaptic
	plasticity. Unfortunately, none of those studies assessed the question of what
	specific parameters are modulated during analog-to-digital facilitation. Bouhours et
	al., 2011 suggested that the effect is mediated by a signaling pathway that involves
	local activation of PKC. However, Rowan and Christie, 2017 reported a facilitation
	of GABA release in PKC knock-out animals. Here we show that subthreshold
	somatic Ca 2+ rises modulates GABA release by increasing the occupancy
	probability of docking sites, thereby producing an increase in the size of the RRP.
	We show that this effect is independent of the Ca 2+ levels reached during the
	suprathreshold Ca 2+ pulse (as long as the RRP is depleted); our Ca 2+ imaging
	experiments show that the conditioning train of 5 low intensity laser pulses
	produces a subthreshold Ca 2+ increase in the nanomolar range (lower than 170
	nM). They additionally tend to suggest that the suprathreshold Ca 2+ rise generated

Table 2 . Peak amplitude and synaptic charge values for control and test condition

 2

		RRP Peak amplitude (pA)			RRP Synaptic charge (pC)	Failures (%)
	Nb	Ctl	CV	Tst	CV	Ratio Tst/Ctl	Ctl	CV	Tst	CV	Ratio Tst/Ctl	Ctl	Tst
	1	216.00 0.20 226.70 0.15	1.05	6.81 0.31 7.18 0.21	1.06	0.00 0.00
	2	220.00 0.71 267.70 0.57	1.22	4.20 0.73 4.77 0.58	1.14	0.27 0.18
	3	110.00 0.62 110.00 0.49	1.00	1.26 0.64 1.23 0.52	0.97	0.23 0.15
	4	161.60 0.44 154.10 0.43	0.95	5.44 0.50 5.45 0.49	1.00	0.13 0.13
	5	145.80 0.73 156.90 0.67	1.08	4.88 0.69 5.54 0.70	1.14	0.29 0.29
	6	97.33 0.88 127.30 0.65	1.31	1.90 1.01 2.93 0.72	1.54	0.40 0.27
	7	69.69 0.81 94.37 0.41	1.35	5.17 0.88 6.92 0.53	1.34	0.31 0.08
	8	35.14 0.72 33.32 0.79	0.95	0.55 0.76 0.61 0.80	1.12	0.29 0.36
	9	81.25 0.58 64.98 0.86	0.80	1.66 0.73 1.36 0.88	0.82	0.20 0.40
	10 148.60 0.48 175.80 0.63	1.18	2.94 0.59 3.73 0.78	1.27	0.05 0.16
	11 103.20 0.78 110.20 0.70	1.07	3.25 0.88 3.54 0.77	1.09	0.35 0.30
	12 622.80 0.18 571.70 0.49	0.92	10.76 0.30 9.72 0.63	0.90	0.00 0.11
	13 273.90 0.19 269.50 0.19	0.98	9.64 0.26 9.71 0.22	1.01	0.00 0.00
	14 206.70 0.85 326.50 0.34	1.58	4.66 0.94 9.13 0.41	1.96	0.38 0.08
	15	41.08 1.14 52.38 1.13	1.28	0.70 1.19 1.21 1.32	1.73	0.50 0.44
	16	72.77 0.91 89.00 0.75	1.22	1.32 0.99 1.62 0.79	1.23	0.45 0.36
	17 149.20 0.37 159.40 0.28	1.07	3.25 0.45 3.77 0.24	1.16	0.08 0.00
	18 566.50 0.31 566.00 0.26	1.00	15.72 0.31 15.17 0.23	0.97	0.00 0.00
	19 536.80 0.35 493.80 0.35	0.92	10.24 0.48 9.06 0.60	0.89	0.00 0.00
	20 300.60 0.18 291.50 0.16	0.97	10.72 0.23 9.56 0.18	0.89	0.00 0.00
	21 226.60 0.77 307.10 0.40	1.36	7.60 0.83 11.19 0.47	1.47	0.33 0.08
	Avg 208.84 0.58 221.35 0.51	1.11	5.36 0.65 5.88 0.57	1.18	0.20 0.16
	SEM 37.14 0.06 34.98 0.06	0.04	0.89 0.06 0.87 0.06	0.06	0.04 0.03

Table 3 . Temporal parameters of PSCs: All latency, first latency and time to peak values for control and test condition All latencies (ms) First latencies (ms) Time to peak (ms)

