## 5.3 Results

The results from investigating the cerebro-olivo-cerebellar projection can be sub-divided into three inter-related lines of study (1) Physiological – study of somatotopical and converging inputs, (2) Anatomical – tracing the neural pathway by which the cerebral responses are conveyed to and from the inferior olive, and (3) Pharmacological – inactivation of a putative pre-olivary motor cortical relay and the effect of this on evoked climbing fibre responses.

### 5.3.1 Physiology

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

Climbing fibre responses were mapped over the full extent of the cerebellar posterior lobe up to the limits of the exposure. This included mapping both sides of the cerebellum, up to and just lateral of the C3 zone, although the main focus was on the C1 zone. This allowed a full comparison of climbing fibre responses evoked from the limb stimulation and by stimulation of the limb representations in the motor cortex. In the physiological investigations, low frequency and low intensity cerebral cortical stimulation (e.g. single pulse at 50 µA) evoked climbing fibre responses at the same recording sites as limb stimulation, but when stimulated at a higher frequency and current (e.g. thirty consecutive pulses at 2 mA), the same cortical areas elicited movements of the limbs.

#### 5.3.1.1 Climbing Fibre Responses

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

At threshold stimulation (T), only a small, transient response was present, characterising the minimum intensity need to evoke a response. The typical T value for peripheral stimulation was 0.05 V and for cerebral stimulation 1 mA. Measurements were then taken at twice this intensity (2T) and three times (3T) to gain clear waveforms from climbing fibre field potentials. At 2T, responses could be readily evoked from all stimulation sites, and at 3T, there was sometimes a slight increase in the amplitude of the response. An intensity of 2T was therefore deemed a sufficient level to stimulate/record although 3T and occasionally 5T were sometimes used to evoke supramaximal responses.

Table 5.1 shows the cases used (n = 28 animals) and details the values found for corresponding peripheral ipsilateral limb stimulation and contralateral and ipsilateral MC stimulation to recording sites in the C1 zone of LS, PML, and CP. Table 5.2 shows the results of testing for statistically significant differences between different combinations of stimulation and recording sites. The onset latencies for the peripheral forelimb and contralateral MC forelimb responses were very similar with average latencies between 12.20 ms and 12.74 ms and table 5.2 shows no significant difference between any of these combinations of stimulation and recording site. However, there were significant differences between the ipsilateral forelimb MC stimulation and other sites and both peripheral hindlimb and contralateral hindlimb MC stimulation showed overall significant differences between most recording sites.

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

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

The responses sometimes also displayed a characteristic waveform with distinct secondary components. The climbing fibre potential typically reversed when the recording electrode was advanced into the cerebellar cortex (Eccles *et al.*, 1967). The climbing fibre responses found in this investigation were therefore similar to those reported in previous work (e.g. Pardoe & Apps, 2002). The response latencies were also very similar when compared to other work on responses evoked in the same regions of the cerebellar cortex in the rat by limb stimulation (see Atkins & Apps, 1997; Pardoe & Apps, 2002). The latency information for the recording sites in the C1 zone for LS, PML and CP is shown later.

In addition, the use of barbiturate anaesthesia has been found to enhance climbing fibre activity but depress mossy fibre activity for both peripheral ascending (e.g. Gordon *et al.*, 1973) and cerebral descending pathways (e.g. Andersson & Nyquist, 1983). In some of the present experiments it was possible sometimes to see early responses of around 5 ms latency when the preparation lightly anaesthetised. These were presumably mediated via mossy fibres and after a top-up injection of
pentobarbitone, such responses decreased in size. This is similar to the findings of Adrian (1943) where at deep levels of pentobarbitone anaesthesia cerebrally evoked mossy fibre potentials were extinguished. Further tests were also carried out to ensure that climbing fibre rather than mossy fibre responses were recorded. This was particularly important for responses evoked by cerebral stimulation. In addition to short latency mossy fibre-related responses, longer latency responses have also been described (Morissette & Bower, 1996). These can have similar onset latencies to climbing fibre responses.

## Paired Pulse Paradigm

Classically, climbing fibre responses have a long refractory period (Eccles et al., 1967). Therefore, applying two pulses from the same stimulation site in close succession should mean the putative climbing fibre response occurs only once, to the initial stimulation. With a progressive increase in the interval between the pulses beyond 40 to 60 ms, there was a progressive recovery of response to the second pulse with distance from the first pulse, as demonstrated in Figure 5.2. Responses evoked by peripheral stimulation started to recover at an interstimulus interval of 80 ms and showed recovery to control values by 100 ms (see graph under waveforms). Note that after 80 ms, there was an increase in the size of the cerebrally evoked responses to 160% of the control. This rebound facilitation has also been characterised previously as a property of climbing fibre responses (Eccles et al., 1967).

## Conditioning Paradigm

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

## Threshold Stimulation Tests

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

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

### 5.3.1.2 Motor Cortex Map

Once responses were identified as mediated via the climbing fibres according to the above criteria, the experiments then focussed on mapping on the cerebellar surface the responses to cerebral and peripheral input. When the stimulating electrode was lowered perpendicularly into the motor cortex, the evoked cerebellar climbing fibre responses increased in amplitude until an optimum depth of ≈1.7 mm was reached. This corresponded to the pyramidal projection neurones of layer V in the agranular motor cortex (see General Introduction). At shallower and deeper sites, climbing fibre responses could still be evoked but the responses were smaller in size and larger
stimulation currents were needed. A depth of 1.7 mm was used in subsequent acute electrophysiological and anatomical tract tracer experiments.

Forelimb motor cortex stimulation evoked twitches typically of the distal muscles of the forearm, and hindlimb motor cortex usually evoked twitches in distal muscles of the leg; although occasionally some digital movements were noted for both. The centre of the forelimb area typically corresponded to Bregma and the centre of the hindlimb approximately 1 mm caudal to Bregma, both approximately 1 mm lateral of midline. The whisker representation was rostral to the forelimb representation and the trunk lateral to the hindlimb area (see sites of injection in Figure 4.1 for locations of forelimb and hindlimb areas). This topographical representation is similar to previous descriptions (e.g. Hall & Lindholm, 1974). However, there were interspecies variations in details and only the gross representations were generalised; finer grain variabilities have been described in previous studies (e.g. Neafsey *et al.*, 1986).

### 5.3.2 Anatomical Labelling

A total of 12 cases investigated projections from the motor cortex to the inferior olive. These are the same cases already considered in the preceding chapter but for cerebro-pontine projections. Directly from the injection site in the motor cortex, labelled axons could be seen coursing towards the corpus callosum, where the fibres continued in the tract to descend in a caudal direction. From there, fibres were seen extending towards the internal capsule, but occasionally seen coursing medially to the contralateral hemisphere; this pattern of fibre labelling has been previously noted by Mihailoff *et al.* (1978).

