# Anatomical and Physiological Studies of Cerebro-Cerebellar Pathways

Rochelle Ackerley

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Rochelle Ackerley. Anatomical and Physiological Studies of Cerebro-Cerebellar Pathways. Neuroscience. University of Bristol, 2006. English. NNT: tel-04218058

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Distributed under a Creative Commons Attribution 4.0 International License
Anatomical and Physiological Studies
of Cerebro-Cerebellar Pathways

Rochelle Ackerley

<figure><img src="image_2.png" /></figure>

A thesis submitted to the

**University of Bristol**

in accordance with the requirements of the degree of

**Doctor of Philosophy**

in the

**Faculty of Medical & Veterinary Sciences**

Department of Physiology

University of Bristol

June 2006

External examiner: Martin Garwicz, Lund University
Internal examiner: Peter Brennan, University of Bristol

Date of defense: 31st August 2006

Word count: 51,551
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
*To my Mum & Dad*
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 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.

This work was supported by the Physiology Department, the Medical Research Council and the Wellcome Trust.
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: ........................
**Bibliography**

**Publications**

Ackerley, R. & Apps, R. (2006). Topographical relations within the pons between motor cortical input & cerebellar output to the C1 climbing fibre zone. In preparation.

Ackerley, R., Pardoe, J. & Apps, R. (2006). A novel site of synaptic relay for climbing fiber pathways relaying signals from the motor cortex to the cerebellar cortical C1 zone. In press.

Odeh, F.*, Ackerley, R.*, Bjaalie, J.G. & Apps, R. (2005). Pontine maps linking somatosensory and cerebellar cortices are in register with climbing fiber somatotopy.
*J. Neurosci.* 25 (24): 5680-5690. *author's contributed equally to this work*

**Poster Presentations**

Ackerley, R. & Apps, R. (2006). Topographical relations within the pons between motor cortical inputs and cerebellar cortical outputs. *Society for Neurosci. Abstract*.

Ackerley, R., Odeh, F., Bjaalie, J.G. & Apps, R. (2004). Ponto-cerebellar projections and their congruence to climbing fibre zones in the rat. *FENS Abstracts Vol. 2*, Abstract number A129.1.

Ackerley, R., Pardoe, J. & Apps, R. (2004). Anatomical and physiological aspects of the cerebro-cerebellar system in the rat. *Society for Neuroscience Abstracts* 535.13.

**Oral Communications**

Ackerley, R. (2005). Brain-map transformation: ponto-cerebellar projections. Joint Postgraduate Symposium of the Medical & Veterinary and Medicine & Dentistry Faculties, University of Bristol.

Ackerley, R. (2005). 'Your Amazing Brain'. Brain Awareness Week, Bristol.

Ackerley, R. & Apps, R. (2005). A putative site of synaptic relay for the motor cortical projection to the inferior olive in rats. *J. Physiol.* 567P, Abstract C63.

**Awards & Prizes**

Prize for best oral communication at University of Bristol Joint Medical & Veterinary and Medicine & Dentistry Postgraduate Symposium (2005).

Travel research grant from The Physiological Society (2003).
Abbreviations

<table><tr><td>AI</td><td>Anterior interpositus cerebellar nucleus</td></tr><tr><td>a.s.g.</td><td>Anterior sigmoid gyrus</td></tr><tr><td>BDA</td><td>Biotinylated dextran amine</td></tr><tr><td>BPN</td><td>Basilar pontine nuclei</td></tr><tr><td>CNS</td><td>Central nervous system</td></tr><tr><td>Co/Contra</td><td>Contralateral</td></tr><tr><td>COCPs</td><td>Cerebro-olivo-cerebellar pathways</td></tr><tr><td>cp</td><td>Cerebral peduncle</td></tr><tr><td>CP</td><td>Copula pyramidis</td></tr><tr><td>Ctx</td><td>Cerebral (motor) cortex</td></tr><tr><td>Cu</td><td>Cuneate nucleus</td></tr><tr><td>DAO</td><td>Dorsal accessory olive</td></tr><tr><td>DCN</td><td>Dorsal column nuclei</td></tr><tr><td>DL</td><td>Dorsal lamella of principal olive</td></tr><tr><td>F</td><td>Fastigial cerebellar nucleus</td></tr><tr><td>FE</td><td>Fluoro-Emerald</td></tr><tr><td>FL</td><td>Forelimb</td></tr><tr><td>FR</td><td>Fluoro-Ruby</td></tr><tr><td>GABA</td><td>γ-aminobutyric acid</td></tr><tr><td>GB</td><td>Green beads</td></tr><tr><td>HL</td><td>Hindlimb</td></tr><tr><td>HRP</td><td>Horseradish peroxidase</td></tr><tr><td>Ip/Ipsil</td><td>Ipsilateral</td></tr><tr><td>KA</td><td>Kynurenic acid</td></tr><tr><td>LA</td><td>Local anaesthetic</td></tr><tr><td>LFP</td><td>Longitudinal fasciculus of the pons</td></tr><tr><td>LS</td><td>Lobulus simplex</td></tr><tr><td>LTD</td><td>Long-term depression</td></tr><tr><td>MI</td><td>Motor cortex</td></tr><tr><td>MAO</td><td>Medial accessory olive</td></tr><tr><td>MC</td><td>Motor cortical</td></tr><tr><td>ml</td><td>Medial lemniscus</td></tr></table>
<table><tr><td>MZMCs</td><td>Multizonal microcomplexes</td></tr><tr><td>n</td><td>Number (of cases)</td></tr><tr><td>NRTP</td><td>Nucleus reticularis tegmenti pontis</td></tr><tr><td>ns</td><td>Not significant</td></tr><tr><td>PB</td><td>Phosphate Buffer</td></tr><tr><td>PI</td><td>Posterior interpositus cerebellar nucleus</td></tr><tr><td>PML</td><td>Paramedian lobule</td></tr><tr><td>PO</td><td>Principal olive</td></tr><tr><td>p.s.g.</td><td>Posterior sigmoid gyrus</td></tr><tr><td>RB</td><td>Red beads</td></tr><tr><td>SI</td><td>(Primary) Somatosensory cortex</td></tr><tr><td>SII</td><td>Secondary somatosensory cortex</td></tr><tr><td>SE</td><td>Standard error</td></tr><tr><td>SOCPs</td><td>Spino-olivo-cerebellar pathways</td></tr><tr><td>T</td><td>Threshold</td></tr><tr><td>VL</td><td>Ventral lamella of principal olive</td></tr><tr><td>WGA</td><td>Wheatgerm-agglutinin</td></tr><tr><td>WM</td><td>White matter (of cerebellum)</td></tr></table>
Table of Contents

