Abstract

This investigation seeks to elucidate the organisation of the cerebro-cerebellar pathways by relating them to the cerebellar climbing fibre zones, using various anatomical, physiological and computational methods. The cerebro-cerebellar pathways link two of the largest structures in the mammalian brain; the cerebral cortex and the cerebellum both of which are associated with motor skills. The two main ways in which afferent information reaches the cerebellar cortex is via climbing fibres and mossy fibres, however, it is not clear how these pathways are linked, especially with regard to the descending motor input.

The cerebro-ponto-cerebellar pathway serves primarily to transform descending signals from the orderly somatotopically organised cerebral cortex to the discontinuous, fractured somatotopy of the cerebellar cortex and the present investigation studies the way these maps are represented in the basilar pontine nuclei (BPN). The projection from BPN to defined climbing fibre zones in the cerebellar cortex was investigated using 3D reconstruction of retrograde labelling in BPN, and also in dual tracer experiments labelling cells and terminal in BPN. A systematic topographical relationship in the ponto-cerebellar projection to the climbing fibre zones was found and was related to the motor cortical input to BPN.

The cerebro-olivo-cerebellar pathway was investigated using physiological and anatomical methods. Previous studies have shown a clear climbing fibre response to cerebral stimulation, although the path by which the cerebro-olivary information travels is less clear. The present results show that ascending and descending information is convergent in the cerebellum and that a nuclear region termed the 'matrix' caudal to the olive is a relay station in cerebro-olivary paths arising from the motor cortex.

The results are presented in three chapters:

(1) 3D Reconstruction of the ponto-cerebellar projection to climbing fibre zones (2) Cerebro-ponto-cerebellar pathway (3) Cerebro-olivo-cerebellar pathway iii

To my Mum & Dad

Pardoe for her help and always being cheerful. The staff in ASU have also been a great help over my time here, especially Gareth who has shown endless patience.

To the NeSys Laboratory in Oslo University; special thanks to Professor Jan Bjaalie for being so hospitable. Finally, a big thanks to all my family and friends -for your love, support, encouragement and confidence in me.

List of Illustrative Material

General Introduction 1.1 Overview

The mammalian sensorimotor system is extremely complex. In this system, both gross and fine movements can be initiated, executed, controlled and completed to an exceptionally high degree. In particular, the cerebro-cerebellar projection constitutes major pathways in the brain linking the cerebral cortex with the cerebellar cortex.

The function of the cerebral cortex is devoted to processes as diverse as movement, thought, visual and auditory processing and it therefore has structure-function localisations. Movements are controlled by the motor cortex and this information is relayed to the cerebellum via climbing fibres and mossy fibres. The cerebellum is the largest sensorimotor structure in the mammalian brain, comprising approximately 10% of the volume of the mammalian brain and half the total number of neurones.

Both the cerebral and cerebellar cortices become increasingly more convoluted with fissures and sulci with the complexity of species [START_REF] Clark | Scalable architecture in mammalian brains[END_REF], thus increasing the processing power. Across all mammals, the basic structures and workings of the brain are therefore fundamentally the same, such as the descending fibre tracts [START_REF] Kuypers | Anatomy of the descending pathways[END_REF]. The present investigation concentrated on motor descending input to the cerebellum, terminating as climbing fibres and mossy fibres. The two main nuclei providing cerebellar afferents are the inferior olive (climbing fibres) and the basilar pontine nuclei (BPN; mossy fibres). The climbing fibres project in an ordered somatotopical arrangement onto the cerebellar cortex, where different parts of the inferior olive project to different zones. However, the mossy fibre projection shows a 'fractured' topography, where body parts are represented non-contiguously (Shambes 1978a, b;[START_REF] Bower | Is the cerebellum sensory for motor's sake, or motor for sensory's sake: the view from the whiskers of a rat?[END_REF]. As the climbing fibres impose a somatotopical order onto the cerebellum, the less-ordered mossy fibre pontine projection was investigated in this framework.

Historical Importance

Two of the largest structures in vertebrate brains are the cerebral cortex and cerebellum, and both have a key role in sensorimotor control. Historically, both the cerebral cortex and cerebellum have been used to investigate structure-function relations. The cerebellum, often termed the 'little brain' (cerebellum = 'little brain' in Latin), has been of particular interest due to the repeated architecture found in the cerebellar cortex, which means that function is dictated to a large extent, by structural input-output connections. Methods to investigate the underlying connectivity of the nervous system have remained essentially unchanged over the years. Through using physiological stimulating and recording, and anatomical tract tracing, both structure and function relations can be effectively investigated. Modern day techniques are, however, highly sensitive so that more detailed and thorough analyses can be made.

The cerebellum has long been associated with motor control and historical work on the cerebellum has shown it to be imperative in the co-ordination of movements (e.g. lesion studies of Florens [1824], cited in [START_REF] Houk | Models of the cerebellum and motor learning[END_REF]). The importance of the cerebellum in humans was highlighted by [START_REF] Holmes | Clinical symptoms of cerebellar disease and their interpretation[END_REF], who studied patients with cerebellar damage due to gun shot wounds in the First World War and cerebellar tumours in civil life. He found severe motor deficits including disturbances in balance and co-ordination of movements, with atonia and ataxic movements produced. Rather than seeking explanations from the impaired system, the present investigation uses the normal functioning model to study fundamental pathways.

The significance of the cerebral cortex, particularly the motor areas, was highlighted by Penfield's work mapping the human cortex during neurosurgery in the 1940s and 1950s. The organisation of the cortex was mapped and the motor area was found to contain a detailed, continuous topographical map of the body, where body parts are on the whole represented next to each other. Also, these constructed body maps helped during further surgical procedures. Reflexes and 'simple' movements can be explained by subcortical structures and networks, but the cerebral cortex is intricately associated with complicated action, acquired skills and learned performance [START_REF] Penfield | The Cerebral Cortex of Man The Macmillan Company[END_REF].

The Cerebellum

The primary focus of the present investigation is on inputs to the cerebellum. The most striking feature of the cerebellum is the repeating nature of its neural circuits: cerebellar functioning is governed by its input and output connections. This intricate organisation critically depends on two inputs, climbing fibres and mossy fibres, and one output, the cerebellar nuclei, although the afferent input is numerically much larger than the efferent output (e.g. humans 40:1 relationship; Brodal, 1992b). This

shows considerable integration of information from different sources to give an output. This implies that the cerebellum receives input from a varied source of modalities, such as sensory and visual and not just motor information, and processes these inputs together.

Although both the cerebral cortex and cerebellum increase in surface area with complexity of species, the ratio of white matter to grey matter is more constant in the cerebellum than the cerebral cortex. This is partly due to the short-range local connectivity of the cerebellum as compared to the longer-range connections of the cerebral cortex [START_REF] Sultan | Analysis of mammalian brain architecture[END_REF]. The cerebellum is located above the brainstem and is connected to it anteriorly by the white matter of the cerebellar peduncles. It receives discrete information from climbing fibres and mossy fibres entering via the cerebellar peduncles and terminating in the cerebellar cortex. Local information is then directed out via inhibitory transmission to specific targets of the cerebellar nuclei. In general, the inferior and middle peduncles convey afferent input and the superior peduncle contains most of the efferent fibres.

The primary function of the cerebellum is the modulation and regulation of movements, such as control of posture [START_REF] Eccles | The Cerebellum as a Neuronal Machine[END_REF][START_REF] Eccles | Mode of operation of the cerebellum in the dynamic loop control of movement[END_REF], rather than the generation or initiation of movements. The cerebellum is not necessary for movement to occur, but when damaged, there are clear motor deficits, such as with cerebellar ataxia where there is atonia in muscles [START_REF] Holmes | Clinical symptoms of cerebellar disease and their interpretation[END_REF]. It is constantly processing and acting on a mass of sensorimotor information and therefore receives information from all over the body. The cerebellum also has an important role in the acquisition of new motor tasks [START_REF] Ghez | The Cerebellum[END_REF]. This structure has been extensively studied over the years yet there are still many hypotheses on the functions of the cerebellum and its projections, such as in learning, memory, conditioning, and acquiring skills, in both motor and sensory aspects.

The cerebellum is mainly associated with motor skills, although inevitably, sensory aspects must be integrated with motor tasks, and there is much evidence that the cerebellum receives somatosensory information (e.g. Brodal, 1968a, b;[START_REF] Brodal | The corticopontocerebellar pathway to crus I in the cat as studied with anterograde and retrograde transport of horseradish peroxidase[END_REF][START_REF] Bower | Is the cerebellum sensory for motor's sake, or motor for sensory's sake: the view from the whiskers of a rat?[END_REF]Leergaard et al., 2000a, b;[START_REF] Leergaard | Clustered and laminar topographic patterns in rat cerebropontine pathways[END_REF]. Damage to the cerebellum primarily affects skilled, co-ordinated voluntary movements (Brodal, 1992a). Typically, fine control is lost and movements become jerky and ataxic. The cerebellum also plays a key role in automation of functioning (Brodal, 1992a) and cerebellar dysfunction has even been implicated in dyslexia [START_REF] Nicolson | Developmental dyslexia: the cerebellar deficit hypothesis[END_REF] with problems in the automation of other higher order cognitive functions. Therefore, the cerebellum acts upon a wide variety of ascending and descending inputs and when impaired is primarily associated with loss of motor co-ordination, although these losses are also tied in with sensory problems.

Anatomy & Physiology

The cerebellum is divided into the anterior and posterior lobes, separated by the primary fissure, and these lobes are further divided into lobules. The nomenclature of [START_REF] Larsell | The morphogenesis and adult pattern of the lobules and fissures of the cerebellum of the white rat[END_REF] dictates that the mammalian cerebellum is divided into ten lobes in total. The cerebellum becomes more convoluted with phylogenetic complexity, which entails an increase in the numbers of lobules and folia, although the structural connectivity of the cerebellum remains fairly constant across species. The cerebellum contains two main parts, the cerebellar nuclei and the surrounding cortex. The white matter divides the cerebellar nuclei from the cortex, and comprises myelinated afferent and efferent axons. The cerebellar nuclei consist of three main divisions, the fastigial, interpositus (anterior and posterior), and dentate nuclei, and are somatotopically related to the zonal organisation imposed on the cerebellar cortex by its climbing fibre input.

The cerebellar cortex can be divided into the vermis, paravermis and hemispheres. In general, the vermis has long been associated with balance and posture and projects to the fastigial (medial) cerebellar nuclei, the paravermis functions in limb movements and projects to the interpositus nuclei, and the hemispheres have been implicated in higher order fine movements, such as eye and finger movements, and project to the dentate nuclei (for an overview see Brodal, 1992a, b). The cytoarchitecture of the cortex is extremely uniform and cannot be used to discriminate between regions of different function; therefore, the differentiation of functions depends on the afferent and efferent connections [START_REF] Oscarsson | Functional units of the cerebellum -sagittal zones and microzones[END_REF].

The main inputs, climbing fibres and mossy fibres, enter the cerebellum through the cerebellar peduncles and terminate in the cortex. Figure 1.1 shows the general architecture of the cerebellum. The cortex can be divided into distinct layers; from external to internal they are: the molecular layer, the Purkinje cell layer, and the granule cell layer (as described in early work by Ramón y Cajal, 1911, cited in [START_REF] Eccles | The Cerebellum as a Neuronal Machine[END_REF]. Climbing fibres terminate in the molecular layer and wind around Purkinje cells on a one-to-one basis, thus making hundreds of synapses with individual Purkinje cells. The Purkinje cell layer contains the cell bodies of Purkinje cells (see Figure 1.1 [A] for an example photomicrograph of a Purkinje cell), which project their massive dendritic trees into the molecular layer and receive synapses also from thousands of parallel fibres. The mossy fibres indirectly contact many Purkinje cells. They terminate in the granule cell layer and synapse with granule cells, which act as a relay and also increase the number of Purkinje cell activated. The axon of the granule cell courses into the molecular layer where it bifurcates and gives rise to a parallel fibre.

There are also various interneurones in the cerebellar cortex, including Golgi cells, basket cells, stellate cells, Lugaro cells and unipolar brush cells (for an overview see [START_REF] Palay | Cerebellar Cortex[END_REF]. The Golgi cell receives input from mossy fibres and from parallel fibres and its main role is to provide inhibitory feedback to the granule cell. Basket cells and stellate cells are located in the molecular layer; both receive input from parallel fibres and send efferents that terminate on different parts of the Purkinje cell. Less is known about the structure or function of Lugaro cells and unipolar brush cells.

The present investigation focuses on the physiological afferent connections of the climbing fibres and mossy fibres. While there is a modular organisation of the output of the cerebellar cortex, the mossy fibre and climbing fibre inputs to the cerebellar cortex are organised very differently. The mossy fibre projection via granule cells displays a patchy representation, where there are multiple representations of the same body part, so-called 'fractured somatotopy' (Shambes, 1978a, b;[START_REF] Bower | Is the cerebellum sensory for motor's sake, or motor for sensory's sake: the view from the whiskers of a rat?[END_REF]. This fractured bodily representation is grossly different from the continuous bodily representation in the cerebral cortex.

Conversely, the olivary climbing fibre projection terminates in a regular somatotopical pattern in the cerebellar cortex, where there are multiple representations but the topography is maintained. In the original work on the longitudinal topographical division of the cerebellum, [START_REF] Jansen | Experimental studies on the intrinsic fibres of the cerebellum. II. The corticonuclear projection[END_REF] proposed that the division was based on the cortico-nuclear organisation. Further work from [START_REF] Voogd | The cerebellum of the cat. Structure and fibre connections[END_REF] demonstrated compartmentation in the white matter. The climbing fibres from the inferior olive were also found to project to the cerebellar cortex in topographically organised longitudinal zones, where certain parts of the olive project to specific organised zones in the cerebellar cortex (e.g. [START_REF] Groenewegen | The parasagittal zonation within the olivocerebellar projection. I. Climbing fiber distribution in the vermis of cat cerebellum[END_REF][START_REF] Groenewegen | The parasagittal zonation within the olivocerebellar projection. II. Climbing fiber distribution in the intermediate and hemispheric parts of cat cerebellum[END_REF][START_REF] Voogd | Topographical Distribution of Olivary and Corticonuclear Fibers in the Cerebellum. A Review[END_REF][START_REF] Brodal | The olivocerebellar projection in the monkey. Experimental studies with the method of retrograde tracing of horseradish peroxidase[END_REF][START_REF] Apps | Columnar organisation of the inferior olive projection to the posterior lobe of the rat cerebellum[END_REF]. This somatotopy is preserved in the olivo-cortico-nuclear projection for various species [START_REF] Brodal | Olivocerebellar projection: a review[END_REF]Garwicz et al., 1998;[START_REF] Voogd | The anatomy of the cerebellum[END_REF][START_REF] Apps | Precise matching of olivo-cortical divergence and cortico-nuclear convergence between somatotopically corresponding areas in the medial C1 and medial C3 zones of the paravermal cerebellum[END_REF]Pardoe & Apps, 2002;[START_REF] Edge | Lateral cerebellum: functional localization within crus I and correspondence to cortical zones[END_REF][START_REF] Pijpers | Topography of olivo-cortico-nuclear modules in the intermediate cerebellum of the rat[END_REF].

The innervation of Purkinje cells from the mossy fibre-parallel fibre system and from climbing fibres means that there are different responses seen to either activation. The response to mossy fibre input is typically of short latency (around 5 ms) and produces a simple spike whereas the climbing fibre response is longer in latency (typically more than 9 ms) (for mossy fibre and climbing fibre responses see Allen et al., 1974a, b;[START_REF] Allen | Cerebrocerebellar communication systems[END_REF]. The characteristically longer latency to firing from climbing fibre activation is in part due to the slow conduction velocity of the action potential along the small fibres [START_REF] Eccles | The Cerebellum as a Neuronal Machine[END_REF].

The climbing fibre terminates on a one-to-one basis on Purkinje cells by making many synaptic connections. This partly characterises the diphasic complex spike produced from climbing fibre innervation as it is thought that the numerous connections give the characteristic spiking waveform of the climbing fibre response [START_REF] Eccles | The Cerebellum as a Neuronal Machine[END_REF]. The responses to mossy fibre and climbing fibre activation of the Purkinje cell can be seen in Figure 1.2, where the mossy fibre potential generates a simple spike and the climbing fibre, a complex spike. The Purkinje cell is the sole output of the cerebellum via inhibitory action over the cerebellar nuclei (also often termed deep cerebellar nuclei) and as mentioned above, the cerebellar cortical projection to the nuclear output cells is topographically organised.

Inputs to the Cerebellum

Climbing fibres have been found to originate solely from the inferior olive [START_REF] Armstrong | Functional significance of connections of the inferior olive[END_REF][START_REF] Desclin | Histological evidence supporting the inferior olive as the major source of cerebellar climbing fibers in the rat[END_REF], however there are various sources of mossy fibres.

The main source is the basilar pontine nuclei (BPN), which contribute around 90% of all mossy fibres [START_REF] Schwarz | Binding of signals relevant for action: towards a hypothesis of the functional role of the pontine nuclei[END_REF]. The capacity of both the climbing fibre and mossy fibre pathways in relaying information to the cerebellum is large, although the BPN mossy fibre projection is approximately 20 times that from the inferior olive (in humans; [START_REF] Tomasch | The numerical capacity of the human cortico-pontocerebellar system[END_REF]. This demonstrates the vast amount of information reaching the cerebellum and thus the massive processing power of the cerebellum. There are also inter-species similarities for general bodily inputs to the cerebellum such as the limb input [START_REF] Adrian | Afferent areas in the cerebellum connected with the limbs[END_REF]in cat, dog, goat & monkey). These similarities also generally hold true for rat, where for example, the climbing fibre zonal organisation of the posterior lobe is similar to that in cat (e.g. [START_REF] Furber | Organization of the olivocerebellar projection in the rat[END_REF].

Climbing Fibres

The olivo-cerebellar pathway provides a discrete, private line of communication; not only do climbing fibres solely originate from the inferior olive, but olivary efferent fibres only terminate as climbing fibres, in the cerebellum (Szentágothai & Rajkovits [1959] cited in [START_REF] Eccles | Functional organization of the cerebellum in relation to its role in motor control[END_REF][START_REF] Eccles | Excitation of cerebellar Purkinje cells by the climbing fibres[END_REF]. The climbing fibre innervation of a Purkinje cell is one of the most powerful in the nervous system due to the vast number of synapses a climbing fibre has on the large dendritic tree of a Purkinje cell. The contact made by the climbing fibre synapses produces a large excitatory post-synaptic potential and a characteristic complex spike is triggered from the Purkinje cell. As seen in Figure 1.2 (part A i), the initial action potential generates a large spike followed by a few high frequency smaller spikes (see [START_REF] Eccles | The Cerebellum as a Neuronal Machine[END_REF] for an in-depth review).

Figure 1.2 (part A ii) also shows a depth profile of the climbing fibre response, where there is a maximal response at the site of synaptic contacts optimally between 150-200 µm in depth. This is the location where the climbing fibre wraps around and contacts most parts of the Purkinje cell. The climbing fibre projection is represented as parasagittal zones in the cerebellar cortex arising from specific rostrocaudally oriented columns of projection neurones within the inferior olive; this organisation is found throughout species (e.g. rat, [START_REF] Atkins | Somatotopical organisation within the climbing fibre projection to the paramedian lobule and copula pyramidis of the rat cerebellum[END_REF]cat, Ekerot and Larson, 1979a, b;ferret, Garwicz, 1997;monkey, Brodal & Brodal, 1981). This implies that the function of the climbing fibre somatotopy is preserved across species.

There is a one-to-one relationship of climbing fibre to Purkinje cell although there are approximately seven Purkinje cells to every one inferior olive cell in the rat [START_REF] Armstrong | A quantitative study of the Purkinje cells in the cerebellum of the albino rat[END_REF][START_REF] Schild | On the inferior olive of the albino rat[END_REF]. Therefore, branching of the climbing fibre occurs where one axon branches on average to innervate seven Purkinje cells [START_REF] Sugihara | Morphology of single olivocerebellar axons labeled with biotinylated dextran amine in the rat[END_REF][START_REF] Sugihara | The entire trajectories of single olivocerebellar axons in the cerebellar cortex and their contribution to Cerebellar compartmentalization[END_REF]. The axon of a climbing fibre often innervates different folia, lobes or lobules of the cerebellum (Armstrong et al., 1973a, d). For example among responses to forelimb stimulation, corresponding responses can be found in Lobule IV/V and in the paramedian lobule (Armstrong et al., 1971a), which are due to climbing fibre branching (Armstrong et al., 1973b). This holds true for both ascending (Armstrong et al., 1973c) and descending (Armstrong et al., 1973b) inputs.

The complex pattern of termination and branching by climbing fibres means that there are equivalent projections to different parts of the cerebellum, thus multiple representations are found arising from the same olivary cells.

The Inferior Olive

The inferior olive is recognised as the sole source of climbing fibres to the cerebellar cortex. It projects throughout the cerebellar cortex and specific parts of the inferior olive project to certain zones in the cerebellum. There are three main regions of the inferior olive, the medial accessory olive (MAO; phylogenetically the oldest part), dorsal accessory olive (DAO), and the principal olive (PO; phylogenetically most recent), which can further be divided into the ventral lamella (VL) and dorsal lamella (DL). A precise topographical organisation within the inferior olivary climbing fibre projection to the cerebellar cortex has been previously demonstrated (e.g. [START_REF] Groenewegen | The parasagittal zonation within the olivocerebellar projection. I. Climbing fiber distribution in the vermis of cat cerebellum[END_REF]Groenewegen et al., 1979, in cat;Apps, 1990, in rat).

The main nuclei of the inferior olive are comparable in various species, although more complex animals have an increasingly developed PO (Jenkins, 1905, cited in Azizi & Woodward, 1987;[START_REF] Kooy | The inferior olive in vertebrates[END_REF]. To understand the function of the inferior olive, the normal structure and morphology must be determined. More in-depth studies such as [START_REF] Gwyn | The inferior olivary nucleus of the rat: a light and electron microscopic study[END_REF], [START_REF] Azizi | Inferior olivary nuclear complex of the rat: morphology and comments on the principles of organization within the olivocerebellar system[END_REF] and [START_REF] Apps | Columnar organisation of the inferior olive projection to the posterior lobe of the rat cerebellum[END_REF] in the rat have shown detailed anatomical relationships between nuclear regions of the inferior olive and the olivocerebellar system (for a review of the general topography of the olivo-cerebellar system, see [START_REF] Brodal | Olivocerebellar projection: a review[END_REF]. Topographical principles of organisation have been found in both the inferior olive and olivocerebellar system in the rat, between the nuclear arrangement and within individual nuclei [START_REF] Azizi | Inferior olivary nuclear complex of the rat: morphology and comments on the principles of organization within the olivocerebellar system[END_REF]. Specialisations in the structure of the inferior olive are also present in the morphology of its cells. For example, synchrony in olivary cell firing has been judged to be due to the functional units found, including gap junctions and electrotonic coupling of cells [START_REF] Gwyn | The inferior olivary nucleus of the rat: a light and electron microscopic study[END_REF][START_REF] Llinás | Electrotonic coupling between neurons in cat inferior olive[END_REF][START_REF] Sotelo | Structural study of inferior olivary nucleus of the cat: morphological correlates of electrotonic coupling[END_REF].

The inferior olive receives convergent inputs from certain segments of the spinal cord and corresponding parts of the motor cortex and therefore has been theorised as acting as a 'comparator' of inputs [START_REF] Andersson | Origin and sagittal termination areas of cerebrocerebellar climbing fibre paths in the cat[END_REF] to project this information in a zonal organisation in the cerebellar cortex [START_REF] Andersson | Origin and sagittal termination areas of cerebrocerebellar climbing fibre paths in the cat[END_REF]. Although there is much physiological evidence that the inferior olive transmits cerebral information to the cerebellum [START_REF] Armstrong | Functional significance of connections of the inferior olive[END_REF]Armstrong et al., 1973b;[START_REF] Andersson | Origin and sagittal termination areas of cerebrocerebellar climbing fibre paths in the cat[END_REF], the past literature generally does not necessarily ascribe a direct cerebral projection to the olive [START_REF] Kuypers | An anatomical analysis of cortico-bulbar connections to the pons and lower brainstem in the cat[END_REF][START_REF] Saint-Cyr | Anatomical organization of cortico-mesencephalo-olivary pathways in the cat as demonstrated by axonal transport techniques[END_REF], and where it does, the evidence is limited [START_REF] Sousa-Pinto | Demonstration of a somatotopical pattern in the cortico-olivary projection in the cat. An experimental-anatomical study[END_REF][START_REF] Saint-Cyr | The projection from the motor cortex to the inferior olive in the cat. An experimental study using axonal transport techniques[END_REF]). Nevertheless, a distinct pattern of (convergent) termination has been found for both the ascending and descending pathways. These matters will be further introduced and discussed in detail in Chapter 5.

Zonal Organisation

As the zonal organisation is of importance in input and output connectivity, it makes sense to further explore this organisation pattern and build on it. Work on the intricacies of the zonal somatotopy has revealed further distinctions and complexities, such as microzones and zebrin (adolase C) banding. Microzones are smaller constituent parts of climbing fibre zones and have been claimed to be the operational 'unit' of the cerebellum [START_REF] Andersson | Climbing fiber microzones in cerebellar vermis and their projection to different groups of cells in the lateral vestibular nucleus[END_REF]. The olivo-cortico-nuclear organisation provides a central organisational principle of the cerebellum, where further details of this olivo-cerebellar projection and the projection of mossy fibres to the zones can be studied.

From detailed anatomical studies on the olivo-cortico-nuclear projection in various species (as mentioned previously), the cerebellar cortex has been divided into zones.

Pioneering work from [START_REF] Voogd | The cerebellum of the cat. Structure and fibre connections[END_REF] into the olivo-cerebellar projection in the cat divided the cerebellum into zones, where the A and B zones were medially located, the C1, C2 and C3 zones were within the intermediate cerebellum, and the D zone located in the hemisphere. These zones have specific olivary inputs and project to specific target regions in the cerebellar nuclei. In the olivo-cerebellar projection, the A and B zones were found to have olivary input from the contralateral caudal poles of MAO and DAO, respectively, the C1 and C3 zones received information from the contralateral rostral DAO, the C2 zone received fibres from the rostral parts of the contralateral MAO, and the D zone from the contralateral PO (see [START_REF] Voogd | Topographical Distribution of Olivary and Corticonuclear Fibers in the Cerebellum. A Review[END_REF] for a review). A distinction between anatomically (use of capital lettering)

and physiologically (use of lower case lettering) defined zones is not maintained in this investigation as these zones have been shown to be essentially equivalent [START_REF] Trott | Lateral and medial sub-divisions within the olivocerebellar zones of the paravermal cortex in lobule Vb/c of the cat anterior lobe[END_REF][START_REF] Trott | Zonal organization within the projection from the inferior olive to the rostral paramedian lobule of the cat cerebellum[END_REF].

Additional work has identified further differentiation in zonal organisation, such as the X zone and the CX in the anterior lobe of the cat [START_REF] Ekerot | Branching of olivary axons to innervate pairs of sagittal zones in the cerebellar anterior lobe of the cat[END_REF][START_REF] Campbell | Origin in the medial accessory olive of climbing fibres to the x and lateral c1 zones of the cat cerebellum: a combined electrophysiological/WGA-HRP investigation[END_REF] and division within the D zone into D1 and D2 (for an overview, see [START_REF] Ito | The Cerebellum and Neuronal Control Raven Press[END_REF]). An additional division was also found in work by [START_REF] Buisseret-Delmas | Sagittal organisation of the olivocerebellonuclear pathway in the rat. III. Connections with the nucleus dentatus[END_REF] with the D0 zone in rat. Figure 1.3 gives an overview of the current main zonal divisions in the cat and rat. There is further detail and organisation in the olivo-cerebellar projection as there are distinguishable 'microzones' within these climbing fibre zones.

Microzonal Organisation

The microzonal organisation was first described by [START_REF] Andersson | Climbing fiber microzones in cerebellar vermis and their projection to different groups of cells in the lateral vestibular nucleus[END_REF], who defined a microzone and its subcortical neuronal network as representing the 'basic computational unit of the cerebellum'. [START_REF] Ito | The Cerebellum and Neuronal Control Raven Press[END_REF][START_REF] Ito | Signal processing in cerebellar Purkinje cells[END_REF] also agreed that the microzone may be the biological unit of functional cerebellar compartmentalization in his hypothesis on learning in the cerebellum, where the climbing fibre potential may signal an error-triggered reorganisation of the cerebellar cortical circuitry.

Microzones have been physiologically described between species, for example in the cat [START_REF] Andersson | Climbing fiber microzones in cerebellar vermis and their projection to different groups of cells in the lateral vestibular nucleus[END_REF] in the B zone; [START_REF] Ekerot | Topography and nociceptive receptive fields of climbing fibres projecting to the cerebellar anterior lobe in the cat[END_REF] and Garwicz et al. [1998] in the C3 zone) and in the rat [START_REF] Jörntell | Functional organization of climbing fibre projection to the cerebellar anterior lobe of the rat[END_REF], where there is an orderly sequence of subclass within the overall climbing fibre zone.

Study of the B zone divided the zone into five smaller microzones, each with a width of approximately 200 µm according to input from the peripheral stimulation of limb nerves. From medial to lateral in the B zone, each specific microzone received purely hindlimb input to mixed latency fore-and hindlimb input, to only forelimb laterally, and each microzone projected to a specific part of the lateral vestibular nucleus.

Therefore, olivo-cortico-nuclear microcomplexes can be defined and that each olivocortico-nuclear unit may form a motor unit with a specific motor function and topographical specificity, such as the control of a specific muscle or musculature [START_REF] Jörntell | Relation between cutaneous receptive fields and muscle afferent input to climbing fibres projecting to the cerebellar C3 zone in the cat[END_REF].

Further work has found that there are between-zone relations in the paravermis. The original microzonal organisation has therefore been revised to include this and more recently been termed 'multizonal microcomplexes' (MZMCs; for a review see [START_REF] Apps | Anatomical and physiological foundations of cerebellar information processing[END_REF]. MZMCs consist of a cerebellar module where one or more microzones converge on a common nuclear cell group, such as in the paravermal microzone in the C1, C3 and Y zones converging on the nucleus interpositus anterior [START_REF] Ekerot | The control of forelimb movements by intermediate cerebellum[END_REF]. Anatomical studies have given evidence towards the existence of microzones in the C3 zone [START_REF] Garwicz | Micro-organization of olivocerebellar and corticonuclear connections of the paravermal cerebellum in the cat[END_REF] and that MZMCs exist between the C1 and C3 zones [START_REF] Apps | Precise matching of olivo-cortical divergence and cortico-nuclear convergence between somatotopically corresponding areas in the medial C1 and medial C3 zones of the paravermal cerebellum[END_REF]. This modular MZMC organisation suggests that a microzonal organisation exists where, in the paravermis, the DAO innervates microzones in the C1 and C3 zone, which converge on similar cerebellar neurones in the nucleus interpositus anterior. Therefore, the feature of microzones and MZMCs has been investigated over only small parts of the longitudinal climbing fibre zones and for general organisational principles to be reached, further work must be conducted into the olivo-cortico-nuclear pathway.

Zebrin Banding

Zebrin banding was first investigated by [START_REF] Hawkes | Purkinje cell axon collateral distributions reflect the chemical compartmentation of the rat cerebellar cortex[END_REF] and was directly linked to the parasagittal organisation of the cerebellum and climbing fibre somatotopy [START_REF] Gravel | Parasagittal organization of the rat cerebellar cortex: direct comparison of Purkinje cell compartments and the organization of the spinocerebellar projection[END_REF], and across species [START_REF] Leclerc | The compartmentalization of the monkey and rat cerebellar cortex: zebrin I and cytochrome oxidase[END_REF].

Since the discovery of this specific Purkinje cell marker, the banding distribution of zebrin positive and negative banding in the cerebellum has been investigated.