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

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

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

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

5.3.2.1 Anterograde Labelling in the Caudal Brainstem

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

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

Figure 5.7 shows a full example case of the distribution of axonal and terminal labelling in the caudal brainstem for a double tracer experiment, where both forelimb and hindlimb regions of motor cortex were injected. The labelling pattern for the Fluoro-Ruby ‘forelimb’ motor cortex injection and the Fluoro-Emerald ‘hindlimb’ motor cortex injection are plotted on standard transverse maps. The labelled fibres (wavy red and green lines) can be seen coursing through the pyramidal decussation, and heading dorsally to the dorsal columns, with some axons terminating in the matrix region.

Figure 5.7 also shows that the terminal labelling was not very different in extent or location between forelimb and hindlimb cases. Overall, the hindlimb labelling was slightly more extensive (see Figure 5.8) but this may have been due to having two more hindlimb cases than forelimb cases and of the hindlimb cases two were very well labelled. In these well labelled hindlimb cases (DP1 and DP11), there was also terminal labelling in the contralateral gracile nucleus (to the injection site), and also in one forelimb case (DP13). In DP11 (hindlimb) and DP13 (forelimb), the gracile labelling was also found to a lesser degree on the ipsilateral side to the injection site.
An overview of the pooled data and a summary of the individual areas of terminal labelling in the matrix can be seen in Figure 5.8. The pooled cases show that the total area covered by the terminal labelling is similar for both projections although the hindlimb projection extends slightly more caudal and rostral to the forelimb projection. As previously mentioned, this may be due to the higher number of hindlimb cases and also the two well-labelled hindlimb cases. Overall, the projection pattern is indistinguishable between forelimb and hindlimb motor cortical projections.

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

## Labelling in the Thalamus

In addition to terminal labelling in BPN (Chapter 4) and the matrix region, anterograde labelling was also noted at the level of the thalamus. Some cell labelling was also present here (n = 5). This pattern of labelling is indicative of thalamic reciprocal circuits with the cerebral cortex, demonstrating, in this case, circuits involving the motor cortex. These thalamo-cerebral and cerebro-thalamic mechanisms exert feed-forward and feed-back control, respectively, to and from the cortex (Rouiller & Welker, 2000). The specific thalamic areas labelled depended upon the injection site but frequently, there was cell and terminal labelling in the reticular thalamic nucleus and surrounding areas such as the anteroventral thalamic nucleus (dorsomedial and ventrolateral parts).

## Labelling in the Substantia Nigra

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

### Labelling in the Spinal cord

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

### 5.3.3 Inactivation of Putative Pre-Olivary Relay

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

Various combinations of stimulation of the limbs ipsilateral to the recording site and the contralateral MC limb areas (and the ipsilateral MC in one forelimb case) were used to investigate the input to cerebellar climbing fibres. Responses in the C1 zone were recorded, typically from the forelimb-receiving area in PML and hindlimb-receiving area in CP, but also in one experiment from the C1 forelimb-receiving zone in LS. Each case was designated by firstly, the drug regime used, secondly, the rostrocaudal site of the injection, and finally whether responses were recorded from forelimb PML and/or hindlimb CP was recorded from (and one case forelimb LS). This meant that a case was considered as a certain recording site in the cerebellum (fore- or hindlimb C1) to which an injection of drug was placed in a position 0.5 to 4 mm caudal from obex.
LA or GABA/KA was injected at different loci spread over a 3.5 mm length of brainstem caudal to the obex to find if there was an optimum site or sites where a decrease in climbing fibre responses could be obtained. In each experiment, climbing fibres responses were evoked in a cyclical sequence by peripheral limb then contralateral motor cortical stimulation and were recorded in the middle of the C1 zone in PML and/or CP (see Methods for further details).

### 5.3.3.1 Injection of Drug into Putative Relay Site

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

This meant that assigning the location of each injection was not possible for all cases. Since injections 2 mm caudal to obex were the most important, special attention was made to recover these injection sites. The general findings from these experiments were that inactivation occurred within 10 minutes and the block occurred for up to 35 minutes. The preferential target was just lateral of the midline, at a depth of 0.6 mm, although depths between 0.5 and 0.7 mm also showed some block.

From all cases at this site (n=10; 5 using LA; 5 using GABA/KA), the average time taken for a decrease in climbing fibres was 4 mins 40 secs and average time to recovery was 19 minutes 30 seconds. For the local anaesthetic cases, the time to a decrease was less (3 minutes 10 seconds) and the duration of the effect was a little longer (average 22 minutes 50 seconds). The time to a decrease in responses to GABA/KA application was longer (average 6 minutes 10 seconds) and the duration shorter overall (average 16 minutes 5 seconds). These values were well within expected time limits of reversible inactivation agents, from studies injecting these
drugs into other CNS structures (e.g. Malpeli, 1999; Martin & Ghez, 1999; Boehnke *et al.*, 2001).

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

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

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

The figure demonstrates this for both forelimb and hindlimb evoked responses recorded simultaneously. Local anaesthetic was injected into the matrix, 2 mm caudal from the obex and within 2 minutes, both fore- and hindlimb cerebrally evoked responses had decreased below the threshold for detection, where the peripherally evoked fore- and hindlimb responses were not affected. These recordings demonstrate that through inactivation of cells, the cerebral input can be selectively and temporarily blocked, whilst the peripheral input remains unchanged. There was no difference between the onset of the effect or the duration for either fore- or hindlimb evoked responses suggesting that the relay for both are close together (perhaps intermingled) in the matrix area. This is consistent with the pattern of terminal labelling found in the anatomical tracing experiments (see previous section). In no case was it possible to block forelimb or hindlimb COCP cases separately.

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

In each case where a successful block was found at 2 mm caudal to obex, the
decrease in cerebrally evoked climbing fibre responses occurred between 1 to 8
minutes after the application of the drug. Both these values and the length of
inactivation were comparable to findings in previous studies, in which the same
drugs were used to selectively inactivate the motor cortex and the red nucleus
(Martin & Ghez, 1999). The following three Figures show the individual case results
at the 2 mm site. Figure 5.12 shows the responses to local anaesthetic injections, Figure 5.13 to GABA/KA injections, and Figure 5.14 shows the pooled data for all cases at 2 mm caudal to obex.