<table><tr><td>Abstract</td><td>ii</td></tr><tr><td>Dedication</td><td>iii</td></tr><tr><td>Acknowledgments</td><td>iv</td></tr><tr><td>Author's Declaration</td><td>v</td></tr><tr><td>Bibliography</td><td>vi</td></tr><tr><td>Abbreviations</td><td>vii</td></tr></table>

<table><tr><td>1. General Introduction</td><td>2</td></tr><tr><td>1.1 Overview</td><td>2</td></tr><tr><td>1.2 Historical Importance</td><td>3</td></tr><tr><td>1.3 The Cerebellum</td><td>4</td></tr><tr><td>1.3.1 Anatomy & Physiology</td><td>5</td></tr><tr><td>1.3.2 Inputs to the Cerebellum</td><td>8</td></tr><tr><td>1.3.2.1 Climbing Fibres</td><td>8</td></tr><tr><td>1.3.2.1.1 The Inferior Olive</td><td>9</td></tr><tr><td>1.3.2.1.2 Zonal Organisation</td><td>10</td></tr><tr><td>1.3.2.1.3 Theories of Climbing Fibre Function</td><td>13</td></tr><tr><td>1.3.2.2 Mossy Fibres</td><td>16</td></tr><tr><td>1.3.2.2.1 Basilar Pontine Nuclei</td><td>18</td></tr><tr><td>1.4 Cerebro-Cerebellar Pathways</td><td>20</td></tr><tr><td>1.4.1 General Structure of the Cerebral Cortex</td><td>20</td></tr><tr><td>1.4.2 Cytoarchitecture of Cerebral Cortex</td><td>21</td></tr><tr><td>1.4.3 Motor Cortical Projections</td><td>22</td></tr><tr><td>1.5 Aims</td><td>24</td></tr><tr><td>2. Methods</td><td>26</td></tr><tr><td>2.1 Surgical Procedures</td><td>26</td></tr><tr><td>2.2 Electrophysiology</td><td>28</td></tr><tr><td>2.2.1 Climbing Fibre Responses</td><td>28</td></tr><tr><td>2.2.2 Peripheral Stimulation</td><td>29</td></tr><tr><td>2.2.3 Motor Cortical Stimulation</td><td>29</td></tr><tr><td>2.2.4 Inactivation of Caudal Brainstem Sites</td><td>30</td></tr><tr><td>2.2.4.1 Analysis of Drug Injection Data</td><td>32</td></tr><tr><td>2.3 Injection & Processing of Material</td><td>33</td></tr></table>
<table>
  <tbody>
    <tr>
      <td>2.3.1 Anterograde Injection into the Motor Cortex</td>
      <td>33</td>
    </tr>
    <tr>
      <td>2.3.1.1 Considerations of the Anterograde Tracer</td>
      <td>33</td>
    </tr>
    <tr>
      <td>2.3.2 Retrograde Tracer Injection into the Cerebellum</td>
      <td>34</td>
    </tr>
    <tr>
      <td>2.3.3 Preparation of Material</td>
      <td>34</td>
    </tr>
    <tr>
      <td>2.4 Mapping of Tracer Material</td>
      <td>36</td>
    </tr>
    <tr>
      <td>2.4.1 Anterograde Terminal Labelling</td>
      <td>36</td>
    </tr>
    <tr>
      <td>2.4.2 Retrograde Cell Labelling</td>
      <td>36</td>
    </tr>
    <tr>
      <td>2.4.3 Injection Sites</td>
      <td>37</td>
    </tr>
    <tr>
      <td>2.4.4 Inferior Olive Mapping</td>
      <td>38</td>
    </tr>
    <tr>
      <td>2.4.5 Basilar Pontine Nuclei Mapping</td>
      <td>38</td>
    </tr>
    <tr>
      <td>2.4.6 Other Areas</td>
      <td>41</td>
    </tr>
    <tr>
      <td>2.5 Colour Format of Data and Figures</td>
      <td>42</td>
    </tr>
    <tr>
      <td>3. Ponto-cerebellar 3D Reconstruction</td>
      <td>44</td>
    </tr>
    <tr>
      <td>3.1 Abstract</td>
      <td>44</td>
    </tr>
    <tr>
      <td>3.2 Introduction</td>
      <td>45</td>
    </tr>
    <tr>
      <td>3.2.1 Aims of This Study</td>
      <td>46</td>
    </tr>
    <tr>
      <td>3.3 Results</td>
      <td>47</td>
    </tr>
    <tr>
      <td>3.3.1 BPN Cell Labelling</td>
      <td>47</td>
    </tr>
    <tr>
      <td>3.4 Discussion</td>
      <td>52</td>
    </tr>
    <tr>
      <td>3.4.1 Implications for Cerebellar Afferent Organisation</td>
      <td>52</td>
    </tr>
    <tr>
      <td>3.4.2 Direct Comparison with Somatosensory Cerebro-pontine Projection</td>
      <td>54</td>
    </tr>
    <tr>
      <td>3.4.3 Further Study</td>
      <td>57</td>
    </tr>
    <tr>
      <td>4. Cerebro-ponto-cerebellar Pathway</td>
      <td>59</td>
    </tr>
    <tr>
      <td>4.1 Abstract</td>
      <td>59</td>
    </tr>
    <tr>
      <td>4.2 Introduction</td>
      <td>60</td>
    </tr>
    <tr>
      <td>4.2.1 Aims of this Study</td>
      <td>62</td>
    </tr>
    <tr>
      <td>4.3 Results</td>
      <td>63</td>
    </tr>
    <tr>
      <td>4.3.1 Injection Sites</td>
      <td>63</td>
    </tr>
    <tr>
      <td>4.3.1.1 Anterograde Tracer</td>
      <td>63</td>
    </tr>
    <tr>
      <td>4.3.1.2 Retrograde Tracer</td>
      <td>64</td>
    </tr>
    <tr>
      <td>4.3.2 Pontine Nuclei Labelling</td>
      <td>65</td>
    </tr>
    <tr>
      <td>4.3.2.1 Labelling from 'Forelimb' Injections</td>
      <td>66</td>
    </tr>
    <tr>
      <td>4.3.2.2 Labelling from 'Hindlimb' Injections</td>
      <td>68</td>
    </tr>
    <tr>
      <td>4.3.2.3 Overlap of Labelling</td>
      <td>69</td>
    </tr>
  </tbody>
</table>
<table>
  <tbody>
    <tr>
      <td>4.3.2.4 NRTP Cell Labelling</td>
      <td>72</td>
    </tr>
    <tr>
      <td>4.4 Discussion</td>
      <td>73</td>
    </tr>
    <tr>
      <td>4.4.1 Methodological Considerations</td>
      <td>73</td>
    </tr>
    <tr>
      <td>4.4.2 Basilar Pontine Nuclei Labelling</td>
      <td>74</td>
    </tr>
    <tr>
      <td>4.4.3 Implications of Labelling in Other Areas</td>
      <td>77</td>
    </tr>
    <tr>
      <td>4.4.4 Further Studies</td>
      <td>78</td>
    </tr>
    <tr>
      <td>5. Cerebro-olivo-cerebellar Pathway</td>
      <td>80</td>
    </tr>
    <tr>
      <td>5.1 Abstract</td>
      <td>80</td>
    </tr>
    <tr>
      <td>5.2 Introduction</td>
      <td>81</td>
    </tr>
    <tr>
      <td>5.2.1 Aims of This Study</td>
      <td>83</td>
    </tr>
    <tr>
      <td>5.3 Results</td>
      <td>84</td>
    </tr>
    <tr>
      <td>5.3.1 Physiology</td>
      <td>84</td>
    </tr>
    <tr>
      <td>5.3.1.1 Climbing Fibre Responses</td>
      <td>84</td>
    </tr>
    <tr>
      <td>5.3.1.2 Motor Cortex Map</td>
      <td>88</td>
    </tr>
    <tr>
      <td>5.3.2 Anatomical Labelling</td>
      <td>89</td>
    </tr>
    <tr>
      <td>5.3.2.1 Anterograde Labelling in the Caudal Brainstem</td>
      <td>90</td>
    </tr>
    <tr>
      <td>5.3.3 Inactivation of Putative Pre-Olivary Relay</td>
      <td>93</td>
    </tr>
    <tr>
      <td>5.3.3.1 Injection of Drug into Putative Relay Site</td>
      <td>94</td>
    </tr>
    <tr>
      <td>5.3.4 Responses to Rostrocaudal Injection</td>
      <td>97</td>
    </tr>
    <tr>
      <td>5.3.4.1 Rostral Injection Sites to Putative Relay</td>
      <td>97</td>
    </tr>
    <tr>
      <td>5.3.4.2 Caudal Injection Sites to Putative Relay</td>
      <td>98</td>
    </tr>
    <tr>
      <td>5.4 Discussion</td>
      <td>99</td>
    </tr>
    <tr>
      <td>5.4.1 Physiology</td>
      <td>99</td>
    </tr>
    <tr>
      <td>5.4.2 Anatomy</td>
      <td>102</td>
    </tr>
    <tr>
      <td>5.4.3 Reversible Inactivation of Putative Relay</td>
      <td>106</td>
    </tr>
    <tr>
      <td>5.4.4 Further Study</td>
      <td>107</td>
    </tr>
    <tr>
      <td>6. References</td>
      <td>181</td>
    </tr>
  </tbody>
</table>
List of Illustrative Material