 3

						Ratio					Ratio				Ratio
	Nb	Ctl	CV	Tst	CV	Tst/Ctl Ctl	CV	Tst	CV	Tst/Ctl Ctl	CV	Tst	CV	Tst/Ctl
	1	1.76 0.78 1.69 1.14	0.96	1.09 0.31 0.97 0.30	0.89	2.90 0.48 2.23 0.19	0.77
	2	3.74 0.58 1.94 0.78	0.52	1.89 0.48 1.49 0.32	0.79	3.10 0.61 2.42 0.41	0.78
	3	3.23 0.73 3.43 0.80	1.06	2.10 0.67 1.80 0.48	0.86	3.12 0.36 2.85 0.96	0.91
	4	2.27 0.71 2.39 1.10	1.05	1.26 0.67 1.13 0.55	0.90	1.77 0.63 1.18 0.58	0.67
	5	2.20 0.69 2.36 0.78	1.07	1.79 0.52 1.57 0.40	0.88	1.17 0.60 1.16 0.52	0.99
	6	2.90 0.79 2.22 0.51	0.76	1.92 0.87 1.79 0.34	0.93	1.63 0.28 1.93 0.55	1.19
	7	2.00 0.37 2.18 0.62	1.09	2.00 0.37 1.83 0.27	0.91	4.21 0.48 5.34 0.34	1.27
	8	2.30 0.47 2.60 0.60	1.13	2.32 0.48 2.17 0.37	0.93	1.44 0.23 1.93 0.26	1.34
	9	1.96 0.41 1.88 0.62	0.96	1.86 0.44 1.39 0.28	0.75	1.43 0.30 1.58 0.58	1.11
	10	2.36 0.52 2.27 0.69	0.96	1.82 0.47 1.55 0.54	0.85	2.40 0.41 2.85 0.41	1.18
	11	2.43 0.50 2.59 0.62	1.07	2.43 0.50 2.09 0.60	0.86	1.19 0.34 1.54 0.42	1.29
	12	2.70 0.51 2.68 0.66	0.99	1.85 0.44 1.68 0.29	0.91	2.39 0.42 2.25 0.51	0.94
	13	1.11 0.28 1.49 1.13	1.35	1.11 0.28 0.98 0.17	0.89	1.98 0.30 2.00 0.24	1.01
	14	2.65 0.54 3.20 0.73	1.21	2.12 0.55 1.92 0.55	0.91	1.67 0.20 2.20 0.38	1.32
	15	4.86 0.56 5.14 0.52	1.06	2.91 0.44 2.98 0.28	1.02	2.04 1.13 3.36 0.80	1.65
	16	2.57 0.68 2.42 0.95	0.94	2.01 0.40 1.69 0.44	0.84	0.96 0.20 0.95 0.28	0.99
	17	1.72 0.64 2.30 0.75	1.34	1.14 0.28 1.33 0.40	1.17	1.84 0.44 2.13 0.58	1.15
	18	2.01 0.93 3.24 0.75	1.61	1.40 0.29 1.17 0.18	0.83	4.58 0.39 4.64 0.34	1.01
	19	2.13 0.94 1.86 1.19	0.88	1.26 0.45 0.96 0.25	0.76	1.95 0.30 1.66 0.24	0.85
	20	3.50 0.69 2.59 0.41	0.74	1.82 0.31 2.07 0.38	1.14	4.16 0.58 2.05 0.42	0.49
	21	2.74 0.58 3.17 0.55	1.16	1.87 0.44 1.98 0.18	1.05	2.23 0.60 3.44 0.79	1.54
	Avg 2.53 0.61 2.55 0.76	1.04	1.81 0.46 1.64 0.36	0.91	2.29 0.44 2.37 0.47	1.07
	SEM 0.18 0.04 0.17 0.05	0.05	0.10 0.03 0.11 0.03	0.02	0.23 0.05 0.24 0.04	0.06

Table 4 . Peak amplitude and synaptic charge values for control and test condition for 2 second delay RRP Peak amplitude (pA) RRP Synaptic charge (pC) (0-150 ms) Failures (%)