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

### 5.3.4 Responses to Rostrocaudal Injection

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

#### 5.3.4.1 Rostral Injection Sites to Putative Relay

Table 5.4 shows for drug injections made 0.5, 1.0 and 1.5 mm caudal to the obex, percentage changes seen in the responses and their degree of statistical significance. Four chosen time points after injection are included. A total of 15 cases were included in the analysis (0.5 mm caudal to obex, n = 6; 1 mm caudal to obex, n = 5; 1.5 mm caudal, n = 4). Note that both increases (green) and decreases (yellow) in the response size were observed. The general conclusion drawn from these data was that there was no consistent effect on the peripherally or cerebrally evoked responses.
5.3.4.2 Caudal Injection Sites to Putative Relay

The farther caudal the drug injection, the more likely it was that both the peripherally
and the cerebrally evoked responses were significantly decreased though there were a
few significant increases, as can be seen in table 5.5. A possible explanation for the
decreases, may have been due to local anaesthetic spread to fibre tracts. Local
anaesthetic may actually have a more potent blocking effect on fibre tracts than on
cells (Sandkühler *et al.*, 1987). From a total of 9 caudal cases, only two (DPE28 LS
& PML) involved injecting GABA/KA. There was little effect on cerebrally evoked
responses and a varied effect on responses evoked from the periphery. With the local
anaesthetic, a higher proportion of responses decreased, especially to peripheral
stimulation, which implied that ascending fibre tracts may have been affected,
especially as these injections were in the rostral spinal cord.
## 5.4 Discussion

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

Using anatomical tracers, the cerebro-olivary projection was found to be indirect, with no terminal labelling from the motor cortical injections ever found in the inferior olive. This negative finding was not entirely unexpected as the literature on cerebro-olivary projections has previously shown conflicting results as to whether there is a direct projection or one that involves at least one pre-olivary synaptic interruption (e.g. anatomical findings of Saint-Cyr & Courville [1980] and Saint-Cyr [1983, 1987]; physiological findings of Baker *et al.*, 2001). Through the use of neuronal blockers, the matrix region in the caudal brainstem was found in the present study to relay cerebrally-evoked, but not peripherally evoked climbing fibre responses. Earlier studies that investigated the cerebro-olivo-cerebellar pathway have shown conflicts between physiological data and anatomical pathway data, and the present data are discussed below in light of these past findings.

### 5.4.1 Physiology

Climbing fibre input imposes a zonal topographical organisation on the cerebellum, thus giving an organisation where zones can be systematically studied. In the present study, the motor cortex was mapped and an ordered representation of body parts was found whereby the fore- and hindlimb areas converged respectively with the corresponding peripheral input to specific cortical zones. The investigation revealed that in the rat, convergence of peripherally and motor cortically evoked climbing fibre responses is at or before the inferior olive. This was found for forelimb-receiving C1 zones in LS and PML, and for the hindlimb-receiving C1 zone in CP. Previous studies have found convergence of climbing fibre ascending and
descending input (e.g. Adrian, 1943; Snider & Eldred, 1952; Armstrong & Harvey, 1966; Provini *et al.*, 1968; Miller *et al.*, 1969a, b; Allen *et al.*, 1974a) and it demonstrates that similar olivary areas must be activated by different inputs.

Previous physiological studies in the monkey and cat of cerebro-olivary projections (e.g. Snider & Barnard, 1949) found a bilateral projection though the pathways were not demonstrated anatomically. The data from Snider & Barnard's study agrees with physiological data such as Armstrong & Harvey (1966) and also with the physiological data gained in the present study, although the bilateral aspect was only found for the forelimb input, not hindlimb, and the ipsilateral MC stimulation showed a significantly longer response latency than that from contralateral MC stimulation, implying different multi-synaptic pathways are involved. The role of the bilateral projection may concern behaviours that are more complex, such as feeding and foraging, as the forelimbs play a further role in behaviours other than locomotion and balance, whereas it is the main function of the hindlimbs.

The convergence in the inferior olive shown by limb-receiving areas was important (cf. Allen *et al.*, 1974a; Andersson & Nyquist, 1983), where weak signals arriving at the inferior olive conjunctively are nevertheless effective in producing a climbing fibre response. Therefore, as in studies in the cat, the principle that cerebral and peripheral inputs to the inferior olive exert a facilitated effect when they arrive concurrently, seems to also hold true in the rat. However, where there were no peripherally evoked climbing fibre responses, there were also no cerebrally evoked responses. Although the absence of responses should be interpreted with caution, this gives weight towards the representations of body parts in the cerebellum receiving only convergent input and shows the specificity of the climbing fibre projection to certain zones. For example, it may take a strong input from either the periphery or motor cortex for an inferior olivary neurone to fire, however when these inputs occur at the same time, a weaker input may be required to fire the same neurone. Therefore, the signals may serve as a coincidence or error detector of SOCP and COCP signals and may tie in with the comparator hypothesis (see Andersson & Nyquist, 1983).

The latency of the onset of all cerebrally evoked climbing fibre responses found in the present study (>10 ms) suggests slow conduction in the pyramidal tract and the
likelihood of a pre-olivary relay. Kitai *et al.* (1969) have previously shown in the cat that the early mossy fibre response in the cerebellum is mediated by fast conducting fibres in the pyramidal tract, whereas the climbing fibre response is mediated by slower conducting fibres, hence the longer latency. Allen *et al.* (1974b) investigated the forelimb and hindlimb input from the motor cortex in the cat and came to the conclusion that the cerebro-olivary pathway for these inputs could not be mono-synaptic due to the long delay.

In the cat, there are fast and slow conducting pathways (Takahashi, 1965), however in the rat, only slow conducting pyramidal tract axons have been found (Landry *et al.*, 1984). As the timing of climbing fibre and mossy fibre latencies are similar between rat and cat, but there only seems to be one class of projection neurone in the pyramidal tract in the rat, then it follows that there could be some system that 'delays' the climbing fibre responses from MC stimulation, i.e. a pre-olivary relay. A longer pathway and additional synapse(s) would mean a longer onset latency of the climbing fibre response and thus maintain differences in mossy and climbing fibre response latencies to the same cortical stimulus.