Various studies have now compared the zebrin compartmentalisation with afferent projections (e.g. tactile projection patterns, [START_REF] Hallem | Spatial correspondence between tactile projection patterns and the distribution of the antigenic Purkinje cell markers anti-zebrin I and anti-zebrin II in the cerebellar folium crus IIA of the rat[END_REF] the climbing fibre zonal organisation, [START_REF] Voogd | The distribution of climbing and mossy fiber collateral branches from the copula pyramidis and the paramedian lobule: congruence of climbing fiber cortical zones and the pattern of zebrin banding within the rat cerebellum[END_REF][START_REF] Voogd | The organization of the corticonuclear and olivocerebellar climbing fiber projections to the rat cerebellar vermis: the congruence of projection zones and the zebrin pattern[END_REF] and have found substantial overlap but not a one-to-one relationship in the patterns. This was a new way of understanding both the climbing fibre somatotopy and the distribution of zebrin +/-parasagittal compartments. In an elegant study by [START_REF] Sugihara | Molecular, topographic, and functional organization of the cerebellar cortex: a study with combined aldolase C and olivocerebellar labeling[END_REF], the climbing fibre somatotopy was contrasted with zebrin banding across the whole cerebellum. The significance of the expression of zebrin was still unclear, yet relations were found between the bands and zones, with five functionally related groups.

Theories of Climbing Fibre Function

The main focus of the present investigation is the climbing fibre projection to the cerebellum and the way in which it interacts with the other main source of input to the cerebellum, the mossy fibres. The climbing fibre system has been studied in many species and from these findings, theories about the function of the climbing fibre input to the cerebellum have been devised. The main theories focus on the timing hypothesis, the comparator hypothesis and the learning hypothesis. Some of the main theories are discussed in the overview below; the reader is referred to the following reviews for further details: [START_REF] Simpson | On climbing fiber signals and their consequence(s)[END_REF], [START_REF] Houk | Models of the cerebellum and motor learning[END_REF], [START_REF] Llinás | The Cerebellum, LTD, and Memory: Alternative views[END_REF], [START_REF] Bloedel | Current concepts of climbing fiber function[END_REF] and [START_REF] Apps | Anatomical and physiological foundations of cerebellar information processing[END_REF].

The timing device hypothesis focuses on the intrinsic properties of olivary cell firing.

Through studying the microcircuitry of the inferior olive and the electrophysiological properties of olivary neurones, the timing hypothesis was formulated based on the rhythmicity of neuronal firing and electrotonic coupling of neurones via gap junctions [START_REF] Llinás | Electrotonic coupling between neurons in cat inferior olive[END_REF][START_REF] Sotelo | Structural study of inferior olivary nucleus of the cat: morphological correlates of electrotonic coupling[END_REF][START_REF] De Zeeuw | Microcircuitry and function of the inferior olive[END_REF]. The oscillatory behaviour and low firing frequency of around 8-10 Hz of olivary neurones (Bloedel & Ebner, 1984;Ebner & Bloedel, 1984) are important for timing of climbing fibre activation of the Purkinje cell, thus have implication in motor learning and timing of actions. This hypothesis focuses on synchronous olivary activity, which can be related to the initiation and performance of different movements whereby the 'repatterning' of synchrony may allow different combinations of muscles to be used for different movements [START_REF] Welsh | Dynamic organization of motor control within the olivocerebellar system[END_REF].

The comparator hypothesis was based upon the inferior olive acting as a comparator of ascending and descending afferent input [START_REF] Oscarsson | The sagittal organisation of the cerebellar anterior lobe as revealed by the projection patterns of the climbing fibre system[END_REF]Miller et al., 1969a, b;[START_REF] Oscarsson | Functional Organization of Spinocerebellar Paths[END_REF]. The basic assumption was that signals that are not congruent result in a climbing fibre error signal being relayed to the cerebellum. Hence, this theory is based upon the detection of events, particularly unexpected events and errors, such as a change in load. Parts of the olive receive convergent input from the periphery and motor cortex, thus providing a way in which intended versus achieved movement can be compared [START_REF] Andersson | Origin and sagittal termination areas of cerebrocerebellar climbing fibre paths in the cat[END_REF]. Further studies have provided supporting evidence for the role of the climbing fibre signal in error detection such as through recording complex spike discharge in the awake behaving cat, where a response was seen to when a rung in a ladder unexpectedly fell when the cat walked over it [START_REF] Andersson | Complex spikes in Purkinje cells in the lateral vermis (b zone) of the cat cerebellum during locomotion[END_REF]; see also similar findings from an unexpected action from [START_REF] Gellman | Inferior olivary neurons in the awake cat: detection of contact and passive body displacement[END_REF].

The gating of climbing fibre input is a similar idea to the theory of error detection. A mechanism by which context-dependent input is gated has been proposed for the climbing fibre system (for overviews see [START_REF] Apps | Movement-related gating of climbing fibre input to cerebellar cortical zones[END_REF][START_REF] Apps | Gating of climbing fibre input to cerebellar cortical zones[END_REF]. The gating mechanism is theorised to work by gating-in errors generated through unexpected occurrences and gating-out 'predictable' self-generated climbing fibre input. This is context or task dependent as these mechanisms have been found to act in specific parts of an active movement (e.g. classical conditioning in motor learning, [START_REF] Apps | Central regulation of cerebellar climbing fibre input during motor learning[END_REF]motor co-ordination, Garwicz, 2002;step-related modulation, Pardoe et al., 2004) and the pattern of gating can also be related to the zonal organisation of the climbing fibres [START_REF] Apps | Gating of climbing fibre input to cerebellar cortical zones[END_REF].

A slightly different viewpoint of the error/event detection hypothesis has been formulated whereby the climbing fibre is viewed as a sensory detector/encoder (see overview of work by [START_REF] Bloedel | Current concepts of climbing fiber function[END_REF]. This work was based on visual input where magnitude and direction of a sensory occurrence can evoke a climbing fibre response, which could constitute a type of error signal in itself. This is similar to the event detection hypothesis, although could account for a further range of sensory stimuli, as well as the classical view of detection of (unexpected) motor events.

The gain change hypothesis focuses on the interaction of climbing fibre and mossy fibre activity at the Purkinje cell, where climbing fibre activity can modulate mossy fibre activity. This hypothesis is based on the findings of [START_REF] Ebner | Role of climbing fiber afferent input in determining responsiveness of Purkinje cells to mossy fiber inputs[END_REF] where the firing of a complex spike through climbing fibre activation of the Purkinje cell results in a short-lasting facilitation of the Purkinje cell responsiveness to mossy fibre input for a period of up to several hundred milliseconds. This consequence of climbing fibre input was further modified in the dynamic selection hypothesis [START_REF] Lou | Responses of sagittally aligned Purkinje cells during perturbed locomotion: relation of climbing fiber activation to simple spike modulation[END_REF] where climbing fibre activation in sagittally aligned Purkinje cells focuses modulation of simple spikes. Therefore, Purkinje cells in close proximity synchronously activated by climbing fibre and mossy fibre input are more dramatically modulated than Purkinje cells activated by mossy fibre input alone, which has consequences for learning and activation in the cerebellum.

The Marr-Albus-Ito theory of cerebellar function is based on the complexity of cerebellar circuitry at a biological level and uses computational methods to describe it (see [START_REF] Marr | A theory of cerebellar cortex[END_REF][START_REF] Albus | A theory of cerebellar function[END_REF][START_REF] Ito | The Cerebellum and Neuronal Control Raven Press[END_REF]. These theories also focus on the interactions of mossy fibre and climbing fibre input at the Purkinje cell. The mossy fibre and climbing fibre input were thought to have different roles of influence over the firing of the Purkinje cell whereby the mossy fibre input relays contextual information and the climbing fibre input acts as a 'teacher' and modifies the parallel fibre-Purkinje cell synaptic weighting. The theory therefore proposes that the longterm depression (LTD) of the Purkinje cell by the coincident interaction of climbing fibre input with parallel fibre activation from mossy fibre input, establishes a memory trace that is stored. This 'memory' functions through changing the strength of the connections of inputs to the Purkinje cell and with repeated activation, new skills are learned and performed correctly.

Although, another role for LTD has been proposed by De Schutter (1995) whereby LTD is part of a mechanism for normalising the excitation of Purkinje cells. This theory argues that the LTD of the parallel fibre synapses with the Purkinje cell are part of a local negative feedback loop to prevent overstimulation of the Purkinje cell through parallel fibre activation, rather than functioning in motor learning. The work of [START_REF] Albus | A theory of cerebellar function[END_REF] though in-part takes into account overstimulation of the Purkinje cell and proposes that the influence of the Golgi cell on the circuitry has a stabilising effect, although the role of LTD is still not clear. As there no general agreements by investigators over climbing fibre function, no one theory accounts for all the activities and properties of the climbing fibre system, although each theory has experimental support. This is often because only certain aspects of climbing fibres have been focussed on and explored. These theories, along with many other different view points have been useful to describe aspects of cerebellar functioning, although much work is still needed due to the complexity of the climbing fibre system.

Mossy Fibres

The excitatory mossy fibre response is different to the climbing fibre response, as a simple spike is elicited from the Purkinje cell. Figure 1.2 (part B) details the responses from mossy fibre activation of the Purkinje cell and shows the typical shape of the responses (see [START_REF] Eccles | The Cerebellum as a Neuronal Machine[END_REF] for an in-depth review). The mossy fibre simple spike is evoked via parallel fibre activation and synaptic contact with the Purkinje cell. The mossy fibre input via the granule cells give a patchy organisation of termination in the cerebellar cortex where non-contiguous parts of the body are represented next to each other. This is shown in Figure 1.4 where there is a clear patchy organisation in the mossy fibre representation of body part input to the cerebellum. This is very different to the topographical relationship of the olivocerebellar projection. The pattern of termination of the mossy fibre projection forms a 'fractured', discontinuous tactile body map (Shambes et al., 1978a, b;[START_REF] Bower | Principles of organization of a cerebro-cerebellar circuit. Micromapping the projections from cerebral (SI) to cerebellar (granule cell layer) tactile areas of rats[END_REF]Welker, 1897;[START_REF] Bower | Variability in tactile projection patterns to cerebellar folia crus IIA of the Norway rat[END_REF][START_REF] Bower | Is the cerebellum sensory for motor's sake, or motor for sensory's sake: the view from the whiskers of a rat?[END_REF].

The olivo-cerebellar projection is unilaterally crossed; however the mossy fibre projection is bilateral to varying degrees. Studies into the bilateral mossy fibre projections have found varying degrees of bilaterality from different nuclei to different parts of the cerebellum. [START_REF] Serapide | Laterality of the pontocerebellar projections in the rat[END_REF] found 82% of BPN cells and 60% of nucleus reticularis tegmenti pontis (NRTP) cells projected contralaterally to the cerebellum, in rat. Other studies have found similar results, in cat such as [START_REF] Brodal | The corticopontocerebellar pathway to crus I in the cat as studied with anterograde and retrograde transport of horseradish peroxidase[END_REF] where 78% of BPN mossy fibres projected contralaterally to crus I, although no topographical representation in BPN could be found. However, some studies have found variability in the projections such as Rosina & Provini (in cat, 1981, 1984) who found overall 5-10% of BPN cells project bilaterally, although in parts of BPN, it may be as high as 50%, especially to lateral areas in crus I and II.

Specifically investigating the forelimb-receiving parts of the C1 zone in rat, [START_REF] Herrero | Pontine and lateral reticular projections to the c1 zone in lobulus simplex and paramedian lobule of the rat cerebellar cortex[END_REF] found BPN to project on average 83% contralaterally and NRTP 55%.

Similarly in rabbit, [START_REF] Bukowska | Axonal ramification of neurons in the nucleus reticularis tegmenti pontis projecting to the paramedian lobule in the rabbit cerebellum[END_REF] found the NRTP projection to be 58% contralateral; these studies generally show that BPN sends approximately 80% of its BPN neurones and 60% of NRTP neurones contralaterally (Cicirata et al., 2005a); therefore there is an ipsilateral aspect to both projections, with perhaps NRTP having a more equal bilateral projection than BPN.

The main source of mossy fibres is BPN, although other nuclei such as the NRTP and the lateral reticular nucleus (LRN) send mossy fibre projections to the cerebellum, although these are numerically much smaller. All of these nuclei can be further divided into separate sub-regions of each nucleus. The cytoarchitecture of BPN has been studied in various investigations and can be divided into four regions [START_REF] Mihailoff | The cytoarchitecture, cytology, and synaptic organization of the basilar pontine nuclei in the rat. I. Nissl and Golgi studies[END_REF]. Mihailoff et al. divided BPN into medial, ventral, lateral and peduncular nuclear regions with respect to their position relative to the cerebral peduncle though the division did not reflect cytoarchitectural differences.

Jansen & Brodal (1958, cited in Ito, 1984) also described similar divisions in BPN.

The subdivisions have been useful when describing regions in BPN, such as the location of labelled areas in tracer studies but there is no apparent functional significance to the divisions. These divisions have mainly been used for ease of mapping the nuclei and do not necessarily imply differences in function. The NRTP can be divided into central and pericentral portions [START_REF] Torigoe | Anatomical studies on the nucleus reticularis tegmenti pontis in the pigmented rat. I. Cytoarchitecture, topography, and cerebral cortical afferents[END_REF].

Basilar Pontine Nuclei

The BPN are a comparatively large brain structure and receives major input from the sensorimotor cortex (for a review see Brodal & Bjaalie, 1997). Descending cerebrofugal axons from the cerebral cortex enter BPN rostrally in the cerebral peduncle, which becomes the longitudinal fasciculus of the pons (LFP) throughout the course of the nuclei, then exits BPN as the pyramidal tract. Very large numbers of axons terminate in BPN which therefore acts as a major synaptic relay for descending motor information from the cerebral cortex to the cerebellar cortex (pons = 'bridge' in Latin). Past studies have examined the neuronal make-up of the pontine nuclei and have shown that there are two general categories of neurones in BPN [START_REF] Mihailoff | The basilar pontine gray of the Opossum: A correlated light and electron microscopic analysis[END_REF]. These are intrinsic neurones and projection neurones. The intrinsic neurones have axons which arborise in the region of their dendrites and are relatively small in size, which may indicate a kind of local circuitry. The projection neurones send their axons into the cerebellum and can vary in size and shape [START_REF] Mihailoff | The cytoarchitecture, cytology, and synaptic organization of the basilar pontine nuclei in the rat. I. Nissl and Golgi studies[END_REF]. The latter make up at least 90% of the total cells in BPN. [START_REF] Brodal | The ponto-cerebellar projection in the rabbit and cat[END_REF] and [START_REF] Brodal | The corticopontocerebellar pathway to crus I in the cat as studied with anterograde and retrograde transport of horseradish peroxidase[END_REF] attempted to quantify the pontocerebellar pathway and although made progress, they could only draw conclusions that the neuronal populations that project to the cerebellum were intricately organised in a manner of convergent and divergent areas from BPN and did not describe topographical patterns to different cerebellar cortical areas or zones (hence in later work, the description of the patchy mossy fibre projection to the cerebellum). One of the main drawbacks to the analysis of these projections was the difficulty in viewing and quantifying the ponto-cerebellar pathway, particularly in BPN. Advances in reconstruction techniques and anatomical tracers have helped in quantifying projections, such as using 3D reconstruction in the cerebro-pontine projection (e.g. Leergaard et al., 1995Leergaard et al., , 2000a[START_REF] Leergaard | Three-dimensional topography of corticopontine projections from rat sensorimotor cortex: comparisons with corticostriatal projections reveal diverse integrative organization[END_REF].

The primary function of BPN is to relay information via mossy fibres to granule and other cells of the cerebellar cortex. It also seems that there may be scope for comparison of inputs by neighbouring regions of the pontine nuclei, which has implications in theories of pontine function [START_REF] Schwarz | Binding of signals relevant for action: towards a hypothesis of the functional role of the pontine nuclei[END_REF], especially considering the diverse afferent information that BPN receives. The importance of the projection is demonstrated by clinical studies on patients who have suffered pontine infarction, where pontine injury can lead to disorders such as cerebellar symptoms (e.g. ataxia) or almost total motor paralysis with locked-in syndrome (LIS) (see Leon-Carrion et al., 2002, for a review). LIS symptoms include very limited movement capabilities (typically only vertical eye movements), although the sensory system remains functional. From this it is evident that damage to BPN, causes major disruption to the body's movement system and thus plays a primary role in motor control.

In investigations into the properties of pontine neurones, there was evidence for a complex synaptic integration thus leading to an intrinsic connectivity within the pontine nuclei supporting the notion that the pontine nuclei is far more than a relay [START_REF] Möck | Electrophysiological properties of rat pontine nuclei neurons In vitro II. Postsynaptic potentials[END_REF]. [START_REF] Schwarz | Binding of signals relevant for action: towards a hypothesis of the functional role of the pontine nuclei[END_REF] hypothesized that the role and function of BPN was in the binding of signals relevant for action. They suggest that this structure must have a role in the generation of skilled movement, as the pontine nuclei are highly developed and complex in synaptic properties, paralleling the development of both the cerebral cortex and cerebellar cortex.

BPN plays a key role in relaying signals from the cerebral cortex to the cerebellum, conveying specific spatially-arranged information (Allen et al., 1975a, b). The continuous bodily representations in the cerebral cortex (see next section for more details) are transformed into the patchy organisation seen in the termination of the mossy fibres in the cerebellar cortex. The pons acts as a relay to transform this body map information, but it is not known whether the map becomes 'fractured' in BPN or in the mossy fibre projection (cf. Bjaalie & Leergaard [2000] and Schwarz & Thier [2000]). It is generally accepted that there is convergence and divergence in the BPN from cerebral input and cerebellar targets, although studies have suggested that there are topographical relations in both the cerebral input (e.g. Leergaard et al., 1995Leergaard et al., , 2000a[START_REF] Leergaard | Three-dimensional topography of corticopontine projections from rat sensorimotor cortex: comparisons with corticostriatal projections reveal diverse integrative organization[END_REF]) and cerebellar output (e.g. [START_REF] Bjaalie | Spatial segregation between populations of ponto-cerebellar neurons: statistical analysis of multivariate spatial interactions[END_REF][START_REF] Nikundiwe | Lamellar organization of pontocerebellar neuronal populations. A multi-tracer and 3-D computer reconstruction study in the cat[END_REF]. This will be covered more in the Results chapters.

Cerebro-Cerebellar Pathways

The cerebro-cerebellar pathway is one of the largest in the mammalian brain and the cerebral cortex influences the cerebellum via relays in both of its main inputs, the mossy fibres and the climbing fibres (for an overview see [START_REF] Allen | Cerebrocerebellar communication systems[END_REF].

A direct pathway from the cerebral cortex to the cerebellum has been suggested in very young animals (e.g. [START_REF] Tolbert | Transient cerebrocerebellar projections in kittens: postnatal development and topography[END_REF][START_REF] Tolbert | Somatotopically organised transient projections from the primary somatosensory cortex to the cerebellar cortex[END_REF], although this has been shown to be transient and therefore eliminated well before maturation. [START_REF] Wild | A direct cerebrocerebellar projection in adult birds and rats[END_REF] gave evidence towards a direct cerebro-cerebellar projection in the adult, although this was limited to birds and has not been found in other adult species.

Thus the relays of cerebro-cerebellar signals, primarily the pontine nuclei for mossy fibres and exclusively the inferior olive for climbing fibres, will be investigated in the present study using both anatomical and physiological methods.

General Structure of the Cerebral Cortex

The cerebral cortex becomes increasingly more convoluted, like the cerebellar cortex, with phylogenetic order, although in the cerebral cortex there are gyri and sulci rather than lobules. In rat, the cerebral cortex is rather smooth, whereas in cat, it develops gyri and sulci, and in humans, there are deep fissures and large convolutions. The more convoluted the cerebral cortex, the more complex the behaviour and in primates higher order abilities can occur, such as verbal capacity and self-awareness.

Non-primate species do nevertheless display some higher order characteristics, such as the rat's spatial capabilities. This allows us to investigate the workings of the brain in other animals such as rats and cats and compare this to our abilities. It is, in fact, an advantage that these animals have less developed brains, so we can use them as models to investigate fundamental and to an extent less complex behaviours and processes to build up to more complex ones. For example, investigating structurefunction relations in a certain framework, such as locomotion, then using these models for further work on locomotion in the awake animal and factors that drive locomotion such as motivation.

The present investigation specifically looks at descending motor information. The motor cortex (MI), (primary) somatosensory cortex (SI), and secondary somatosensory cortex (SII) constitute the sensorimotor areas in the rat brain. In other species there are similar areas, and often multiple representations (e.g. the motor cortex and the supplementary motor and pre-motor areas in humans). The sensorimotor cortex has a specific cytoarchitecture, depending on region and function.

Cytoarchitecture of Cerebral Cortex

The cerebral cortex is divided into six main layers, depending on the cell type found.

These layers have different functions and the cells found differ in their density according to the speciality of the region. In rat, the cortex is grossly divided into the agranular motor cortex and granular somatosensory cortices, with a transitional dysgranular interface between them. For example, the main task of the motor (agranular) cortex is to direct movements, thus information-projecting neurones in layer III and V are abundant and the granular layers are less pronounced [START_REF] Donoghue | The motor cortex of the rat: cytoarchitecture and microstimulation mapping[END_REF]. Whereas, in somatosensory (granular) areas, the informationreceiving cells in layers II and IV are more abundant than the pyramidal projection cells of layers III and V [START_REF] Donoghue | The motor cortex of the rat: cytoarchitecture and microstimulation mapping[END_REF][START_REF] Moore | Dynamics of neuronal processing in rat somatosensory cortex[END_REF]. Also, there are indistinct areas between the agranular (projection) cortex and granular (receiving) cortex, the so-called dysgranular cortex, where there are no predominant cell types [START_REF] Donoghue | The motor cortex of the rat: cytoarchitecture and microstimulation mapping[END_REF][START_REF] Sanderson | Reevaluation of motor cortex and of sensorimotor overlap in cerebral cortex of albino rats[END_REF]. Figure 1.5 displays different maps of the rat cerebral cortex. The main divisions can be clearly seen for MI, SI and SII.

The motor cortex is involved in producing bodily movements whereas the somatosensory cortices are more concerned with interpreting incoming information.

Each of these cerebral areas have ordered representations of the body, where parts are arranged in a continuous fashion with neighbouring sites on the body represented with neighbouring sites in the cortex. These form sensory and motor homunculi in humans (cf. 'rattunculi' relating to similar representations in rat [START_REF] Welker | Receptive fields of barrels in the somatosensory neocortex of the rat[END_REF]).

Figure 1.5 also shows different rattunculi, for MI, SI and SII, and the continuous, topographical representation of all the maps can be seen.

Motor Cortical Projections

With the specific cytoarchitecture of the sensorimotor cortex comes specialisation within different areas. Meaningful topographic representations where contiguous body parts are represented next to each other have been demonstrated in many species, although the exact motor representation often depends on specialisations of the animal. For example, rats have a large whisker representation as they use these to explore the environment, whereas humans do not have vibrissae as such, but do have a large oral representation presumably relating to our specialisation for speech and therefore need for fine control of tongue and lips (Brodal, 1992b). The motor cortex can be identified by both physiological and histological means. The motor cortex can be physiologically mapped via stimulating probe microelectrodes on the surface of the cortex and observing a bodily twitch. The type of cortex (agranular/granular) can be distinguished by histologically analysing the layers using counter-staining.

The main output of the motor cortex is through the composite descending fibre tract, namely the cerebro-fugal pathway, which includes cerebro-cerebral, cerebro-striatal, cerebro-thalamic, cerebro-pontine, cerebro-bulbar and cerebro-spinal connections (see [START_REF] Brown | Projections and termination of the corticospinal tract in rodents[END_REF] and [START_REF] Biedenbach | Origin of the pyramidal tract determined with horseradish peroxidase[END_REF] for details of the tract pathway).

Typically, the descending information from the motor cortex includes information about movements and involves direct projection neurones, such as those from layer V to the spinal cord [START_REF] Landry | Morphological and electrophysiological characteristics of pyramidal tract neurons in the rat[END_REF]. There is also a substantial input from the cerebral cortex to the cerebellum, relayed as climbing fibres and mossy fibres (e.g. [START_REF] Curtis | Cerebellar action potentials in response to stimulation of cerebral cortex[END_REF][START_REF] Jansen | Afferent impulses to the cerebellar hemispheres from the cerebral cortex and certain subcortical nuclei; an electroanatomical study in the cat[END_REF][START_REF] Jansen | Observations of cerebrocerebellar evoked potentials in the cat[END_REF][START_REF] Provini | Mossy and climbing fibre organization on the anterior lobe of the cerebellum activated by forelimb and hindlimb areas of the sensorimotor cortex[END_REF][START_REF] Brodal | The corticopontocerebellar pathway to crus I in the cat as studied with anterograde and retrograde transport of horseradish peroxidase[END_REF].

Historically, the cerebral input to the cerebellum has been viewed as being relayed by BPN, although physiological studies have shown there is a cerebral input via the inferior olive and climbing fibres (e.g. [START_REF] Armstrong | Responses in the inferior olive to stimulation of the cerebellar and cerebral cortices in the cat[END_REF]. BPN receives direct input from many parts of the cerebral cortex and projects to the cerebellum as mossy fibres (Brodal, 1968a, b;Brodal 1972, a, b;Wiesendanger & Wiesendanger, 1982a, b;[START_REF] Brodal | The corticopontocerebellar pathway to crus I in the cat as studied with anterograde and retrograde transport of horseradish peroxidase[END_REF]. The cerebro-ponto-cerebellar pathway transforms the continuous bodily representation of the motor cortex into the discontinuous, fractured somatotopy in the cerebellar cortex and the present investigation seeks to elucidate the patterns of the cerebro-ponto-cerebellar projection and comprehend how the information is transformed.

Much remains to be discovered about the cerebro-olivo-cerebellar pathways as well.

There is a well-known physiological pathway (e.g. [START_REF] Adrian | Afferent areas in the cerebellum connected with the limbs[END_REF][START_REF] Andersson | Origin and sagittal termination areas of cerebrocerebellar climbing fibre paths in the cat[END_REF][START_REF] Armstrong | Responses in the inferior olive to stimulation of the cerebellar and cerebral cortices in the cat[END_REF] but there has been dispute as to the route and extent to which the inferior olive receives direct cerebral input (cf. [START_REF] Swenson | The afferent connections of the inferior olivary complex in rats. An anterograde study using autoradiographic and axonal degeneration techniques[END_REF] who found a direct cerebro-olivary projection and Saint-Cyr [1987] who found it to be indirect). The specific cerebro-olivo-cerebellar and cerebro-pontocerebellar pathways under investigation are introduced in more detail in the individual Results chapters.

Aims

The main aims of this investigation are firstly to reconstruct the ponto-cerebellar pathway to defined climbing fibre zones using anatomical tract tracing and 3D

computer-sided reconstruction techniques to study brain-map transformations in the mossy fibre projection (termed the ponto-cerebellar 3D reconstruction experiments;

Chapter 3). Secondly, to anatomically trace the cerebro-ponto-cerebellar pathway in relation to limb input, to elucidate whether topographical relationships exist in the inputs and outputs of BPN (termed cerebro-ponto-cerebellar experiments; Chapter 4).

Thirdly, the cerebro-olivo-cerebellar pathways will be investigated in relation to the convergence of ascending and descending inputs to defined climbing fibre zones, using anatomical, pharmacological and physiological techniques to understand the underlying structure-function relations (termed cerebro-olivo-cerebellar experiments;

Chapter 5). For all studies, the zonal projection from the inferior olive to the cerebellum will be used as a framework for organisation.

----------------------------------------

Chapter 2: Methods ---------------------------------------- Sodium pentobarbitone (50 mg/kg, Sagatal, Rhône Mérieux, Harlow, UK) was injected intraperitoneally (i.p.) and supplementary doses were given i.p. as required to maintain surgical levels of anaesthesia. In two of the Sprague-Dawley cases, the animals were initially sedated with fentanyl-fluanisone, (0.4 ml/kg, Hypnorm, High Wycombe, UK), intramuscularly (i.m.). Once drowsy, Propofol (Rapinovet, Schering-Plough, Welwyn Garden City, UK) was given intravenously through a continuous flow pump (flow rate variable between 0.006 ml/minute to 0.05 ml/minute), via a cannula inserted into a tail vein to achieve surgical levels of anaesthesia. During surgery, limb-withdrawal and corneal reflexes were checked to ensure a sufficient depth of anaesthesia.

Methods

Surgical Procedures

In the electrophysiological components of the investigations, barbiturate anaesthesia was used because climbing fibre responses were required to define the olivocerebellar zones. This type of anaesthesia has been shown previously to reduce mossy fibre activity [START_REF] Andersson | Origin and sagittal termination areas of cerebrocerebellar climbing fibre paths in the cat[END_REF] and increase the amplitude of the climbing fibre response [START_REF] Gordon | Effect of pentothal on the activity evoked in the cerebellar cortex[END_REF] in the cerebellum. Climbing fibre responses have been shown to be more stable and fluctuate less under pentobarbitone, as compared to other anaesthetics (Oscarsson & Sjölund, 1977). This stability was important for long periods of recording, as required in some of the experiments performed here.

Previously, the effects of Propofol on climbing fibre responses have been shown to be similar to those of barbiturate anaesthesia [START_REF] Glen | Animal studies of the anaesthetic activity of ICI 35 868[END_REF][START_REF] Pardoe | Changes in excitability of ascending and descending inputs to cerebellar climbing fibers during locomotion[END_REF]. In [START_REF] Pardoe | Changes in excitability of ascending and descending inputs to cerebellar climbing fibers during locomotion[END_REF], both Sagatal and Propofol anaesthesia were used on separate occasions during surgery for recovery, chronic-implantation experiments. The climbing fibre responses found were equivalent using either anaesthesia and similar climbing fibre responses were recorded from peripheral input to a defined zone in the awake behaving cat. In the present experiments, Propofol was used to anaesthetise Sprague-Dawley rats as it was better tolerated than Sagatal, and had a quicker recovery time. In all other experiments, Sagatal was used for its long lasting, stable anaesthetic effects and ease of administration.

Atropine sulphate (0.6 mg/kg, Atrocare, Animalcare Ltd., Dunnington, UK) was administered either subcutaneously (s.c.) or i.p. to reduce mucosal secretions, as required. Rats were placed in a stereotaxic frame and secured using atraumatic ear bars coated with a topical local anaesthetic (Xylocaine, Astra Pharmaceuticals, Kings Langley, UK or Xylonor gel, Septodont, Saint-Maur-des-Fosses, France) and a nose bar. The temperature of the rat was maintained within physiological limits using a heated blanket controlled by a probe measuring rectal temperature.

An incision was made down the midline of the scalp to expose the dorsal surface of the skull. A craniotomy was carried out to expose either the posterior cerebellum (unilaterally or bilaterally), or the cerebellum and the surface of the motor cortex (unilaterally or bilaterally). The dura was carefully removed and the exposed brain surfaces periodically flushed with saline. All animals were humanely sacrificed, with an overdose of barbiturate anaesthesia (i.p.) at the termination of the experiment.

Animals in recovery experiments (BR and DP series) were routinely given an injection (i.m. or s.c.) of buprenorphine (Temgesic, 0.1 mg/kg i.m. or s.c., buprenorphine, Schering-Plough, Welwyn Garden City, UK) for post-operative analgesia.

Electrophysiology

Climbing fibre field potentials were recorded in the cerebellar cortex evoked by peripheral and/or motor cortical stimulation. The body parts or cerebral representations stimulated were forelimb, hindlimb and/or facial areas. Figure 2.1

shows the general experimental setup for the electrophysiological stimulation and recording sites, and also for the main nuclei being investigated.