 4

	Nb	Ctl	CV	Tst	CV	Ratio Tst/Ctl	Ctl	CV	Tst	CV	Ratio Tst/Ctl	Ctl	Tst
	1	63.90 0.89 56.55 1.09	0.88	1.13 1.01 0.84 1.18	0.74	0.40 0.50
	2	220.30 0.15 216.10 0.38	0.98	3.74 0.32 4.31 0.47	1.15	0.00 0.10
	3	120.29 0.46 119.71 0.47	1.00	2.47 0.50 2.19 0.66	0.88	0.14 0.14
	4	139.54 0.45 102.18 0.85	0.73	1.57 0.71 1.62 0.96	1.03	0.09 0.36
	5	223.00 0.80 220.91 0.85	0.99	4.86 0.80 4.88 0.96	1.00	0.36 0.36
	6	537.90 0.21 406.70 0.61	0.76	7.03 0.36 5.68 0.71	0.81	0.00 0.20
	7	428.20 0.29 416.60 0.18	0.97	9.12 0.38 8.25 0.15	0.90	0.00 0.00
	8	229.56 0.42 220.89 0.40	0.96	7.48 0.49 6.13 0.55	0.82	0.11 0.11
	9	161.82 0.51 180.58 0.35	1.12	2.65 0.76 3.14 0.53	1.18	0.18 0.00
	Avg 236.06 0.46 215.58 0.58	0.93	4.45 0.59 4.11 0.68	0.95	0.14 0.20
	SEM 50.88 0.08 41.76 0.10	0.04	0.95 0.08 0.80 0.10	0.05	0.05 0.06

Table 5 . Temporal parameters of PSCs for 2 second delay: All latency, first latency and time to peak values for control and test condition All latencies (ms) First latencies (ms) Time to peak (ms)

 5

						Ratio					Ratio				Ratio
	Nb	Ctl	CV	Tst	CV	Tst/Ctl Ctl	CV	Tst	CV	Tst/Ctl Ctl	CV	Tst	CV	Tst/Ctl
	1	2.59 0.17 2.03 0.26	0.78	2.59 0.17 2.03 0.26	0.78	0.79 0.29 1.00 0.23	1.26
	2	2.52 0.57 2.35 0.57	0.93	1.82 0.54 1.37 0.53	0.75	2.13 0.77 2.88 0.57	1.35
	3	1.99 0.34 2.50 0.71	1.26	1.71 0.33 1.56 0.32	0.91	1.51 0.46 1.75 0.43	1.16
	4	1.24 0.50 1.27 0.53	1.03	1.24 0.50 1.21 0.58	0.98	0.90 0.28 1.20 0.50	1.33
	5	2.38 0.72 1.91 0.36	0.80	1.62 0.40 1.52 0.22	0.94	2.30 0.61 2.31 0.40	1.00
	6	2.08 0.54 2.72 0.59	1.31	1.78 0.52 2.21 0.57	1.24	1.61 0.62 1.91 0.82	1.19
	7	1.91 0.70 2.31 0.75	1.21	1.05 0.22 1.14 0.24	1.09	1.93 0.28 2.13 0.53	1.10
	8	3.41 0.54 3.07 0.53	0.90	2.14 0.38 2.31 0.28	1.08	3.30 0.52 2.16 0.30	0.65
	9	2.83 0.43 2.98 0.51	1.06	2.47 0.48 2.30 0.44	0.93	1.94 0.65 2.11 0.49	1.09
	Avg 2.33 0.50 2.35 0.53	1.03	1.82 0.39 1.74 0.38	0.97	1.82 0.50 1.94 0.47	1.13
	SEM 0.21 0.06 0.19 0.05	0.07	0.17 0.04 0.16 0.05	0.05	0.25 0.06 0.19 0.06	0.07

Table 6 . Distribution of docking sites among the synaptic terminals evaluated in this section.

 6

	Number of DS	% for 200 ms delay	% for 2 s delay	% for total n=30
	1	0.0	11.1	3.3
	2	19.0	11.1	16.7
	3	52.4	44.4	50.0
	4	23.8	33.3	26.7
	5	4.8	0.0	3.3

Table 7 . Number of docking sites, RRPavg and docking site occupancy probability values for 200 ms delay

 7

	Nb NDS	RRP avg Control RRP Test RRP	Ctrl		Test
	1	4	2.57	2.67	0.64		0.67
	2	4	1.64	2.00	0.41		0.50
	3	4	2.00	1.85	0.50		0.46
	4	3	1.73	1.87	0.58		0.62
	5	3	0.94	0.94	0.31		0.31
	6	3	0.93	1.20	0.31		0.40
	7	4	1.92	2.62	0.48		0.65
	8	2	0.64	0.79	0.32		0.39
	9	3	2.00	1.67	0.67		0.56
	10	3	1.42	1.68	0.47		0.56
	11	2	0.78	1.00	0.39		0.50
	12	3	2.39	2.00	0.80		0.67
	13	2	1.50	1.60	0.75		0.80
	14	3	1.15	2.08	0.39		0.69
	15	3	0.88	1.07	0.29		0.36
	16	2	0.91	0.91	0.46		0.46
	17	3	1.75	2.17	0.58		0.72
	18	4	3.50	3.62	0.88		0.91
	19	5	3.33	3.11	0.67		0.62
	20	3	2.40	2.40	0.80		0.80
	21	3	1.25	1.83	0.42		0.61
	Avg	3.1	1.70	1.86	0.53		0.58
	SEM 0.2	0.18	0.16	0.04		0.03