The anatomical findings support a mono-synaptic cerebro-cerebellar pathway via BPN (as expected, see Chapter 4) and when seen, mossy fibre responses were much shorter in latency than the climbing fibre responses. Conversely, the physiological data here supports the notion of a pre-olivary relay in the rat (cf. Baker *et al.*, 2001), where the latency of even the shortest forelimb MC climbing fibre response suggests a disynaptic relay. Furthermore there was a longer latency from ipsilateral forelimb MC stimulation, suggesting a poly-synaptic relay for this input. Even with the slow conduction velocities in COCPs, the findings show that there would still be sufficient time for at least one additional pre-olivary synaptic interruption. Also, the fastest conducting SOCPs targeting forelimb parts of the C1 zone have a similar latency to the corresponding COCPs (approximately 12 ms), implying that precise timing of the integration of ascending and descending climbing fibre inputs is important for cerebellar contributions to the control of forelimb movements.
### 5.4.2 Anatomy

Controversy exists as to whether there is a direct cerebro-olivary projection. In the present study, no direct anatomical projection was found for the motor cerebro-olivary projection for fore- or hindlimb motor cortical areas in any experiment. Even in cases where there was a significant amount of terminal labelling in BPN, no detectable terminal fibres were ever found in the olive; when viewed in transverse section only short lengths of labelled axons were seen ventral to the olive. Wherever there was terminal labelling in the CNS, it was clear from the plane of section to see the labelled fibres getting longer as they came into the same transverse plane as the sectioned material. The short fibres were typical of axons only, coursing down the pyramidal tract where the plane of sectioning was perpendicular to the plane of axonal labelling. The lack of terminal labelling in the olive was not thought to be due to methodological or tracer limitations (see also Chapter 4), especially as other studies have used similar survival times to label cerebro-spinal connections with the tracer (Coleman et al., 1997, in the rat) and as the evidence for a direct projection from the motor cortex to the inferior olive is varied.

A direct cerebro-olivary projection may exist from other cerebral areas that the present investigation did not study. For example, such direct cerebro-olivary projections may arise mainly from somatosensory cortex (e.g. Swenson & Castro, 1983; Swenson et al., 1989). An additional possibility is that they entail diffusely scattered projection cells in the cerebral cortex (e.g. Bishop et al., 1976), therefore the small microinjections made in the present study may not have been effective in labelling sufficient numbers of these neurones to produce a detectable level of terminal labelling in the inferior olive. However, a direct cerebro-olivary pathway from diffusely organised pyramidal cells would suggest that the cerebro-olivo-cerebellar pathway was not substantial or functionally powerful, contrary to the findings from the present and previous physiological studies (e.g. Provini et al., 1968, Miller et al., 1969a, b; Anderson & Nyquist, 1983; Baker et al., 2001).

There have been many anatomical studies investigating cerebro-olivary connections. Some studies have shown a direct cerebro-olivary projection in the rat (Brown et al., 1977; Swenson & Castro, 1983; Swenson et al., 1989) and the cat (Walberg, 1956; Sousa-Pinto & Brodal, 1969; Bishop et al., 1976; Berkley and Worden 1978; Saint-
Cyr, 1983). However, one of the main drawbacks of these earlier studies is the large quantity of cerebro-fugal fibres that traverse the olive and send axons to other brainstem nuclei. In particular, the use of degeneration studies can be ambiguous in this regard as degenerating axons and terminals can be difficult to distinguish from one another (Kuypers, 1981). Also, due to the close proximity of fibres of passage, injection of retrograde tracer into the inferior olive makes it difficult to draw any reliable conclusions about direct projections, particularly given the complex shape of the olive – it is almost impossible to be sure that injections are confined to this structure.

However, the labelling found in the olive from cerebral (sensory and motor) injections in the study by Swenson *et al.* (1989) cannot be disputed for the same reasons. In their study, a direct ipsilateral projection was found from fore- and hindlimb motor cortex to the inferior olive using WGA-HRP as an anterograde tracer. The disagreement with the present findings may relate to the possibility that different areas of the sensorimotor cortex were injected and that their injections were more superficial and larger (c. 1mm diameter), thus targeting different projection neurones. In the present study, microinjections were placed into regions of the motor cortex that elicited more proximal muscle movements than the digit movements evoked in Swenson *et al.*'s study. This raises the possibility that some parts of the rat motor cortex have direct pathways to the olive, while others do not; perhaps the control of limbs for locomotion requires a pre-olivary relay, while control of distal fine movements in exploratory tasks requires a direct cerebro-olivary link.

Similar differences may also be present in cats. In this species, varied results for cerebro-olivary projections have been reported (Kuypers, 1958; Saint-Cyr, 1980, 1983; Sousa-Pinto & Brodal, 1969), with either no direct cerebro-olivary projection found, or only certain parts of the motor cortex projecting to the inferior olive. In cats, the motor cortex is located in the anterior sigmoid gyrus (a.s.g.) and rostral part of the posterior sigmoid gyrus (p.s.g.), collectively known as the pericruciate cortex. The a.s.g. has been found to provide a discrete projection to the inferior olive (Sousa-Pinto & Brodal, 1969; Saint-Cyr & Courville, 1980; Saint-Cyr, 1983), while no direct projection has been reported from the p.s.g. Bishop *et al.* (1976) also found
from retrograde labelling, many cells in a.s.g. and very few in p.s.g., although
injections into the olive have interpretative difficulties as discussed earlier.

If we accept that there is no direct p.s.g.-olive projection, it does not necessarily
imply that this motor area does not contribute input to the cerebellum via climbing
fibres. Sousa-Pinto & Brodal (1969) presume that the p.s.g. has no influence over the
cerebellum via the olive. However, Andersson & Nyquist (1983) showed that the
p.s.g. projects to the forelimb-receiving (anterior lobe) C1 zone in cat. Therefore, the
C1 (forelimb) zone may receive input from p.s.g. (presumably from indirect
pathways) and not the a.s.g. As the cat has these different areas of representations in
the pericruciate cortex, this cannot be directly compared to the rat, which has no
equivalent differentiation of its motor cortex. This may reflect specialisation related
to phylogenetic complexity.

Some studies in cats have emphasized the role of mesodiencephalic structures that
relay cerebral information to the inferior olive (Saint-Cyr & Courville, 1980;
Nakamura *et al.*, 1983a, b; Saint-Cyr, 1987; Rutherford *et al.*, 1989). However, there
was no obvious terminal labelling in these structures in rat, suggesting there may be
other important differences between species in terms of the anatomical organisation
of cerebro-olivary projections.

Due to the lack of terminal labelling in the inferior olive, a pre-olivary relay was
sought. Labelled cells and presumed terminal fibres were found in the thalamus
(mainly ventrolateral thalamic regions), from the injections into the motor cortex.
However, this labelling was not thought to be a likely candidate for a relay in the
cerebro-olivary pathway, as it is considered to be mainly involved in reciprocal
connections with the cerebral cortex and olivary projections from this part of the
thalamus have not been reported. Terminal labelling was also found in the substantia
nigra, pars reticular, although again, this structure is not thought to have a substantial
projection to the olive. The substantia nigra is part of the basal ganglia and is
typically involved in the initiation (rather than overseeing/monitoring/correction) of
movement. There have been no previous reports of either thalamo-olivary or nigral-
olivary projections (for overviews see Armstrong, 1974; Bloedel & Courville, 1981).
The pontine nuclei appeared to be the only other possible rostral relay in the cerebro-
olivary pathway due to the large amount of terminal labelling found there. From the
literature, though, there is little evidence of a ponto-olivary projection. Some
electrophysiological data has been reported in which pontine stimulation was found
to evoke climbing fibre responses in the cerebellum (e.g. Sasaki et al., 1969).
However, Sasaki and colleagues concluded that a direct ponto-olivary projection was
unlikely and suggested that their results were probably due to activation of fibres of
passage.