Climbing Fibre Responses

The climbing fibre field potentials evoked by stimulation were recorded extracellularly from the surface of the cerebellum, using tungsten-in-glass microelectrodes (tip diameter ≈ 50 µm) or a 0.1 MΩ electrode (World Precision Instruments (WPI), Florida, USA). Each cerebellar zone was defined in terms of the peripheral stimulation site that evoked the largest climbing fibre responses within that zone, with the largest responses indicating the physiological centre of the zone, as in previous studies (e.g. peripherally evoked responses found by [START_REF] Atkins | Somatotopical organisation within the climbing fibre projection to the paramedian lobule and copula pyramidis of the rat cerebellum[END_REF]Pardoe & Apps, 2002).

One or two recording electrodes were used, depending on the aims of the experiment.

Peripheral Stimulation

Pairs of percutaneous needle electrodes were used to stimulate peripheral body parts to evoke climbing fibre responses mediated through spino-olivary pathways.

Routinely, single, square pulses of 0.1 ms duration, once every 1.5 to 2.5 seconds (s)

(or up to 4.5 s for pharmacological experiments), were applied from an isolated stimulating unit driven by a Digitimer unit. Stimulus intensity was sufficient to evoke a visible reflex twitch in the body part stimulated. In the 3D reconstruction experiments the body parts stimulated to locate certain climbing fibre zones were the ipsilateral fore-and hindlimbs and the contralateral facial/furry buccal pad areas.

With the other anatomical and physiological experiments, the body parts stimulated were primarily the ipsilateral and contralateral forelimbs and the ipsilateral hindlimb.

Motor Cortical Stimulation

In the cerebro-cerebellar experiments, the motor cortex was also stimulated, to evoke climbing fibre responses mediated by cerebro-cerebellar pathways. Concentric bipolar electrodes (WPI, Florida, USA) were inserted through the pia matter to a depth of 1.6-1.8 mm to stimulate the pyramidal projection neurones in the cerebral cortex. Single or double, square pulses (0.1 ms) of varying intensity (typically between 30 µA to 2 mA) were delivered to the motor cortex when recording responses and trains of thirty pulses were used to evoke bodily movements to determine the site of stimulation relative to the somatotopy in the motor cortex.

Cortical stimulation was delivered cyclically every 1.5 to 2.5 s, and in one pharmacological experiment using forelimb stimulation, the cycle was 4.5 s with peripheral stimulation delivered at 0 s, contralateral motor cortex at 1.5 s, then ipsilateral motor cortex at 3 s.

The motor cortical map of [START_REF] Donoghue | The motor cortex of the rat: cytoarchitecture and microstimulation mapping[END_REF] was used as a starting point for locating limb responsiveness. The stimulating electrode was used to roam the motor cortex to produce a map of areas in the cerebellum responsive to cerebral stimulation;

the stimulating electrode was also positioned in the centre of body part areas and the recording electrode was then moved around the cerebellum. In the acute physiological experiments, two bipolar stimulating electrodes were often used simultaneously depending on the aims of the experiment. This was so that different bodily representations e.g. contralateral forelimb and hindlimb areas, or contralateral and ipsilateral forelimb areas in relation to the recording electrode, could be compared in the same experiment. This method also meant that more data was gained per animal used.

Inactivation of Caudal Brainstem Sites

Where anterograde tracer was injected into the motor cortex to trace the cerebroolivary projection, a putative synaptic relay (see Chapter 5 for results) was found and inactivated using drugs to observe whether there was a selective decrease in cerebrally-evoked, but not peripherally-evoked climbing fibre field potentials.

Reversible pharmacological inactivation of this putative relay site was used as the target area was small and control sites further rostral and caudal needed to be inactivated to seek a specific effect. Different drugs were used to inactivate the identified putative relay area and were chosen carefully. A recovery of the climbing fibre responses was considered desirable, as was a short period of inactivation (up to an hour) because multiple sites in one animal could then be studied and ensured that it was not simply tissue damage that caused any inactivation of responses. Also, using multiple injections in the same animal was a clear advantage in providing an internal control comparing different rostrocaudal areas, therefore increasing the reliability and repeatability of the data as well as using fewer animals.

Two main classes of drugs are typically used to reversibly inactivate neuronal areas; sodium channel blockers and inhibitory neurotransmitters. Sodium channel blockers e.g. local anaesthetic, work by preventing conduction of an action potential along an axon, and at the cell body by preventing initiation of an action potential. A commonly used local anaesthetic, Xylocaine liquid (10% lignocaine; Astra Pharmaceuticals Ltd., Kings Langley, UK), was used in small quantities, between 25 nl and 150 nl per injection at a depths of 0.5-0.7 mm into the caudal brainstem and the effects were recorded for off-line analysis.

The use of inhibitory neurotransmitters (also their agonists) was deemed advantageous as these work by increasing inhibition, therefore selectively hyperpolarising neuronal cell bodies. The most prevalent inhibitory neurotransmitter in the CNS is γ-aminobutyric acid (GABA) and it has been widely used as a drug of choice to inhibit neurones [START_REF] Hupé | Spatial and temporal parameters of cortical inactivation by GABA[END_REF]. A combination of GABA and kynurenic acid in solution were used to increase inhibition and decrease excitation (from blocking excitatory glutamate receptors), respectively. The double effect was used to facilitate the reversible neuronal block. The GABA solid (Tocris, Bristol, UK) and kynurenic acid solid (4-hydroxyquinoline-2-carboxylic acid; Sigma, Missouri, USA) were weighed out and made up to 0.1 M concentration and 0.001 M concentration respectively in 10 ml artificial cerebrospinal fluid (Harvard, Massachusetts, USA). Small injections of between 100-150 nl of the GABA/kynurenic acid solution were injected into the caudal brainstem at a depth of 0.5 -0.6 mm and the effects recorded for off-line analysis.

The extent of effective spread was also taken into consideration. Local anaesthetics have been shown before to inactivate an area of approximately 500 µm diameter around the injection site [START_REF] Malpeli | Response properties of single cells in monkey striate cortex during reversible inactivation of individual lateral geniculate laminae[END_REF]) using 100 nl 2% lidocaine. [START_REF] Boehnke | Time course and effective spread of lidocaine and tetrodotoxin delivered via microdialysis: an electrophysiological study in cerebral cortex[END_REF] found that significant inactivation showed up to 1 mm spread but they used far greater volumes of 10% lidocaine (between 40 to 110 µl compared to up to 150 nl per injection in the present study). GABA was chosen as it produces a short block in neuronal responses, similar to that of local anaesthetics (see [START_REF] Martin | Pharmacological inactivation in the analysis of the central control of movement[END_REF] where lidocaine and GABA were compared). The effective spread of GABA has been investigated in previous studies (e.g. [START_REF] Hupé | Spatial and temporal parameters of cortical inactivation by GABA[END_REF]Nealy & Maunsell, 1994). Hupé et al. showed that a pressure injection of 60 nl, 100 mM GABA inactivated cells in a radius of between 250 to 500 µm. Nealy & Maunsell found that using 100 nl, 25 mM GABA produced a blockade of approximately 500 µm radius. Therefore, with the present regime, it was hoped that area of inactivation would be large enough to encompass the putative relay site, without encroaching significantly into other nuclei or tracts.

For the caudal brainstem drug injections into the putative relay site, the distance from the obex to the site of injection was found by using stereotaxic co-ordinates from a manipulator, and by using a 1 x 5 mm 2 piece of graph paper placed at the obex, running along the rostrocaudal axis, on the side of the caudal brainstem opposite to the injection site. Pressure injections using a glass micropipette (≈ 20 µm tip diameter) attached to a 1 µl Hamilton syringe (Precision Sampling Corporation, Louisiana, USA) were made into the caudal brainstem on the side ipsilateral to the recording electrode(s). Small amounts of pontamine sky blue were used as a dye to colour the drug solutions injected. This served as a way of identifying the pipette tip during the experiment and as a marker of the injection site for histological investigations. Climbing fibre responses to forelimb and/or hindlimb peripheral and cerebral stimulation were recorded for at least 2 minutes prior to drug injection and for up to approximately 70 minutes after microinjection of drug.

Analysis of Drug Injection Data

Climbing fibre responses to peripheral and motor cortical stimulation were recorded using the Spike 2 program then analysed off-line. The custom-made script 'Locomot(new)' was used to compare relative climbing fibre field response sizes.

The measurement taken depended upon two moveable cursors, which were positioned at the beginning and end of each waveform. For each analysed waveform after a certain, defined stimulus, the area under the curve (waveform) was measured over time (in mV x ms), thus producing a numerical value for each response. Values for the onset latency of the response and the duration of the waveform were also obtained and averages were calculated. Every 10 individual waveform area values were binned to give an average value for every 30 s (in one case every 45 s). Graphs were drawn using a control value (pre-drug application) and the data post-drug application were compared to this (100% value).

Injection & Processing of Material

Axonal tracing methods were employed in ponto-cerebellar 3D reconstruction experiments to chart the mossy fibre projection from BPN to defined climbing fibre zones and in cerebro-cerebellar experiments to trace the cerebro-pontine, cerebroolivary and ponto-cerebellar projections. The centre of each zone was taken to be the part yielding the largest evoked responses. All injections were made using a glass micropipette (≈ 20 µm tip diameter) attached to a 1 µl Hamilton syringe.

Anterograde Injection into the Motor Cortex

When anterograde tracers were injected into the motor cortex, Fluoro-Ruby (FR)

and/or Fluoro-Emerald (FE) (Molecular Probes, OR, USA) were used. In some cases, biotinylated dextran amine (BDA) was also injected, although none of these injections were successful, and therefore not considered further. The area injected into was located by physiologically identifying it from responses evoked in the cerebellum as a result of cerebral stimulation. Also, using a train of thirty stimuli, limb movements could be evoked to help identify the sub-region within the motor cortex. The pyramidal projection neurones located in the agranular cortex, were the target neurones for the uptake of tracer material (layer V, see section 2.3.1.1 below).

The stimulating electrode was slowly lowered into the motor cortex to gauge the optimum depth for evoking climbing fibre field potentials; the injections were targeted on these physiologically-defined cortical areas.

Pressure injections of tracer material were made using 20% solutions (1 mg in 5 µl saline) of FE or FR (also the same for BDA). Between 100-400 nl of any one tracer was injected into the motor cortex. No more than 300 nl was injected at any one site, and often multiple sites (up to three) in close proximity in the same bodily representation were injected to increase the likelihood of tracer uptake (the anterograde tracer is taken up and transported by damaged axons [START_REF] Köbbert | Current concepts in neuroanatomical tracing[END_REF]). The tracer was injected into the most effective stimulation site, typically where the threshold for evoking cerebellar responses was lowest.

Considerations of the Anterograde Tracer

There were some methodological considerations, particularly concerning the anterograde tracer. There were difficulties in gaining both axonal transport of and terminal labelling by the anterograde tracer, injected into the motor cortex. The tracer uptake to gain terminal labelling was not without difficulties. It is relevant to note that in a study by [START_REF] Mihailoff | Projections of the sensorimotor cortex to the basilar pontine nuclei in the rat: an autoradiographic study[END_REF], there was also a fairly low success rate using titrated amino acid methods where only 13 out of 21 injections into the motor cortex were judged to be successful. No reasons for this were given, although it seems likely that it may have been due to similar factors outlined above. This may in part explain the divergent results in previous studies tracing the afferent connections to pre-cerebellar nuclei. This has been especially so for olivary afferent connections arising from the motor cortex and is discussed later in Chapter 5.

Retrograde Tracer Injection into the Cerebellum

Peripheral and/or motor cortical stimulation was used to determine the centre of the face A2 zone, FL (C1) PML zone or HL (C1) CP zone. For cerebellar injections, retrograde tracer material (40-100 nl, green beads (GB) or red beads (RB),

Lumafluor Inc., New York, USA) was delivered hydraulically via a micropipette.

Each microinjection was made at a depth of 0.3 -0.4 mm, perpendicular to the pial surface of the cerebellum. In the cerebro-cerebellar experiments (DP series), forelimb and/or hindlimb receiving areas in the cerebellum were injected (up to 100 nl per site), whereas in the ponto-cerebellar experiments (BR series), facial, forelimb and/or hindlimb receiving areas were injected (up to 50 nl per site).

Preparation of Material

For the anatomical experiments, the animals were kept for a survival period of 7-10 days to allow for sufficient axonal transport of tracer material. The rats were then deeply re-anaesthetised using sodium pentobarbitone (60 mg/kg, i.p., Sagatal, Rhône Mérieux, Harlow, UK) and transcardially perfusion-fixed. A heparinised saline flush (approximately 500 ml, 0.9% saline) was immediately followed by fixative (approximately 500-1000 ml 4% paraformaldehyde in phosphate buffered solution, PBS), and finally with sucrose PBS (approximately 500 ml, 10% sucrose) for cryoprotection. The brain and spinal cord were dissected out, stored in 30% sucrose PBS at 4°C and left to sink until sectioned. At the end of the non-recovery experiments (DPE series), where drugs mixed with pontamine sky blue were injected into the caudal brainstem, the animals were humanely killed and the brainstem was carefully removed and post-fixed in 4% paraformaldehyde. After this, 100 µm sections were cut using a freezing microtome and mounted on glass slides for inspection using light microscopy.

The tissue was cut into cerebellar, brainstem, cerebral and spinal cord blocks, although for the 3D reconstruction experiments, the brainstem block was divided into separate olivary and pontine parts. All blocks were then sectioned on a freezing microtome at 50 µm, apart from some cerebellar and cerebral blocks which were sectioned at 100 µm. Cerebral blocks were routinely embedded in gelatine so that the cerebrum and midbrain parts did not separate. In one case (DP11), the whole brain was embedded in gelatine and sectioned as a single block for full rostrocaudal anatomical mapping. The tracers needed no further processing and were temporarily coverslipped using phosphate buffer to be viewed under a fluorescent microscope.

Mapping of Tracer Material

Sections were examined and mapped using a Leica DMRB microscope (Wetzlar, Germany) or a Zeiss Axioskop II Mot microscope (Oberkochen, Germany). Both microscopes used a mercury UV light source for illuminating fluorescent material.

On the Leica microscope green fluorescent labelling produced from injections of green beads or Fluoro-Emerald was scrutinised using an H3 filter block (dichroic mirror (DM) 510 nm, band pass (BP) 420-490 nm, long pass (LP) 520 nm). Red fluorescent labelling from red beads or Fluoro-Ruby was viewed with an N2.1 filter block (DM 580 nm, BP 515-560 nm, LP 580 nm). On the Zeiss microscope, green fluorescence was viewed using filter set no. 09 (DM 510 nm, BP450-490 nm, LP 515 nm), and red fluorescence using filter set no. 15 (DM 580 nm, BP 546/12 nm, LP 590 nm). The same (light) microscopes were used for histologically verifying the caudal brainstem injection sites in the pharmacological inactivation experiments (DPE series).

Anterograde Terminal Labelling

Anterograde labelling was mapped by tracing the axonal pathways throughout the rostrocaudal axis of the brain. The density of any terminal labelling was recorded in a semi-quantitative manner (very light -light -medium -dense). Nuclear regions were defined more quantitatively by splitting the region mediolaterally and dorsoventrally into tenths and coding each square with a number (0 = no labelling; 1 = very light; 2 = light; 3 = medium; 4 = dense). From this, the total extent and overlapping areas of terminal labelling could be compared between modalities and also with the cerebellar injection labelling in nuclear regions with overlapping terminal and cell labelling.

Also, for a further quantitative approach, the pontine transverse maps were divided into a matrix of 150 µm 2 squares at each rostro-caudal level (each level spaced approximately every 300 µm). Each square was given a coding depending on the amount of terminal labelling (as above). This could then be quantified and represented graphically, as single and averaged cases, and as the merged total extent.

Retrograde Cell Labelling

Cell labelling from the retrograde tracer was easier to quantify as cell bodies could be counted and summed for different areas where labelling was found. Only whole or nearly-whole cells from only one series were counted; this was to minimise over-estimation of cell counts. Cell labelling produced from injections of red and/or green beads in the cerebellar cortex was evident in various brainstem nuclei. Retrograde labelling was quantified by mapping and counting the numbers of labelled cells present at selected levels of each nucleus studies. Typically, cell labelling was mapped onto integrated BPN and NRTP standard maps, and onto inferior olive standard maps. These maps were of transverse levels and each level could be equated to around 200-300 µm, in the rostrocaudal plane. The numbers of cells were noted for each nucleus, per section, and mapped onto the allocated level on the standard maps, to then be further analysed and converted to a horizontal plane of view, for ease of comparison, if required.

Cell body labelling was also present in the thalamus and substantia nigra from the injections of tracer into the motor cortex and although the labelling was noted, the nuclei were not studied further. This was due to the labelling of projection neurones to the motor cortex, as the dextran amines are bi-directional and can produce retrograde labelling. However they do not produce retrograde labelling with the same efficiency as the beads (for a review see [START_REF] Köbbert | Current concepts in neuroanatomical tracing[END_REF].

Injection Sites

The injections of tracer material caused small lesions in the brain tissue injected and left remnants of tracer that was not taken up. These sites in the cerebellum and motor cortex could thus be easily identified and quantified as to size.

Motor Cortex Injection Site

In the cerebro-cerebellar recovery experiments, tracers for anterograde transport were pressure injected into the motor cortex (FE and/or FR) to be taken up by projection neurones, labelling axons and terminal fibres/boutons. Injections were made perpendicular to the surface at a depth of 1.6-1.8 mm. Histological analysis included identifying the section containing most of the tracer material. This was considered as the centre of the injection and the surrounding sections that contained solid tracer gave the rostro-caudal extent of the core injection site. Measurements were also taken to gauge the depth and medio-lateral location of the core, and to confirm the tracer was in layer V of the agranular cortex. Each injection site was photographed using a Zeiss digital camera attachment on the microscope. The depth to the core of the injection site, the mediolateral distance to the core, and the area of the injection site, were all measured using the digital camera attachment to the microscope and the software program Axiovision (Zeiss, Oberkochen, Germany).

Cerebellar Injection Site

RB and/or GB were injected perpendicular to the cerebellar surface. The core injectate area was located and the medio-lateral spread of the beads recorded. The injection site was photographed and the length of the spread over the white matter/granular layer interface was determined from the photograph using the Axiovision program. A curved line was drawn digitally on the photograph along the length of the interface spread and the scale adjusted to the lens power so that the length over the core number of sections was measured accurately in µm 2 .

Caudal Brainstem Injection Site

The sections were mapped onto standard caudal brainstem maps (based on [START_REF] Paxinos | The Rat Brain in Stereotaxic Co-ordinates[END_REF] and the injection sites located where possible. Due to the number of injections and sometimes to a failure of pontamine sky blue dye colouring, many injection sites were difficult to map.

Inferior Olive Mapping

Cell labelling was mapped onto standard olivary transverse maps (based on [START_REF] Apps | Columnar organisation of the inferior olive projection to the posterior lobe of the rat cerebellum[END_REF]) and the numbers of labelled cells in each main sub-nucleus of the olive, per section, were recorded. Any anterograde labelling was also plotted onto these maps.

Labelling was transferred to horizontal maps, constructed from the transverse maps, to view the labelling in a different plane. This served as another assessment as to the extent to which the labelling from an injection into a specified zone, was restricted to the expected area in the inferior olive. Cell counts were summed per sub-nuclear region.

Basilar Pontine Nuclei Mapping

The cell labelling in BPN (and NRTP) was mapped using standard pontine maps.

These were constructed by taking a low power photomicrograph of both sides of the pons and drawing round the nuclei in Corel Draw graphics program. Three different animals were used to construct the standard maps and adjustments were made after looking at numerous pontine sections. Any anterograde labelling was also drawn onto the maps, indicating the labelled fibres and the density of terminal labelling using different cross-hatching. The terminal labelling was graded and its location noted.

The standard views of BPN used in publications from the NeSys Laboratory at Oslo

University have been adopted in this investigation as they are useful in displaying data. The views consist of the ventral, rostral, and medial views, often used to visualise 3D labelling compressed into 2D planes. Figure 2.2 shows the orientation of the defined planes (for reference in all BPN maps). Additionally, in the 3D pontine experiments, the retrograde cell labelling seen in BPN, in selected cases, was reconstructed using the custom-made software Micro 3D (Neural Systems and Graphics (NeSys) Computing Laboratory, University of Oslo, Oslo, Norway; see Leergaard & Bjaalie [1995] for reference to original software).

For the reconstruction, labelled cells found in BPN were mapped with the aid of a given tags, either 'red' or 'green' or, if double labelled, a 'double' overlying tag and landmarks were drawn as lines and given a specific (colour) coding for each boundary so they could be identified easily. Alternate sections containing pontine material were drawn round and digitised.

Cell locations and structural information were transferred to a specialist computer system, where the data was uploaded into the software program Micro3D using a Silicon Graphics Octane 2 workstation. This system was used to view and analyse the data in three dimensions. The 3D reconstruction facilitated the process of comparing the data within and between cases, and the investigation of spatial relationships in the distribution of cell labelling. Each section in turn, rostral to caudal, was assigned a z (depth) value in accordance to section thickness and serial order. This was performed for the entire rostrocaudal axis of BPN. Serial sections were visually aligned interactively on the computer screen using the colour-coded landmarks, especially the midline and ventral boundary, to gain a realistic reconstruction of the pontine area of the brainstem. Another advantage of using Micro3D is that it allowed the sections to be inspected using real time rotation, thus viewing the sections from different angles ensuring accurate alignment.

Once all the sections were uploaded and aligned, a local pontine co-ordinate system was used to define BPN and the cell labelling located within BPN, as introduced by The dimensions of the bounding box ran in the rostrocaudal axis (rostral 0% to caudal 100%), mediolaterally (medial 0% to lateral 100%), and horizontally in the dorsoventral plane (ventral 100% to 0% centrally then to dorsal -100%). The origin of the co-ordinate system was therefore the intersection between all of these boundaries at 0% (rostral-medial-central). The cell labelling and anatomical landmarks within the box are given relative co-ordinates in the bounding box space.

The local pontine co-ordinate system has been used for previous studies (Leergaard et al., 2000a, b;[START_REF] Brevik | Threedimensional computerised atlas of the rat brain stem precerebellar system: approaches for mapping, visualization, and comparison of spatial distribution data[END_REF] and affine transformations [START_REF] Ashburner | Incorporating prior knowledge into image registration[END_REF][START_REF] Ashburner | Nonlinear spatial normalization using basis functions[END_REF] take into account this prior information (e.g. variability in BPN shape) using Bayesian reasoning. Thus, data from BPN labelling in previous studies are comparable with this present investigation. . The data could also be re-sliced in a user-defined plane and rotated and these facilities were used for detailed inspection of the data. Analyses of pontine areas were also performed for the cerebro-cerebellar tracer experiments.

Other Areas

In other areas where labelling was found, the extent and area was recorded. The rostral block containing the cerebral cortex and midbrain was mapped using standard diagrams adapted from [START_REF] Paxinos | The Rat Brain In Stereotaxic Co-ordinates[END_REF] where anterogradely labelled fibres and terminal labelling from the motor cortex injection could be found. From the anterograde injection, fibres could be traced down the spinal cord. The standard maps of [START_REF] Brichta | Cytoarchitectural organization of the spinal cord[END_REF] were used to map the levels of the spinal cord. Maps were also constructed for the brainstem, which were based around the pontine and olivary standard maps and included structures such as the LRN and NRTP.

Colour Format of Data and Figures

There is a theme to the colour coding of all the figures and data in this thesis.

Ponto-cerebellar 3D Reconstruction

Abstract

These experiments were carried out in collaboration with the Neural Systems and Graphics Computing Laboratory (NeSys) at the University of Oslo, Norway. The aim of the experiments was to investigate the underlying anatomical connectivity of the ponto-cerebellar mossy fibre projection in relation to climbing fibre zones, as this has not been systematically studied. This was of interest as the parasagittal organisation of the climbing fibre zones imposes a degree of somatotopical order on the inputs and outputs of the cerebellum. By contrast, previous studies suggest a less organised, patchy arrangement to the mossy fibre projection, with varying degrees of bilaterality present.

Retrograde tracer material was injected into the cerebellar cortex to label cells in the inferior olive and in BPN. Injections were centred on the vermal A2 zone of PML (face-receiving), and the paravermal C1 zones in PML (forelimb-receiving) and CP (hindlimb-receiving). BPN was reconstructed in 3D to aid the analysis of the data and to reveal relationships between cases. The double tracer method provided strong evidence for a topographically arranged relationship of the mossy fibre projection to climbing fibre zones. The cases that were analysed and reconstructed in 3D can also be found in the NeSys database at www.nesys.uio.no/Database, where each case can be compared with other cases and inspected in 3D.

The results were best viewed from a ventral position and gave evidence towards a topographical organisation of the ponto-cerebellar pathway to the climbing fibre zones under investigation. The face A2 zone was represented centrally and bilaterally in BPN, whereas the labelling from fore-and hindlimb injections into the C1 zone were respectively centred more caudally with a contralateral preponderance. The ponto-cerebellar projection was also compared to the cerebro-pontine projection from the somatosensory cortex as previously described by Leergaard et al. (2000b).

As both pathways were mapped using the local co-ordinate system in 3D (for cerebro-pontine data see Leergaard et al. (2000b) and www.nesys.uio.no/Database), volume and spatial overlap could be compared.

Introduction

This part of the thesis specifically investigates the ponto-cerebellar pathway to establish whether there is an order in the BPN mossy fibre projection to defined climbing fibre zones. Computerised 3D brain mapping was utilised as a system for the reconstruction of BPN and for mapping the subsequent labelling. Such an approach has been used particularly successfully in mapping the cerebro-pontine projection in rat and has shown topographical representations of body parts in BPN [START_REF] Bjaalie | Corticopontine terminal fibres form small scale clusters and large scale lamellae in the cat[END_REF]Leergaard et al., 1995Leergaard et al., , 2000a[START_REF] Leergaard | Clustered and laminar topographic patterns in rat cerebropontine pathways[END_REF][START_REF] Leergaard | Three-dimensional topography of corticopontine projections from rat sensorimotor cortex: comparisons with corticostriatal projections reveal diverse integrative organization[END_REF]. By contrast, previous studies have failed to identify a clear topographical organisation in the next link in the cerebro-cerebellar pathway: the ponto-cerebellar projection [START_REF] Brodal | The ponto-cerebellar projection in the rabbit and cat[END_REF]Brodal & Bjaalie, 1997;[START_REF] Schwarz | Modular organization of the pontine nuclei: dendritic fields of identified pontine projection neurons in the rat respect the borders of cortical afferent fields[END_REF][START_REF] Leergaard | Clustered and laminar topographic patterns in rat cerebropontine pathways[END_REF].

Mapping BPN in the standard transverse plane has not shown to any great degree a topographical order in the ponto-cerebellar projection (see General Introduction).

The climbing fibres, originating solely from the inferior olive, target all parts of the cerebellar cortex and terminate in parasagittally oriented zones, thus imposing a topographical framework that is thought to be key in cerebellar information processing (e.g. [START_REF] Voogd | The anatomy of the cerebellum[END_REF]. The present study takes into account the climbing fibre somatotopy in regard to the ponto-cerebellar projection, whereas previous studies have typically studied one type of cerebellar afferent in isolation.

Therefore, combining the climbing fibre zonal organisation and the projection pattern of specific cells in BPN would give evidence towards a more unified model of cerebellar functioning, by integrating the anatomical organisation of the two main sources of input to the cerebellum.

A large amount of BPN afferent information arises from the cerebral cortex.

Projections from various cerebral areas including motor, sensory and visual areas all innervate BPN [START_REF] Snider | Cerebrocerebellar relationships in the monkey[END_REF]Brodal, 1968aBrodal, , b, 1972a, b;, b;[START_REF] Schmahmann | The cerebrocerebellar system[END_REF]. These pathways have been previously found to preserve the topographical arrangement of the cerebral maps in the 3D reconstruction work of Leergaard et al. (2000a[START_REF] Leergaard | Clustered and laminar topographic patterns in rat cerebropontine pathways[END_REF][START_REF] Leergaard | Three-dimensional topography of corticopontine projections from rat sensorimotor cortex: comparisons with corticostriatal projections reveal diverse integrative organization[END_REF]. Therefore, the way in which this large volume of descending information is processed and relayed by BPN to the cerebellum is of great interest. The newer 3D technology available encompasses older, traditional anatomical tracer techniques to gain a more complete view of individual maps and how these combine together. A clear advantage of using new technology in bioinformatics is that more data can be shared. [START_REF] Chicurel | Databasing the brain[END_REF] outlined the advantages of bioinformatics, specifically in neuroscience, including the many anatomical and physiological standardised brain databases (e.g. see rat brain database at http://www.nesys.uio.no). Although brain-map 3D transformations have been useful in elucidating the bodily representations in different areas in the brain, they nevertheless depend on well-established fundamental anatomical and physiological techniques.

The 3D representations and databases take the experimental data further to reveal additional information and relationships. The newer technology, means that mapping brain structures is easier and brain atlases can be re-shaped and sliced in a userdefined plane to reveal nuclei in a selected view and labelling in different ways. Also, the actual outline of nuclei and position of labelling can be accurately mapped to display real relationships that can be standardised and used within and across experimental series. Thus, a critical aspect of this work is to assign location of function to anatomical (and physiological) data (see [START_REF] Bjaalie | Opinion: Localization in the brain: new solutions emerging[END_REF]. The database associated with some of this work uses a standardised co-ordinate system to gain a relational database. This system purposely allows flexibility, dynamism, and scalability in the data presented and provides for future expansion (see [START_REF] Bjaalie | Advances in computational neuroanatomy[END_REF][START_REF] Bjaalie | Database and tools for analysis of topographic organization and map transformations in major projection systems of the brain[END_REF] for overviews).

Aims of This Study

This chapter firstly seeks to elucidate the ponto-cerebellar projection to defined climbing fibre zones and then in part, relate it to the somatosensory cerebral input to BPN. This is attempted by using physiological and anatomical methods, in combination with specialist 3D reconstruction software to model the ponto-cerebellar projection in BPN and to further view and quantify any relationships present.

Chapter 4 takes this investigation further by studying cerebro-pontine and pontocerebellar pathways in the same animal and relating the BPN labelling to the climbing fibre somatotopy.

Results

Results were obtained from 3 Sprague Dawley and 5 adult Wistar rats (weights 230-285 g). The results gained from each strain of rat were very similar, therefore the data are considered together. Two microinjections of retrograde tracer (red or green beads) were made into the A2 face, C1 forelimb or C1 hindlimb regions of the posterior lobe of the cerebellum. A total of 16 cases were obtained with 7 'face' cases, 6 'forelimb' cases and 3 'hindlimb' cases ('case' defined as a single injection site; there were 2 cases per animal and the prefix 'BR' was used to denote experimental series with the animal number).

BPN Cell Labelling

Table 3.1 details all the cases including the details of the injection site location and the cell counts for labelled neurones in the brainstem structures studied. Cell labelling was found in the inferior olive, contralateral to the injection site, although bilateral cell labelling was found in BPN to varying degrees. There was a highly significant positive correlation between the number of cells found in the inferior olive and in BPN (r = 0.77, p < 0.001, n = 16; see graph below Table 3.1 for correlations), indicating that there was effective uptake and transport of the retrograde tracer from the cerebellar injection site for both mossy fibre and climbing fibre afferents.

For internal consistency, the numbers of labelled cells in BPN displayed in Table 3.1 were all counted by the author, as was the olivary cell labelling, and the cell distributions were mapped onto standard transverse maps of each structure. However, the 3D reconstruction figures of BPN (e.g. Figure 3.2) cells show numbers counted independently by a second investigator (Francis Odeh) in Oslo. There was a good correspondence between both sets of values from each independent investigator, as there was no significant difference between BPN cell counts (paired t test p=0.65 n.s.

n=16) and a strong positive correlation was found between the paired cases (r=0.97, p<0.0001).