<table>
  <tbody>
    <tr>
      <td>1.1 Cerebellar circuitry</td>
      <td>110</td>
    </tr>
    <tr>
      <td>1.2 Responses of the Purkinje cell to mossy fibre and climbing fibre activation</td>
      <td>111</td>
    </tr>
    <tr>
      <td>1.3 Climbing fibre zonal organisation in the cat and rat</td>
      <td>112</td>
    </tr>
    <tr>
      <td>1.4 Mossy fibre patchy representation in the cerebellar cortex</td>
      <td>113</td>
    </tr>
    <tr>
      <td>1.5 Cerebral cortical somatotopical body representations in the rat</td>
      <td>114</td>
    </tr>
    <tr>
      <td>2.1 Diagram of the experimental stimulation and recording areas in the rat brain</td>
      <td>115</td>
    </tr>
    <tr>
      <td>2.2 Pontine orientation maps</td>
      <td>116</td>
    </tr>
    <tr>
      <td>3.1 Physiological responses and anatomical labelling of two cases in the same animal</td>
      <td>119</td>
    </tr>
    <tr>
      <td>3.2 Reconstruction of two cases in the same animal</td>
      <td>120</td>
    </tr>
    <tr>
      <td>3.3 Inferior olive and BPN cell labelling in three cases, representative of each injection site</td>
      <td>121</td>
    </tr>
    <tr>
      <td>3.4 Surface representation reconstructions of accumulated cases of BPN labelling from cerebellar and SI injections</td>
      <td>124</td>
    </tr>
    <tr>
      <td>3.5 3D dot map reconstructions of accumulated cases</td>
      <td>125</td>
    </tr>
    <tr>
      <td>4.1 Schematic map of reconstruction of injection sites in cerebral cortex</td>
      <td>126</td>
    </tr>
    <tr>
      <td>4.2 Examples of photomicrographs of motor cortex injection sites</td>
      <td>127</td>
    </tr>
    <tr>
      <td>4.3 Examples of photomicrographs of cerebellar cortex injection sites</td>
      <td>128</td>
    </tr>
    <tr>
      <td>4.4 Location of retrogradely labelled cells in the inferior olive</td>
      <td>131</td>
    </tr>
    <tr>
      <td>4.5 Terminal labelling in BPN from a motor cortical injection of tracer</td>
      <td>133</td>
    </tr>
    <tr>
      <td>4.6 Cell labelling in BPN from an injection into the cerebellum</td>
      <td>134</td>
    </tr>
    <tr>
      <td>4.7 Average and individual forelimb cases showing terminal labelling in BPN</td>
      <td>135</td>
    </tr>
    <tr>
      <td>4.8 Full documented case of example 'forelimb' BPN labelling</td>
      <td>136</td>
    </tr>
    <tr>
      <td>4.9 Total extent of 'forelimb' BPN terminal labelling</td>
      <td>137</td>
    </tr>
    <tr>
      <td>4.10 Total extent of 'forelimb' BPN cell labelling</td>
      <td>138</td>
    </tr>
    <tr>
      <td>4.11 Average and individual hindlimb cases showing terminal labelling in BPN</td>
      <td>139</td>
    </tr>
    <tr>
      <td>4.12 Full documented case of example 'hindlimb' BPN labelling</td>
      <td>140</td>
    </tr>
    <tr>
      <td>4.13 Total extent of 'hindlimb' BPN terminal labelling</td>
      <td>141</td>
    </tr>
    <tr>
      <td>4.14 Total extent of 'hindlimb' BPN cell labelling</td>
      <td>142</td>
    </tr>
    <tr>
      <td>4.15 Overlap of 'forelimb' terminal and 'forelimb' cell labelling in BPN</td>
      <td>145</td>
    </tr>
    <tr>
      <td>4.16 Overlap of 'hindlimb' terminal and 'hindlimb' cell labelling in BPN</td>
      <td>146</td>
    </tr>
  </tbody>
</table>
<table>
  <tbody>
    <tr>
      <td>4.17</td>
      <td>Overlap of 'forelimb' terminal and 'hindlimb' cell labelling in BPN</td>
      <td>147</td>
    </tr>
    <tr>
      <td>4.18</td>
      <td>Overlap of 'hindlimb' terminal and 'forelimb' cell labelling in BPN</td>
      <td>148</td>
    </tr>
    <tr>
      <td>4.19</td>
      <td>Areas of total overlap in BPN</td>
      <td>149</td>
    </tr>
    <tr>
      <td>4.20</td>
      <td>Photomicrographs and ventral map of NRTP cell labelling</td>
      <td>150</td>
    </tr>
    <tr>
      <td>5.1</td>
      <td>Evoked climbing fibre field potentials to peripheral and central limb stimulation</td>
      <td>151</td>
    </tr>
    <tr>
      <td>5.2</td>
      <td>Paired pulse tests for climbing fibre responses to repeated stimulation sites</td>
      <td>154</td>
    </tr>
    <tr>
      <td>5.3</td>
      <td>Conditioning tests for climbing fibre responses to different stimulation</td>
      <td>155</td>
    </tr>
    <tr>
      <td>5.4</td>
      <td>Convergent responses from stimulation at threshold</td>
      <td>156</td>
    </tr>
    <tr>
      <td>5.5</td>
      <td>Labelled fibres in pyramidal tract under inferior olive</td>
      <td>157</td>
    </tr>
    <tr>
      <td>5.6</td>
      <td>Photomicrograph and camera lucida drawing of labelling in the matrix area</td>
      <td>158</td>
    </tr>
    <tr>
      <td>5.7</td>
      <td>Double anterograde labelling in the caudal brainstem</td>
      <td>159</td>
    </tr>
    <tr>
      <td>5.8</td>
      <td>Overview of pooled and individual cases giving terminal labelling in the matrix</td>
      <td>160</td>
    </tr>
    <tr>
      <td>5.9</td>
      <td>Photomicrograph of an example injection site made to inactivate the matrix area</td>
      <td>161</td>
    </tr>
    <tr>
      <td>5.10</td>
      <td>Bar graphs of binned raw cerebellar responses to peripheral and cerebral stimulation from an injection of LA into the matrix</td>
      <td>163</td>
    </tr>
    <tr>
      <td>5.11</td>
      <td>Selective decrease in climbing fibre responses to cerebral stimulation from an injection of GABA/KA into the matrix</td>
      <td>164</td>
    </tr>
    <tr>
      <td>5.12</td>
      <td>Effects of injecting local anaesthetic into matrix on climbing fibre field responses</td>
      <td>165</td>
    </tr>
    <tr>
      <td>5.13</td>
      <td>Effects of injecting GABA/KA into matrix on climbing fibre field responses</td>
      <td>166</td>
    </tr>
    <tr>
      <td>5.14</td>
      <td>Pooled cases to demonstrate the overall effect of inactivating the matrix on climbing fibre field responses</td>
      <td>167</td>
    </tr>
    <tr>
      <td>5.15</td>
      <td>Potential relay sites for bilateral forelimb motor cortical projection to cerebellar forelimb-receiving areas via inferior olive</td>
      <td>170</td>
    </tr>
  </tbody>
</table>
List of Tables