Therefore, in rat, there may not be a rostral relay, but only the possibility of a caudal
relay. Through physiological evidence Baker *et al.* (2001) by stimulating the
pyramidal tract and selectively lesioning parts of it, found that if there was any relay
involved in the (rat) MC projection to the inferior olive, it would not be mediated by
relays in the rostral brainstem as lesions of the pyramidal tract rostral to the inferior
olive substantially reduced responses evoked from stimulation of cerebro-fugal fibres.
The dorsal column nuclei (DCN) have also previously been identified as receiving a
cerebro-fugal projection (Kuypers, 1958; Gordon & Jukes, 1964; Cheema *et al.*,
1985). The DCN, in turn, are well known to provide direct projections to the inferior
olive (Walberg, 1957; Tomasulo & Emmers, 1972; Boesten & Voogd, 1975; Berkley
& Hand, 1978; Martinez *et al.*, 1995; Molinari *et al.*, 1996; Apps, 1998) and
therefore could be a possible target to receive MC information and relay it onto the
inferior olive.

The matrix region may be a distinct part of the DCN, where in the past it may have
been grouped with the cuneate or gracile nuclei. In a degeneration study, Kuypers
(1958) found terminal labelling near cuneate and gracile as a result of lesions into the
cat motor cortex; this is likely to have included the matrix region. Also, Sousa-Pinto
& Brodal (1969) using degeneration techniques found a heavy projection to the DCN
from motor cortical areas, which was bilateral with a mainly contralateral
predominance. The results from the present study have found a contralateral
projection to the matrix region (n=10) and on occasion, a partly bilateral projection
to the gracile nucleus (n=3). The available anatomical evidence therefore suggests
that the matrix region is a potential site for an initial synaptic interruption in the
cerebro-olivary pathway (there may well be additional synapses before information
reaches the olive, especially due to the finding that forelimb MC stimulation evoked
climbing fibre responses in both sides of the cerebellum).

5.4.3 Reversible Inactivation of Putative Relay

Little is known about the matrix region but it was identified in relation to the human
pericuneate and peritrigeminal matrices (identified in Paxinos & Huang, 1995). A
similar structure was observed later in the rat (Paxinos & Watson, 2005) and is
thought to function as a residual region relating to the dorsal column nuclei, when all
the surrounding nuclei have been accounted for. Reversible pharmacological
inactivation of the matrix region selectively abolished climbing fibre responses
evoked by MC stimulation. This effect was obtained with both types of blocker (LA
or GABA/KA). Taken together with the anatomical evidence, these inactivation
experiments give considerable evidence towards the matrix region being a synaptic
relay in the cerebro-olivary projection from the motor cortex to the C1 zone. The
selective inactivation of COCPs was localised to a site 2 mm caudal from the obex.
This corresponds closely to where terminal labelling was found in the matrix.
Injections of blocker further rostrally or caudally failed to produce a selective effect.
The differential effect on COCPs as compared to SOCPs indicated that the synaptic
relay was prior to the convergence of the ascending and descending information to
the olive.

Both local anaesthetics and GABA have approximate areas of inactivation of up to
500 µm radius for the volumes injected in this study. As the region of the terminal
labelling in the putative relay was estimated to be approximately 1 mm in
rostrocaudal length and 300 µm in width, the area of inactivation should have
encompassed much of the region, but possibly not all of the rostrocaudal length,
although in most cases this was enough to give a full abolition of evoked cerebral
responses. Also, consistent with the anatomical findings was the similarity in the
time course and effect of inactivation on both forelimb and hindlimb pathways. The
territories occupied by terminals for fore- and hindlimb paths overlapped extensively
in the matrix. However, since these paths are functionally independent this implies
that either both fore- and hindlimb relay cells are intermingled in the matrix or that
higher resolution mapping will be required to identify any topographical organisation.
Andersson (1984) found that in the cat, topical application of local anaesthetic to the caudal cuneate nucleus abolished SOCP and short-latency COCP responses in the C1 and C3 zones and therefore that COCPs were at least partly relayed through the cuneate nucleus. The results from the present study raise the possibility that there are inter-animal differences in anatomical connectivity and structural differences between the rat and cat caudal brainstem nuclei, but also that the local anaesthetic injected into cuneate may have actually spread deeper into the brainstem and inactivated the matrix, to gain similar results as the present investigation.

This notion is supported by McCurdy *et al.* (1992) where a rostrocaudal column of cells in the ventral cuneate nucleus was shown to project to rostral DAO, through anatomical tracer injections into the inferior olive and the cuneate nucleus, in the cat. Furthermore, it was found that with cerebral injections into the forelimb area of the pericruciate cortex, the consequent terminal labelling in the cuneate nucleus was located in a similar area to the cell labelling from retrograde tracer injections into the DAO. This suggests that there is a convergence of forelimb ascending and descending climbing fibre paths to the C1 and C3 zones in the cat. Also, in the present investigation, injections of to inactivate the more caudal parts of the matrix (i.e. 2.5-4 mm caudal to obex) reduced both SOCP and COCP transmission. Thus, the matrix may comprise of at least two rostrocaudally separated regions: one region at approximately 2mm caudal to the obex, which relays COCPs but not SOCPs targeting the same parts of the C1 zone, and a second more caudal region (2.5-4 mm caudal to obex) where SOCPs and COCPs conveying climbing fibre signals to the C1 zone converge in the rat.

### 5.4.4 Further Study

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

In addition, a different anaesthetic could be used (such as a ketamine/xylezene mixture) so that both climbing fibre and mossy fibre activity could be recorded simultaneously. By recording both climbing and mossy fibre activity to peripheral and cerebral stimulation, it would be possible to investigate the effect of drug injection (blockers and excitants) into the matrix area on climbing fibre activity, but also on mossy fibre activity. Similarly, stimulation (electrical or chemical) within the matrix region and recording in the olive would help determine the extent to which the projection is direct. In conjunction with these physiological methods, injection of bidirectional tracer may be carried out to look at the input-output connections of the matrix, specifically where direct olivary connections exist. Figure 5.15 shows the potential pathways for both the contralateral and ipsilateral forelimb motor input to the C1 zones in LS and PML. The potential relay pathways are shown for each side of the brain and as deduced from the present results. In the figure, the information is shown as targeting the matrix and then projecting to the inferior olive, although there may be other synaptic interruptions before the termination of signals from the matrix to the olive, especially taking into consideration bilateral projections.