Cases were chosen for reconstruction on the basis of injection site and olivary labelling. The inferior olive was inspected for cell labelling and the regions in the inferior olive, defined in past work (e.g. [START_REF] Apps | Columnar organisation of the inferior olive projection to the posterior lobe of the rat cerebellum[END_REF][START_REF] Atkins | Somatotopical organisation within the climbing fibre projection to the paramedian lobule and copula pyramidis of the rat cerebellum[END_REF] as providing climbing fibre afferents to the zones in question were: for the A2 face zone, caudal MAO, specifically, the subnucleus b1 and the ventro-lateral outgrowth; for the C1 forelimb zone in PML, mid-rostral MAO and rostral DAO; and for the C1 hindlimb CP, rostrolateral DAO. If the labelling found was not located in the expected territory or was spread over other areas, the case was rejected. or olive. In the cases selected, over 94.4% of the cell labelling in the inferior olive was restricted to the expected territory for a given zone. Using the above selection criteria, a total of 9 cases from 7 animals were selected for giving 3 cases per injected zonal region (see table 3.2 for details). The low cell count in case BR13 HL, was probably due to the injection being rather superficial and not quite reaching the Purkinje cell layer. Although the amount of labelling found in the olive was minimal (only 10 cells were found), it was nevertheless in the expected region. In the two double tracer experiments, no double labelled cells were found in the brainstem, providing strong evidence for the topographical organisation of different ponto-cerebellar projection regions, in the same animal. For the 9 cases (apart from BR05 face where the physiological width of the A2 zone was not established) all the histologically defined injection sites were within the electrophysiological width of the zone, as defined before the tracer injection, which was sited at the approximate centre of the zone. For the remaining cases that were not included in the analysis due to reasons such as spread of tracer into neighbouring zones (as shown by patterns of olivary cell labelling), the BPN labelling were nevertheless inspected and similar patterns and regions of cell labelling were found compared to the cases used in the analysis.

A case from each zonal injection was chosen in Compared to the labelling from the A2 face injection, the C1 forelimb cell labelling was clustered more caudally, but also with a small scatter of cells in the rostromedial half of BPN, most clearly viewed ventrally. There were cells on the side ipsilateral to the injection site, mirroring the pattern on the contralateral side but the contralateral dominance was strong (86.7% of cell labelling was on the contralateral side).

For the C1 hindlimb injection, a large, dense cluster of cell labelling was in caudal BPN spanning most of the mediolateral width as can be seen in the ventral and rostral views. Similar to the C1 forelimb labelling, a scatter of cells was also found rostral to the main region. These cells were slightly more rostral, and central in the mediolateral plane, compared to the corresponding 'forelimb' scatter. The C1 hindlimb injection consistently produced the least labelling ipsilateral to the injection site (96.5% of cell labelling was on the contralateral side), as can most readily be seen in the ventral and rostral views. A cluster of cells was also seen in the medial and rostral views of BPN for the C1 hindlimb injection, located dorsomedially to BPN, more so on the contralateral side. These cells are in fact part of the dense, main area of labelling in BPN but are located in the region dorsal to the pontine fasciculus, which transects each side of BPN, therefore not visible in the ventral view.

Differences in the bilaterality of the cell labelling in BPN were a consistent finding that was related to the injected zone. Cell labelling from the A2 zone injections was essentially bilateral with the average cell count contralateral to the injection site making up 54.5% of the total number of labelled cells in BPN. There was no significant difference between the cell counts contralateral and ipsilateral to the injection site (paired t test, p = 0.27, n = 3). For the C1 forelimb data, there was an average predominance of 86.7% of labelled cells contralateral to the injection site and the difference between the two sides was statistically significant (paired t test, p<0.01, n = 3). For the C1 hindlimb cases resulted in little labelling on the side ipsilateral to the injection site, with an average of 96.5% of labelled cells found on the contralateral side to the injection site.

Discussion

Injections of retrograde tracer into A2 face-, C1 forelimb-and C1 hindlimbreceiving zones gave rise to internal to external lamella-like labelling, respectively, in BPN. This topographical pattern of labelling is similar to that described originally by Leergaard et al. (1995) in the development of the cerebro-pontine projection. In the present experiments where two retrograde tracers were used, no double labelled cells were ever found, thus indicating that these neuronal populations are distinct from one another. From the literature, it is not clear how the continuous topography of the cerebral cortex is transformed into the fractured somatotopy seen in the mossy fibre projection to the cerebellar cortex. The site of 'fracturing' is either in BPN and/or in the mossy fibre projection itself (cf. Bjaalie & Leergaard [2000] with Schwarz & Thier [2000]). The present study gives evidence for a topographical organisation of body parts in BPN projecting to corresponding body-part receiving zones in the cerebellum, thus the fracturing is not likely to take place in BPN.

Implications for Cerebellar Afferent Organisation

The results of this chapter give new insight into the organisation of cerebellar afferents in that the mossy fibre BPN projection is now seen to be related to the somatotopy imposed on the cerebellum by its climbing fibre input. The labelling produced from the A2 face-, C1 forelimb-, and C1 hindlimb-receiving zonal injections gave an internal to external lamella arrangement in BPN, in that the face projecting neurones were located centrally, the forelimb more externally and the hindlimb projecting neurones more in outer, caudal BPN, as best viewed in the ventral views. Furthermore, there was a progressive difference in the bilaterality of the projection in that the face projecting neurones were bilateral; forelimb projecting neurones were more contralateral; and hindlimb projecting neurones were almost completely contralateral.

Overall, there was therefore a distinct organisation in BPN for the spatial representation of cells projecting to different body part-receiving zones in the cerebellar cortex. Previously, little topographic organisation has been attributed to the ponto-cerebellar pathway, especially due to the fractured representation of body parts in the mossy fibre projection (e.g. the projection to Crus I [START_REF] Brodal | The corticopontocerebellar pathway to crus I in the cat as studied with anterograde and retrograde transport of horseradish peroxidase[END_REF]). Through the use of 3D reconstruction, a topographical arrangement of body parts projecting to certain zones was found in the present study. The labelling appeared very patchy when viewed in transverse section but when reconstructed, particularly in the ventral view, the spatial arrangement was clear. This may not have been found in previous studies due to the cell labelling being difficult to visualise without the aid of these specialist computer methods.

By contrast, previous studies investigating the cerebro-pontine projection have found strikingly similar results, in that the body part representation arising from the somatosensory cortex is also arranged in a lamellae-like internal (vibrissae) to external (hindlimb) arrangement within BPN (Leergaard et al., 2000b). The volumes occupied by terminal labelling in BPN have been termed 'lamellae' by Leergaard et al. in this internal-external organisation as the component body part representations were arranged in shell-like concentric sub-regions. The volumes occupied by the cell labelling in the present study were very similar in shape to those found previously.

The rostral cell scatter, particularly seen with the cells projecting to the C1 forelimband hindlimb-receiving zones may reflect the development of this arrangement in BPN (Leergaard et al., 1995).

However, these studies have on the whole focussed on the ipsilateral projection and ignored its bilateral aspect (Leergaard et al., 2000a, b). Similarly, studies on the ponto-cerebellar projection have acknowledged a bilateral pathway, with contralateral predominance (usually around 80% of fibres projecting contralaterally, e.g. [START_REF] Brodal | The corticopontocerebellar pathway to crus I in the cat as studied with anterograde and retrograde transport of horseradish peroxidase[END_REF][START_REF] Herrero | Pontine and lateral reticular projections to the c1 zone in lobulus simplex and paramedian lobule of the rat cerebellar cortex[END_REF][START_REF] Serapide | Laterality of the pontocerebellar projections in the rat[END_REF]. The results from the present investigation have shown a high correlation in the bilaterality of the ponto-cerebellar projection with regard to injecting into different climbing fibre zones. The extent to which the projection is bilateral may have functional implications, such as independent usage of body parts and the amount to which the co-ordination of movements on both sides of the body is important.

Consistent with this possibility, [START_REF] Rosina | Pontine projections to crus I and crus II of the cat cerebellum. A horseradish peroxidase study[END_REF], 1984) found varying degrees to which the ponto-cerebellar pathway was bilateral and they found a particularly high incidence of bilateral projections in lateral areas of crus I and II.

These lateral regions of the cerebellum have been implicated in complex movements, such as in eye pursuit of moving objects [START_REF] Mano | Saccade-related Purkinje cells in the cerebellar hemispheres of the monkey[END_REF]. The face and whisker region are of great significance in the rat for orientation and inquisitive behaviours [START_REF] Berg | Vibrissa movement elicited by rhythmic electrical microstimulation to motor cortex in the aroused rat mimics exploratory whisking[END_REF] and therefore may need a larger bilateral pontine representation. Also in rat, the forelimbs play a key role with the face and whiskers in exploratory behaviour. The weak bilateral hindlimb-receiving projection may reflect the rat not needing to use its feet in similar types of behaviour.

For the topographical pattern evident in the ponto-cerebellar projection described in the present results to be regarded as a general feature of cerebellar afferent organisation, the BPN projection to other zones must also be taken into consideration.

This would be a useful and interesting next step in this line of investigation, but there is already evidence in the literature to suggest this general organisational principle.

For example, [START_REF] Herrero | Pontine and lateral reticular projections to the c1 zone in lobulus simplex and paramedian lobule of the rat cerebellar cortex[END_REF] found a similar spatial organisation of the BPN projection to the C1 forelimb-receiving zones in PML and lobulus simplex (LS).

Along a different, yet related line of inquiry, [START_REF] Lillehaug | Ponto-cerebellar projections to electrophysiologically defined patches in the granule cell layer of rat CrusIIa[END_REF] found that retrograde tracer injections into Crus IIa produced a similar pattern of cell labelling in BPN to that found here in the mossy fibre projection to the A2 zone. Their injections were focussed on the mossy fibre patch that receives upper lip input (corresponding to the zebrin band 5b-, thus equivalent to the C2 zone [see Voogd et al., 2003 for comparison]) and their results therefore imply that the centrally located labelling in BPN may be related to facial input to the cerebellum. Therefore taking these studies into account, the general topographical organisation of body parts in BPN is likely to hold true for pontine projections to the rest of the cerebellum.

Direct Comparison with Somatosensory Cerebro-pontine Projection

The data in the present study were compared and related to the projection from the primary somatosensory (SI) cerebral cortical area in the 3D co-ordinate system. In To gain a more quantitative measure of the extent of the correspondence in the cerebro-ponto-cerebellar pathway, the 3D reconstruction system was used to analyse the overlap of the cerebro-pontine and ponto-cerebellar projections of the accumulated cases, at the level of BPN. This was done by comparing the voxels (3D unit in the reconstructed BPN) containing representative terminal boutons with those containing cells. Table 3.3 below shows the overlap, comparing each projection as a percentage of the other in terms of the total volume occupied by the labelling, as calculated using the Micro3D program.

These figures show that although there is some convergence, especially between somatotopically corresponding cerebro-pontine and ponto-cerebellar projections, only modest overlaps were present. The highest amounts of overlap were obtained when comparing the corresponding 'hindlimb' areas, with lesser extents of overlap present with 'face' and 'forelimb' areas. In fact, the SI face terminals showed a better correspondence with the C1 forelimb cells (11.2 %) than the A2 face cells (4.7 %).

Also, the SI forelimb terminals showed a higher percentage of correspondence with the C1 hindlimb cells (7.0 %) compared to the C1 forelimb cells (4.0 %). When inspecting the dot maps in Figure 3.4, it can be seen that, although the shapes and areas made by the respective injections are similar, the corresponding SI injection terminals appear to intermingle with and surround the cell labelling in a shell-like manner. This is compelling evidence for the use of the 3D reconstruction system and the need for normalising the data, to facilitate comparisons within and between experiments.

Further Study

The first area of further investigation should be to look at ponto-cerebellar projections to parts of the cerebellar cortex that are known to contain equivalent body-receiving climbing fibre zones e.g. the forelimb-receiving C1 zone in LS.

Similarly, it would be of interest to study the ponto-cerebellar projection to the Crus Cell labelling in other brainstem targets could also be mapped using the 3D co-

Cerebro-ponto-cerebellar Pathway

Abstract

The previous chapter identified the ponto-cerebellar projection in relation to climbing fibre zones and found a topographical relationship in BPN. This relationship was found to be very similar to the topography in cerebro-pontine projections arising from the somatosensory cortex (SI). The present chapter aims to build on this knowledge and extend investigation of cerebro-ponto-cerebellar projections to pathways arising from the fore-and hindlimb areas of the motor cortex (MI). Electrophysiological mapping techniques were used to guide injection of anterograde tracer into the motor cortex and also retrograde tracer into the cerebellum, so that cerebro-pontine and ponto-cerebellar projections could be directly compared in the same animals.

An ordered projection from the motor cortex to BPN and from BPN to the cerebellum was found: forelimb-related pathways were centred rostral to hindlimb pathways. There were overlaps between the core terminal and cell territories for somatotopically-corresponding cerebro-pontine and ponto-cerebellar projections.

However there was also some overlap (although to a lesser extent) between somatotopically different cell and terminal territories but there was a significant increase in the overlap of labelling from corresponding limb injections than noncorresponding ones. Overall, the results suggest a complex interaction at the level of BPN between cerebro-pontine inputs from the motor cortex and ponto-cerebellar outputs to different regions of the cerebellar cortex defined by climbing fibre somatotopy.

Introduction

Previous studies of BPN and the cerebro-ponto-cerebellar pathway have often concentrated on one aspect, such as cerebro-pontine input. However, to fully understand functional links between cerebro-pontine and ponto-cerebellar connections both inputs and outputs of BPN need to be studied in relation to each other in the same experiment. As BPN has no clear structural distinctions (i.e. no obvious anatomical nuclear subdivisions), the basis for relating structure and function must come from tracer studies. Work on the cerebro-pontine projection has previously shown that the cerebral cortex sends many afferents to BPN, especially from motor and sensory areas [START_REF] Kuypers | An anatomical analysis of cortico-bulbar connections to the pons and lower brainstem in the cat[END_REF]Brodal, 1968;Leergaard et al., 2000a[START_REF] Leergaard | Three-dimensional topography of corticopontine projections from rat sensorimotor cortex: comparisons with corticostriatal projections reveal diverse integrative organization[END_REF] [see General Introduction for further details]). Concerning the pontocerebellar pathway, less knowledge is available as most studies have concentrated on the nature of the mossy fibre termination in the cerebellar cortex [START_REF] Bower | Is the cerebellum sensory for motor's sake, or motor for sensory's sake: the view from the whiskers of a rat?[END_REF], rather than which cells provide the source of these mossy fibres, often due to the seemingly unorganised projection neurones in BPN (e.g. [START_REF] Brodal | The corticopontocerebellar pathway to crus I in the cat as studied with anterograde and retrograde transport of horseradish peroxidase[END_REF].

Studies such as those by [START_REF] Mihailoff | Projections of the sensorimotor cortex to the basilar pontine nuclei in the rat: an autoradiographic study[END_REF], Leergaard et al. (1995), Leergaard et al. (2000), and [START_REF] Leergaard | Clustered and laminar topographic patterns in rat cerebropontine pathways[END_REF] and that the transformation of information therefore takes place primarily in the mossy fibre ponto-cerebellar projection.

BPN Representation Transformation

Bjaalie et al.

Orderly topography

In the mossy fibre projection

Schwarz & Thier

Pontine modules In the pontine nuclei Table 4.1: Hypothesised organisation in the ponto-cerebellar projection. Leergaard et al. (1995) base their hypothesis on the development of the early cerebro-pontine projection, as they believe that this pathway has a specific continuous projection where the orderly topographic relationship is preserved. With development, they hypothesise that the projection becomes more complex and some segregation occurs but the internal-to-external lamellae continuous organisation is preserved. Opposingly, [START_REF] Schwarz | Modular organization of the pontine nuclei: dendritic fields of identified pontine projection neurons in the rat respect the borders of cortical afferent fields[END_REF] hypothesise that the topography is not preserved in the cerebro-pontine projection and that the pontine nuclei contain compartments or 'pontine modules' that resemble the fractured nature of the pontocerebellar mossy fibre projection.

Typically, the cerebral cortex and cerebellum are viewed as 'computational units' and the pontine nuclei as a relay. [START_REF] Schwarz | Binding of signals relevant for action: towards a hypothesis of the functional role of the pontine nuclei[END_REF] describe the pontine nuclei as the interface needed to accommodate the differing representational topographies of the cerebral cortex and cerebellar cortex. This has led to the idea that the BPN must play a role in the transformation of the information rather than simply relaying it, although it is possible still that the ponto-cerebellar projection is where the transformation of information takes place.

Both theories acknowledge that there is a change and increase in complexity in the representation of information from the cerebral cortex to the pontine nuclei and that the pontine representation does receive overlapping and segregated cerebral projections. Bjaalie & Leergaard (2000) accept that there is much evidence for the segregation of pontine inputs but they have observed overlapping projections thus implying that there is an integrative role for BPN.

Aims of this Study

The aim of the present investigation was to follow on from the previous chapter where topographical relations were found between ponto-cerebellar projections from BPN and certain climbing fibre zones that closely matched cerebro-pontine projections from SI. If this topography is a general principle of organisation in cerebro-ponto-cerebellar circuits, then a similar relationship should also be found in other major cerebral sources of cerebellar input. The present chapter therefore specifically investigates the cerebro-ponto-cerebellar pathway arising from the motor cortex and its organisation in BPN using a combination of anatomical and electrophysiological techniques.

The experiments in this chapter are linked with the results in the next chapter; the present chapter investigates cerebro-ponto-cerebellar pathways whereas Chapter 4

focuses on cerebro-olivo-cerebellar pathways, although the results were gained from the same animals. The aim of the present chapter is therefore to investigate structurefunction relations of the motor cortical projection to BPN, in correspondence to the electrophysiological responses found in climbing fibre zones, through cerebral stimulation. Two types of input to the cerebellum will be compared: forelimb and the hindlimb, and the spatial representations of the input and output will be mapped, (semi-)quantified, and compared in BPN.

Results

Injection Sites

Retrograde tracer was injected into fore-and/or hindlimb receiving areas of the C1 zone in the cerebellum, and anterograde tracer into fore-and/or hindlimb projecting areas in layer V of the motor cortex in the same animals. The sites of injection were defined physiologically: the cerebellar injection was defined by the responsiveness of climbing fibre field potentials to fore-or hindlimb input; the cerebral injection site was defined both by the location of the bodily twitches evoked through higher frequency stimulation and by the responsiveness of climbing fibre potentials in the corresponding part of the C1 zone to fore-or hindlimb motor cortex stimulation (see Methods for details). Therefore the climbing fibre zonal organisation defined injection sites in both the motor cortex and the cerebellar cortex.

A total of 23 animals were used in the cerebro-ponto-cerebellar tracer experiments.

Table 4.2 details all of the experiments where histological analyses were conducted.

From this, only some of the experiments were used in the analysis as either the tracer injection was not successful (note use of biotinylated dextran amine for 5 anterograde injections, which never yielded successful anterograde tracer labelling), or were not on target (i.e. the retrograde cell labelling in the inferior olive was not confined to the expected region projecting to the zone injected into). The results are divided into the anterograde and retrograde tracer injections and the forelimb or hindlimb data for each injection. Therefore a 'case' is termed by its injection site and whether it related to forelimb or hindlimb. The successful cases (highlighted in blue in Table 4.2) were considered in the analysis. In 7 cases it was possible to chart the full cerebro-ponto-cerebellar pathway in the same animal.

Anterograde Tracer

From the 23 experiments conducted, 9 experiments provided evidence of terminal labelling. Out of these, 3 were successful double anterograde tracer studies, therefore a total of 12 cases were yielded. In some experiments, there was evidence of labelled fibres coursing down the cerebro-fugal tract with no evidence of terminations in the brain (n = 7). on the same plane or rostral to Bregma, whereas all of the injections into the hindlimb motor cortex were caudal to Bregma. Also, the table details the small injection site size: all of the injections were less than 0.01 mm 3 .

Retrograde Tracer

There were more cases with successful retrograde cell labelling than with anterograde tracing, and the retrograde tracers used consistently and robustly labelled cells projecting to the cerebellum, although these were not necessarily all 'on target', as the olivary labelling sometimes indicated spread into different cerebellar zones.

The cerebellar injections were electrophysiologically guided (see Chapter 5 for indepth physiology). The factor in deciding whether the retrograde cell labelling was confined to the intended part of the C1 zone (fore-or hindlimb in PML and CP, respectively) was determined by the labelling pattern found in the inferior olive. Thus, labelled cells in the olive were counted and mapped to determine the zone in which the retrograde injection was centred. These zonal patterns were compared to previous work (e.g.

Atkins [START_REF] Atkins | Somatotopical organisation within the climbing fibre projection to the paramedian lobule and copula pyramidis of the rat cerebellum[END_REF]Pardoe & Apps, 2002) to assess any spread of injected material into neighbouring zones and cases where there was little or no spread were termed 'pure' cases and included in the analysis. Table 4.4 shows the injection site details for the cases where the inferior olivary labelling was over 80% on target in the anticipated area of the olive ('pure' cases). From this table, it can also be seen that these injections were relatively small with all injection sites less than 0.15 mm 2 in area covering the white matter interface.

Figure 4.4 shows the individual cases for inferior olivary cell labelling in the 'pure' cases (n = 6 for 'forelimb' and n = 6 for 'hindlimb'). For the forelimb-receiving C1 PML zone, the projection areas in the olive have been previously defined as midrostral MAO and medial parts of mid-rostral DAO, whereas the hindlimb projecting areas extends to slightly more rostral and caudal parts of lateral DAO [START_REF] Apps | Columnar organisation of the inferior olive projection to the posterior lobe of the rat cerebellum[END_REF][START_REF] Atkins | Somatotopical organisation within the climbing fibre projection to the paramedian lobule and copula pyramidis of the rat cerebellum[END_REF]Pardoe & Apps, 2002). Cases that were not classed as 'pure'

were not included in the analysis. Cases that were pure were included in the analysis for pontine cell labelling as these were defined as having labelled cells from BPN projecting to the intended part of the defined climbing fibre C1 zone.

Table 4.5 gives the cell counts for the nuclei mapped in the brainstem for the 'pure' C1-projecting cases. It details the olivary and pontine labelling, including the bilateral labelling found in BPN and NRTP (compared to the contralateral only labelling in the inferior olive). The labelling in BPN was predominantly contralateral for C1-hindlimb injections than C1-forelimb (as found in Chapter 3), although a surprising finding was that labelled cells in NRTP were only found in relation to the C1-forelimb retrograde tracer injections (these data will be presented later in the present chapter).

Pontine Nuclei Labelling

In every case, the full rostrocaudal extent of BPN was mapped for anterograde and retrograde labelling. In all cases where there was a histologically confirmed injection site of retrograde tracer in the cerebellum, there was cell labelling in BPN (and the inferior olive) whereas, the success of the uptake of anterograde tracer was more variable. shows examples of typical cell labelling found in BPN, again at lower and higher power, where the higher power view shows the typical cells found in BPN. The retrograde tracer labels mainly the outer cell body and these cells were of similar shapes and sizes throughout BPN, with an approximate cell diameter of 15-20 µm.

This estimate of diameter was comparable to sizes found in previous studies (e.g. [START_REF] Mihailoff | The cytoarchitecture, cytology, and synaptic organization of the basilar pontine nuclei in the rat. I. Nissl and Golgi studies[END_REF].

Labelling from 'Forelimb' Injections

The labelling from injections into the cerebral forelimb area in MI and into the C1 The main aim of the present chapter was not just to investigate the motor cortical projection to BPN, but also relate the cerebro-ponto-cerebellar pathway in the same animal, in relation to climbing fibre zones. Figure 4.10 shows the cell labelling from the C1-forelimb cerebellar injection. The labelling here was densest in level 6, thus having a slightly more rostral core than the terminal labelling to the same corresponding limb. Cell labelling was located throughout most transverse levels of BPN, although not in far rostral regions. The labelled cells were found bilaterally, with predominance contralateral to the injection site. The ipsilateral cell labelling was located in mid-ventral BPN and was more lateral to the terminal labelling. On average for the 'pure forelimb' cases, 84.4% of the cell labelling was found contralateral to the injection site whereas 15.6% was found ipsilaterally. The actual case by case percentages varied contralaterally from 62.7% contralaterally to 96.4%, and ipsilaterally from 3.6% to 37.3%, but overall the labelling pattern was similar.

Labelling from 'Hindlimb' Injections

The labelling from injections into the cerebral hindlimb area and into the C1 hindlimb-receiving climbing fibre zone in CP in the cerebellum yielded cell and terminal labelling in similar areas in BPN. demonstrates the extent and density of terminal labelling in BPN from the motor cortical injection. The pattern of the terminal labelling was patchy and densest in caudal level 3 in BPN, on the ipsilateral side to the cerebral injection. Labelling was found throughout the rostrocaudal axis of BPN but was consistently heaviest at the far caudal levels. There was terminal labelling found on the contralateral side to the cerebral injection but it was far less prevalent and dense than the ipsilateral labelling and was located mainly in ventral regions.

Figure 4.14 gives the pooled cell labelling cases for the C1-hindlimb receiving cerebellar injection. Again, the labelling is located in caudal BPN and predominantly contralateral to the cerebellar injection site. On average among the cases 95.9% of the cell labelling was found contralateral to the injection site whereas 4.1% was found ipsilaterally. The actual percentages on a case by case varied contralaterally from 89.5% to 100% and ipsilaterally from 0% to 10.5%. The labelling had a far more contralateral predominance than the 'forelimb' cases, with even less variability.

The majority of the labelling was found at level 3 in far caudal contralateral BPN, where the cells filled nearly the whole of BPN. At further rostral levels, the labelling pattern became increasingly more patchy, with a few single cells found from levels 6 to 9. Labelled cells on the ipsilateral side were sparse but occurred in the caudal half of BPN.

Overlap of Labelling

Overlap analyses were performed for locations of tracer injection to compare the overlap of labelling between corresponding and non-corresponding limb projections in the cerebro-ponto-cerebellar pathway. Table 4.6 provides an overview of the percentages of overlap between corresponding (e.g. forelimb cerebral and forelimb cerebellar injections) and non-corresponding (e.g. forelimb cerebral and hindlimb cerebellar) injections in individual cases where the whole cerebro-ponto-cerebellar pathway was mapped. As in Chapter 3 (Table 3.3), the areas occupied by cell and terminal labelling were expressed as overlaps of each other. Therefore, two values for each case were obtained; the overlap was expressed as a percentage of the total areas of overlap occupied by cell labelling and also as the percentage of the total area occupied by the terminal labelling. From these cases, the results of the overlap analyses suggest that there is a higher overlap of cell and terminal labelling in corresponding cases as compared to non-corresponding cases.

Statistical analyses were used to seek whether there was a significant increase in overlap in the corresponding to non-corresponding limb projections. Firstly, as the overlaps were expressed as two different values, a two-tailed t-test was performed between all of the cases to see whether the percentage overlap of cells compared to terminal labelling was greater or the opposite of terminal labelling compared to cell labelling. The results of the t-test showed that these values for each expression of overlap were similar as there was no significant difference (p=0. 6783, n=11) between any of the corresponding and non-corresponding expressions and therefore using each value gave equivalent expressions of overlap.

From this, the 'matched' cases in the same animal were compared for corresponding and non-corresponding projection overlaps (e.g. DP25 corresponding values compared to matched DP25 non-corresponding values). This was to determine whether there was a significant difference through directly comparing results

obtained from experiments with corresponding and non-corresponding injections in the same animal. A significant increase in overlap was found for corresponding cases as compared to non-corresponding matched cases (p=0.0230, one-tailed paired t-test, n=8). As this showed a significantly more overlap for corresponding whole cerebroponto-cerebellar projections, a further separate t-test was carried out on all of the values comparing all corresponding and all non-corresponding values. The previous t-test was more specific, whereas this test increased the number of cases and was more conservative to test whether there was a general significant difference. There was significantly more overlap again found for the corresponding cases (p=0.0303, one-tailed unpaired t-test) compared to the overlap in the non-corresponding cases.

The present chapter now will compare the pooled data for overlaps and show rostrocaudal transverse maps of overlapping areas for corresponding and noncorresponding combinations of injections. Table 4.7 details the overlaps for the pooled data, as before expressing two values for each compared overlap. The table

shows that there were complex patterns of overlap as at times, the overlaps were patchy. There is a trend for corresponding limb injections to have higher overlaps, but in some cases, there are also high non-corresponding limb projection overlaps.

For example, due to the area occupied by labelling, the percentage overlap of 'forelimb' cells and 'hindlimb' terminal labelling appears to be high when taken as the cell labelling expressed as a percentage of the overlap (30.3%), but conversely, where the same terminal labelling is expressed as a percentage of overlap, the value is very low (0.003%).

Therefore, as these interactions are complicated, the overlaps between pooled data are shown diagrammatically. From these, it is best seen that the places of overlap differ in corresponding and non-corresponding comparisons. The corresponding interactions show more overlaps of the core areas of labelling for the cerebro-pontine and ponto-cerebellar projections, whereas the non-corresponding projection areas of overlap tended to be at peripheral areas of labelling (i.e. where the labelling was adjacent in the ventral plane).

Overlap Between Forelimb Cell and Forelimb Terminal Labelling

There was overlap in the core regions occupied by the cerebro-pontine and pontocerebellar forelimb projections. Figure 4.15 shows the overlap between the 'forelimb' corresponding labelling. These values show an overlap but only partial, between the regions of labelling. The regions of overlap were in levels 3-8, mainly in mid-BPN, near the centres of gravity for both projections.

Overlap Between Hindlimb Cell and Hindlimb Terminal Labelling

The overlap for corresponding hindlimb injections showed a large overlap in caudal BPN, as can be seen in Figure 4.16. The overlap occurred mainly in levels 2-4 and nearly all in the right BPN (ipsilateral to the motor cortical injection site and contralateral to the cerebellar injection site). Therefore, the overlap between 'hindlimb' cell and terminal labelling was confined (even more so than the 'forelimb') to the centres of gravity for each projection.

Overlap Between Forelimb Terminal and Hindlimb Cell Labelling

There was a very patchy organisation of overlap over levels 3-5 in BPN when comparing the overlap from the non-corresponding forelimb motor cortical injection and hindlimb cerebellar injection, as seen in Figure 4.17. The overlap was nearly all confined to the right BPN (ipsilateral to the motor cortical injection site and contralateral to the cerebellar injection site).

Overlap Between Hindlimb Terminal and Forelimb Cell Labelling

Again, with the non-corresponding comparison of the injections into the hindlimb area of the motor cortex and forelimb C1 zone in PML in the cerebellum, there was a very patchy overlap arrangement. In Figure 4.18 it can be seen that in the transitional regions between caudal BPN and mid-BPN, there are patches of overlap for this noncorresponding comparison, mainly at level 5. There was also some minimal overlap in the middle of the left BPN (contralateral to the motor cortical injection site and ipsilateral to the cerebellar injection site).

Areas of Total Overlap

There was minimal overlap for all of the combinations of injections and labelling,

shown in Figure 4.19. The patchy overlap was situated in levels 4 and 5, and only on the side ipsilateral to the motor cortex injection and contralateral to the cerebellar injection. The overlap of all types of labelling shows a complicated pattern of segregation and integration of labelling and representations found in BPN and was located in the transitional region between caudal BPN (typically occupied by 'hindlimb' labelling) and mid-BPN (typically occupied by 'forelimb' labelling).

NRTP Cell Labelling

Many retrogradely labelled cells (average diameter = 20 µm) were found in NRTP from injections into the forelimb C1 zone in PML. No labelled fibres or terminal labelling from the anterograde injection, were found near or in NRTP. Also, no cell labelling from the cerebellar injection into the hindlimb-receiving part of the C1 zone in CP was found either. Figure 4.20 shows the pattern of cell labelling as found in the 6 'pure' forelimb cases. Overall, the labelling was bilateral with the percentage of cells found in NRTP contralateral to the forelimb injection was 54.3% and ipsilateral to the injection site was 45.7%. The cells were spread along the rostrocaudal axis of NRTP and were arranged throughout the nuclei.

Discussion

Methodological Considerations

Of the 23 experiments conducted, 9 provided evidence of terminal labelling. The absence of terminal labelling, but presence of labelled fibres in the cerebro-fugal tract for some of the experiments, has been attributed to the method of tracer injection. From histological comparison of injection sites between cases with and without terminal labelling, but having labelled fibres present, the factors determining degree of labelling appeared to include depth of injection site and the amount of damage to the tissue. The depth was crucial, and earlier experiments had injections too shallow in the motor cortex, whereas the layer V pyramidal projection neurones were located deeper in the cortex, around 1.75 mm down.