Table 8 . Number of docking sites, RRPavg and docking site occupancy probability values for 2 second delay

 8

			RRP avg		
	Nb NDS	Control RRP Test RRP	Ctrl	Test
	1	1	0.60	0.50	0.60	0.50
	2	4	2.80	2.70	0.70	0.68
	3	4	2.29	2.29	0.57	0.57
	4	2	1.09	1.00	0.55	0.50
	5	3	1.09	1.00	0.36	0.33
	6	3	2.20	1.80	0.73	0.60
	7	4	2.80	2.80	0.70	0.70
	8	3	2.33	2.11	0.78	0.70
	9	3	1.27	1.50	0.42	0.50
	Avg 3.00	1.83	1.74	0.60	0.56
	SEM 0.33	0.27	0.27	0.05	0.04

Table 9 . PSC peak amplitude values for the 3 different delays tested

 9

					Peak amplitude (pA)
			No delay		200 ms delay		2 s delay
	Nb Control	Test	Ratio Test/Control	Control	Test	Ratio Test/Control	Control Test	Ratio Test/Control
	1	47.19	50.77	1.08	38.58	44.67	1.16	55.80	60.40	1.08
	2	214.00 265.00	1.24	174.00 264.00	1.52	181.00 230.00	1.27
	3	79.00 128.00	1.62	83.00	81.00	0.98	103.00 74.00	0.72
	4	170.00 161.00	0.95	161.00 140.00	0.87	149.00 161.00	1.08
	5	56.00	46.00	0.82	46.00	44.00	0.96	70.00	66.00	0.94
	6	70.50	66.00	0.94	48.00	77.50	1.61	75.00	61.00	0.81
	7	47.50	81.00	1.71	39.50	98.30	2.49	63.50	44.30	0.70
	8	-	-	-	199.00 264.00	1.33	229.00 247.00	1.08
	Avg	97.7	114.0	1.2	98.6	126.7	1.4	115.8	118.0	1.0
	SEM	25.2	29.8	0.1	24.0	31.8	0.2	22.5	29.2	0.1

Table 10 . ARC peak amplitude values for the 3 different delays tested.

 10

					ARC Peak amplitude (pA)
			No delay		200 ms delay		2 s delay
	Nb Control	Test	Ratio Test/Control	Control	Test	Ratio Test/Control	Control Test	Ratio Test/Control
	1	238.92 290.22	1.21	232.98 251.16	1.08	236.99 245.52	1.04
	2	136.87 139.89	1.02	126.12 139.45	1.11	126.47 153.80	1.22
	3	25.35	38.86	1.53	72.76	68.77	0.95	52.97	56.93	1.07
	4	137.70 153.45	1.11	118.09 145.42	1.23	138.58 123.33	0.89
	5	204.05 233.56	1.14	211.43 203.50	0.96	202.90 219.20	1.08
	6	155.56 176.29	1.13	161.08 157.35	0.98	169.82 163.60	0.96
	7	88.43	88.39	1.00	79.38	79.38	1.00	73.19	76.82	1.05
	8	-	-	-	16.20	15.82	0.98	10.18	8.34	0.82
	Avg 140.98 160.09	1.17	127.26 132.61	1.03	126.39 130.94	1.02
	SEM 26.70	31.95	0.07	25.73	26.88	0.03	27.34	28.66	0.04

Table 11 . Synaptic latency values for the 3 different delays tested

 11

						PSC latency (ms)
			No delay		200 ms delay		2 s delay
	Nb	Control Test	Ratio Test/Control	Control Test	Ratio Test/Control	Control Test	Ratio Test/Control
	1	1.3	1.41	1.08	1.42	1.49	1.05	1.18	1.44	1.22
	2	1.49	1.55	1.04	1.61	1.45	0.90	1.61	1.45	0.90
	3	2.26	2.42	1.07	2.19	2.07	0.95	2.14	2.5	1.17
	4	1.38	1.86	1.35	1.66	1.82	1.10	1.76	1.86	1.06
	5	2.02	2.1	1.04	1.56	1.59	1.02	1.6	1.56	0.98
	6	1.56	1.58	1.01	1.525 1.52	1.00	1.47	1.495	1.02
	7	1.82	2.04	1.12	1.88	1.84	0.98	1.84	1.98	1.08
	8	-	-	-	2.07	2.07	1.00	2.16	2.05	0.95
	Avg	1.69	1.85	1.10	1.74	1.73	1.00	1.72	1.79	1.05
	SEM	0.13	0.14	0.04	0.10	0.09	0.02	0.12	0.13	0.04