In further study, recovery experiments could be used to investigate the contribution the putative relay area provides to behaviour. Recording electrodes (e.g. tetrodes for multiple recordings) could be chronically implanted into the matrix region and single unit activity recorded while an animal performs voluntary movements (thus engaging the motor cortex), such as pressing a lever for a reward. Also, if a cannula was placed in a headpiece over the caudal brainstem, to deliver drugs into the putative relay area, the effect of these drugs in the awake animal, in response to such an experimental paradigm could provide insights into the contribution(s) of this cerebro-olivo-cerebellar pathway during movements.
## Figures & Tables
A

<figure><img src="image_3.png" /></figure>

High power photomicrograph
of a Purkinje cell

B

<figure><img src="image_4.png" /><figcaption>**Figure 1.1: Cerebellar circuitry.**</figcaption></figure>

(A) A photomicrograph of a Purkinje cell, the output neurone of the cerebellum.

(B) The above diagram represents the main components of the circuitry of the cerebellum. The layers of the cerebellum are shown (from inner white matter to outer molecular layer), the two main inputs of mossy fibres from the pontine nuclei and climbing fibres from the olive, and the major projection cell types included in this circuit (interneurones not included for clarity).
<figure><img src="image_5.png" /><figcaption>(B) Mossy fibre potentials</figcaption></figure>

<figure><img src="image_6.png" /></figure>

**Figure 1.2:** Responses of the Purkinje cell to mossy fibre and climbing fibre activation.

(A) The diagram shows the climbing fibre activation response where (i) shows the complex spike response of the Purkinje cell to climbing fibre activation and (ii) shows the depth profile to climbing fibre activation through advancing the recording electrode into the cerebellum. (B) A diagrammatic illustrations of mossy fibre potentials; the upper responses taken at a depth of 200 µm and the lower responses at 500 µm. Diagrams adapted from Eccles *et al.* (1967).
<figure><img src="image_7.png" /></figure>

**(B) Rat cerebellar zones**

<figure><img src="image_8.png" /><figcaption>**Figure 1.3: Climbing fibre zonal organisation in the cat and rat.**</figcaption></figure>

(A) The zonal organisation of the olivo-cortico-nuclear projection in cat (from Apps & Garwicz, 2005) showing the main zonal divisions in the cat, with the corresponding afferent olivary input and the efferent projection to the cerebellar nuclei. (B) The climbing fibre zonal organisation in the posterior rat cerebellum as defined by peripheral input (from Voogd *et al.*, 2003, adapted from work by Atkins & Apps, 1997).
<figure><img src="image_9.png" /><figcaption>Figure 1.4: Mossy fibre patchy representation in the cerebellar cortex.</figcaption></figure>

From the view of the posterior lobe of the rat cerebellum and the corresponding body representations, it can be seen that the mossy fibre representation in the cerebellar cortex is patchy where body parts are represented in a fractured manner. For example, the coloured forelimb in the rat is represented next to other non-contiguous body parts in the cerebellum (adapted from Bower [1997], from original work by Bower *et al.*, 1981).
<figure><img src="image_10.png" /><figcaption>**Figure 1.5: Cerebral cortical somatotopical body representations in the rat.**</figcaption></figure>

(A) The diagram shows a dorsal view of agranular (motor) and granular (sensory) regions in the cortex (diagram summated from various previous studies in Coleman *et al.*, 1997). (B) A map is of the continuous representation of the rattunculus in SI (from Neafsey *et al.*, 1985). (C) The diagram details the motor (MI) and sensory (SI and SII) areas of the cerebral cortex, as represented (A) on the cerebral surface and (B) together in scale (from Leergaard *et al.*, 2004). Scale represented in millimetres in all diagrams.
<figure><img src="image_11.png" /><figcaption>Figure 2.1: Diagram of the experimental stimulation and recording areas in the rat brain.</figcaption></figure>

The ascending peripheral body part stimulation and descending motor cortex body representation stimulation evoked climbing fibre responses in the cerebellum. The major structures investigated are shown in colour (BPN, cerebellum, inferior olive, motor cortex). Tracer injections were also made into the cerebral stimulation areas (DP experiments) and cerebellar recording sites (BR and DP experiments). Sagittal diagram at 1.4 mm lateral. Scale bar = 1 mm. Adapted from Paxinos & Watson (1998). AI: Anterior interpositus cerebellar nucleus; cp: cerebral peduncle; F: Fastigial cerebellar nucleus; Lfp: Longitudinal fasciculus of the pons; ml: medial lemniscus; PI: Posterior interpositus cerebellar nucleus.
Caudal ∞

<figure><img src="image_12.png" /></figure>

Ventral View

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>

Rostral View

<figure><img src="image_15.png" /></figure>

Medial View

<figure><img src="image_16.png" /><figcaption>**Figure 2.2: Pontine orientation maps.**</figcaption></figure>

Rostro-caudal standard maps for mapping cell and terminal labelling in BPN and NRTP (levels 1-10; scale bar = 500 µm). Ventral, rostral and medial views constructed to show BPN in compressed 3D form (in line with the previous and collaborative work from Oslo University); see specific map for orientation.
<table><thead><tr><th>Case</th><th>Zone Injected</th><th>Tracer Injected</th><th>IO Cells</th><th>Doubles IO</th><th>Cells Ipsil BPN</th><th>Cells Contra BPN</th><th>Doubles BPN</th></tr></thead><tbody><tr><td>BR03</td><td>A2 Face C1 FL</td><td>Red Green</td><td>794 76</td><td>38</td><td>1940 23</td><td>1778 394</td><td>57</td></tr><tr><td>BR05</td><td>A2 Face C1 FL</td><td>Green Red</td><td>108 318</td><td>0</td><td>359 112</td><td>171 503</td><td>7</td></tr><tr><td>BR06</td><td>A2 Face C1 FL</td><td>Green Red</td><td>246 140</td><td>1</td><td>1183 108</td><td>817 604</td><td>30</td></tr><tr><td>BR12</td><td>A2 Face C1 FL</td><td>Green Red</td><td>63 1109</td><td>15</td><td>331 487</td><td>388 1593</td><td>96</td></tr><tr><td>BR13</td><td>A2 Face C1 HL</td><td>Red Green</td><td>53 10</td><td>0</td><td>250 0</td><td>152 4</td><td>0</td></tr><tr><td>BR14</td><td>C1 FL C1 HL</td><td>Red Green</td><td>100 392</td><td>0</td><td>58 109</td><td>421 1222</td><td>0</td></tr><tr><td>BR15</td><td>A2 Face C1 FL</td><td>Green Red</td><td>38 427</td><td>12</td><td>220 799</td><td>292 1263</td><td>176</td></tr><tr><td>BR16</td><td>A2 Face C1 HL</td><td>Red Green</td><td>275 51</td><td>0</td><td>611 9</td><td>1702 377</td><td>0</td></tr></tbody></table>