In later experiments more tracer material was injected into the brain (e.g. 400 nl) but some injections were once again too shallow. From careful inspection of cases where terminal labelling had occurred, it became clear that the amount of tracer injected was not a strong determining factor as to whether terminal labelling would be found.

The main factor appeared to be the amount of damage caused at the injection site.

Microstimulation of the motor cortex, even with the larger bipolar electrode did not cause significant damage to the brain, visible on histological reconstruction.

However, the track of the tracer injection was usually evident, although damage was often minimal. In the absence of sufficient damage to projecting fibres, the tracer could not be taken up and therefore produced no detectable terminal labelling and little or no labelling of fibres. This is because the fluorescent dextran amines used for the anterograde tracing are taken up by damaged axons [START_REF] Köbbert | Current concepts in neuroanatomical tracing[END_REF].

In later experiments, injections were centred on the forelimb and hindlimb defined areas of the motor cortex but instead of one injection, three smaller injections were made in very close proximity to one another (no more than 0.2 mm apart) at the depth of the pyramidal projection layer V neurones, as defined by vernier depth and of bodily twitch to stimulation and electrophysiological recordings of climbing fibre field responses in the cerebellar cortex. It is assumed that these injections caused sufficient damage to surrounding projection neurones so that uptake of tracer produced labelled fibres in the cerebro-fugal tract and terminal labelling, but within a small, defined region. As a result, all three double tracer experiments of this type provided results.

Basilar Pontine Nuclei Labelling

There is some evidence in the literature of topographical organisation in BPN for both cerebro-pontine and ponto-cerebellar projections (see General Introduction), although these have not been previously directly compared in regard to climbing fibre zones. In the present study, both the injections into the motor cortex and cerebellum gave rise to plenty of terminal and cell labelling, respectively. This labelling was distributed bilaterally in BPN. Previous studies have on the whole concentrated on the ipsilateral cerebro-pontine projection (Leergaard et al., 2000a, b), but from the present study there is a significant bilateral projection. This must be taken into consideration when interpreting the data, as there is likely to be bilateral interactions in the cerebro-ponto-cerebellar pathway.

Overlapping Projections

The pattern of labelling in BPN for all injections was similar to the respective corresponding labelling produced in other studies from injections into SI (e.g. Leergaard et al., 2000a, b). The ipsilateral motor cortical projection to BPN was far less dense than that to the contralateral side. The pathway of the contralateral projection and terminal labelling was not from the contralateral descending fibre tract, but rather was an extension of the ipsilateral projection as labelled fibres were traced coursing from the ipsilateral longitudinal fasciculus of the pons into ipsilateral BPN, crossing the midline, and terminating in various, specific regions in contralateral BPN. [START_REF] Mihailoff | Projections of the sensorimotor cortex to the basilar pontine nuclei in the rat: an autoradiographic study[END_REF] considered this crossed projection but did not assign function to it, and Leergaard et al. (2000a, b) noted that there was a slight projection to the contralateral BPN from SI but did not map or quantify it. The contralateral labelling must be considered in analysis and hypotheses about BPN structure and function in the cerebro-ponto-cerebellar pathway.

From the overlap analysis of the labelling produced from injecting into different areas in the cerebellum and motor cortex, a striking pattern of overlap and segregation exists in BPN. The overlap itself was analysed with the help of a grid system, where the terminal and cell labelling could be compared between and within

The comparison of projections in the same animal is a strong method to compare the two projections, although to generalise the findings, it was best to also compare pooled data sets. This is especially so due to the differences of tracer uptake. In some cases, there was more uptake of tracer than others, therefore possibly leading to biased comparisons in the overlap analysis. To prevent biases, the pooled data was used to obtain an overview of typical overlaps and generalise the findings.

The location of overlap has implications for structure-function relationships. The results imply that the cerebral labelling in BPN is more likely to contact pontine cells projecting to corresponding climbing fibre limb-receiving zones in the cerebellar cortex. This suggests an integrated (within-modality) and segregated (betweenmodality) nature to the cerebro-ponto-cerebellar projection. There was also another overlap centred more rostrally which was present in labelling from all injection sites but showed a much smaller representation than the main core. This more rostral region of both cell and terminal labelling has been attributed to BPN early development.

Studies have previously shown that descending cerebro-fugal fibres arrive (and migrate in BPN) in a temporal sequence. That is, the first fibres from the cerebral cortex innervate the middle of BPN and subsequent projections innervate the area around the inner part (Leergaard et al., 1995); these patterns of labelling give rise to lamellae-like subspaces in BPN. Furthermore, a specific pattern of labelling was found with labelling shifting 'externally', depending on the site of injection in the cerebral cortex. [START_REF] Altman | Development of the precerebellar nuclei in the rat: IV. The anterior precerebellar extramural migratory stream and the nucleus reticularis tegmenti pontis and the basal pontine gray[END_REF] also showed the ponto-cerebellar projection develops in a similar concentric pattern in BPN.

Past studies show the lamella-like, internal to external organisation of body part representations found in the present study for both the cerebro-pontine and pontocerebellar projections in BPN. There has been a consistent appearance of internal to external wrapping around of labelling from different areas of the somatosensory cortex where the main core of the labelling is found in the middle to caudal half of BPN and there is a second smaller representation found more rostrally. This has been implicated as being due to pruning in development in BPN postnatally and is a remnant body part representation (Leergaard et al., 1995(Leergaard et al., , 2000b)).

The results gained in the present study support this general scheme, but extend them to cerebro-pontine and ponto-cerebellar pathways, in the adult rat. Moreover, the data were investigated in relation to the climbing fibre zonal framework. This suggests that the BPN afferent and efferent projections are somatotopically related to climbing fibre zones in the cerebellar cortex. In other words, the cerebro-pontine motor cortical projection can be somatotopically related to the ponto-cerebellar projection in the framework of climbing fibre cerebellar input.

Implications of Labelling in Other Areas

Both cell and terminal labelling was found in other areas, apart from the inferior olive and BPN. The labelling pattern in NRTP showed a clear difference between injecting into the fore-or hindlimb part of the C1 zone in that labelled cells were only ever found in NRTP projecting to the forelimb-receiving C1 zone in PML and this projection was uniformly bilateral. This shows further specialisation in mossy fibres targeting different parts of a climbing fibre zone (these discrete differences in projection have not been previously recognised e.g. Cicirata et al., 2005b described similar projection patterns onto the cerebellar cortex for BPN and NRTP projections).

Also, terminal labelling was never found in NRTP, where previous studies have described sensorimotor projections to NRTP (Cicirata et al., 2005b). This difference may also be similar to the lack of finding terminals in the inferior olive also; there may be only discrete cerebral projections to NRTP and the olive that were not detected in the present study investigating only fore-and hindlimb motor pathways.

In addition, the axonal labelling in the cerebro-fugal tract was more spatially organised between fore-and hindlimb injection sites at rostral levels as found previously in the literature [START_REF] Coleman | Topography of fibre organisation in the corticofugal pathways of rats[END_REF]. In Coleman et al.'s study, they found that increasingly caudally, the cerebro-fugal tract became less organised, although the present study found it to be spatially organised even after the level of the inferior olive, with the 'forelimb' fibres located in slightly different areas to the 'hindlimb' fibres. The 'forelimb' fibres were slightly further ventral to the 'hindlimb' fibres.

Further Studies

Further experiments injecting tracer into the motor cortex could be carried out to include other areas, such as the facial/vibrissae representation, and to directly compare projections from different cerebral areas. The cerebro-pontine projection could be investigated using different anterograde tracers to establish finer grain somatotopical differences in the motor cortical projection to BPN, and also in sensory projections. The labelling on the side ipsilateral to the motor cortex injection, in BPN is quantitatively larger than the contralateral component, but there is a definite bilateral characteristic to the cerebro-pontine projection. Studies could be carried out to identify the possible interactions between the input each BPN gets from both ipsilateral and contralateral sides of the cortex. This could also be related to the ponto-cerebellar projection. One approach would be to inject different tracers into both sides of the cerebral cortex to chart in the same animal, the bilateral aspect of the projection to BPN.

There is a 3D representation of body parts in BPN, which differs to the 2D continuous bodily representations in the cerebral cortex. The pontine arrangement may allow more connections and integration of inputs. The work of [START_REF] Schwarz | Binding of signals relevant for action: towards a hypothesis of the functional role of the pontine nuclei[END_REF] suggests that there may be a further level of processing that takes place in BPN, as it receives inputs from the periphery and from various areas of the cerebral cortex and thus 'binds' these incoming signals before projecting the information to the cerebellum. Further work is needed to clarify the level of transformation in BPN and this could be done by directly recording physiologically evoked responses from the periphery and cerebral cortex in BPN. This method could also be combined with intra-cellular filling to visualise location and structure of the cell(s) recorded from.

Cerebro-olivo-cerebellar Pathway

Abstract

These experiments in rats investigated input from motor cortical areas to the C1 zone in the posterior lobe of the cerebellum, relayed via climbing fibres. A combination of electrophysiological stimulation and recording techniques, and physiologicallyguided injections of tracer material were used to chart cerebro-olivo-cerebellar pathways (COCPs). Both peripheral and motor cortical stimulation readily evoked climbing fibre field potentials in the forelimb-and hindlimb-receiving parts of the C1 zone and there was convergence between the two inputs.

The cerebral cortical projection was further investigated as there is controversy in the literature as to whether there is a direct pathway to the inferior olive. From anterograde tracer injections into the motor cortex, terminal labelling was never found in the inferior olive, but labelling was found in other more rostral and more caudal sites. A relay for the cerebro-olivary projection was sought and a potential site in the caudal brainstem was found where discrete terminal labelling was identified just ventral to the gracile nucleus. The labelling was in an area termed the 'matrix region'. To obtain evidence that this was a synaptic relay for COCPs, drugs were used to reversibly inactivate the area. Cerebrally evoked climbing fibre field responses were decreased and in some cases abolished, then recovered, whereas the peripherally evoked climbing fibre potentials remained unchanged at the same cerebellar recording sites.

Overall, the results show a powerful projection from limb areas in the motor cortex to somatotopically-corresponding parts of the C1 zone in the cerebellum. A direct cerebro-olivary projection was not found, although the matrix area seemed to be a likely pre-olivary candidate. Therefore, from both anatomy and physiology, there is evidence of convergence between peripheral and cerebral information to the inferior olive and that there is a pre-olivary synaptic relay in the COCP projection to the C1 zone in rats.

Introduction

For many years now, it has been known that there is a projection from the cerebral cortex to the cerebellum, via the climbing fibre system (e.g. [START_REF] Armstrong | Responses to a spino-olivo-cerebellar pathway in the cat[END_REF][START_REF] Andersson | Origin and sagittal termination areas of cerebrocerebellar climbing fibre paths in the cat[END_REF][START_REF] Baker | Activation of cerebellar climbing fibres to rat cerebellar posterior lobe from motor cortical output pathways[END_REF]. This has been mainly investigated using physiological stimulation and recording techniques. The exact pathway of the evoked responses in the cerebellum from cerebral stimulation is less well characterised. Climbing fibre responses have been found in the cerebellum to cerebral stimulation and through stimulation of descending fibre tracts (e.g. Allen et al., 1974a, b;[START_REF] Baker | Activation of cerebellar climbing fibres to rat cerebellar posterior lobe from motor cortical output pathways[END_REF]. Different parts of the cerebral cortex have been implicated in projecting to the inferior olive [START_REF] Snider | Electro-anatomical studies on the afferent projection to the inferior olive[END_REF][START_REF] Swenson | Organization of cerebral cortico-olivary projections in the rat[END_REF][START_REF] Walberg | Descending connections to the inferior olive; an experimental study in the cat[END_REF], although the areas most documented have been motor areas [START_REF] Baker | Activation of cerebellar climbing fibres to rat cerebellar posterior lobe from motor cortical output pathways[END_REF][START_REF] Fennell | Sensorimotor cortical influences on the climbing fibre input to cerebellar Purkyne cells[END_REF]Nakamura, 1983a, b;[START_REF] Saint-Cyr | The projection from the motor cortex to the inferior olive in the cat. An experimental study using axonal transport techniques[END_REF], 1987;[START_REF] Sousa-Pinto | Demonstration of a somatotopical pattern in the cortico-olivary projection in the cat. An experimental-anatomical study[END_REF].

There have been various studies that claim a direct cerebro-olivary projection [START_REF] Brown | A study of afferent input to the inferior olivary complex in the rat by retrograde axonal transport of horseradish peroxidase[END_REF][START_REF] Saint-Cyr | The projection from the motor cortex to the inferior olive in the cat. An experimental study using axonal transport techniques[END_REF][START_REF] Swenson | The afferent connections of the inferior olivary complex in rats. An anterograde study using autoradiographic and axonal degeneration techniques[END_REF][START_REF] Walberg | Descending connections to the inferior olive; an experimental study in the cat[END_REF], although even in these studies, difficulty in interpretation of labelling near the olive has confounded interpretation of results. For this reason, some studies have queried the existence of a direct cerebro-olivary projection (e.g. [START_REF] Kuypers | An anatomical analysis of cortico-bulbar connections to the pons and lower brainstem in the cat[END_REF] and there have been suggestions that the projection has an indirect pathway involving one or more pre-olivary relays (Allen et al., 1974a, b;Nakamura, 1983a, b;[START_REF] Saint-Cyr | Anatomical organization of cortico-mesencephalo-olivary pathways in the cat as demonstrated by axonal transport techniques[END_REF]. Modern fluorescent tracers have overcome many of the problems encountered using older tracer methods (such as those associated with degeneration methods).

However, there has been little anatomical work on the topography of cerebrocerebellar pathways using these more sensitive methods.

There have been sites identified in the midbrain (rostral to the inferior olive) and the brainstem (caudal to the inferior olive) as relays in the cerebro-olivary pathway. The former includes mesodiencephalic structures (Nakamura et al., 1983b;[START_REF] Saint-Cyr | Anatomical organization of cortico-mesencephalo-olivary pathways in the cat as demonstrated by axonal transport techniques[END_REF][START_REF] Kyuhou | Cerebro-cerebellar projections from the ventral bank of the anterior ectosylvian sulcus in the cat[END_REF]. For instance, it is well recognised that the nucleus of Darkschewitsch conveys signals from the cerebral cortex to the inferior olive (Nakamura et al., 1983a;[START_REF] Kitao | An electron microscope study of the cortico-pretecto-olivary projection in the cat by a combined degeneration and horseradish peroxidase tracing technique[END_REF][START_REF] Rutherford | A comparison of the distribution of the cerebellar and cortical connections of the nucleus of Darkschewitsch (ND) in the cat: a study using anterograde and retrograde HRP tracing techniques[END_REF], at least in cat.

This pathway was found to relay information from a discrete part of the cat motor cortex to the nucleus of Darkschewitsch and then to rostral MAO. Physiological studies also suggest there may be caudal relays such as at the level of the dorsal column nuclei (DCN; e.g. [START_REF] Andersson | Demonstration of a cuneate relay in a cortico-olivo-cerebellar pathway in the cat[END_REF].

Electrophysiological studies in rat suggest that only caudal relay(s) may be present in COCPs in this species. Through stimulating and lesioning the pyramidal tract [START_REF] Baker | Activation of cerebellar climbing fibres to rat cerebellar posterior lobe from motor cortical output pathways[END_REF], concluded that if there was any pre-olivary relay involved in the (rat) cerebro-cerebellar projection to the inferior olive, it was likely to be at the level of the olive or caudal to it. Also, from stimulation of the cat fore-and hindlimb motor cortex and taking into account pyramidal tract conduction latencies, Allen et al. (1974a, b) proposed that there was not a monosynaptic cerebro-olivary projection arising from these parts of the motor cortex.

The dorsal column nuclei have previously been identified as playing a role in the cerebro-fugal projection [START_REF] Kuypers | An anatomical analysis of cortico-bulbar connections to the pons and lower brainstem in the cat[END_REF][START_REF] Gordon | Descending influences on the exteroceptive organizations of the cat's gracile nucleus[END_REF][START_REF] Cheema | Sensorimotor cortical projections to the primate cuneate nucleus[END_REF]. It is well known that the DCN relay ascending limb information to the inferior olive [START_REF] Walberg | Corticofugal fibres to the nuclei of the dorsal columns; an experimental study in the cat[END_REF][START_REF] Tomasulo | Activation of neurons in the gracile nucleus by two afferent pathways in the rat[END_REF][START_REF] Martinez | Pyramidal tract and corticospinal neurons with branching axons to the dorsal column nuclei of the cat[END_REF][START_REF] Molinari | Gracile, cuneate, and spinal trigeminal projections to inferior olive in rat and monkey[END_REF][START_REF] Apps | Input-output connections of the "hindlimb" region of the inferior olive in cats[END_REF] and are targeted by descending motor cortical input [START_REF] Walberg | Corticofugal fibres to the nuclei of the dorsal columns; an experimental study in the cat[END_REF][START_REF] Kuypers | An anatomical analysis of cortico-bulbar connections to the pons and lower brainstem in the cat[END_REF][START_REF] Gordon | Descending influences on the exteroceptive organizations of the cat's gracile nucleus[END_REF][START_REF] Andersson | Demonstration of a cuneate relay in a cortico-olivo-cerebellar pathway in the cat[END_REF][START_REF] Cheema | Sensorimotor cortical projections to the primate cuneate nucleus[END_REF] Afferents from the forelimbs and hindlimbs have been found to project respectively to the cuneate and gracile nucleus. The gracile and cuneate nuclei both send projections to MAO, although the cuneate projection is the more extensive. Also, both nuclei innervate DAO to similar extents, but in these projections, the afferent input from gracile is located in lateral DAO, whereas from cuneate, the labelling is more medial [START_REF] Boesten | Projections of the dorsal column nuclei and the spinal cord on the inferior olive in the cat[END_REF][START_REF] Berkley | Projections to the inferior olive of the cat. II. Comparisons of input from the gracile, cuneate and the spinal trigeminal nuclei[END_REF][START_REF] Molinari | Gracile, cuneate, and spinal trigeminal projections to inferior olive in rat and monkey[END_REF].

Spino-olivocerebellar pathways (SOCPs) have been found to extensively overlap in the inferior olive with inputs from DCN, these areas in the olive have been found to be in caudal MAO and DAO [START_REF] Boesten | Projections of the dorsal column nuclei and the spinal cord on the inferior olive in the cat[END_REF][START_REF] Berkley | Projections to the inferior olive of the cat. I. Comparisons of input from the dorsal column nuclei, the lateral cervical nucleus, the spino-olivary pathways, the cerebral cortex and the cerebellum[END_REF].

Also, both the DCN and the direct spinal input have been found to be somatotopically organised (DCN: [START_REF] Berkley | Projections to the inferior olive of the cat. II. Comparisons of input from the gracile, cuneate and the spinal trigeminal nuclei[END_REF][START_REF] Molinari | Gracile, cuneate, and spinal trigeminal projections to inferior olive in rat and monkey[END_REF][START_REF] Apps | Input-output connections of the "hindlimb" region of the inferior olive in cats[END_REF]spinal: Miller et al., 1969a, b;[START_REF] Oscarsson | Functional Organization of Spinocerebellar Paths[END_REF]Oscarsson & Sjölund, 1977).

In summary, investigating ascending and descending 'limb' inputs to the cerebellum via climbing fibres is an important step towards a full understanding of these paths in the field of motor control. Combining physiological and modern anatomical tracer techniques can further our knowledge of afferents to the inferior olive. For example, [START_REF] Adrian | Afferent areas in the cerebellum connected with the limbs[END_REF] long ago showed physiologically that there is convergence of peripheral and cerebral inputs to the limb receiving areas in the cerebellum, for both forelimb and hindlimb afferent information, and the present study aims to extend knowledge of such convergence.

Aims of This Study

This study aims to clarify and investigate aspects of the cerebro-olivo-cerebellar pathway. This is especially in relation to the following points:

 The extent to which limb input from the periphery and motor cortex coincide in the cerebellar posterior lobe.

 The extent to which cerebro-olivo-cerebellar pathways arising from the motor cortex involve direct projections to the olive or pre-olivary synaptic relays.

Results

The results from investigating the cerebro-olivo-cerebellar projection can be subdivided into three inter-related lines of study (1) Physiological -study of somatotopical and converging inputs, (2) Anatomical -tracing the neural pathway by which the cerebral responses are conveyed to and from the inferior olive, and (3)

Pharmacological -inactivation of a putative pre-olivary motor cortical relay and the effect of this on evoked climbing fibre responses.

Physiology

In acute physiological experiments investigating the climbing fibre responses to peripheral and motor cortical stimulation, data were gathered primarily from three different areas of the cerebellar cortex. These were all located in the C1 zone and responses were recorded in the forelimb-receiving areas in LS and PML, and in the hindlimb-receiving area in CP. Both the peripheral and cerebral stimulation consistently evoked large climbing fibre responses in all three areas. Climbing fibre field potentials (rather than unitary activity) were recorded as this allowed a more comprehensive survey of different cerebellar areas in each experiment. As no significant difference was found between the C1 forelimb receiving sites in LS and PML, their data are considered together.

Climbing fibre responses were mapped over the full extent of the cerebellar posterior lobe up to the limits of the exposure. This included mapping both sides of the cerebellum, up to and just lateral of the C3 zone, although the main focus was on the C1 zone. This allowed a full comparison of climbing fibre responses evoked from the limb stimulation and by stimulation of the limb representations in the motor cortex.

In the physiological investigations, low frequency and low intensity cerebral cortical stimulation (e.g. single pulse at 50 µA) evoked climbing fibre responses at the same recording sites as limb stimulation, but when stimulated at a higher frequency and current (e.g. thirty consecutive pulses at 2 mA), the same cortical areas elicited movements of the limbs.

Climbing Fibre Responses

Climbing fibre responses were identified by a number of criteria. Typically, surface recordings were positive going (e.g. Figure 5.1) and progressively increased in size with stimulus intensity, indicating the responses were recorded from populations of neurones. In each of LS, PML and CP, the C1 zone was approximately 1 mm wide mediolaterally and responses to limb inputs were at their largest in the middle of the zone for each respective stimulation and recording site. The same pattern of responses was found to motor cortical (MC) stimulation, again for tracking across LS, PML and CP. Figure 5.1 shows climbing fibre field responses to fore-and hindlimb peripheral and MC stimulation. In this case, responses to ipsilateral limb stimulation and contralateral MC stimulation were recorded in the C1 zone of PML or CP.

At threshold stimulation (T), only a small, transient response was present, characterising the minimum intensity need to evoke a response. The typical T value for peripheral stimulation was 0.05 V and for cerebral stimulation 1 mA.

Measurements were then taken at twice this intensity (2T) and three times (3T) to gain clear waveforms from climbing fibre field potentials. At 2T, responses could be readily evoked from all stimulation sites, and at 3T, there was sometimes a slight increase in the amplitude of the response. An intensity of 2T was therefore deemed a sufficient level to stimulate/record although 3T and occasionally 5T were sometimes used to evoke supramaximal responses.

Table 5.1 shows the cases used (n = 28 animals) and details the values found for corresponding peripheral ipsilateral limb stimulation and contralateral and ipsilateral MC stimulation to recording sites in the C1 zone of LS, PML, and CP. Table 5.2

shows the results of testing for statistically significant differences between different combinations of stimulation and recording sites. The onset latencies for the peripheral forelimb and contralateral MC forelimb responses were very similar with average latencies between 12.20 ms and 12.74 ms and table 5.2 shows no significant difference between any of these combinations of stimulation and recording site.

However, there were significant differences between the ipsilateral forelimb MC stimulation and other sites and both peripheral hindlimb and contralateral hindlimb MC stimulation showed overall significant differences between most recording sites.

The hindlimb peripherally evoked responses had an average onset latency of 16.29 ms and showed significant differences in latency to all stimulating/recording sites apart from the latencies from stimulation of the ipsilateral MC giving responses in PML and LS. The latencies of hindlimb area cerebrally evoked responses were significantly different from those for all other stimulation/recording sites and had an overall average latency of 21.21 ms. The responses evoked to forelimb area ipsilateral MC stimulation were not significantly different for PML and LS responses, but there was a different pattern with the other combinations of stimulation/recording site. The average response latency for ipsilateral MC evoked responses in LS was 14.35 ms but there were only 4 experiments where these responses were recorded.

The ipsilateral forelimb motor cortex was not investigated in detail as the responses fluctuated making it difficult to obtain longer term stable recordings. The latency was longer than responses from stimulating either the periphery or contralateral motor cortex for LS and PML. Also, the current needed to produce a threshold response in both of these recording zones was typically much higher than that needed to produce responses from stimulating the contralateral motor cortex (threshold 3-5 mA, as compared to thresholds often less than 1 mA). This suggests that the ipsilateral projection is likely to involve a more complex multi-synaptic route to the olive than the contralateral projection.

The responses sometimes also displayed a characteristic waveform with distinct secondary components. The climbing fibre potential typically reversed when the recording electrode was advanced into the cerebellar cortex [START_REF] Eccles | The Cerebellum as a Neuronal Machine[END_REF]. The climbing fibre responses found in this investigation were therefore similar to those reported in previous work (e.g. Pardoe & Apps, 2002). The response latencies were also very similar when compared to other work on responses evoked in the same regions of the cerebellar cortex in the rat by limb stimulation (see [START_REF] Atkins | Somatotopical organisation within the climbing fibre projection to the paramedian lobule and copula pyramidis of the rat cerebellum[END_REF]Pardoe & Apps, 2002). The latency information for the recording sites in the C1 zone for LS, PML and CP is shown later.

In addition, the use of barbiturate anaesthesia has been found to enhance climbing fibre activity but depress mossy fibre activity for both peripheral ascending (e.g. [START_REF] Gordon | Effect of pentothal on the activity evoked in the cerebellar cortex[END_REF] and cerebral descending pathways (e.g. [START_REF] Andersson | Origin and sagittal termination areas of cerebrocerebellar climbing fibre paths in the cat[END_REF]. In some of the present experiments it was possible sometimes to see early responses of around 5 ms latency when the preparation lightly anaesthetised. These were presumably mediated via mossy fibres and after a top-up injection of pentobarbitone, such responses decreased in size. This is similar to the findings of [START_REF] Adrian | Afferent areas in the cerebellum connected with the limbs[END_REF] where at deep levels of pentobarbitone anaesthesia cerebrally evoked mossy fibre potentials were extinguished. Further tests were also carried out to ensure that climbing fibre rather than mossy fibre responses were recorded. This was particularly important for responses evoked by cerebral stimulation. In addition to short latency mossy fibre-related responses, longer latency responses have also been described [START_REF] Morissette | Contribution of somatosensory cortex to responses in the rat cerebellar granule cell layer following peripheral tactile stimulation[END_REF]. These can have similar onset latencies to climbing fibre responses.

Paired Pulse Paradigm

Classically, climbing fibre responses have a long refractory period [START_REF] Eccles | The Cerebellum as a Neuronal Machine[END_REF]. Therefore, applying two pulses from the same stimulation site in close succession should mean the putative climbing fibre response occurs only once, to the initial stimulation. With a progressive increase in the interval between the pulses beyond 40 to 60 ms, there was a progressive recovery of response to the second pulse with distance from the first pulse, as demonstrated in Figure 5.2. Responses evoked by peripheral stimulation started to recover at an interstimulus interval of 80 ms and showed recovery to control values by 100 ms (see graph under waveforms). Note that after 80 ms, there was an increase in the size of the cerebrally evoked responses to 160% of the control. This rebound facilitation has also been characterised previously as a property of climbing fibre responses [START_REF] Eccles | The Cerebellum as a Neuronal Machine[END_REF].

Conditioning Paradigm

The conditioning paradigm was similar to the paired pulse test in that two stimuli were applied at varying time intervals. In these tests however, the stimuli used were combinations of peripheral and cerebral pulses. This test was carried out to see whether the peripheral and cerebral stimuli could be paired in a similar way to the paired pulse tests and that both the peripheral and cerebral responses in combination, shared the same climbing fibre properties as in the previous tests. In addition to providing further evidence that the responses were climbing fibre in origin, this test also provided evidence that the peripheral and cerebral stimuli were eliciting a response from neurones located in similar areas in the inferior olive. Figure 5.3 shows example data. After an interstimulus interval of more than 60 ms, there is a visible response to the second stimulus and by an interstimulus interval of 80 ms, there was complete recovery, with rebound facilitation of the second response at this interval and higher.

Threshold Stimulation Tests

The paired pulse paradigm showed the type of responsiveness characteristic of climbing fibre potentials. The conditioning paradigm also showed that it was highly likely that the responses were elicited from overlapping, perhaps identical, populations of neurones in the inferior olive. The threshold test provided further evidence that the responses elicited from peripheral and cerebral stimulation were evoked via a similar set of inferior olivary neurones. The two pathways were stimulated at threshold intensity (i.e. at an intensity where little or no visible response was present for either stimulus applied alone). When the two paths were stimulated in conjunction, a sizeable response was evoked, indicating mutual facilitation and strongly suggesting a convergence of the two pathways at the olivary, or perhaps at a pre-olivary level (Figure 5.4). This effect was found in responses for two C1 forelimb-receiving areas (in LS and PML) and in the hindlimb-receiving area in CP (n = 4 LS; n = 10 PML; n = 7 CP).

Collectively, the response characteristics and tests described above give good evidence that climbing fibre responses (rather than responses mediated via mossy fibres) were being recorded and that there was a convergence of limb peripheral ascending and cerebral descending motor input at the level of the inferior olive or earlier in the respective pathways.

Motor Cortex Map

Once responses were identified as mediated via the climbing fibres according to the above criteria, the experiments then focussed on mapping on the cerebellar surface the responses to cerebral and peripheral input. When the stimulating electrode was lowered perpendicularly into the motor cortex, the evoked cerebellar climbing fibre responses increased in amplitude until an optimum depth of ≈1.7 mm was reached.

This corresponded to the pyramidal projection neurones of layer V in the agranular motor cortex (see General Introduction). At shallower and deeper sites, climbing fibre responses could still be evoked but the responses were smaller in size and larger stimulation currents were needed. A depth of 1.7 mm was used in subsequent acute electrophysiological and anatomical tract tracer experiments.

Forelimb motor cortex stimulation evoked twitches typically of the distal muscles of the forearm, and hindlimb motor cortex usually evoked twitches in distal muscles of the leg; although occasionally some digital movements were noted for both. The centre of the forelimb area typically corresponded to Bregma and the centre of the hindlimb approximately 1 mm caudal to Bregma, both approximately 1 mm lateral of midline. The whisker representation was rostral to the forelimb representation and the trunk lateral to the hindlimb area (see sites of injection in Figure 4.1 for locations of forelimb and hindlimb areas). This topographical representation is similar to previous descriptions (e.g. [START_REF] Hall | Organization of motor and somatosensory neocortex in the albino rat[END_REF]. However, there were interspecies variations in details and only the gross representations were generalised; finer grain variabilities have been described in previous studies (e.g. [START_REF] Neafsey | The organization of the rat motor cortex: a microstimulation mapping study[END_REF].

Anatomical Labelling

A total of 12 cases investigated projections from the motor cortex to the inferior olive. These are the same cases already considered in the preceding chapter but for cerebro-pontine projections. Directly from the injection site in the motor cortex, labelled axons could be seen coursing towards the corpus callosum, where the fibres continued in the tract to descend in a caudal direction. From there, fibres were seen extending towards the internal capsule, but occasionally seen coursing medially to the contralateral hemisphere; this pattern of fibre labelling has been previously noted by [START_REF] Mihailoff | Projections of the sensorimotor cortex to the basilar pontine nuclei in the rat: an autoradiographic study[END_REF].