Considering that the effect of SSD is evidenced in the "digital" AP-dependent neurotransmitter release.

test this idea, we next measured the frequency of GABAergic miniature events between control and test conditions during the time window comprehended from the start of the train of subthreshold laser pulses and the start of the suprathreshold RRP-releasing laser pulse (-2400 to 0 ms). The results (Figure 7-7) show that there was a significant increase in the frequency of miniature events after applying the conditioning train, thus confirming the partial emptying of the RRP previous to its experimental interrogation (see the cumulative histogram of Figure 7-7c).

Additionally, there was no significant change for all the resting parameters evaluated in the previous set of experiments (peak amplitude CV: 0.46 ± 0.08 vs. 0.58 ± 0.10, p=0.1000; synaptic charge CV: 0.59 ± 0.08 vs. 0.68 ± 0. Nevertheless, there was a negative correlation between the strength of the effect and the control value for this new set of experiments, and interestingly the During vesicular release, each docking site undergoes a cycle starting from an occupied state, followed by exocytosis and the docking of a new synaptic vesicle.

Considering this, we took advantage of the methodology described so far to calculate the following parameters: 1) Average value of RRP (RRP avg ), corresponding to the mean value obtained from the sequence of repetitions in each condition.

2) The number of docking site (NDS), corresponding to the maximal value of the RRP observed in the sequence and 3) the docking site occupancy probability (), corresponding to the ratio between RRP avg and the NDS (see equations 1 and 2, bellow). A representative experiment is presented in Figure 7-10. In order to test whether the synapse-specific initial values of RRP avg and docking site occupancy probability can account for real differences in the outcome after the conditioning protocol (facilitation vs. depression), we decided to compare the differences between the "extreme points" along the distribution of the absolute difference values (test-ctrl) for RRP avg and  values of Interestingly, in relation with the autoreceptor current it is possible to see that in this case the larger and only significant effect was obtained when there is no delay between the end of the SSD and the presynaptic AP, and the effect is gone for 200 ms and 2 s delays. This suggests that the effect of SSD on GABA release depends on the specific type of GABA A receptors, affecting the pre-and postsynaptic cells in different ways, probably due to the type and number of synapses that PSC and ARC reports.

Erratum:

The results from this last section should be taken carefully because there was a mistake in the composition of the internal solution, which resulted in a high concentration of free Ca 2+ (~ 80 µM). This allows to obtain a reliable estimate of the number of vesicles that fused with the plasma membrane upon Ca 2+ photorelease. Because we can control the amount of photoreleased Ca 2+ , this approach can be used to deplete the terminal and directly estimate the number of vesicles composing the RRP. The major challenge to accurately measure the RRP by using more physiological approaches (like prolonged trains of presynaptic APs) is to release the RRP while accounting for the replenishment contribution [START_REF] Kaeser | The readily releasable pool of synaptic vesicles[END_REF]). Here we avoided this problematic issue by releasing the RRP in a time window which is significantly shorter (within 10 ms; see Fig. 7-2) than the reported value for the replenishment time constant (1.7 s under resting conditions and 300-400 ms during the application of an AP train (Takeshi [START_REF] Sakaba | Two Ca( 2+ )-dependent steps controlling synaptic vesicle fusion and replenishment at the cerebellar basket cell terminal[END_REF]. Additionally, we have confirmed the emptying of the RRP by testing laser-evoked release 30 ms after the first high Ca 2+ pulse (see methods); this time window is large enough to permit the diffusion of the calcium cage to the presynaptic terminal [START_REF] Trigo | Readily releasable pool of synaptic vesicles measured at single synaptic contacts[END_REF]) and short enough to permit the full replenishment of an empty docking site.

In this work we have assumed that the RRP reflects the number of vesicles in the docked state in a single active zone. This has been suggested by previous functional and electron microscopy studies of the Calyx of Held and neuromuscular junction (see introduction) and where the RRP has been interpreted