<table><tr><td>2.1 Key for 3D Reconstruction Experiments (Chapter 3)</td><td>42</td></tr><tr><td>2.2 Key for Cerebro-olivo-cerebellar Physiological Experiments (Chapter 5)</td><td>42</td></tr><tr><td>3.1 Numbers of labelled cells in the ponto-cerebellar tracer cases</td><td>117</td></tr><tr><td>3.2 Details of cases used in 3D reconstruction and analysis</td><td>118</td></tr><tr><td>3.3 Cell labelling and terminal overlap analysis, as determined using the local pontine co-ordinate system</td><td>125</td></tr><tr><td>4.1 Hypothesised organisation in the ponto-cerebellar projection</td><td>61</td></tr><tr><td>4.2 Overview of anatomical tract tracer experiments</td><td>125</td></tr><tr><td>4.3 Details of successful anterograde injection sites in the motor cortex that gave terminal labelling in BPN</td><td>130</td></tr><tr><td>4.4 Details of successful retrograde injection sites in the cerebellar cortex</td><td>132</td></tr><tr><td>4.5 Cell counts for the successful retrograde tracer cases</td><td>133</td></tr><tr><td>4.6 Individual overlaps in BPN in cases with both successful cerebral and cerebellar injections</td><td>143</td></tr><tr><td>4.7 Pooled overlaps in BPN in cases with successful cerebral and cerebellar injections</td><td>144</td></tr><tr><td>5.1 Evoked latencies per case for climbing fibre field recordings</td><td>152</td></tr><tr><td>5.2 Significant differences between onset latencies comparing stimulation and recording sites</td><td>153</td></tr><tr><td>5.3 Significant differences and percentage changes in responses from injections into the matrix area</td><td>162</td></tr><tr><td>5.4 Significant differences and percentage changes in recordings at injection sites rostral to the labelling in the matrix relay area</td><td>168</td></tr><tr><td>5.5 Significant differences and percentage changes in recordings at injection sites caudal to the labelling in the matrix relay area</td><td>169</td></tr></table>
Chapter 1:
General Introduction
# 1. 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 (Clark *et al.*, 2001), 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 (Kuypers, 1981). 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; Bower, 1997). As the climbing fibres impose a somatotopical order onto the cerebellum, the less-ordered mossy fibre pontine projection was investigated in this framework.
## 1.2 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 Houk *et al.* [1997]). The importance of the cerebellum in humans was highlighted by Holmes (1922), 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 (Penfield & Rasmussen, 1950).
1.3 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 (Sultan, 2002). 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 (Eccles *et al.*, 1967; Eccles *et al.*, 1972),
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 (Holmes, 1922). 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 (Ghez, 1991). 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; Brodal & Steen, 1983; Bower, 1997; Leergaard *et al.*, 2000a, b; Leergaard, 2003). 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 (Nicolson *et al.*, 2001) 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.

### 1.3.1 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 Larsell (1952) 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 (Oscarsson, 1979).