Fibres were easily followed caudally through the internal capsule and continued down to the cerebral peduncle, then entering the LFP in the pontine section of the brainstem. In the medullary section of the brainstem, long fibres were occasionally traced heading dorsally towards the superior colliculus, although these fibres were of very fine calibre and no terminal labelling could be established due to the sparse nature of the fibres. Fibres could also be traced further caudally, always on the ipsilateral side of the injection site, and past the inferior olive in the pyramidal tract.

Caudal to the inferior olive, the fibres crossed the midline through the decussation of the pyramid to head dorsally on the side contralateral to the injection site. From there, fibres were traced down the pyramidal tract to the spinal cord.

For all anterograde injections in the motor cortex, no detectable terminal labelling was ever found in the inferior olive, even in those cases where substantial pontine labelling was found. Since injections of retrograde tracer were also made in the same cases into the cerebellar cortex where the COCP responses were recorded, the pattern of cell labelling in the olive served as a useful marker for where any direct cerebroolivary terminations might be found. In no case was any terminal labelling found in the vicinity of these labelled cells (Figure 5.5).

As there was no detectable terminal labelling in the inferior olive, a pre-olivary relay was sought and a site in the caudal brainstem was identified as a potential target. In 12 cases where there was terminal labelling in BPN (n = 5 forelimb; n = 7 hindlimb), the majority also gave rise to terminal labelling in a putative relay (n = 4 forelimb; n = 6 hindlimb). This was located just caudal to the olive at the level of the pyramidal tract decussation (further details below). In the cases where terminal labelling could not be detected in the putative relay, there was less BPN labelling and labelled descending fibres were also sparse by the time they reached the putative relay site, therefore there may have been insufficient transport of tracer in these areas.

In some cases (n = 7) fibres could be seen coursing down the descending cerebrofugal tract but there was no terminal labelling in the brain. In the cases where there was pontine labelling but no detectable putative nucleus labelling, very small, fine labelled fibres could still be traced down the caudal pyramidal tract into the spinal cord. The tracer had therefore reached the putative relay but was insufficient to give rise to detectable terminal labelling. The injection site data for this chapter is the same as detailed for Chapter 4 for the cerebral and cerebellar injection sites.

Anterograde Labelling in the Caudal Brainstem

An area in the caudal brainstem with terminal labelling was identified as being a potential pre-olivary relay in the cerebro-olivo-cerebellar projection. The terminal labelling was located on the contralateral side to the motor cortex injection site, slightly caudal to the pyramidal decussation. The nuclear region for the location of the terminal labelling was determined as being in the matrix. The labelling occurred approximately 2 mm caudal to the obex and was approximately 1 mm in rostrocaudal length. It was ventral to the gracile nucleus and approximately 1 mm lateral to the midline. In 10 cases from the motor cortical injection, terminal labelling was found in the matrix; 4 cases from forelimb injections and 6 cases from hindlimb injections. (wavy red and green lines) can be seen coursing through the pyramidal decussation, and heading dorsally to the dorsal columns, with some axons terminating in the matrix region.

Figure 5.7 also shows that the terminal labelling was not very different in extent or location between forelimb and hindlimb cases. Overall, the hindlimb labelling was slightly more extensive (see Figure 5.8) but this may have been due to having two more hindlimb cases than forelimb cases and of the hindlimb cases two were very well labelled. In these well labelled hindlimb cases (DP1 and DP11), there was also terminal labelling in the contralateral gracile nucleus (to the injection site), and also in one forelimb case (DP13). In DP11 (hindlimb) and DP13 (forelimb), the gracile labelling was also found to a lesser degree on the ipsilateral side to the injection site.

An overview of the pooled data and a summary of the individual areas of terminal labelling in the matrix can be seen in Figure 5.8. The pooled cases show that the total area covered by the terminal labelling is similar for both projections although the hindlimb projection extends slightly more caudal and rostral to the forelimb projection. As previously mentioned, this may be due to the higher number of hindlimb cases and also the two well-labelled hindlimb cases. Overall, the projection pattern is indistinguishable between forelimb and hindlimb motor cortical projections.

The approximate length of the labelled region was calculated as 1 mm, taking into account all cases, with the highest density of labelling in the middle of this area and was centred 2 mm caudal to the obex (see Figure 5.8). There was little difference in the dorsoventral or mediolateral position or size of the labelling for forelimb or hindlimb cases, with the area being an elongated oval in shape. The terminal labelling was centred approximately 0.6 mm in depth from the dorsal surface and 0.5 mm lateral from the midline.

Labelling in the Thalamus

In addition to terminal labelling in BPN (Chapter 4) and the matrix region, anterograde labelling was also noted at the level of the thalamus. Some cell labelling was also present here (n = 5). This pattern of labelling is indicative of thalamic reciprocal circuits with the cerebral cortex, demonstrating, in this case, circuits involving the motor cortex. These thalamo-cerebral and cerebro-thalamic mechanisms exert feed-forward and feed-back control, respectively, to and from the cortex [START_REF] Rouiller | A comparative analysis of the morphology of corticothalamic projections in mammals[END_REF]. The specific thalamic areas labelled depended upon the injection site but frequently, there was cell and terminal labelling in the reticular thalamic nucleus and surrounding areas such as the anteroventral thalamic nucleus (dorsomedial and ventrolateral parts).

Labelling in the Substantia Nigra

At the level of the substantia nigra, where the internal capsule becomes the cerebral peduncle, fibres could be traced heading caudally and ventrally. There was some terminal labelling medially of the cerebro-fugal tract in the substantia nigra pars reticularis (n = 4).

Labelling in the Spinal cord

Axonal labelling could be followed as far caudally as the spinal cord coursing in the ventral part of the dorsal funiculus. In all cases with BPN terminal labelling (Chapter 4), fibres were found in the rostral spinal cord (usually part of the sectioned brainstem block). Terminal labelling was not found in any case in the spinal cord because the sections usually did not progress caudal enough to localise cortico-spinal terminations.

Inactivation of Putative Pre-Olivary Relay

A total of 8 adult male Wistar rats were used in the inactivation experiments to reversibly inactivate the matrix region of the preceding section. Two different types of drug were used: local anaesthetic (n = 5 animals; experiments DPE22 to DPE26) or a mixture of GABA and KA (n = 3 animals; experiments DPE28, DPE29 and DPE32). Lignocaine was used because it was an accessible drug and blocked neuronal activity (including fibres of passage) whereas the GABA and kynurenic acid (KA) mixture was used because of its selective neuronal inactivation properties (see Methods for further details). From previous studies, these two drug regimes have been shown to be similar in the latencies and durations of their effects.

Various combinations of stimulation of the limbs ipsilateral to the recording site and the contralateral MC limb areas (and the ipsilateral MC in one forelimb case) were used to investigate the input to cerebellar climbing fibres. Responses in the C1 zone were recorded, typically from the forelimb-receiving area in PML and hindlimbreceiving area in CP, but also in one experiment from the C1 forelimb-receiving zone in LS. Each case was designated by firstly, the drug regime used, secondly, the rostrocaudal site of the injection, and finally whether responses were recorded from forelimb PML and/or hindlimb CP was recorded from (and one case forelimb LS).

This meant that a case was considered as a certain recording site in the cerebellum (fore-or hindlimb C1) to which an injection of drug was placed in a position 0.5 to 4 mm caudal from obex.

LA or GABA/KA was injected at different loci spread over a 3.5 mm length of brainstem caudal to the obex to find if there was an optimum site or sites where a decrease in climbing fibre responses could be obtained. In each experiment, climbing fibres responses were evoked in a cyclical sequence by peripheral limb then contralateral motor cortical stimulation and were recorded in the middle of the C1 zone in PML and/or CP (see Methods for further details).

Injection of Drug into Putative Relay Site

Lignocaine and a mixture of GABA/KA both produced reversible inactivation of climbing fibre responses evoked by cerebral stimulation when injections were made 2 mm caudal to obex. Figure 5.9 shows an example injection site (from a case where GABA/KA was injected) into the matrix, 2 mm caudal from the obex. Histological reconstructions of the caudal brainstem injection sites were gained wherever possible, to confirm the location of the drug injections. In 6 experiments, the brainstem was successfully removed. The material was examined for both pontamine sky blue dye and track damage from the injection pipette. Matching of injections to location was difficult due to the lack of dye found in the brainstem and also because the sections were often damaged as the material was only post-fixed.

This meant that assigning the location of each injection was not possible for all cases.

Since injections 2 mm caudal to obex were the most important, special attention was made to recover these injection sites. The general findings from these experiments were that inactivation occurred within 10 minutes and the block occurred for up to 35 minutes. The preferential target was just lateral of the midline, at a depth of 0.6 mm, although depths between 0.5 and 0.7 mm also showed some block.

From all cases at this site (n=10; 5 using LA; 5 using GABA/KA), the average time taken for a decrease in climbing fibres was 4 mins 40 secs and average time to recovery was 19 minutes 30 seconds. For the local anaesthetic cases, the time to a decrease was less (3 minutes 10 seconds) and the duration of the effect was a little longer (average 22 minutes 50 seconds). The time to a decrease in responses to GABA/KA application was longer (average 6 minutes 10 seconds) and the duration shorter overall (average 16 minutes 5 seconds). These values were well within expected time limits of reversible inactivation agents, from studies injecting these drugs into other CNS structures (e.g. [START_REF] Malpeli | Reversible inactivation of subcortical sites by drug injection[END_REF][START_REF] Martin | Pharmacological inactivation in the analysis of the central control of movement[END_REF][START_REF] Boehnke | Time course and effective spread of lidocaine and tetrodotoxin delivered via microdialysis: an electrophysiological study in cerebral cortex[END_REF].

Table 5.3 shows an overview of the statistical and percentage differences for the four time points tested after drug application against the control. In all but one case, a statistically significant decrease was found at least two of the four time points tested for responses to cerebral stimulation. The most common finding was a decrease of the cerebrally evoked responses (except for one initial increase at 5 minutes for DPE28 GABA/KA FL PML), whereas in marked contrast, for the peripherally evoked responses, the pattern was more mixed, although for the majority of time points there was no significant difference from control. In all statistical comparisons to investigate changes from control over time (at 5, 10, 15 and 20 minutes postinjection) for each case an ANOVA repeated measures analysis was used with Dunnett's multiple comparison post test.

From inspection of all the data and consideration of average time to drug effect and duration, the 10 minute time point was used as a gauge of effect for all the peripheral and cerebrally evoked responses. This allowed comparison of all the data obtained at different injection sites to gain a global overview of whether a significant effect was present in any one case for peripherally and cerebrally evoked responses. From the data gained at 10 minutes after drug injection into the putative site 2 mm caudal to obex (Table 5.3), there was a significant decrease of either p<0.05 or p<0.001 for 8 out of 10 of the sites, whereas for the same time point of peripheral responses, 7 out of 10 showed no significant change and of the 3 that did, there was a significant increase.

The data therefore shows a selective block of only the cerebrally evoked responses compared to the control but not the peripherally evoked responses at this rostrocaudal location. Table 5.3 shows that the significant decrease ranged between 19.2% and 77.5% compared to the control, whereas in the majority of the same cases there was no significant difference to drug application for the peripherally evoked responses. Figure 5.10 shows original data, presented graphically in binned responses (bins of 30 climbing fibre field potentials for each bar) from one example double stimulating/recording case to demonstrate the selective inactivation and recovery of cerebrally evoked climbing fibre responses, and that the peripherally evoked responses do not follow this pattern but remain stable.

The figure demonstrates this for both forelimb and hindlimb evoked responses recorded simultaneously. Local anaesthetic was injected into the matrix, 2 mm caudal from the obex and within 2 minutes, both fore-and hindlimb cerebrally evoked responses had decreased below the threshold for detection, where the peripherally evoked fore-and hindlimb responses were not affected. These recordings demonstrate that through inactivation of cells, the cerebral input can be selectively and temporarily blocked, whilst the peripheral input remains unchanged.

There was no difference between the onset of the effect or the duration for either fore-or hindlimb evoked responses suggesting that the relay for both are close together (perhaps intermingled) in the matrix area. This is consistent with the pattern of terminal labelling found in the anatomical tracing experiments (see previous section). In no case was it possible to block forelimb or hindlimb COCP cases separately. In each case where a successful block was found at 2 mm caudal to obex, the decrease in cerebrally evoked climbing fibre responses occurred between 1 to 8 minutes after the application of the drug. Both these values and the length of inactivation were comparable to findings in previous studies, in which the same drugs were used to selectively inactivate the motor cortex and the red nucleus [START_REF] Martin | Pharmacological inactivation in the analysis of the central control of movement[END_REF]. The following three Figures show the individual case results at the 2 mm site. Figure 5.12 shows the responses to local anaesthetic injections, Figure 5.13 to GABA/KA injections, and Figure 5.14 shows the pooled data for all cases at 2 mm caudal to obex.

In Figure 5.13, case DPE28 shows the similarity between the cerebral and peripheral input to forelimb receiving sites (in LS and PML). There was no difference in the onset latency of reduction in COCP responses or the duration of the effect between recording sites (like when comparing forelimb PML and hindlimb CP). In some experiments, further injections were placed in the putative relay site to investigate whether the reversible inactivation could be repeated. There were 2 experiments (DPE22 LA and DPE29 GABA/KA) where the drug was applied later in the experiment and in each experiment a second reversible block was found, although in both experiments it lasted 4-5 minutes and had an onset latency of 1.5 minutes (not shown).

Responses to Rostrocaudal Injection

Injections were also made at sites rostral and caudal to the putative relay to investigate whether the effect of the drugs was localised. Microinjections at intervals of 0.5 mm were made along the rostrocaudal axis of the caudal brainstem. Sites just rostral and caudal to 2 mm caudal from obex produced no changes in peripherally or cerebrally evoked responses. More rostral sites showed some variability in cerebrally and peripherally evoked responses, although no consistent differences were found for responses evoked by peripheral or motor cortical stimulation. Further caudally, there was a trend towards decreases in the responses, especially to peripheral stimuli.

Rostral Injection Sites to Putative Relay

Table 5.4 shows for drug injections made 0.5, 1.0 and 1.5 mm caudal to the obex, percentage changes seen in the responses and their degree of statistical significance.

Four chosen time points after injection are included. A total of 15 cases were included in the analysis (0.5 mm caudal to obex, n = 6; 1 mm caudal to obex, n = 5;

1.5 mm caudal, n = 4). Note that both increases (green) and decreases (yellow) in the response size were observed. The general conclusion drawn from these data was that there was no consistent effect on the peripherally or cerebrally evoked responses.

Caudal Injection Sites to Putative Relay

The farther caudal the drug injection, the more likely it was that both the peripherally and the cerebrally evoked responses were significantly decreased though there were a few significant increases, as can be seen in table 5.5. A possible explanation for the decreases, may have been due to local anaesthetic spread to fibre tracts. Local anaesthetic may actually have a more potent blocking effect on fibre tracts than on cells [START_REF] Sandkühler | The use of local anaesthetic microinjections to identify central pathways: a quantitative evaluation of the time course and extent of the neuronal block[END_REF]. From a total of 9 caudal cases, only two (DPE28 LS & PML) involved injecting GABA/KA. There was little effect on cerebrally evoked responses and a varied effect on responses evoked from the periphery. With the local anaesthetic, a higher proportion of responses decreased, especially to peripheral stimulation, which implied that ascending fibre tracts may have been affected, especially as these injections were in the rostral spinal cord.

Discussion

The physiological, anatomical and pharmacological data from these experiments provides evidence for a pre-olivary relay in the cerebro-olivo-cerebellar pathway, from the motor cortex to the C1 zone. The relay is in a region of the caudal brainstem termed the matrix. A clear, topographical limb input from the motor cortex was found targeting areas in the cerebellar C1 zone that corresponded closely to peripheral limb inputs. Climbing fibre responses were readily evoked from both descending and ascending pathways, demonstrating that the climbing fibre somatotopy imposed on the cerebellum holds true for descending inputs arising from the motor cortex.

Using anatomical tracers, the cerebro-olivary projection was found to be indirect, with no terminal labelling from the motor cortical injections ever found in the inferior olive. This negative finding was not entirely unexpected as the literature on cerebro-olivary projections has previously shown conflicting results as to whether there is a direct projection or one that involves at least one pre-olivary synaptic interruption (e.g. anatomical findings of Saint-Cyr & [START_REF] Saint-Cyr | Projections from the Motor Cortex, Midbrain and Vestibular Nuclei to the Inferior Olive in the Cat. Anatomical Organization and Functional Correlates[END_REF] and [START_REF] Saint-Cyr | The projection from the motor cortex to the inferior olive in the cat. An experimental study using axonal transport techniques[END_REF], 1987]; physiological findings of [START_REF] Baker | Activation of cerebellar climbing fibres to rat cerebellar posterior lobe from motor cortical output pathways[END_REF]. Through the use of neuronal blockers, the matrix region in the caudal brainstem was found in the present study to relay cerebrally-evoked, but not peripherally evoked climbing fibre responses. Earlier studies that investigated the cerebro-olivo-cerebellar pathway have shown conflicts between physiological data and anatomical pathway data, and the present data are discussed below in light of these past findings.

Physiology

Climbing fibre input imposes a zonal topographical organisation on the cerebellum, thus giving an organisation where zones can be systematically studied. In the present study, the motor cortex was mapped and an ordered representation of body parts was found whereby the fore-and hindlimb areas converged respectively with the corresponding peripheral input to specific cortical zones. The investigation revealed that in the rat, convergence of peripherally and motor cortically evoked climbing fibre responses is at or before the inferior olive. This was found for forelimbreceiving C1 zones in LS and PML, and for the hindlimb-receiving C1 zone in CP.

Previous studies have found convergence of climbing fibre ascending and descending input (e.g. [START_REF] Adrian | Afferent areas in the cerebellum connected with the limbs[END_REF][START_REF] Snider | Cerebrocerebellar relationships in the monkey[END_REF][START_REF] Armstrong | Responses in the inferior olive to stimulation of the cerebellar and cerebral cortices in the cat[END_REF][START_REF] Provini | Mossy and climbing fibre organization on the anterior lobe of the cerebellum activated by forelimb and hindlimb areas of the sensorimotor cortex[END_REF]Miller et al., 1969a, b;Allen et al., 1974a) and it demonstrates that similar olivary areas must be activated by different inputs.

Previous physiological studies in the monkey and cat of cerebro-olivary projections (e.g. [START_REF] Snider | Electro-anatomical studies on the afferent projection to the inferior olive[END_REF] found a bilateral projection though the pathways were not demonstrated anatomically. The data from Snider & Barnard's study agrees with physiological data such as [START_REF] Armstrong | Responses in the inferior olive to stimulation of the cerebellar and cerebral cortices in the cat[END_REF] and also with the physiological data gained in the present study, although the bilateral aspect was only found for the forelimb input, not hindlimb, and the ipsilateral MC stimulation showed a significantly longer response latency than that from contralateral MC stimulation, implying different multi-synaptic pathways are involved. The role of the bilateral projection may concern behaviours that are more complex, such as feeding and foraging, as the forelimbs play a further role in behaviours other than locomotion and balance, whereas it is the main function of the hindlimbs.

The convergence in the inferior olive shown by limb-receiving areas was important (cf. Allen et al., 1974a;[START_REF] Andersson | Origin and sagittal termination areas of cerebrocerebellar climbing fibre paths in the cat[END_REF], where weak signals arriving at the inferior olive conjunctively are nevertheless effective in producing a climbing fibre response. Therefore, as in studies in the cat, the principle that cerebral and peripheral inputs to the inferior olive exert a facilitated effect when they arrive concurrently, seems to also hold true in the rat. However, where there were no peripherally evoked climbing fibre responses, there were also no cerebrally evoked responses. Although the absence of responses should be interpreted with caution, this gives weight towards the representations of body parts in the cerebellum receiving only convergent input and shows the specificity of the climbing fibre projection to certain zones. For example, it may take a strong input from either the periphery or motor cortex for an inferior olivary neurone to fire, however when these inputs occur at the same time, a weaker input may be required to fire the same neurone. Therefore, the signals may serve as a coincidence or error detector of SOCP and COCP signals and may tie in with the comparator hypothesis (see [START_REF] Andersson | Origin and sagittal termination areas of cerebrocerebellar climbing fibre paths in the cat[END_REF].

The latency of the onset of all cerebrally evoked climbing fibre responses found in the present study (>10 ms) suggests slow conduction in the pyramidal tract and the likelihood of a pre-olivary relay. [START_REF] Kitai | Cerebro-cerebellar connections mediated by fast and slow conducting pyramidal tract fibres of the cat[END_REF] have previously shown in the cat that the early mossy fibre response in the cerebellum is mediated by fast conducting fibres in the pyramidal tract, whereas the climbing fibre response is mediated by slower conducting fibres, hence the longer latency. Allen et al. (1974b) investigated the forelimb and hindlimb input from the motor cortex in the cat and came to the conclusion that the cerebro-olivary pathway for these inputs could not be monosynaptic due to the long delay.

In the cat, there are fast and slow conducting pathways [START_REF] Takahashi | Slow and fast groups of pyramidal tract cells and their respective membrane properties[END_REF], however in the rat, only slow conducting pyramidal tract axons have been found [START_REF] Landry | Morphological and electrophysiological characteristics of pyramidal tract neurons in the rat[END_REF]. As the timing of climbing fibre and mossy fibre latencies are similar between rat and cat, but there only seems to be one class of projection neurone in the pyramidal tract in the rat, then it follows that there could be some system that 'delays' the climbing fibre responses from MC stimulation, i.e. a pre-olivary relay. A longer pathway and additional synapse(s) would mean a longer onset latency of the climbing fibre response and thus maintain differences in mossy and climbing fibre response latencies to the same cortical stimulus.

The anatomical findings support a mono-synaptic cerebro-cerebellar pathway via BPN (as expected, see Chapter 4) and when seen, mossy fibre responses were much shorter in latency than the climbing fibre responses. Conversely, the physiological data here supports the notion of a pre-olivary relay in the rat (cf. [START_REF] Baker | Activation of cerebellar climbing fibres to rat cerebellar posterior lobe from motor cortical output pathways[END_REF],

where the latency of even the shortest forelimb MC climbing fibre response suggests a disynaptic relay. Furthermore there was a longer latency from ipsilateral forelimb MC stimulation, suggesting a poly-synaptic relay for this input. Even with the slow conduction velocities in COCPs, the findings show that there would still be sufficient time for at least one additional pre-olivary synaptic interruption. Also, the fastest conducting SOCPs targeting forelimb parts of the C1 zone have a similar latency to the corresponding COCPs (approximately 12 ms), implying that precise timing of the integration of ascending and descending climbing fibre inputs is important for cerebellar contributions to the control of forelimb movements.

Anatomy

Controversy exists as to whether there is a direct cerebro-olivary projection. In the present study, no direct anatomical projection was found for the motor cerebroolivary projection for fore-or hindlimb motor cortical areas in any experiment. Even in cases where there was a significant amount of terminal labelling in BPN, no detectable terminal fibres were ever found in the olive; when viewed in transverse section only short lengths of labelled axons were seen ventral to the olive. Wherever there was terminal labelling in the CNS, it was clear from the plane of section to see the labelled fibres getting longer as they came into the same transverse plane as the sectioned material. The short fibres were typical of axons only, coursing down the pyramidal tract where the plane of sectioning was perpendicular to the plane of axonal labelling. The lack of terminal labelling in the olive was not thought to be due to methodological or tracer limitations (see also Chapter 4), especially as other studies have used similar survival times to label cerebro-spinal connections with the tracer [START_REF] Coleman | Topography of fibre organisation in the corticofugal pathways of rats[END_REF], in the rat) and as the evidence for a direct projection from the motor cortex to the inferior olive is varied.

A direct cerebro-olivary projection may exist from other cerebral areas that the present investigation did not study. For example, such direct cerebro-olivary projections may arise mainly from somatosensory cortex (e.g. [START_REF] Swenson | The afferent connections of the inferior olivary complex in rats. An anterograde study using autoradiographic and axonal degeneration techniques[END_REF][START_REF] Swenson | Organization of cerebral cortico-olivary projections in the rat[END_REF]). An additional possibility is that they entail diffusely scattered projection cells in the cerebral cortex (e.g. [START_REF] Bishop | A horseradish peroxidase study of the cortico-olivary projection in the cat[END_REF], therefore the small microinjections made in the present study may not have been effective in labelling sufficient numbers of these neurones to produce a detectable level of terminal labelling in the inferior olive. However, a direct cerebro-olivary pathway from diffusely organised pyramidal cells would suggest that the cerebro-olivocerebellar pathway was not substantial or functionally powerful, contrary to the findings from the present and previous physiological studies (e.g. [START_REF] Provini | Mossy and climbing fibre organization on the anterior lobe of the cerebellum activated by forelimb and hindlimb areas of the sensorimotor cortex[END_REF], Miller et al., 1969a, b;Anderson & Nyquist, 1983;[START_REF] Baker | Activation of cerebellar climbing fibres to rat cerebellar posterior lobe from motor cortical output pathways[END_REF].

There have been many anatomical studies investigating cerebro-olivary connections.

Some studies have shown a direct cerebro-olivary projection in the rat [START_REF] Brown | A study of afferent input to the inferior olivary complex in the rat by retrograde axonal transport of horseradish peroxidase[END_REF][START_REF] Swenson | The afferent connections of the inferior olivary complex in rats. An anterograde study using autoradiographic and axonal degeneration techniques[END_REF][START_REF] Swenson | Organization of cerebral cortico-olivary projections in the rat[END_REF] and the cat [START_REF] Walberg | Descending connections to the inferior olive; an experimental study in the cat[END_REF][START_REF] Sousa-Pinto | Demonstration of a somatotopical pattern in the cortico-olivary projection in the cat. An experimental-anatomical study[END_REF][START_REF] Bishop | A horseradish peroxidase study of the cortico-olivary projection in the cat[END_REF][START_REF] Berkley | Projections to the inferior olive of the cat. I. Comparisons of input from the dorsal column nuclei, the lateral cervical nucleus, the spino-olivary pathways, the cerebral cortex and the cerebellum[END_REF][START_REF] Saint-Cyr | The projection from the motor cortex to the inferior olive in the cat. An experimental study using axonal transport techniques[END_REF]). However, one of the main drawbacks of these earlier studies is the large quantity of cerebro-fugal fibres that traverse the olive and send axons to other brainstem nuclei. In particular, the use of degeneration studies can be ambiguous in this regard as degenerating axons and terminals can be difficult to distinguish from one another [START_REF] Kuypers | Anatomy of the descending pathways[END_REF]. Also, due to the close proximity of fibres of passage, injection of retrograde tracer into the inferior olive makes it difficult to draw any reliable conclusions about direct projections, particularly given the complex shape of the olive -it is almost impossible to be sure that injections are confined to this structure.

However, the labelling found in the olive from cerebral (sensory and motor)

injections in the study by [START_REF] Swenson | Organization of cerebral cortico-olivary projections in the rat[END_REF] cannot be disputed for the same reasons. In their study, a direct ipsilateral projection was found from fore-and hindlimb motor cortex to the inferior olive using WGA-HRP as an anterograde tracer.

The disagreement with the present findings may relate to the possibility that different areas of the sensorimotor cortex were injected and that their injections were more superficial and larger (c. 1mm diameter), thus targeting different projection neurones.

In the present study, microinjections were placed into regions of the motor cortex that elicited more proximal muscle movements than the digit movements evoked in Swenson et al.'s study. This raises the possibility that some parts of the rat motor cortex have direct pathways to the olive, while others do not; perhaps the control of limbs for locomotion requires a pre-olivary relay, while control of distal fine movements in exploratory tasks requires a direct cerebro-olivary link.

Similar differences may also be present in cats. In this species, varied results for cerebro-olivary projections have been reported [START_REF] Kuypers | An anatomical analysis of cortico-bulbar connections to the pons and lower brainstem in the cat[END_REF][START_REF] Saint-Cyr | Projections from the Motor Cortex, Midbrain and Vestibular Nuclei to the Inferior Olive in the Cat. Anatomical Organization and Functional Correlates[END_REF][START_REF] Saint-Cyr | The projection from the motor cortex to the inferior olive in the cat. An experimental study using axonal transport techniques[END_REF][START_REF] Sousa-Pinto | Demonstration of a somatotopical pattern in the cortico-olivary projection in the cat. An experimental-anatomical study[END_REF], with either no direct cerebro-olivary projection found, or only certain parts of the motor cortex projecting to the inferior olive. In cats, the motor cortex is located in the anterior sigmoid gyrus (a.s.g.) and rostral part of the posterior sigmoid gyrus (p.s.g.), collectively know as the pericruciate cortex. The a.s.g. has been has been found to provide a discrete projection to the inferior olive [START_REF] Sousa-Pinto | Demonstration of a somatotopical pattern in the cortico-olivary projection in the cat. An experimental-anatomical study[END_REF][START_REF] Saint-Cyr | Projections from the Motor Cortex, Midbrain and Vestibular Nuclei to the Inferior Olive in the Cat. Anatomical Organization and Functional Correlates[END_REF][START_REF] Saint-Cyr | The projection from the motor cortex to the inferior olive in the cat. An experimental study using axonal transport techniques[END_REF], while no direct projection has been reported from the p.s.g. [START_REF] Bishop | A horseradish peroxidase study of the cortico-olivary projection in the cat[END_REF] also found from retrograde labelling, many cells in a.s.g. and very few in p.s.g., although injections into the olive have interpretative difficulties as discussed earlier.

If we accept that there is no direct p.s.g.-olive projection, it does not necessarily imply that this motor area does not contribute input to the cerebellum via climbing fibres. [START_REF] Sousa-Pinto | Demonstration of a somatotopical pattern in the cortico-olivary projection in the cat. An experimental-anatomical study[END_REF] presume that the p.s.g. has no influence over the cerebellum via the olive. However, [START_REF] Andersson | Origin and sagittal termination areas of cerebrocerebellar climbing fibre paths in the cat[END_REF] showed that the p.s.g. projects to the forelimb-receiving (anterior lobe) C1 zone in cat. Therefore, the C1 (forelimb) zone may receive input from p.s.g. (presumably from indirect pathways) and not the a.s.g. As the cat has these different areas of representations in the pericruciate cortex, this cannot be directly compared to the rat, which has no equivalent differentiation of its motor cortex. This may reflect specialisation related to phylogenetic complexity.

Some studies in cats have emphasized the role of mesodiencephalic structures that relay cerebral information to the inferior olive [START_REF] Saint-Cyr | Projections from the Motor Cortex, Midbrain and Vestibular Nuclei to the Inferior Olive in the Cat. Anatomical Organization and Functional Correlates[END_REF]Nakamura et al., 1983a, b;[START_REF] Saint-Cyr | Anatomical organization of cortico-mesencephalo-olivary pathways in the cat as demonstrated by axonal transport techniques[END_REF][START_REF] Rutherford | A comparison of the distribution of the cerebellar and cortical connections of the nucleus of Darkschewitsch (ND) in the cat: a study using anterograde and retrograde HRP tracing techniques[END_REF]. However, there was no obvious terminal labelling in these structures in rat, suggesting there may be other important differences between species in terms of the anatomical organisation of cerebro-olivary projections.

Due to the lack of terminal labelling in the inferior olive, a pre-olivary relay was sought. Labelled cells and presumed terminal fibres were found in the thalamus (mainly ventrolateral thalamic regions), from the injections into the motor cortex.

However, this labelling was not thought to be a likely candidate for a relay in the cerebro-olivary pathway, as it is considered to be mainly involved in reciprocal connections with the cerebral cortex and olivary projections from this part of the thalamus have not been reported. Terminal labelling was also found in the substantia nigra, pars reticular, although again, this structure is not thought to have a substantial projection to the olive. The substantia nigra is part of the basal ganglia and is typically involved in the initiation (rather than overseeing/monitoring/correction) of movement. There have been no previous reports of either thalamo-olivary or nigralolivary projections (for overviews see [START_REF] Armstrong | Functional significance of connections of the inferior olive[END_REF][START_REF] Bloedel | Cerebellar afferent systems[END_REF].