<figure><img src="image_17.png" /><figcaption>**Table 3.1: Numbers of labelled cells in the ponto-cerebellar tracer cases.**</figcaption></figure>

The table shows the experiments where red and green beads were injected per case into the cerebellum. The cell counts are absolute numbers. The numbers of doubles are included in the actual cell counts. The graph shows the positive correlation of number of cells counted in BPN to number of cell in the inferior olive.
<table><caption>**Table 3.2: Details of cases used in 3D reconstruction and analysis.**</caption><thead><tr><th>Case</th><th>Physiology Width</th><th>Injection Width</th><th>Injection Site size</th><th>Distance between cores</th><th>% IO Cells</th></tr></thead><tbody><tr><td>BR05 Face</td><td>-</td><td>300</td><td>0.082</td><td>600</td><td>94.4</td></tr><tr><td>BR13 Face</td><td>300</td><td>200</td><td>0.017</td><td>300</td><td>100</td></tr><tr><td>BR15 Face</td><td>400</td><td>200</td><td>0.040</td><td>400</td><td>94.7</td></tr><tr><td>BR03 FL</td><td>800</td><td>200</td><td>0.066</td><td>350</td><td>100</td></tr><tr><td>BR06 FL</td><td>500</td><td>100</td><td>0.007</td><td>600</td><td>98.6</td></tr><tr><td>BR14 FL</td><td>600</td><td>200</td><td>0.041</td><td>600</td><td>100</td></tr><tr><td>BR13 HL</td><td>400</td><td>100</td><td>0.004</td><td>300</td><td>100</td></tr><tr><td>BR14 HL</td><td>700</td><td>400</td><td>0.125</td><td>600</td><td>100</td></tr><tr><td>BR16 HL</td><td>1400</td><td>300</td><td>0.094</td><td>200</td><td>100</td></tr></tbody></table>

The table shows data used in determining the cases to be analysed. Physiological width, injection width and distance between cores (per case in each animal) in µm; injection site size in mm²; % IO cells denotes the percentage of cells in the inferior olive identified in the expected area for the given zonal injection; -, not able to determine.
<figure><img src="image_18.png" /><figcaption>**Figure 3.1: Physiological responses and anatomical labelling of two cases in the same animal. (BR14)**</figcaption></figure>

The left column represents an injection of red beads into the C1 forelimb-receiving zone and right column represents an injection of green beads into the C1 hindlimb-receiving zone in the same animal. (i) Physiological recordings of climbing fibre field potentials (average of n=4 sweeps) in the approximate centre of each zone; arrow denotes time of peripheral stimulation. (ii) Outline view of the posterior right cerebellum displaying location of tracer injections. (iii) Photomicrographs of sagittal cerebellar section at the level of the injection site core. (iv) Camera lucida drawings of transverse sections of BPN (approximately -8 mm bregma) where the boxes indicate the location of the (v) cell labelling in the high power photomicrographs. Scale bar = (i) 5 ms; 0.1 mV and 0.05 mV for fore- and hindlimb responses respectively; (ii) 1 mm (iii) 500 µm (iv) 200 µm (v) 20 µm.
<figure><img src="image_19.png" /><figcaption>**Figure 3.2: Reconstruction of two cases in the same animal. (BR14)**</figcaption></figure>

(A) Reconstructions of BPN projection to C1 forelimb injection (red dots) and C1 hindlimb injection (green dots). (i) Ventral view; (ii) rostral view; (iii) medial view; (B) 3D reconstruction with BPN shown as transparent grey surfaces and LFP shown as grey solids. Scale bar = 500 µm in A, 1 mm in B.
<figure><img src="image_20.png" /><figcaption>Figure 3.3: Inferior olive and BPN cell labelling in three cases, representative of each injection site.</figcaption></figure>

Standard horizontal maps of the right inferior olive showing the areas occupied by retrogradely labelling (A = A2 face BR15; B = C1 forelimb BR06; C = C1 hindlimb BR14). Ventral, medial and rostral views of BPN constructed using the local coordinate system showing individually labelled cells. Outline of cerebellum indicates the approximate location of each injection, with the below figurine showing the physiologically defined mediolateral width of the zone and the coloured box, the anatomically measured extent of injection site. Scale bar = 0.5 mm BPN and inferior olive; 3 mm cerebellum; 0.2 mm injection site.
<figure><img src="image_21.png" /><figcaption>Figure 3.4: Surface representation reconstructions of accumulated cases of BPN labelling from cerebellar and SI injections.</figcaption></figure>

(A) The top diagrams show the cases included for each zone injected into, with the far right displaying how the labelling fits together in BPN. (B) The bottom labelling from injections into SI (primary somatosensory cortex) show comparison similarly labelled areas in BPN and how they fit together. Scale bar = 0.5 mm.
<table><thead><tr><th></th><th>A2 Face Cells</th><th>C1 FL Cells</th><th>C1 HL Cells</th></tr></thead><tbody><tr><th>SI Face Terminals</th><td>4.7%</td><td>11.2%</td><td>0.6%</td></tr><tr><th>SI FL Terminals</th><td>0.6%</td><td>6.2%</td><td>6.9%</td></tr><tr><th>SI HL Terminals</th><td>0.6%</td><td>0.1%</td><td>11.4%</td></tr></tbody></table>

<table><caption>Table 3.3: Cell labelling and terminal overlap analysis, as determined using the local pontine co-ordinate system.</caption><thead><tr><th></th><th>SI Face Terminals</th><th>SI FL Terminals</th><th>SI HL Terminals</th></tr></thead><tbody><tr><th>A2 Face Cells</th><td>5.3%</td><td>1.1%</td><td>0.09%</td></tr><tr><th>C1 FL Cells</th><td>2.6%</td><td>4.0%</td><td>0.07%</td></tr><tr><th>C1 HL Cells</th><td>0.4%</td><td>7.0%</td><td>9.3%</td></tr></tbody></table>

(A) Pontine cell and terminal overlap expressed as a percentage of the total BPN volume occupied by labelled cells from a given climbing fibre zone. (B) Overlap expressed as a percentage of the total BPN volume occupied by axon terminals from a given SI cerebral body representation.