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 Eccles *et al.*, 1967). 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 Palay & Chan-Palay, 1974). 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; Bower, 1997). 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, Jansen & Brodal (1940) proposed that the division was based on the cortico-nuclear organisation. Further work from Voogd (1964) 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. Groenewegen & Voogd, 1977; Groenewegen *et al.*, 1979; Voogd & Bigare, 1980; Brodal & Brodal, 1981; Apps, 1990). This somatotopy is preserved in the olivo-cortico-nuclear projection for various species (Brodal & Kawamura, 1980; Garwicz *et al.*, 1998; Voogd & Glickstein, 1998; Apps & Garwicz, 2000; Pardoe & Apps, 2002; Edge *et al.*, 2003; Pijpers *et al.*, 2005).

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; Allen & Tsukahara, 1974). 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 (Eccles *et al.*, 1967).

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
(Eccles *et al.*, 1967). 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.

### 1.3.2 Inputs to the Cerebellum

Climbing fibres have been found to originate solely from the inferior olive (Armstrong, 1974; Desclin, 1974), 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 (Schwarz & Thier, 1999). 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; Tomasch, 1969). 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 (Adrian, 1943; 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. Furber & Watson, 1983).

#### 1.3.2.1 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 Eccles *et al.*, 1965; Eccles *et al.*, 1964). 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 Eccles *et al.* [1967] 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, Atkins & Apps, 1997; 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 (Armstrong & Schild, 1970; Schild, 1970). Therefore, branching of the climbing fibre occurs where one axon branches on average to innervate seven Purkinje cells (Sugihara et al., 1999, 2001). 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.

### 1.3.2.1.1 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. Groenewegen & Voogd, 1977; 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; Kooy, 1917). To understand the function of the inferior olive, the normal structure and morphology must be determined. More in-depth studies such as Gwyn *et al.* (1977), Azizi & Woodward (1987) and Apps (1990) 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 Brodal & Kawamura, 1980). Topographical principles of organisation have been found in both the inferior olive and olivo-cerebellar system in the rat, between the nuclear arrangement and within individual nuclei (Azizi & Woodward, 1987). 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 (Gwyn *et al.*, 1977; Llinás *et al.*, 1974; Sotelo *et al.*, 1974).

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 (Andersson & Nyquist, 1983) to project this information in a zonal organisation in the cerebellar cortex (Andersson & Nyquist, 1983). Although there is much physiological evidence that the inferior olive transmits cerebral information to the cerebellum (Armstrong, 1974; Armstrong *et al.*, 1973b; Andersson & Nyquist, 1983), the past literature generally does not necessarily ascribe a direct cerebral projection to the olive (Kuypers, 1958; Saint-Cyr, 1987), and where it does, the evidence is limited (Sousa-Pinto & Brodal, 1969; Saint-Cyr, 1983). 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.

### 1.3.2.1.2 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 (Andersson & Oscarsson, 1978). 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 Voogd (1964) 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 Voogd & Bigare
[1980] 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 (Trott
& Apps, 1991, 1993).

Additional work has identified further differentiation in zonal organisation, such as
the X zone and the CX in the anterior lobe of the cat (Ekerot & Larson, 1982;
Campbell & Armstrong, 1985) and division within the D zone into D1 and D2 (for
an overview, see Ito, 1984). An additional division was also found in work by
Buisseret-Delmas & Angaut (1989) 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 Andersson & Oscarsson (1978),
who defined a microzone and its subcortical neuronal network as representing the
‘basic computational unit of the cerebellum’. Ito (1984, 1987) 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 (Andersson & Oscarsson [1978] in the B zone; Ekerot et al. [1991] and Garwicz et al. [1998] in the C3 zone) and in the rat (Jörntell et al., 2000), 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 olivo-cortico-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 (Jörntell et al., 1996).

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 Apps & Garwicz, 2005). 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 (Ekerot et al., 1997). Anatomical studies have given evidence towards the existence of microzones in the C3 zone (Garwicz et al., 1996) and that MZMCs exist between the C1 and C3 zones (Apps & Garwicz, 2000). 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 Hawkes & Leclerc (1989) and was directly linked to the parasagittal organisation of the cerebellum and climbing fibre somatotopy (Gravel & Hawkes, 1990), and across species (Leclerc et al., 1990). 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, Hallem et al., 1999; the climbing fibre zonal organisation, Voogd et al., 2003; Voogd & Ruigrok, 2004) 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 Sugihara & Shinoda (2004), 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.

### 1.3.2.1.3 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: Simpson et al. (1996), Houk et al., (1997), Llinás et al. (1997), Bloedel & Bracha (1998) and Apps & Garwicz (2005).

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 (Llinás et al., 1974; Sotelo et al., 1974; De Zeeuw et al., 1998). 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 (Welsh et al., 1995).

The comparator hypothesis was based upon the inferior olive acting as a comparator of ascending and descending afferent input (Oscarsson, 1969; Miller et al., 1969a, b; Oscarsson, 1973). 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 (Andersson & Nyquist, 1983). 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 (Andersson & Armstrong, 1987; see also similar findings from an unexpected action from Gellman et al., 1985).

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 Apps, 1999, 2000). 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, Apps & Lee, 2002; 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 (Apps, 2000).

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 Bloedel & Bracha, 1998). 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 Ebner & Bloedel (1981) 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 (Lou & Bloedel, 1992) 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 Marr, 1969; Albus, 1971; Ito, 1984). 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 long-term 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 Albus (1971) 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.

### 1.3.2.2 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 Eccles et al. [1967] 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 olivo-cerebellar projection. The pattern of termination of the mossy fibre projection forms a ‘fractured’, discontinuous tactile body map (Shambes et al., 1978a, b; Bower et al., 1981; Welker, 1987; Bower & Kassel, 1990; Bower, 1997).

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. Serapide et al. (2002) 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 Brodal & Steen (1983) 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, Herrero *et al.* (2002) found BPN to project on average 83% contralaterally and NRTP 55%. Similarly in rabbit, Bukowska *et al.* (2005) 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 (Mihailoff *et al.*, 1981). 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 (Torigoe *et al.*, 1986).
1.3.2.2.1 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 cerebro-
fugal 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 (Mihailoff &
King, 1975). 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 (Mihailoff et al.,
1981). The latter make up at least 90% of the total cells in BPN.

Brodal & Jansen (1946) and Brodal & Steen (1983) attempted to quantify the ponto-cerebellar 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., 1995, 2000a, b, 2004).