The pontine nuclei appeared to be the only other possible rostral relay in the cerebroolivary pathway due to the large amount of terminal labelling found there. From the literature, though, there is little evidence of a ponto-olivary projection. Some electrophysiological data has been reported in which pontine stimulation was found to evoke climbing fibre responses in the cerebellum (e.g. [START_REF] Sasaki | Responses evoked in the cerebellar cortex by pontine stimulation[END_REF].

However, Sasaki and colleagues concluded that a direct ponto-olivary projection was unlikely and suggested that their results were probably due to activation of fibres of passage.

Therefore, in rat, there may not be a rostral relay, but only the possibility of a caudal relay. Through physiological evidence [START_REF] Baker | Activation of cerebellar climbing fibres to rat cerebellar posterior lobe from motor cortical output pathways[END_REF] by stimulating the pyramidal tract and selectively lesioning parts of it, found that if there was any relay involved in the (rat) MC projection to the inferior olive, it would not be mediated by relays in the rostral brainstem as lesions of the pyramidal tract rostral to the inferior olive substantially reduced responses evoked from stimulation of cerebro-fugal fibres.

The dorsal column nuclei (DCN) have also previously been identified as receiving a cerebro-fugal projection [START_REF] Kuypers | An anatomical analysis of cortico-bulbar connections to the pons and lower brainstem in the cat[END_REF][START_REF] Gordon | Descending influences on the exteroceptive organizations of the cat's gracile nucleus[END_REF][START_REF] Cheema | Sensorimotor cortical projections to the primate cuneate nucleus[END_REF]. The DCN, in turn, are well known to provide direct projections to the inferior olive [START_REF] Walberg | Corticofugal fibres to the nuclei of the dorsal columns; an experimental study in the cat[END_REF][START_REF] Tomasulo | Activation of neurons in the gracile nucleus by two afferent pathways in the rat[END_REF][START_REF] Boesten | Projections of the dorsal column nuclei and the spinal cord on the inferior olive in the cat[END_REF][START_REF] Berkley | Projections to the inferior olive of the cat. II. Comparisons of input from the gracile, cuneate and the spinal trigeminal nuclei[END_REF][START_REF] Martinez | Pyramidal tract and corticospinal neurons with branching axons to the dorsal column nuclei of the cat[END_REF][START_REF] Molinari | Gracile, cuneate, and spinal trigeminal projections to inferior olive in rat and monkey[END_REF][START_REF] Apps | Input-output connections of the "hindlimb" region of the inferior olive in cats[END_REF]) and therefore could be a possible target to receive MC information and relay it onto the inferior olive.

The matrix region may be a distinct part of the DCN, where in the past it may have been grouped with the cuneate or gracile nuclei. In a degeneration study, [START_REF] Kuypers | An anatomical analysis of cortico-bulbar connections to the pons and lower brainstem in the cat[END_REF] found terminal labelling near cuneate and gracile as a result of lesions into the cat motor cortex; this is likely to have included the matrix region. Also, Sousa-Pinto & Brodal (1969) using degeneration techniques found a heavy projection to the DCN from motor cortical areas, which was bilateral with a mainly contralateral predominance. The results from the present study have found a contralateral projection to the matrix region (n=10) and on occasion, a partly bilateral projection to the gracile nucleus (n=3). The available anatomical evidence therefore suggests that the matrix region is a potential site for an initial synaptic interruption in the cerebro-olivary pathway (there may well be additional synapses before information reaches the olive, especially due to the finding that forelimb MC stimulation evoked climbing fibre responses in both sides of the cerebellum).

Reversible Inactivation of Putative Relay

Little is known about the matrix region but it was identified in relation to the human pericuneate and peritrigeminal matrices (identified in [START_REF] Paxinos | Atlas of the Human Brainstem Academic Press[END_REF]. A similar structure was observed later in the rat [START_REF] Paxinos | The Rat Brain in Stereotaxic Co-ordinates[END_REF] and is thought to function as a residual region relating to the dorsal column nuclei, when all the surrounding nuclei have been accounted for. Reversible pharmacological inactivation of the matrix region selectively abolished climbing fibre responses evoked by MC stimulation. This effect was obtained with both types of blocker (LA or GABA/KA). Taken together with the anatomical evidence, these inactivation experiments give considerable evidence towards the matrix region being a synaptic relay in the cerebro-olivary projection from the motor cortex to the C1 zone. The selective inactivation of COCPs was localised to a site 2 mm caudal from the obex.

This corresponds closely to where terminal labelling was found in the matrix.

Injections of blocker further rostrally or caudally failed to produce a selective effect.

The differential effect on COCPs as compared to SOCPs indicated that the synaptic relay was prior to the convergence of the ascending and descending information to the olive.

Both local anaesthetics and GABA have approximate areas of inactivation of up to 500 µm radius for the volumes injected in this study. As the region of the terminal labelling in the putative relay was estimated to be approximately 1 mm in rostrocaudal length and 300 µm in width, the area of inactivation should have encompassed much of the region, but possibly not all of the rostrocaudal length, although in most cases this was enough to give a full abolition of evoked cerebral responses. Also, consistent with the anatomical findings was the similarity in the time course and effect of inactivation on both forelimb and hindlimb pathways. The territories occupied by terminals for fore-and hindlimb paths overlapped extensively in the matrix. However, since these paths are functionally independent this implies that either both fore-and hindlimb relay cells are intermingled in the matrix or that higher resolution mapping will be required to identify any topographical organisation. [START_REF] Andersson | Demonstration of a cuneate relay in a cortico-olivo-cerebellar pathway in the cat[END_REF] found that in the cat, topical application of local anaesthetic to the caudal cuneate nucleus abolished SOCP and short-latency COCP responses in the C1 and C3 zones and therefore that COCPs were at least partly relayed through the cuneate nucleus. The results from the present study raise the possibility that there are inter-animal differences in anatomical connectivity and structural differences between the rat and cat caudal brainstem nuclei, but also that the local anaesthetic injected into cuneate may have actually spread deeper into the brainstem and inactivated the matrix, to gain similar results as the present investigation.

This notion is supported by [START_REF] Mccurdy | Spatial overlap of rubrospinal and corticospinal terminals with input to the inferior olive[END_REF] where a rostrocaudal column of cells in the ventral cuneate nucleus was shown to project to rostral DAO, through anatomical tracer injections into the inferior olive and the cuneate nucleus, in the cat.

Furthermore, it was found that with cerebral injections into the forelimb area of the pericruciate cortex, the consequent terminal labelling in the cuneate nucleus was located in a similar area to the cell labelling from retrograde tracer injections into the DAO. This suggests that there is a convergence of forelimb ascending and descending climbing fibre paths to the C1 and C3 zones in the cat. Also, in the present investigation, injections of to inactivate the more caudal parts of the matrix (i.e. 2.5-4 mm caudal to obex) reduced both SOCP and COCP transmission. Thus, the matrix may comprise of at least two rostrocaudally separated regions: one region at approximately 2mm caudal to the obex, which relays COCPs but not SOCPs targeting the same parts of the C1 zone, and a second more caudal region (2.5-4 mm caudal to obex) where SOCPs and COCPs conveying climbing fibre signals to the C1 zone converge in the rat.

Further Study

Further physiological studies could be carried out to include projections from other areas of the cerebral cortex and also recording from other zones and lobules. For example, the primary and secondary somatosensory cortices could be stimulated to investigate the sensory input to zones in the cerebellum. This could be done in relation to both the peripheral and MC inputs, and to gauge any convergence between these other projections on areas in the inferior. A more in-depth way of investigating climbing fibre responses to the cerebellum would be to use single unit recording from individual Purkinje cells, in response to peripheral and cerebral stimulation.

In addition, a different anaesthetic could be used (such as a ketamine/xylezene mixture) so that both climbing fibre and mossy fibre activity could be recorded simultaneously. By recording both climbing and mossy fibre activity to peripheral and cerebral stimulation, it would be possible to investigate the effect of drug injection (blockers and excitants) into the matrix area on climbing fibre activity, but also on mossy fibre activity. Similarly, stimulation (electrical or chemical) within the matrix region and recording in the olive would help determine the extent to which the projection is direct. In conjunction with these physiological methods, injection of bidirectional tracer may be carried out to look at the input-output connections of the matrix, specifically where direct olivary connections exist. In further study, recovery experiments could be used to investigate the contribution the putative relay area provides to behaviour. Recording electrodes (e.g. tetrodes for multiple recordings) could be chronically implanted into the matrix region and single unit activity recorded while an animal performs voluntary movements (thus engaging the motor cortex), such as pressing a lever for a reward. Also, if a cannula was placed in a headpiece over the caudal brainstem, to deliver drugs into the putative relay area, the effect of these drugs in the awake animal, in response to such an experimental paradigm could provide insights into the contribution(s) of this cerebro-olivocerebellar pathway during movements. From the view of the posterior lobe of the rat cerebellum and the corresponding body representations, it can be seen that the mossy fibre representation in the cerebellar cortex is patchy where body parts are represented in a fractured manner. For example, the coloured forelimb in the rat is represented next to other non-contiguous body parts in the cerebellum (adapted from [START_REF] Bower | Is the cerebellum sensory for motor's sake, or motor for sensory's sake: the view from the whiskers of a rat?[END_REF], from original work by [START_REF] Bower | Principles of organization of a cerebro-cerebellar circuit. Micromapping the projections from cerebral (SI) to cerebellar (granule cell layer) tactile areas of rats[END_REF]. The table shows a summary of the injection details for all the cases where cell labelling was found in BPN and in the expected area(s) of the inferior olive for the injection site. From left to right, the columns show case number (HL and FL refer respectively to injections into the forelimb C1 zone in PML or the hindlimb C1 zone in CP in the cerebellum); type of tracer (GB, green beads; RB, red beads); volume of tracer injected (nl); the mediolateral (m-l) size of the core of the injectate (µm); the distance to the anatomical midline from the middle of the core (mm); the length of the white matter (WM) interface (µm); and the injection site size (mm 2 ). The area of the injectate for the injection site size calculation was found from multiplying the values together for the area of an ellipse (see [START_REF] King | Numerical aspects of pontine, lateral reticular, and inferior olivary projections to two paravermal cortical zones of the cat cerebellum[END_REF] for further details). The table show the raw cell counts for the cases where the injection in the cerebellum was in the target (as defined as having over 80% of the olivary labelling in the expected region for the injection site), where olivary data is shown in (A) and pontine in (B). The olivary projection was contralateral, whereas the pontine projection was bilateral. The pontine cell counts are broken down into contralateral side and ipsilateral side labelling from injections into the right side of the cerebellum, and show data for both BPN and NRTP. Key: BPN -basilar pontine nuclei; Contracontralateral; DAO -dorsal accessory olive; MAO -medial accessory olive; NRTP -nucleus reticularis tegmenti pontis. The figure displays the consistent lack of terminal labelling in the olive from anterograde tracer injections into the motor cortex. This photomicrograph shows short, labelled axons in the pyramidal tract in case DP11 from an injection of Fluoro-Ruby into the hindlimb area of the motor cortex (scale bar = 200 µm). Some cells labelled from an injection of red beads into the forelimb-receiving C1 zone in PML in the cerebellum can also be seen, which are located in the expected part of DAO that projects to that zone. -16.5 mm Bregma Table 5.4: Significant differences and percentage changes in recordings at injection sites rostral to the labelling in the matrix area.

Figures & Tables

----------------------------------------
i) (ii) (iii) (iv) (v) (ii) A B (i) (iii)
The table shows a summary of individual cases and injections at sites 0.5 mm, 1 mm and 1.5 mm caudal to obex (putative relay at 2 mm caudal to obex). The responses are to peripheral and cerebral stimulation and values are taken at 5, 10, 15 and 20 minutes, compared to pre-drug baseline. Shaded boxes indicate significant differences from Dunnett's multiple comparison post test in conjunction with repeated measures ANOVA; yellow = decrease and green = increase, to baseline. C1 zone recording site is also indicated (CP -hindlimb receiving copula pyramidis; LS -forelimb receiving lobulus simplex; PML -forelimb receiving paramedian lobule). DPE22, 23, 24, 25 and 26 were injected with local anaesthetic, DPE28, 29 and 32 were injected with GABA/KA. From the results, the cortical forelimb pathway seemed to be conveyed along different paths, depending upon whether contralateral or ipsilateral to the C1 recording site. Taking into account the physiological and anatomical data gained, the above suggested pathway would be feasible to interpret the findings.

  This work was supported by the Physiology Department, the Medical Research Council and the Wellcome Trust. v Author's Declaration I declare that the work submitted in this thesis was carried out in accordance with the Regulations of the University of Bristol. The work is original, except where indicated by special reference in the text. The ponto-cerebellar 3D reconstruction experiments were carried out in collaboration with the NeSys Laboratory at Oslo University. Some of the early anatomical tracer experiments were carried out with Dr. Joanne Pardoe, although all the data analysis was completed by the author. The acute physiology experiments were of independent work and analysis. No part of the thesis has been submitted for any other academic award. Any views expressed in this thesis are those of the author. SIGNED:…………………………………………… DATE:…………………

1. 1

 1 Cerebellar circuitry ............................................................................................110 1.2 Responses of the Purkinje cell to mossy fibre and climbing fibre activation ...111 1.3 Climbing fibre zonal organisation in the cat and rat .........................................112 1.4 Mossy fibre patchy representation in the cerebellar cortex ...............................113 1.5 Cerebral cortical somatotopical body representations in the rat .......................114 2.1 Diagram of the experimental stimulation and recording areas in the rat brain .115 2.2 Pontine orientation maps ...................................................................................116 3.1 Physiological responses and anatomical labelling of two cases in the same animal ............................................................................................................... 119 3.2 Reconstruction of two cases in the same animal ...............................................120 3.3 Inferior olive and BPN cell labelling in three cases, representative of each injection site ...................................................................................................... 121 3.4 Surface representation reconstructions of accumulated cases of BPN labelling from cerebellar and SI injections ........................................................ 124 3.5 3D dot map reconstructions of accumulated cases ............................................125 4.1 Schematic map of reconstruction of injection sites in cerebral cortex ..............126 4.2 Examples of photomicrographs of motor cortex injection sites ........................127 4.3 Examples of photomicrographs of cerebellar cortex injection sites ..................128 4.4 Location of retrogradely labelled cells in the inferior olive ..............................131 4.5 Terminal labelling in BPN from a motor cortical injection of tracer ................133 4.6 Cell labelling in BPN from an injection into the cerebellum ............................134 4.7 Average and individual forelimb cases showing terminal labelling in BPN ....135 4.8 Full documented case of example 'forelimb' BPN labelling ............................136 4.9 Total extent of 'forelimb' BPN terminal labelling ............................................137 4.10 Total extent of 'forelimb' BPN cell labelling ...................................................138 4.11 Average and individual hindlimb cases showing terminal labelling in BPN ....139 4.12 Full documented case of example 'hindlimb' BPN labelling ...........................140 4.13 Total extent of 'hindlimb' BPN terminal labelling ...........................................141 4.14 Total extent of 'hindlimb' BPN cell labelling ...................................................142 4.15 Overlap of 'forelimb' terminal and 'forelimb' cell labelling in BPN ...............145 4.16 Overlap of 'hindlimb' terminal and 'hindlimb' cell labelling in BPN ..............146 xiii 4.17 Overlap of 'forelimb' terminal and 'hindlimb' cell labelling in BPN ..............147 4.18 Overlap of 'hindlimb' terminal and 'forelimb' cell labelling in BPN ..............148 4.19 Areas of total overlap in BPN ...........................................................................149 4.20 Photomicrographs and ventral map of NRTP cell labelling ..............................150 5.1 Evoked climbing fibre field potentials to peripheral and central limb stimulation ......................................................................................................... 151 5.2 Paired pulse tests for climbing fibre responses to repeated stimulation sites ...154 5.3 Conditioning tests for climbing fibre responses to different stimulation ..........155 5.4 Convergent responses from stimulation at threshold ........................................156 5.5 Labelled fibres in pyramidal tract under inferior olive .....................................157 5.6 Photomicrograph and camera lucida drawing of labelling in the matrix area ...158 5.7 Double anterograde labelling in the caudal brainstem ...................................... 159 5.8 Overview of pooled and individual cases giving terminal labelling in the matrix .................................................................................................................160 5.9 Photomicrograph of an example injection site made to inactivate the matrix area ....................................................................................................................161 5.10 Bar graphs of binned raw cerebellar responses to peripheral and cerebral stimulation from an injection of LA into the matrix .........................................163 5.11 Selective decrease in climbing fibre responses to cerebral stimulation from an injection of GABA/KA into the matrix ........................................................ 164 5.12 Effects of injecting local anaesthetic into matrix on climbing fibre field responses ...........................................................................................................165 5.13 Effects of injecting GABA/KA into matrix on climbing fibre field responses .166 5.14 Pooled cases to demonstrate the overall effect of inactivating the matrix on climbing fibre field responses ...........................................................................167 5.15 Potential relay sites for bilateral forelimb motor cortical projection to cerebellar forelimb-receiving areas via inferior olive .......................................170 xiv List of Tables 2.1 Key for 3D Reconstruction Experiments (Chapter 3) ..........................................42 2.2 Key for Cerebro-olivo-cerebellar Physiological Experiments (Chapter 5) ..........42 3.1 Numbers of labelled cells in the ponto-cerebellar tracer cases ............................117 3.2 Details of cases used in 3D reconstruction and analysis ......................................118 3.3 Cell labelling and terminal overlap analysis, as determined using the local pontine co-ordinate system ................................................................................... 125 4.1 Hypothesised organisation in the ponto-cerebellar projection .............................61 4.2 Overview of anatomical tract tracer experiments .................................................125 4.3 Details of successful anterograde injection sites in the motor cortex that gave terminal labelling in BPN ..................................................................................... 130 4.4 Details of successful retrograde injection sites in the cerebellar cortex ...............132 4.5 Cell counts for the successful retrograde tracer cases ..........................................133 4.6 Individual overlaps in BPN in cases with both successful cerebral and cerebellar injections .............................................................................................. 143 4.7 Pooled overlaps in BPN in cases with successful cerebral and cerebellar injections .............................................................................................................. 144 5.1 Evoked latencies per case for climbing fibre field recordings .............................152 5.2 Significant differences between onset latencies comparing stimulation and recording sites ....................................................................................................... 153 5.3 Significant differences and percentage changes in responses from injections into the matrix area ............................................................................................... 162 5.4 Significant differences and percentage changes in recordings at injection sites rostral to the labelling in the matrix relay area .....................................................168 5.5 Significant differences and percentage changes in recordings at injection sites caudal to the labelling in the matrix relay area ....................................................169

  The acute physiological experiments often utilised two electrodes to record simultaneously from different areas in the cerebellum e.g. face A2 zone in the paramedian lobule (PML), forelimb C1 zone in PML (hereon termed FL PML), hindlimb C1 zone in copula pyramidis (CP; hereon termed HL CP), or forelimb C1 zones in lobulus simplex (LS; hereon termed FL LS) and PML, because of the longer time course of the experiments where the physiology was investigated in detail. This was an advantage as different zonal sites could be directly compared and more data could be obtained per animal used.Responses were recorded differentially, amplified and band pass filtered (30 Hz -5 kHz); a Humbug device (Quest Scientific, North Vancouver, Canada) was used to eliminate any 50 Hz electrical interference. Records of data (climbing fibre responses and stimulation triggers) were printed out from the oscilloscope and/or digitized online (sampling rate 10 kHz) using a CED (Cambridge Electronic Design, Cambridge, UK) 1401 A/D converter, and customized trigger-sampled software (Spike 2, CED, Cambridge, UK). Responses were evoked at either twice threshold (2T) stimulus intensity or three times threshold (3T), and occasionally supramaximally at five times threshold (5T).

  Märzhäuser motorised stage (Märzhäuser GmbH & Co. KG, Wetzlar-Steindorf, Germany) with a Zeiss fluorescent microscope (filters as before). The computer program Neurolucida (MicroBrightField Inc., Williston, VT, USA) was used in conjunction with the motorised stage to digitise the location of labelled cells in BPN and of the main structural landmarks (ventral surface of the brainstem, midline, outline surfaces of the left and right BPN, outline of the left and right longitudinal fasciculus of the pons (rostrally the cerebral peduncle and caudally the pyramidal tract), and the fourth ventricle) for orientation and alignment of sections. Cells were

  Each cell is represented by a small dot in the local co-ordinate system and the cells were normally viewed in either a 3D rotation or in 2D views. The standard 2D views were 'rostral', 'ventral' and 'medial' (for more details see Chapter 3 [3D reconstruction experiments] and Chapter 4 [anatomical cerebro-ponto-cerebellar experiments]

Figure 3 .

 3 Figure 3.1 documents an example double tracer experiment (BR14), which satisfied the criteria for 3D reconstruction. Cases were selected where the cerebellar injectate did not spread significantly into the white matter (see example injection site photomicrographs in Figure 3.1) or overlap with an adjacent injection site, and where the olivary cell labelling was confined to the expected area(s) corresponding to the zonal region injected into. Mapping of climbing fibre field responses was used to guide microinjections of red beads (FL) and green beads (HL) into the forelimb and hindlimb parts of the C1 zone in the same animal.

Figure 3

 3 Figure 3.1 (part i) shows examples of climbing fibre field responses (average of 4 consecutive sweeps) recorded in each zone to ipsilateral limb stimulation. Forelimbresponses had an onset latency of 11.5 ms and the hindlimb 15.5 ms. The approximate location of the cerebellar injection site and a photomicrograph of the injection core for each case is shown below; the parasagittal sectioning of the injection site allowed the different lobules to be easily identified. Figure3.1 (part v) also a high power view of cells in BPN shows that only the cell bodies were well labelled with the retrograde tracer and from inspection of the cells, there were no differences in the morphology and sizes of cells between cases (average diameter of cell body = 14 µm, n = 9 cases).

Figure 3 .

 3 Figure 3.2 demonstrates the cell labelling found in BPN corresponding to the double tracer experiment in Figure 3.1 (cases BR14 FL and HL). The distribution of cell labelling found in BPN is plotted in the local co-ordinate system. The standard ventral (i), rostral (ii) and medial (iii), views are shown (Figure 3.2 part A). Part (B) displays the 3D reconstruction of the labelling in relation to its topographical placement in BPN, with the LFP dorsally. From these views, especially the ventral 2D orientation, the topographical organisation of the ponto-cerebellar projection becomes clearer as the arrangement of cells for each case is centred in different locations.

  Figure 3.3 to show representative areas of cell labelling in the inferior olive and BPN (pooled are cases shown later as part of the Discussion). For each case is shown a small drawing of the zone injected in the cerebellum below which, is a figurine detailing the mediolateral extent of the evoked responses recorded on the surface of the cerebellum in each experiment, as a thin horizontal line, with the histologically defined width of the injection site placed as a thick line on top.In each case, the spread of the injectate was restricted within the boundaries of the responses for the zone in question, so that as the injection was placed in the approximate centre of each zone, the cell labelling gained was most likely to be confined to the climbing fibre zone being investigated, which was confirmed when the inferior olive was mapped. On the left in each case are standard maps of the inferior olive in a horizontal view, where the cell labelling for each zonal injection site can be seen to lie in the expected regions. On the right in each case are shown the corresponding cell labelling in BPN in ventral, medial and rostral views of BPN, constructed from the local co-ordinate system.The overall pattern of labelling showed clusters of labelled cells with the face, forelimb and hindlimb injections producing labelling centred in progressively more caudal parts of BPN. These clusters of cells displayed an internal to external insideout sequence of body part representations. The centrally located face region was enveloped caudally by the forelimb labelling, which in turn was surrounded caudally by the hindlimb labelling. In Figure3.3, the A2 face pontine data clearly shows the centrality and bilaterality of the cell population labelling, most prominently seen in the ventral view. The labelling was skewed somewhat medially and formed crescent shapes on both sides of BPN (54.5% of cell labelling was on the contralateral side).

Figure 3

 3 Figure3.4, the left column shows the pooled ponto-cerebellar cell labelling whereas the right shows cerebro-pontine axon termination as dots (fromLeergaard et al., 2000b). Five cases were selected from the archive at www.nesys.uio.no/Database from injections made into the somatosensory cortex, to compare to the current pontocerebellar cases. To compare the mossy fibre projection to the A2 face receiving zone, case R103 was used from the database, where five injections of anterograde tracer were made into the face/whisker responsive area of SI. The C1 forelimb zonal

Figure 3 .

 3 Figure 3.5 demonstrates the 'filled in' ponto-cerebellar (A) and cerebro-pontine (B) projections, reconstructed in 3D (contralateral view only). The similarities in the distribution patterns of the ponto-cerebellar (top) and cerebro-pontine (bottom) projections in BPN are remarkable. It is also clear from both reconstructions that when the accumulated cases are taken together and reconstructed in 3D in BPN, there are clear non-overlapping territories for each of the body representations.

  IIa facial receiving area to directly compare the mossy fibre input arising from a different (hemispheric) zone with the pattern of cell labelling from the present (vermal) A2 zone results. Other equivalent climbing fibre zones and mossy fibre patches should be studied and attempts made to take into account zebrin banding to further map the topography in the ponto-cerebellar projection.

  ordinate system in relation to climbing fibre zones e.g. mossy fibre projections from the lateral reticular nuclei. Caution must however be exercised when comparing the cerebro-ponto-cerebellar pathway (also applicable in Chapter 4) in that spatial overlaps rather than actual synaptic connections have been demonstrated. Studies at higher resolution (electron microscope level) could be a useful and relevant line of further investigation, such as injecting anterograde tracer into the cerebral cortex and retrograde tracer into the cerebellum thus labelling terminals and fibres in BPN to demonstrate actual synaptic connections. This may then show that overlapping areas of cell and terminal labelling do actually imply the existence of synaptic contacts.

  have shown topography in the cerebro-pontine projection, in relation to descending limb projections to BPN. For example,[START_REF] Mihailoff | Projections of the sensorimotor cortex to the basilar pontine nuclei in the rat: an autoradiographic study[END_REF] using autoradiographic techniques showed a topographical organisation in BPN arising from forelimb and hindlimb injections into the rat motor cortex, whilst the studies fromLeergaard et al. showed topographical relations between somatosensory-pontine projections (see General Introduction for further details).More recently,[START_REF] Leergaard | Three-dimensional topography of corticopontine projections from rat sensorimotor cortex: comparisons with corticostriatal projections reveal diverse integrative organization[END_REF] showed a correspondence and topography from whisker representations in MI, SI and SII, to BPN. There are two competing theories fromBjaalie & Leergaard (2000) andSchwarz & Thier (2000) of how BPN serves to transform the continuous, orderly projection from the cerebral cortex into the discontinuous, fractured representation present in the cerebellar cortex as summarized on the next page.Leergaard et al. (2000b) hypothesise that the spatial relationships found in the cerebral cortex are largely preserved in the pontine nuclei

  figures together show the localisation in the motor cortex of the injections into foreand hindlimb areas and the typical core transverse section of the injectate.

  Figure 4.3 displays example cerebellar injection sites for an injection into PML and CP, showing examples of the two types of fluorescent retrograde tracer used.

  Figure 4.5 demonstrates examples of terminal labelling in BPN, in lower power and high power views. The higher power photomicrograph show the terminal labelling with pre-synaptic varicosities, presumably terminal boutons.

Figure

  forelimb-receiving climbing fibre zone in PML in the cerebellum yielded cell and terminal labelling in similar areas in BPN. This was located mainly in more caudal regions of BPN, bilaterally. As the cell labelling was quantified by counting individual cells, the terminal labelling required some level of quantification. The terminal labelling was therefore semi-quantified by rating the density of labelling in squares. A grid was placed over the rostrocaudal levels, covering the full extent of BPN. The total area of this cuboid shape extended over 9 squares rostrocaudally, 18 squares mediolaterally (from left BPN to right BPN), and 14 squares dorsoventrally (from the ventral surface of the brainstem) giving a total of 9x18x14 subdivisions (=2268 'boxes').This data was inputted into a spreadsheet per level then taken to the analysis program SigmaPlot (Systat Software, Hounslow, UK) where the equivalent ventral, rostral and medial views of BPN were calculated to enable semi-quantitative evaluation of the density of labelling in BPN, as comparable to the labelling in these views in Chapter 3. This can be seen in Figure4.7 where ventral views are shown for the labelling from the forelimb motor cortical injection (n = 5) for each case and the average of all the cases. From this, it can be see that the location of the terminal labelling from the forelimb motor cortical injection in located in similar spatial volumes as the labelling from forelimb cerebral and cerebellar injections as found in Chapter 3 (e.g. compare ventral BPN views in Figure4.7 with the labelling for SI and cerebellar forelimb injections seen in Figure3.5). The bilaterality of the labelling found in BPN was also similar for the motor cerebro-pontine and ponto-cerebellar injections. There was a predominance of the ipsilateral cerebro-pontine projection and conversely, contralateral predominance in the ponto-cerebellar projection, with the lesser side labelling mirroring the core location of labelling on the predominant side.

  Figure 4.8 documents a representative case of labelling found in BPN from an injection of anterograde tracer into the forelimb motor cortex and an injection of retrograde tracer into the C1 forelimbreceiving zone in PML in the cerebellum. The figure shows the physiological responses recorded in C1 PML cerebellum to forelimb motor cortical and peripheral forelimb stimulation and photomicrographs of the corresponding injections sites into the stimulation site in MI and the recording site in the cerebellum.The labelling in the inferior olive and BPN are shown in horizontal views. Cell labelling was found in the expected region of MAO and DAO in the olive on the contralateral side to the cerebellar injection; no terminal labelling was found in the inferior olive. Cell and terminal labelling were found on both sides of BPN, in caudal regions, as evidently seen in the horizontal views. Although the overlap of the cell and terminal labelling in BPN was patchy at times, the location of both types of labelling was in similar regions and is compared later in this chapter.

Figure 4 .

 4 Figure 4.9 and 4.10 show the extent of the terminal and cell labelling, respectively, in transverse levels through BPN for the pooled cases.Figure 4.9 shows the extent and density of terminal labelling in BPN from the forelimb motor cortical injection. The labelling is patchy and mostly on the ipsilateral side to the injection. The terminal labelling was heaviest at levels 4 and 5, just caudal to mid-BPN, although labelling was found throughout the rostrocaudal axis of BPN. The labelling on the contralateral side was far less than that ipsilaterally and occurred mainly in medial and ventral areas of BPN.

  Figure 4.11 demonstrates the individual views and average view for the hindlimb motor cortical injection (n = 7). The terminal labelling is located in far caudal regions of BPN, bilaterally. The hindlimb labelling was quantified in the same way as the forelimb labelling, and thus represented semi-quantitatively. These views again demonstrate the topographical location of the terminal labelling, to the slightly more rostral labelling from the forelimb MI injection, and the similarity of location to the hindlimb cell and terminal labelling shown in Chapter 3 (e.g. compare ventral BPN views in Figure 4.10 with the labelling for SI and cerebellar hindlimb injections seen in Figure 3.5).

Figure 4 .

 4 Figure 4.12 shows a representative case of the tracing of the whole cerebro-pontocerebellar pathway for the hindlimb injections. The figure demonstrates the physiological responses recorded in the C1 zone of CP to both motor cortical stimulation of the hindlimb area and of the peripheral hindlimb. The corresponding injection sites in the motor cortex and cerebellum are also shown. The olivaryhorizontal map shows that although in this case, there was little cell labelling, it was nevertheless in the expected region in the contralateral DAO. Again, no terminal labelling was found in the inferior olive. Cell and terminal labelling were found bilaterally in BPN to both injections. The terminal labelling was located primarily ipsilateral to the injection site and in far caudal BPN. The cell labelling was also located in caudal BPN; the retrograde tracer uptake was less in this case than in others and therefore only some cell labelling was found, and this was mainly in more lateral regions in caudal BPN, contralateral to the cerebellar injection site. Only a few cells were found ipsilaterally, and these were in caudal BPN.

Figures 4 .

 4 Figures 4.13 and 4.14 show the extent of the terminal and cell labelling, respectively, in transverse levels through BPN for the pooled hindlimb cases. Figure 4.13

Figure 5 .