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 (Schwarz & Thier, 1999), 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
(Möck et al., 1997). Schwarz & Thier (1999) 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., 1995,
2000a, b, 2004) and cerebellar output (e.g. Bjaalie et al., 1991; Nikundiwe et al.,
1994). This will be covered more in the Results chapters.
## 1.4 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 Allen & Tsukahara, 1974). A direct pathway from the cerebral cortex to the cerebellum has been suggested in very young animals (e.g. Tolbert & Panneton, 1983; Tolbert, 1989), although this has been shown to be transient and therefore eliminated well before maturation. Wild & Williams (2000) 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.

### 1.4.1 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 structure-function 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.

### 1.4.2 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 (Donoghue & Wise, 1982). Whereas, in somatosensory (granular) areas, the information-receiving cells in layers II and IV are more abundant than the pyramidal projection cells of layers III and V (Donoghue & Wise, 1982; Moore *et al.*, 1999). 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 (Donoghue & Wise, 1982; Sanderson *et al.*, 1984). 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 [Welker, 1976]). Figure 1.5 also shows different rattunculi, for MI, SI and SII, and the continuous, topographical representation of all the maps can be seen.
### 1.4.3 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 Brown [1971] and Biedenbach & Devito [1980] 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 (Landry et al., 1984). There is also a substantial input from the cerebral cortex to the cerebellum, relayed as climbing fibres and mossy fibres (e.g. Curtis, 1940; Jansen, 1957; Jansen & Fangel, 1961; Provini et al., 1968; Brodal & Steen, 1983).

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. Armstrong & Harvey, 1966). 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; Brodal & Steen, 1983). 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. Adrian, 1943; Andersson & Nyquist, 1983; Armstrong & Harvey, 1966) but there has been dispute as to the route and extent to which the inferior olive receives direct cerebral input (cf. Swenson & Castro [1983] who found a direct cerebro-olivary projection and Saint-Cyr [1987] who found it to be indirect). The specific cerebro-olivo-cerebellar and cerebro-ponto-cerebellar pathways under investigation are introduced in more detail in the individual Results chapters.
## 1.5 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
## 2. Methods

### 2.1 Surgical Procedures

All experimental procedures were carried out in accordance with the UK Animals Scientific Procedures Act (1986) and were approved by the University of Bristol institutional animal licence advisory group. There were three series of experiments (i) injections of retrograde tracer into the cerebellar cortex, where the subsequent pontine labelling was reconstructed in 3D (ii) anatomical tract tracer experiments where injections of anterograde tracer were made into the motor cortex and retrograde tracer into the cerebellar cortex in the same animal, and (iii) detailed electrophysiological investigations of the cerebro-olivo-cerebellar pathway. Wistar rats were routinely used for experiments, although Sprague-Dawley rats were also used for three out of eight of the 3D pontine reconstruction, which were carried out in collaboration with Professor Jan Bjaalie and Dr Francis Odeh from Oslo University.

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.

In the electrophysiological components of the investigations, barbiturate anaesthesia was used because climbing fibre responses were required to define the olivo-cerebellar zones. This type of anaesthesia has been shown previously to reduce mossy fibre activity (Andersson & Nyquist, 1983) and increase the amplitude of the climbing fibre response (Gordon et al., 1973) 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 (Glen, 1980; Pardoe *et al.*, 2004). In Pardoe *et al*. (2004), 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.
## 2.2 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.

### 2.2.1 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 Atkins & Apps, 1997; Pardoe & Apps, 2002).

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

2.2.2 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.

**2.2.3 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 Donoghue & Wise (1982) 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.

### 2.2.4 Inactivation of Caudal Brainstem Sites

Where anterograde tracer was injected into the motor cortex to trace the cerebro-olivary 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 (Hupé *et al.*, 1999). 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 (Malpeli *et al.* 1981) using 100 nl 2% lidocaine. Boehnke & Rasmusson (2001) 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 Martin & Ghez, 1999 where lidocaine and GABA were compared). The effective spread of GABA has been investigated in previous studies (e.g. Hupé *et al.*, 1999; 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 mm² 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.

### 2.2.4.1 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).
## 2.3 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, cerebro-olivary 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.

### 2.3.1 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 [Köbbert et al., 2000]). The tracer was injected into the most effective stimulation site, typically where the threshold for evoking cerebellar responses was lowest.

#### 2.3.1.1 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 Mihailoff *et al*. (1978), 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.

2.3.2 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).

**2.3.3 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.
## 2.4 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, BP 450-490 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).

### 2.4.1 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 µm² 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.

### 2.4.2 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 Köbbert *et al.*, 2000).

**2.4.3 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 µm².

Caudal Brainstem Injection Site

The sections were mapped onto standard caudal brainstem maps (based on Paxinos
& Watson, 2005) 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.

**2.4.4 Inferior Olive Mapping**

Cell labelling was mapped onto standard olivary transverse maps (based on Apps,
1990) 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.

**2.4.5 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
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
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 Brevik *et al.* (2001) (see Chapter 3 for more information on the local pontine co-ordinate system). A cuboid ‘bounding box’ was applied to each side of BPN allowing each single case to be normalised to a template (the bounding box) and thus aid comparisons. This bounding box encompasses the rostrocaudal, dorsoventral and mediolateral boundaries, with the ventral surface landmark serving as an internal boundary.

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; Brevik et al., 2001) and affine transformations (Ashburner et al.,
1997; Ashburner & Friston, 1999) 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.

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]. 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.

2.4.6 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 Paxinos & Watson (1998) 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 Brichta & Grant (1985) 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.
## 2.5 Colour Format of Data and Figures

There is a theme to the colour coding of all the figures and data in this thesis. 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><caption>Table 2.1: Key for 3D Reconstruction Experiments (Chapter 3)</caption><thead><tr><th>Representation of BPN Labelling</th><th>Colour</th></tr></thead><tbody><tr><td>From 'forelimb' cerebellar injection</td><td>Red</td></tr><tr><td>From 'hindlimb' cerebellar injection</td><td>Green</td></tr><tr><td>From 'face' cerebellar injection</td><td>Blue</td></tr><tr><td>Comparing SI injection 'forelimb'</td><td>Magenta</td></tr><tr><td>Comparing SI injection 'hindlimb'</td><td>Dark green</td></tr><tr><td>Comparing SI injection 'face'</td><td>Dark blue</td></tr></tbody></table>

<table><caption>Table 2.2: Key for Cerebro-olivo-cerebellar Physiological Experiments (Chapter 5)</caption><thead><tr><th>Physiological Stimulation/Recording Site</th><th>Colour</th></tr></thead><tbody><tr><td>'Forelimb' C1 PML – Peripheral Stimulation</td><td>Red</td></tr><tr><td>'Forelimb' C1 LS – Peripheral Stimulation</td><td>Blue</td></tr><tr><td>'Hindlimb' C1 CP – Peripheral Stimulation</td><td>Green</td></tr><tr><td>'Forelimb' C1 PML – Cerebral Stimulation</td><td>Magenta</td></tr><tr><td>'Forelimb' C1 LS – Cerebral Stimulation</td><td>Dark Blue</td></tr><tr><td>'Hindlimb' C1 CP – Cerebral Stimulation</td><td>Dark Green</td></tr><tr><td>Pooled Cases – Peripheral Stimulation</td><td>Light purple</td></tr><tr><td>Pooled Cases – Cerebral Stimulation</td><td>Dark purple</td></tr></tbody></table>

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.
Chapter 3:

Ponto-cerebellar 3D Reconstruction
# 3. Ponto-cerebellar 3D Reconstruction

## 3.1 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.
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 (Bjaalie *et al.*, 1997; Leergaard *et al.*, 1995, 2000a, b, 2003, 2004). 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 (Brodal & Jansen, 1946; Brodal & Bjaalie, 1997; Schwarz & Thier, 1995; Leergaard, 2003). 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. Voogd and Glickstein, 1998). 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 (Snider & Eldred, 1952; Brodal, 1968a, b, 1972a, b; Schmahmann & Pandya, 1997). 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, b, 2003, 2004). 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. Chicurel (2000)
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 user-defined 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 Bjaalie, 2002). 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 Bjaalie, 2001; Bjaalie *et al.*, 2005 for overviews).

**3.2.1 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 ponto-
cerebellar pathways in the same animal and relating the BPN labelling to the
climbing fibre somatotopy.
## 3.3 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).

### 3.3.1 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. Apps, 1990; Atkins & Apps, 1997) 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.

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 3.1 (part i) shows examples of climbing fibre field responses (average of 4 consecutive sweeps) recorded in each zone to ipsilateral limb stimulation. Forelimb responses 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. Figure 3.1 (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).

Double labelling was found in the olive and BPN in experiments where the injection sites were in close proximity. The incidence of double labelled cells varied between cases and only occurred in animals where injections were made into the same lobule i.e. in A2 face PML and C1 FL PML cases. Inter-lobule injections i.e. those made in PML and CP in the same animal, never produced double labelled cells indicating that there are no climbing fibre or mossy fibre collateral branches between the zones under investigation in PML and CP. Typically, where the injection site cores in PML were less than 200 µm apart, an increase in double labelled cells was seen. Cores of
more than 400 µm distance in PML never gave rise to double labelled cells in BPN 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).

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.

The forelimb (red) cells are located mainly on the side contralateral to the injection and are centred caudal to the middle of BPN, whereas the hindlimb cells are mainly located in the caudal aspect of BPN. Some cells are also located on the side ipsilateral to the injection site and these will be discussed later. No double labelled cells were found in BPN and the regions of cell labelling in the inferior olive for each case did not overlap and were restricted to the defined territories. The other double tracer experiment (BR13) selected for analysis, also had cells that occupied different parts of BPN with respect to the zonal injection. In this experiment, a microinjection was placed into the A2 face zone, giving cell labelling in the central region of BPN and a second microinjection was made into C1 hindlimb region. The latter resulted in only a small amount of cell labelling in BPN, nevertheless in the same caudal area of BPN as found in BR14.

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 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 inside-
out 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 Figure 3.3, 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).
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.
## 3.4 Discussion

Injections of retrograde tracer into A2 face-, C1 forelimb- and C1 hindlimb-receiving 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.

### 3.4.1 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 [Brodal & Steen, 1983]). 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 forelimb-
and 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. Brodal & Steen, 1983; Herrero et al, 2002; Serapide et al., 2002). 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, Rosina & Provini (1981, 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 (Mano et al., 1991). The face and whisker
region are of great significance in the rat for orientation and inquisitive behaviours (Berg & Kleinfeld, 2003) 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, Herrero *et al.* (2002) 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, Lillehaug *et al.* (2004) 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.

### 3.4.2 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 Figure 3.4, the left column shows the pooled ponto-cerebellar cell labelling whereas the right shows cerebro-pontine axon termination as dots (from Leergaard *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 ponto-cerebellar 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
injection data were compared with the combined results of cases R115 and R117
from the database where single injections were made into the forelimb responsive
area of SI. The C1 hindlimb climbing fibre zonal mossy fibre projection data were
compared with a combination of cases R123 and R124 from the database, where
single injections were made into the hindlimb SI cortex. For methods, mapping
procedures and semi-quantitative analyses of these database cases see Leergaard *et al.*
(2000b) and the online archive.

When the cases for each limb injection site were compared, striking similarities were
found, revealing further topographical correspondence and relationships in the
cerebro-ponto-cerebellar pathway, as shown in Figure 3.3. The isodensity surface
gradients through the rostrocaudal axis of right BPN also show for each case where
the densest areas of cell and terminal labelling occurred in the accumulated cases.
These were generated in the 3D reconstruction program. Once again, a
correspondence is displayed in these areas, although the cell labelling tended to be
more dense but this may have been due to a limitation of the semi-quantification of
the terminal labelling. Nevertheless, close relationships between A2 face and SI face,
C1 forelimb and SI forelimb, and C1 hindlimb and SI hindlimb injections are clearly
apparent.

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.

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.

In summary, all these results suggest a clear topographical organisation in the ponto-cerebellar projection to defined climbing fibre zones (or more strictly to different regions of the cerebellar cortex defined by the somatotopy of their climbing fibre input) and this somatotopical pattern is related to the descending input from the somatosensory cortex. The double tracer experiments especially provide strong evidence to support the topographical pattern of cell labelling, where with no overlap of the injection sites and restricted regions of cell labelling in the inferior olive, no double labelled cells were found in BPN.

The local pontine co-ordinate system provided a good means for comparing individual ponto-cerebellar cases, merged pontine cases from the same zonal injections, and also for relating the cerebro-pontine and ponto-cerebellar pathways. The ventral view displayed the most evident pattern of topographical organisation in BPN, although inspection of the 3D rotation and medial and rostral views proved essential to comprehending the full extent of the spatial distribution of the labelling. 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.
### 3.4.3 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 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.