 5 Figure 5.6 shows a camera lucida drawing of approximately the centre of the terminal labelling in the putative relay area and also a high power photomicrograph of the terminal labelling from an injection of tracer into the hindlimb motor cortex. The extent and location of the terminal labelling was mapped on to standard maps of the caudal brainstem. The maps were adapted from Paxinos & Watson's (2005) atlas to accurately record and chart the labelling. The latest version of the rat brain atlas extends further caudal than previous versions, and the transverse maps included the matrix region.

Figure 5 .

 5 Figure 5.7 shows a full example case of the distribution of axonal and terminal labelling in the caudal brainstem for a double tracer experiment, where both forelimb and hindlimb regions of motor cortex were injected. The labelling pattern for the Fluoro-Ruby 'forelimb' motor cortex injection and the Fluoro-Emerald 'hindlimb' motor cortex injection are plotted on standard transverse maps. The labelled fibres

  Figure 5.8 displays the individual cases plotted in graphical relation to the rostrocaudal axis. This shows the extent of the terminal labelling per case, and the similarity between all cases.

Figure 5 .

 5 Figure 5.11 shows physiologically recorded responses for a different case where GABA/KA was injected. Consecutive waveforms (3 overlaid for each) for cerebrally and peripherally evoked forelimb responses are shown pre-drug injection, 10 minutes after the drug was added and 35 minutes after drug application. The selective effects of the drug application can clearly be seen where there is a substantial reduction of cerebrally evoked responses at 10 minutes post-drug injection, as compared to predrug application and also compared to the peripherally evoked responses at 10 minutes post-drug. Forelimb cerebrally evoked responses reduced in size to below detection threshold (background noise was approximately 15% of control size) and then recovered after about 35 minutes.

  Figure 5.15 shows the potential pathways for both the contralateral and ipsilateral forelimb motor input to the C1 zones in LS and PML. The potential relay pathways are shown for each side of the brain and as deduced from the present results. In the figure, the information is shown as targeting the matrix and then projecting to the inferior olive, although there may be other synaptic interruptions before the termination of signals from the matrix to the olive, especially taking into consideration bilateral projections.

Figure 1 . 1 :

 11 Figure 1.1: Cerebellar circuitry. (A) A photomicrograph of a Purkinje cell, the output neurone of the cerebellum. (B) The above diagram represents the main components of the circuitry of the cerebellum. The layers of the cerebellum are shown (from inner white matter to outer molecular layer), the two main inputs of mossy fibres from the pontine nuclei and climbing fibres from the olive, and the major projection cell types included in this circuit (interneurones not included for clarity).

  Figure 1.4: Mossy fibre patchy representation in the cerebellar cortex.From the view of the posterior lobe of the rat cerebellum and the corresponding body representations, it can be seen that the mossy fibre representation in the cerebellar cortex is patchy where body parts are represented in a fractured manner. For example, the coloured forelimb in the rat is represented next to other non-contiguous body parts in the cerebellum (adapted from[START_REF] Bower | Is the cerebellum sensory for motor's sake, or motor for sensory's sake: the view from the whiskers of a rat?[END_REF], from original work by[START_REF] Bower | Principles of organization of a cerebro-cerebellar circuit. Micromapping the projections from cerebral (SI) to cerebellar (granule cell layer) tactile areas of rats[END_REF].

  Figure 1.5: Cerebral cortical somatotopical body representations in the rat. (A) The diagram shows a dorsal view of agranular (motor) and granular (sensory) regions in the cortex (diagram summated from various previous studies in Colemanet al., 1997). (B) A map is of the continuous representation of the rattunculus in SI (fromNeafsey et al., 1985). (C) The diagram details the motor (MI) and sensory (SI and SII) areas of the cerebral cortex, as represented (A) on the cerebral surface and (B) together in scale (from[START_REF] Leergaard | Three-dimensional topography of corticopontine projections from rat sensorimotor cortex: comparisons with corticostriatal projections reveal diverse integrative organization[END_REF]. Scale represented in millimetres in all diagrams.

Figure 3 . 1 :

 31 Figure 3.1: Physiological responses and anatomical labelling of two cases in the same animal. (BR14) The left column represents an injection of red beads into the C1 forelimb-receiving zone and right column represents an injection of green beads into the C1 hindlimbreceiving zone in the same animal. (i) Physiological recordings of climbing fibre field potentials (average of n=4 sweeps) in the approximate centre of each zone; arrow denotes time of peripheral stimulation. (ii) Outline view of the posterior right cerebellum displaying location of tracer injections. (iii) Photomicrographs of sagittal cerebellar section at the level of the injection site core. (iv) Camera lucida drawings of transverse sections of BPN (approximately -8 mm bregma) where the boxes indicate the location of the (v) cell labelling in the high power photomicrographs. Scale bar = (i) 5 ms; 0.1 mV and 0.05 mV for fore-and hindlimb responses respectively; (ii) 1 mm (iii) 500 µm (iv) 200 µm (v) 20 µm.

(

Figure 3 . 2 :

 32 Figure 3.2: Reconstruction of two cases in the same animal. (BR14) (A) Reconstructions of BPN projection to C1 forelimb injection (red dots) and C1 hindlimb injection (green dots). (i) Ventral view; (ii) rostral view; (iii) medial view; (B) 3D reconstruction with BPN shown as transparent grey surfaces and LFP shown as grey solids. Scale bar = 500 µm in A, 1 mm in B.

Figure 3 . 3 :

 33 Figure 3.3: Inferior olive and BPN cell labelling in three cases, representative of each injection site. Standard horizontal maps of the right inferior olive showing the areas occupied by retrogradely labelling (A = A2 face BR15; B = C1 forelimb BR06; C = C1 hindlimb BR14). Ventral, medial and rostral views of BPN constructed using the local coordinate system showing individually labelled cells. Outline of cerebellum indicates the approximate location of each injection, with the below figurine showing the physiologically defined mediolateral width of the zone and the coloured box, the anatomically measured extent of injection site. Scale bar = 0.5 mm BPN and inferior olive; 3 mm cerebellum; 0.2 mm injection site.

Figure 4 . 4 :

 44 Figure 4.4: Location of retrogradely labelled cells in the inferior olive. The figure shows the individual cases for retrogradely labelled cells for injections into forelimb-receiving C1 PML (red; n = 6) and for injections into hindlimbreceiving C1 CP (green; n = 6). Key: DL -dorsal lamella; PO -principal olive; VLventral lamella. Each case is denoted by its case number (DPxx) and the C1 injection site (forelimb [FL] or hindlimb [HL] receiving). Scale = 500 µm.

Figure 4 . 5 :

 45 Figure 4.5: Terminal labelling in BPN from a motor cortical injection of tracer. These photomicrographs show anterograde labelling from an injection of Fluoro-Ruby into the hindlimb motor cortex. (A) Low power view of BPN with heavy terminal labelling in the medial part of the right BPN, with some medially left. (B) Photomicrograph shows a high power view of the terminal labelling above. Scale bars, A = 500 µm; B = 50 µm (DP11). LFP -longitudinal fasciculus of the pons.

Figure 4 . 7 :

 47 Figure 4.7: Average and individual cases of forelimb terminal labelling in BPN. These figures show both sides of BPN from the ventral angle and the density of labelling for every 150 µm 2 , where blue shading indicates light terminal labelling through to red which indicates dense terminal labelling.

Figure 4 . 8 :

 48 Figure 4.8: Full documented case of example 'forelimb' BPN labelling. (DP27) The figure shows a full cerebro-ponto-cerebellar case for injections into the forelimb motor cortex and forelimb-receiving C1 zone in PML in the cerebellum. The cerebellum figurine demonstrates the recording site, with corresponding evoked physiological responses (3 overlaid) for both cerebral and forelimb stimulation (at 3 times threshold). Photomicrographs of the injection sites for both tracer injections are also shown (Fluoro-Ruby into the cerebral cortex; green beads in the cerebellum). The horizontal maps show labelling in the left inferior olive and bilaterally in BPN. The cell labelling in the olive was in the expected regions for the injection. The right and left BPN are shown for spread of terminal labelling and cell labelling. Scale: 500 µm for photomicrographs and horizontal maps; 2 mm cerebellar figurine.

Figure 4 . 9 :

 49 Figure 4.9: Total extent of 'forelimb' BPN terminal labelling. (n = 5) Terminal labelling found in BPN from injections of anterograde tracer into the forelimb projecting area in the motor cortex. The figure is coded to the density of the terminal labelling (none; very light; light; medium; dense). Scale bar = 500 µm.

Figure 4

 4 Figure 4.11: Average and individual cases of hindlimb terminal labelling in BPN. These figures show both sides of BPN from the ventral angle and the density of labelling for every 150 µm 2 , where blue shading indicates light terminal labelling through to red which indicates dense terminal labelling.

Figure 4 .

 4 Figure 4.12: Full documented case of example 'hindlimb' BPN labelling. (DP25) The figure shows a full cerebro-ponto-cerebellar case for injections into the hindlimb motor cortex and hindlimb-receiving C1 zone in CP in the cerebellum. The cerebellum figurine demonstrates the recording site, with corresponding evoked physiological responses (3 overlaid) for both cerebral and forelimb stimulation (at 3 times threshold). Photomicrographs of the injection sites for both tracer injections are also shown (Fluoro-Emerald into the cerebral cortex; green beads in the cerebellum). The horizontal maps show labelling in the left inferior olive and bilaterally in BPN. The cell labelling in the olive was in the expected regions for the injection. The right and left BPN are shown for spread of terminal labelling and cell labelling. Scale: 500 µm for photomicrographs and horizontal maps; 2 mm cerebellar figurine.

Figure 4 .

 4 Figure 4.13: Total extent of 'hindlimb' BPN terminal labelling. (n = 7) Terminal labelling found in BPN from injections of anterograde tracer into the hindlimb projecting area in the motor cortex. The figure is coded to the density of the terminal labelling (none; very light; light; medium; dense). Scale bar = 500 µm.

  Figure 4.15: Overlap of 'forelimb' terminal labelling and 'forelimb' cell labelling in BPN. The figure shows the overlap from labelling produced by injections of anterograde tracer into forelimb motor cortex area and retrograde tracer into the forelimbreceiving C1 PML zone in the cerebellum. Scale bar = 500 µm.

Figure 5 . 4 :

 54 Figure 5.4: Convergent responses from stimulation at threshold. Threshold stimulation recorded in PML, LS and CP, column (A) peripheral threshold, (B) cerebral threshold and (C) simultaneous peripheral and cerebral at threshold. (see stimulation artefact). Sets of three consecutive responses, scale bars = 0.025 s horizontal; 0.15 mV vertical (Case DPE8 PML and DPE29 for LS and CP recordings).

Figure 5 . 6 :

 56 Figure 5.6: Photomicrograph and camera lucida drawing of labelling in the matrix area. (DP11) The high power photomicrograph shows terminal labelling in the matrix labelled with Fluoro-Ruby (scale bar = 50 µm) from an injection into the hindlimb motor cortex. The camera lucida drawing displays the path of the labelled fibres along the pyramidal tract (small black lines) and the location of the terminal labelling (square blue box). This plane of section was approximately 2 mm caudal to the inferior olive and 16.5 mm caudal to bregma (scale bar = 1 mm).

Figure 5 . 7 :

 57 Figure 5.7: Double anterograde labelling in the caudal brainstem. (DP25) Anatomical and physiological documentation of a full double tracer experiment. (A) The dorsal view of the cerebral cortex shows the sites of stimulation (red -forelimb; green -hindlimb) where the corresponding climbing fibre responses were recorded in the cerebellum. Terminal labelling was found in the matrix from an injection of Fluoro-Ruby into the forelimb motor cortex (injection site in B and labelling in red in D) and an injection of Fluoro-Emerald into the hindlimb motor cortex (injection site in C and labelling in green in E). The axonal labelling is shown in C and D as coloured lines, where axons could be traced crossing the midline to the matrix. Calibration (A): 5 ms, 0.1 mV. Scale bars: A, 2 mm, B-F, 0.5 mm.

Figure 5 . 8 :

 58 Figure 5.8: Overview of pooled and individual cases giving terminal labelling in the matrix. (A) The extent of all the labelling in the matrix from injections into the motor cortex is shown, where blue indicates locations where terminal labelling from both fore-and hindlimb cortical injections was found and green indicates terminal labelling from hindlimb motor cortical injections only, which extended slightly further rostral and caudal than the forelimb labelling. Distance from Bregma is shown on the right of the diagram. Scale bar = 0.5 mm. (B) The extent of terminal labelling is shown diagrammatically for individual cases (n=10) for hindlimb (green) and forelimb (red) injections. The extent of labelling is shown to scale, in graphical format for its rostrocaudal location in the matrix. The cases are identified by their case number and injection site (see Table4.2 for motor cortical injection site details).

Figure 5 .

 5 Figure 5.10: Bar graphs of binned raw cerebellar responses to peripheral and cerebral stimulation from an injection of LA into the matrix. The graphs show the bins of 30 raw climbing fibre responses per stimulation/recording site in mV x ms, over time measured in minutes. The responses to cerebral stimulation decrease by 2.5 minutes post-injection of LA and recover after approximately 30 minutes, where the peripherally evoked responses remain essentially unchanged. The forelimb and hindlimb responses were recorded simultaneously (DPE26).

Figure 5 . 15 :

 515 Figure 5.15: Potential relay sites for bilateral forelimb motor cortical projection to cerebellar forelimb-receiving areas via inferior olive.From the results, the cortical forelimb pathway seemed to be conveyed along different paths, depending upon whether contralateral or ipsilateral to the C1 recording site. Taking into account the physiological and anatomical data gained, the above suggested pathway would be feasible to interpret the findings.

Table of Contents

 of Abstract ..................................................................................................................... ..ii Dedication ................................................................................................................. .iii

Table 2 .2: Key for Cerebro-olivo-cerebellar Physiological Experiments (Chapter 5) Physiological Stimulation/Recording Site Colour

 2 The basic coding is 'forelimb' data is red, 'hindlimb' green and 'face' blue, with overlap being purple in colour. There are further distinctions as in the physiological drug

	inactivation data, 'forelimb' PML responses are graphically represented in red,
	whereas 'forelimb' LS responses in blue. The below table gives an overview to the
	divisions:
	Table 2.1: Key for 3D Reconstruction Experiments (Chapter 3)
	Representation of BPN Labelling	Colour
	From 'forelimb' cerebellar injection	Red
	From 'hindlimb' cerebellar injection	Green
	From 'face' cerebellar injection	Blue
	Comparing SI injection 'forelimb'	Magenta
	Comparing SI injection 'hindlimb'	Dark green
	Comparing SI injection 'face'	Dark blue
	'Forelimb' C1 PML -Peripheral Stimulation	Red
	'Forelimb' C1 LS -Peripheral Stimulation	Blue
	'Hindlimb' C1 CP -Peripheral Stimulation	Green
	'Forelimb' C1 PML -Cerebral Stimulation	Magenta
	'Forelimb' C1 LS -Cerebral Stimulation	Dark Blue
	'Hindlimb' C1 CP -Cerebral Stimulation	Dark Green
	Pooled Cases -Peripheral Stimulation	Light purple
	Pooled Cases -Cerebral Stimulation	Dark purple
	Experimental codes were also used to identify different animal cases in the different
	series; 'BR' refers to the ponto-cerebellar 3D reconstruction experiments in Chapter

3, 'DP' to cerebro-cerebellar tract tracer experiments in Chapters 4 and 5, and 'DPE' to acute electrophysiological and pharmacological experiments in Chapter 5.

Table 4 .

 4 3 gives the injection site data for each case that gave terminal labelling in BPN and was histologically reconstructed. The cases in this table correspond to the case numbers in the schematic diagram in Figure 4.1 and the individual dimensions of each injection site are detailed. All injections into the forelimb motor cortex were

Table 3 .1: Numbers of labelled cells in the ponto-cerebellar tracer cases.

 3 The table shows the experiments where red and green beads were injected per case into the cerebellum. The cell counts are absolute numbers. The numbers of doubles are included in the actual cell counts. The graph shows the positive correlation of number of cells counted in BPN to number of cell in the inferior olive.

	Case	Zone Injected	Tracer Injected	IO Cells	Doubles IO	Cells Ipsil BPN	Cells Contra BPN	Doubles BPN
	BR03	A2 Face C1 FL	Red Green	794 76		38	1940 23	1778 394	57
	BR05	A2 Face C1 FL	Green Red	108 318		0		359 112	171 503	7
	BR06	A2 Face C1 FL	Green Red	246 140		1	1183 108	817 604	30
	BR12	A2 Face C1 FL	Green Red	63 1109	15		331 487	388 1593	96
	BR13	A2 Face C1 HL	Red Green	53 10		0		250 0	152 4	0
	BR14	C1 FL C1 HL	Red Green	100 392		0		58 109	421 1222	0
	BR15	A2 Face C1 FL	Green Red	38 427		12		220 799	292 1263	176
	BR16	A2 Face C1 HL	Red Green	275 51		0		611 9	1702 377	0
			Labelled Cell Count Correlation
			500
			0
			0	200	400	600	800	1000 1200
				Number

of Cells in Olive Number of Cells in BPN

Table 4 .2: Overview of anatomical tract tracer experiments.

 4

	The table shows the experimental series of the injection of tracer material (and
	volume injected) into the cerebral and cerebellar cortices. These experiments were
	histologically investigated and those highlighted in blue were deemed to have
	'successful' transport of tracer. This was determined for the anterograde cerebral
	injection by terminal labelling in BPN, and for the retrograde cerebellar injection, at
	least 80% of the cell labelling was in the expected area(s) in the inferior olive. Key:
	BDA -biotinylated dextran amine; CP -copula pyramidis; FE -Fluoro-Emerald;
	FL -forelimb; FR -Fluoro-Ruby; HL -hindlimb; LHS -left hand side (of the
	cerebellum); PML -paramedian lobule; RHS -right hand side (of the cerebellum); ?
	-volume of tracer injected not recorded.

Table 4 .4: Details of successful retrograde injection sites in the cerebellar cortex.

 4

Table 4 .5: Cell counts for the successful retrograde tracer cases.

 4

		(A) Inferior Olive Cell Counts
	Case	Olive (Contra)	MAO	DAO	% Correct in Olive
	DP2 FL	3	2	1	100
	DP2 FL	678	577	71	100
	DP10 FL	30	4	26	100
	DP11 FL	266	55	211	100
	DP13 FL	75	24	51	100
	DP27 FL	11	2	13	100
	DP1 HL	37	6	31	83.8
	DP11 HL	79	0	79	97.4
	DP12 HL	119	4	115	95.5
	DP14 HL	120	0	120	100
	DP25 HL	1	0	1	100
	DP26 HL	11	0	11	100
		(B) Pontine Cell Counts
	Case	BPN Ipsilateral	BPN Contra	NRTP Ipsilateral	NRTP Contra
	DP2 FL	4	103	3	3
	DP2 FL	63	946	15	8
	DP10 FL	137	230	13	34
	DP11 FL	137	400	33	32
	DP13 FL	47	286	21	22
	DP27 FL	8	185	0	2
	DP1 HL	0	171	0	0
	DP11 HL	13	272	0	0
	DP12 HL	23	823	0	0
	DP14 HL	32	547	0	0
	DP25 HL	2	17	0	0
	DP26 HL	0	87	0	0

Table 5 .2: Significant differences between onset latencies comparing stimulation and recording sites.

 5 The table shows comparisons between all combinations of stimulation and recording sites, showing the probability (p) value if there is a significant difference between onset latency, or 'ns' if not significant difference. See previous Table (5.1) for abbreviations.

		Ip PML	Co FL Ctx PML	Ip FL Ctx PML	Ip FL LS	Co FL Ctx LS	Ip FL Ctx LS	Ip HL CP	Co HL Ctx CP
	Ip FL PML		ns	p<0.001	ns	ns	ns	p<0.001 p<0.001
	Co FL Ctx PML	ns		p<0.001	ns	ns	ns	p<0.001 p<0.001
	Ip FL Ctx PML	p<0.001 p<0.001		p<0.001 p<0.001	ns	ns	p<0.001
	Ip FL LS	ns	ns	p<0.001		ns	ns	p<0.001 p<0.001
	Co FL Ctx LS	ns	ns	p<0.001	ns		ns	p<0.001 p<0.001
	Ip FL Ctx LS	ns	ns	ns	ns	ns		ns	p<0.001
	Ip HL CP	p<0.001 p<0.001	ns	p<0.001 p<0.001	ns		p<0.001
	Co HL Ctx CP	p<0.001 p<0.001 p<0.001 p<0.001 p<0.001 p<0.001 p<0.001

Table 5 .3: Significant differences and percentage changes in responses from injections into the matrix area.

 5 The table shows a summary of individual cases and injections into the putative relay, 2 mm caudal to obex. The responses are to peripheral and cerebral stimulation and values are taken at 5, 10, 15 and 20 minutes, compared to pre-drug baseline. Shaded boxes indicate significant differences from Dunnett's multiple comparison post test in conjunction with repeated measures ANOVA; yellow = decrease and green = increase, to control. C1 zone recording site is also indicated (CP -hindlimb receiving copula pyramidis; LS -forelimb receiving lobulus simplex; PML -forelimb receiving paramedian lobule). DPE22, 23, 24, 25 and 26 were injected with local anaesthetic, DPE28, 29 and 32 were injected with GABA/KA.

				Statistical Significant Difference to Control
	Case	Peripherally Evoked Responses	Cerebrally Evoked Responses
		5 mins	10 mins 15 mins 20 mins	5 mins 10 mins 15 mins 20 mins
	DPE22 PML	p<0.001 p<0.001	ns	p<0.001	ns	p<0.001 p<0.001	ns
	% Change	132.2	130.0	99.8	140.5	100.6	48.6	72.9	81.6
	DPE24 PML	ns	ns	p<0.05	-	p<0.001 p<0.001	ns	-
	% Change	83.6	91.0	120.4	-	25.2	75.2	104.3	-
	DPE26 PML	p<0.001	ns	p<0.05 p<0.001	p<0.001 p<0.001 p<0.001 p<0.001
	% Change	76.4	88.3	86.4	74.4	34.0	31.6	34.1	36.2
	DPE25 CP	ns	p<0.001 p<0.001 p<0.001	p<0.001 p<0.001 p<0.001 p<0.001
	% Change	108.4	120.3	123.7	129.7	22.6	25.5	32.4	69.5
	DPE26 CP	ns	ns	ns	p<0.001	p<0.001 p<0.001 p<0.001 p<0.001
	% Change	100.7	107.3	107.6	112.7	21.2	19.2	21.5	27.7
	DPE28 LS	p<0.001	ns	ns	ns	ns	p<0.05	ns	p<0.001
	% Change	128.7	111.6	102.6	114.0	128.8	63.5	102.3	57.1
	DPE28 PML	p<0.001 p<0.001 p<0.001 p<0.001	p<0.001	ns	p<0.001 p<0.001
	% Change	127.4	142.1	124.9	140.8	140.5	86.9	40.0	40.3
	DPE32 PML	p<0.05	ns	p<0.05	ns	p<0.001 p<0.001 p<0.001 p<0.001
	% Change	85.0	85.2	84.7	86.3	61.6	65.0	64.8	79.0
	DPE29 CP	ns	ns	p<0.001	-	ns	ns	ns	-
	% Change	92.1	94.3	88.2	-	108.6	110.3	94.7	-
	DPE32 CP	ns	ns	ns	p<0.05	ns	p<0.05 p<0.001	ns
	% Change	95.0	101.5	100.0	111.1	92.4	77.5	67.4	85.6

Cerebrally Evoked Responses Peripherally Evoked Responses Pre-drug 35 mins Post-drug 10 mins Post-drug Case Statistical Significant Difference to Control Peripherally Evoked Responses Cerebrally Evoked Responses 5 mins 10 mins 15 mins 20mins 5 mins 10 mins 15 mins 20 mins 0.5 mm Caudal to Obex DPE22 PML

		ns	ns	p<0.001 p<0.001	ns	ns	p<0.001 p<0.001
	% Change	99.8	104.3	80.5	66.0	100.0	111.2	72.9	73.8
	DPE23 PML p<0.001 p<0.001 p<0.001 p<0.001 p<0.001	ns	ns	ns
	% Change	84.0	68.5	54.3	29.7	135.0	107.8	88.0	85.8
	DPE24 PML	ns	ns	ns	p<0.001 p<0.05	ns	ns	p<0.05
	% Change	103.0	101.2	92.9	67.2	85.6	89.1	91.8	86.6
	DPE23 CP	ns	p<0.001 p<0.001 p<0.001 p<0.001 p<0.001 p<0.001 p<0.001
	% Change	101.1	70.0	12.9	13.6	68.7	37.7	27.2	26.6
	DPE28 LS	p<0.001 p<0.001 p<0.001 p<0.001	ns	ns	p<0.001 p<0.001
	% Change	115.7	132.2	137.2	154.0	96.0	99.6	125.7	142.7
	DPE28 PML p<0.05	p<0.05	ns	p<0.001	ns	ns	ns	ns
	% Change	85.1	81.9	91.1	81.5	96.5	88.3	105.4	103.8
	1 mm Caudal to Obex
	DPE23 PML	ns	ns	p<0.05	-	ns	ns	ns	-
	% Change	94.7	111.4	119.4	-	96.6	97.7	89.6	-
	DPE24 PML	ns	ns	ns	p<0.001 p<0.001 p<0.001 p<0.001 p<0.001
	% Change	85.6	103.8	95.4	76.4	59.8	58.3	49.6	57.9
	DPE25 PML p<0.001	ns	ns	p<0.001	ns	ns	p<0.001 p<0.001
	% Change	60.8	91.5	122.3	133.0	98.9	139.5	208.3	266.5
	DPE26 PML	ns	p<0.001 p<0.001 p<0.05 p<0.001 p<0.001	ns	ns
	% Change	116.7	140.3	137.5	123.3	130.4	149.1	116.9	86.1
	DPE26 CP	ns	p<0.001 p<0.001 p<0.001	ns	ns	ns	ns
	% Change	128.0	171.5	174.7	31.0	88.2	105.8	114.5	116.6
	1.5 mm Caudal to Obex
	DPE23 PML p<0.05 p<0.001 p<0.001	-	ns	p<0.05	ns	-
	% Change	86.3	74.1	75.6	-	95.0	126.2	113.2	-
	DPE23 CP	ns	ns	p<0.001	-	ns	ns	ns	-
	% Change	98.7	83.1	68.0	-	109.6	105.4	93.2	-
	DPE28 LS	ns	ns	ns	p<0.001	ns	ns	ns	ns
	% Change	95.2	92.3	96.3	60.3	89.7	94.3	93.6	92.3
	DPE28 PML	ns	ns	ns	p<0.001 p<0.001 p<0.001 p<0.001	ns
	% Change	100.9	101.3	101.6	62.6	125.7	124.2	140.3	97.1

LS LS Left Forelimb Motor Cortex Right Forelimb Motor Cortex Inferior Olive Matrix Midline Left Cerebellum Right Cerebellum

PML PML

Acknowledgments

Thank you to Dr. Richard Apps, my PhD supervisor, for guidance and insight, as well as his never ending enthusiasm over the cerebellum. Also, many thanks to Professor David Armstrong for his intellectual guidance during my PhD and comments on this thesis, and to Dr. Judy Harris for always being available for advice and for her enthusiasm in Physiology.

Many thanks to all past and present members of the Motor Control Group.

Particularly to Rachel and Clare for expert technical assistance and to Dr. Joanne

Abbreviations

AI

Anterior interpositus cerebellar nucleus a.s.g.

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Chapter 1: General Introduction

--------------------------------------------------------------------------------Chapter 3:

Ponto-cerebellar 3D Reconstruction

--------------------------------------------------------------------------------Chapter 4:

Cerebro-ponto-cerebellar Pathway

----------------------------------------each other. This overlap was detected from mapping BPN at a light microscopic level, thus actual boutons or synaptic connections could not be visualised; their presence would have to be confirmed through electron microscopy. The 'overlap' itself does not necessarily imply actual connectivity, but it is presumed that terminal endings and cells in the same general region will make some connections. Small beaded varicosities were seen in the terminal labelling plexuses and electron microscopy have shown that such structures correspond to synaptic contacts (e.g. [START_REF] Kincaid | Connectivity and convergence of single corticostriatal axons[END_REF]. As the dendritic field of a typical pontine cell is around 300 µm [START_REF] Mihailoff | The cytoarchitecture, cytology, and synaptic organization of the basilar pontine nuclei in the rat. I. Nissl and Golgi studies[END_REF], it seems reasonable to assume that a cell in close proximity to terminal labelling is likely to have some synaptic contact.

In the overlap analysis, labelling produced with corresponding limb injection sites i.e.

forelimb-forelimb and hindlimb-hindlimb, produced labelling in BPN where the core regions showed substantial overlap. Conversely, in the non-corresponding overlap analysis (all other combinations), the 'type' of overlap found was very different.

Rather than core regions overlapping, the highest areas of overlapping labelling occurred mostly at peripheral locations around the cores. For both motor cortical and cerebellar injection sites, subsequent hindlimb labelling was centred in far caudal BPN with forelimb labelling more rostrally; the overlap areas for all other combinations of unmatched-modality injections was therefore highest in the border region between these two core territories.

A clear advantage of tracing the whole cerebro-ponto-cerebellar pathway in the same animal meant that comparisons could be made for integration and segregation of the cerebro-pontine and ponto-cerebellar projections, directly in BPN. From the statistical analyses, the corresponding limb projections (e.g. forelimb motor cortical injections and forelimb cerebellar injections) showed a significantly greater overlap in BPN than the non-corresponding limb projections. This gives evidence towards the integration of body parts in BPN, with topographical representations of body parts for afferent and efferent information. Although there was a complex pattern of integration and segregation of labelling from the different injections, overall there was a topographical organisation and overlap of corresponding limb projections.

-

Chapter 5: The schematic cerebral cortex map is modified from [START_REF] Coleman | Topography of fibre organisation in the corticofugal pathways of rats[END_REF]. Key: AGC -agranular (motor) cortex; GC -granular (somatosensory) cortex. The table shows a summary of the injection details for all the cases where terminal labelling was found in BPN. From left to right, the columns show case number (HL and FL refer respectively to injections into hindlimb and forelimb parts of the motor cortex); type of tracer (FE, Fluoro-Emerald, FR, Fluoro-Ruby); volume of tracer injected (nl); then details of the dimensions of the injectate rostrocaudally (r-c), mediolaterally (m-l), and dorsoventral (d-v), all in µm; the depth of the centre of the injection site from the cerebro cortical surface (mm); the distance of the injection from Bregma (mm); and the total calculated volume of injectate (mm 3 ); -, histological data not available. The volume of the injectate was calculated by multiplying values together for volume of an ellipsoid. The graph shows the pooled cases (with standard error bars) for all the cases where either local anaesthetic or the GABA/KA mixture was injected into the matrix and responses were recorded from limb-receiving areas in the C1 zone in the cerebellar cortex. This graphs demonstrates the overall effect that within a few minutes after injection of the inactivating agent, there is a clear selective block of cerebrally evoked responses, with a full recovery of response by 40 minutes post-injection. Table 5.5: Significant differences and percentage changes in recordings at injection sites caudal to the labelling in the matrix relay area.

Cerebro-olivo-cerebellar Pathway

The table shows a summary of individual cases and injections at sites 2.5 mm, 3 mm, 3.5 mm and 4 mm caudal to obex (putative relay at 2 mm caudal to obex). The responses are to peripheral and cerebral stimulation and values are taken at 5, 10, 15 and 20 minutes, compared to pre-drug baseline. Shaded boxes indicate significant differences from Dunnett's multiple comparison post test in conjunction with repeated measures ANOVA; yellow = decrease and green = increase, to control. C1 zone recording site is also indicated (CP -hindlimb receiving copula pyramidis; LS -forelimb receiving lobulus simplex; PML -forelimb receiving paramedian lobule). DPE22, 23, 24, 25 and 26 were injected with local anaesthetic, DPE28, 29 and 32 were injected with GABA/KA.

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