Cell labelling in other brainstem targets could also be mapped using the 3D co-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.
Chapter 4:

Cerebro-ponto-cerebellar Pathway
# 4. Cerebro-ponto-cerebellar Pathway

## 4.1 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 non-corresponding 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.
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 (Kuypers, 1958; Brodal, 1968; Leergaard et al., 2000a, b, 2004 [see General Introduction for further details]). Concerning the ponto-cerebellar pathway, less knowledge is available as most studies have concentrated on the nature of the mossy fibre termination in the cerebellar cortex (Bower, 1997), rather than which cells provide the source of these mossy fibres, often due to the seemingly unorganised projection neurones in BPN (e.g. Brodal & Steen, 1983).

Studies such as those by Mihailoff *et al.* (1978), Leergaard *et al.* (1995), Leergaard *et al.* (2000), and Leergaard (2003) have shown topography in the cerebro-pontine projection, in relation to descending limb projections to BPN. For example, Mihailoff *et al.* (1978) using autoradiographic techniques showed a topographical organisation in BPN arising from forelimb and hindlimb injections into the rat motor cortex, whilst the studies from Leergaard *et al.* showed topographical relations between somatosensory-pontine projections (see General Introduction for further details).

More recently, Leergaard *et al.* (2004) showed a correspondence and topography from whisker representations in MI, SI and SII, to BPN. There are two competing theories from Bjaalie & Leergaard (2000) and Schwarz & 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 and that the transformation of information therefore takes place primarily in the mossy fibre ponto-cerebellar projection.
<table><caption>**Table 4.1: Hypothesised organisation in the ponto-cerebellar projection.**</caption><thead><tr><th></th><th>BPN Representation</th><th>Transformation</th></tr></thead><tbody><tr><td>Bjaalie <em>et al.</em></td><td>Orderly topography</td><td>In the mossy fibre projection</td></tr><tr><td>Schwarz &amp; Thier</td><td>Pontine modules</td><td>In the pontine nuclei</td></tr></tbody></table>

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, Schwarz & Thier (1995) 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 ponto-cerebellar mossy fibre projection.

Typically, the cerebral cortex and cerebellum are viewed as ‘computational units’ and the pontine nuclei as a relay. Schwarz & Thier (1999) 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.
4.2.1 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 structure-
function 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.
## 4.3 Results

### 4.3.1 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.

#### 4.3.1.1 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). Figure 4.1 shows a schematic reconstruction of all of the motor cortical injection sites (to scale) in a dorsal view of the cerebral cortex that gave terminal
labelling in BPN (although it was not possible to histologically confirm the site of injection case DP13). Figure 4.2 gives example injection sites into the forelimb and hindlimb areas of the motor cortex for each type of fluorescent tracer used. These figures together show the localisation in the motor cortex of the injections into fore- and hindlimb areas and the typical core transverse section of the injectate.

Table 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 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 mm³.

### 4.3.1.2 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 in-depth physiology). 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.

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 & Apps, 1997; 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 mm²
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 mid-rostral MAO and medial parts of mid-rostral DAO, whereas the hindlimb projecting areas extends to slightly more rostral and caudal parts of lateral DAO (Apps, 1990; Atkins & Apps, 1997; 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).

4.3.2 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. 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 4.6
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.
Mihailoff *et al.*, 1981).

**4.3.2.1 Labelling from ‘Forelimb’ Injections**

The labelling from injections into the cerebral forelimb area in MI and into the C1
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 Figure 4.7 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 seen 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 Figure 4.7 with the labelling for SI
and cerebellar forelimb injections seen in Figure 3.5). 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.
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 4.8 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 forelimb-receiving 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 4.9 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 4.10 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.

### 4.3.2.2 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. 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 4.12 shows a representative case of the tracing of the whole cerebro-ponto-cerebellar 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 olivary horizontal 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 4.13 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 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.

### 4.3.2.3 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 cerebro-
ponto-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 non-
corresponding 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 ponto-cerebellar 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 non-corresponding 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).

### 4.3.2.4 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.
## 4.4 Discussion

### 4.4.1 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 (Köbbert *et al.*, 2000).

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.

4.4.2 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. Mihailoff *et al*. (1978) 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
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.
Kincaid et al., 1998). As the dendritic field of a typical pontine cell is around 300 µm
(Mihailoff et al., 1981), 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.
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 (between-modality) 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. Altman & Bayer (1987) 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 ponto-cerebellar 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, 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.

### 4.4.3 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 (Coleman *et al.*, 1997). 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.
#### 4.4.4 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 Schwarz & Thier (1999) 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.
Chapter 5:

Cerebro-olivo-cerebellar Pathway
# 5. Cerebro-olivo-cerebellar Pathway

## 5.1 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 physiologically-guided 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.
## 5.2 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. Armstrong & Harvey, 1968; Andersson & Nyquist, 1983; Baker et al., 2001). 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; Baker et al., 2001). Different parts of the cerebral cortex have been implicated in projecting to the inferior olive (Snider & Barnard, 1949; Swenson et al., 1989; Walberg, 1956), although the areas most documented have been motor areas (Baker et al., 2001; Fennell & Rowe, 1973; Nakamura, 1983a, b; Saint-Cyr, 1983, 1987; Sousa-Pinto & Brodal, 1969).

There have been various studies that claim a direct cerebro-olivary projection (Brown et al., 1977; Saint-Cyr, 1983; Swenson & Castro, 1983; Walberg, 1956), 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. Kuypers, 1958) 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; Saint-Cyr, 1987). 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 cerebro-cerebellar 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 mesencephalic structures (Nakamura et al., 1983b; Saint-Cyr, 1987; Kyuhou, 1992). 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; Kitao et al., 1983; Rutherford et al., 1989), 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. Andersson, 1984).

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 Baker *et al.* (2001), 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 (Kuypers, 1958; Gordon & Jukes, 1964; Cheema *et al.*, 1985). It is well known that the DCN relay ascending limb information to the inferior olive (Walberg, 1957; Tomasulo & Emmers, 1972; Martinez *et al.*, 1995; Molinari *et al.*, 1996; Apps, 1998) and are targeted by descending motor cortical input (Walberg, 1957; Kuypers, 1958; Gordon & Jukes, 1964; Andersson, 1984; Cheema *et al.*, 1985) 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 (Boesten & Voogd, 1975; Berkley & Hand, 1978; Molinari *et al.*, 1996).

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 (Boesten & Voogd, 1975; Berkley & Worden, 1978). Also, both the DCN and the direct spinal input have been found to be somatotopically organised (DCN: Berkley & Hand, 1978; Molinari *et al.*, 1996; Apps, 1998; spinal: Miller *et al.*, 1969a, b; Oscarsson, 1973; 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, Adrian (1943) 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.

**5.2.1 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.