Ponto-cerebellar

Cerebro-pontine
<figure><img src="image_22.png" /><figcaption>Face SI</figcaption></figure>

<figure><img src="image_23.png" /><figcaption>FL SI</figcaption></figure>

<figure><img src="image_24.png" /></figure>
<figure><img src="image_25.png" /><figcaption>Figure 3.5: 3D dot map reconstructions of accumulated cases.</figcaption></figure>

On the left, dot map accumulated cases for each of the cerebellar injection sites. On the right, dot maps of cerebro-pontine labelling into SI (from Leergaard *et al.*, 2000b) using the same local pontine co-ordinate system, for comparison of the cerebro-ponto-cerebellar pathway. Scale bar = 0.5 mm BPN maps; 3 mm cerebellum.
<table><caption>**Table 4.2: Overview of anatomical tract tracer experiments.**</caption><thead><tr><th>Expt</th><th>FL or HL</th><th>Motor Cortex (RHS)</th><th>Cerebellum</th></tr></thead><tbody><tr><td>DP1</td><td>HL</td><td>FR HL (100nl)</td><td>GB CP HL (?nl) LHS</td></tr><tr><td>DP2</td><td>FL</td><td>FE caudal FL (100nl)<br>FR rostral FL (100nl)</td><td>GB PML FL(100nl) RHS<br>RB PML FL(100nl) LHS</td></tr><tr><td>DP3</td><td>FL</td><td>FR FL (125nl)</td><td>GB PML FL(150nl) LHS</td></tr><tr><td>DP8</td><td>FL</td><td>FE caudal FL (175nl)<br>FR rostral FL (250nl)</td><td>GB PML FL(100nl) RHS<br>RB PML FL(100nl) LHS</td></tr><tr><td>DP9</td><td>FL</td><td>FR FL (200nl)</td><td>GB PML FL (100nl) RHS</td></tr><tr><td>DP10</td><td>FL</td><td>FR FL (150nl)</td><td>GB PML FL (75nl) RHS</td></tr><tr><td>DP11</td><td>FL + HL</td><td>FE FL (3x100nl)<br>FR HL (3x100nl)</td><td>RB PML FL (50nl) LHS<br>GB CP HL (50nl) LHS</td></tr><tr><td>DP12</td><td>HL</td><td>FR HL (3x100nl)</td><td>GB CP HL (75nl) LHS</td></tr><tr><td>DP13</td><td>FL</td><td>FR FL (1x300nl &amp; 1x100nl)</td><td>GB PML FL (50nl) LHS</td></tr><tr><td>DP14</td><td>HL</td><td>FR HL (1x350nl &amp; 1x150nl)</td><td>GB CP HL (75nl) LHS</td></tr><tr><td>DP15</td><td>FL + HL</td><td>FR HL (250nl)</td><td>GB PML FL (50nl) LHS<br>RB CP HL (50nl) LHS</td></tr><tr><td>DP16</td><td>FL + HL</td><td>BDA FL (300nl)<br>FR HL (200nl)</td><td>RB PML FL (50nl) LHS<br>GB CP HL (50nl) LHS</td></tr><tr><td>DP17</td><td>FL + HL</td><td>FR FL (300nl @ 1.5mm)<br>BDA HL (300nl @ 1.5mm)</td><td>GB PML FL (50nl) LHS<br>RB CP HL (50nl) LHS</td></tr><tr><td>DP18</td><td>FL + HL</td><td>BDA FL (?nl @ 1.5mm)<br>FR HL (300nl @ 1.5mm)</td><td>RB PML FL (50nl) LHS<br>GB CP HL (50nl) LHS</td></tr><tr><td>DP19</td><td>FL + HL</td><td>BDA HL (?nl @ 1.5mm)<br>FR FL (100nl @ 1.5 mm)</td><td>GB CP HL (75nl) LHS</td></tr><tr><td>DP20</td><td>FL + HL</td><td>FR FL (?nl @ 1.7mm)<br>BDA HL (300nl @ 1.7mm)</td><td>GB PML FL (60nl) LHS<br>RB CP HL (60nl) LHS</td></tr><tr><td>DP21</td><td>FL</td><td>FR FL (150nl @ 1.5mm)</td><td>GB PML FL (50nl) LHS</td></tr><tr><td>DP22</td><td>HL</td><td>FR HL (200nl @ 1.5mm)</td><td>GB CP HL (50nl) LHS</td></tr><tr><td>DP23</td><td>FL + HL</td><td>FR FL (3 x 100nl)<br>BDA HL (3 x 100nl)</td><td>GB CP HL (50nl) LHS</td></tr><tr><td>DP24</td><td>FL + HL</td><td>BDA FL (3 x 100nl)<br>FR HL (3 x 100nl)</td><td>GB CP HL (50nl) LHS</td></tr><tr><td>DP25</td><td>FL + HL</td><td>FR FL (3 x 100nl)<br>FE HL (3 x 100nl)</td><td>GB CP HL (70nl) LHS</td></tr><tr><td>DP26</td><td>FL + HL</td><td>FE FL (3 x 100nl)<br>FR HL (3 x 100nl)</td><td>GB CP HL (50nl) LHS</td></tr><tr><td>DP27</td><td>FL + HL</td><td>FR FL (3 x 100nl)<br>FE HL (3 x 100nl)</td><td>GB PML FL (50nl) LHS</td></tr></tbody></table>

The table shows the experimental series of the injection of tracer material (and volume injected) into the cerebral and cerebellar cortices. These experiments were histologically investigated and those highlighted in blue were deemed to have 'successful' transport of tracer. This was determined for the anterograde cerebral injection by terminal labelling in BPN, and for the retrograde cerebellar injection, at least 80% of the cell labelling was in the expected area(s) in the inferior olive. Key: BDA – biotinylated dextran amine; CP – copula pyramidis; FE – Fluoro-Emerald; FL – forelimb; FR – Fluoro-Ruby; HL – hindlimb; LHS – left hand side (of the cerebellum); PML – paramedian lobule; RHS – right hand side (of the cerebellum); ? – volume of tracer injected not recorded.
<figure><img src="image_26.png" /><figcaption>**Figure 4.1: Schematic map of reconstruction of injection sites in cerebral cortex.**</figcaption></figure>

This histological reconstruction of successful anterograde injection site cases (N.B. DP13FL was not confirmed histologically). The circle and horizontal grey line indicate Bregma. The injections were located in the forelimb (rostral of Bregma) and hindlimb (caudal of Bregma) left motor cortex. The below denote the cases (see Tables 4.1 and 4.2 for further injection site information):

<table><tr><td>1 = DP1 (HL)</td><td>5 = DP25 (HL)</td><td>9 = DP25 (FL)</td></tr><tr><td>2 = DP11 (HL)</td><td>6 = DP26 (HL)</td><td>10 = DP26 (FL)</td></tr><tr><td>3 = DP14 (HL)</td><td>7 = DP27 (HL)</td><td>11 = DP27 (FL)</td></tr><tr><td>4 = DP24 (HL)</td><td>8 = DP23 (FL)</td><td></td></tr></table>

The schematic cerebral cortex map is modified from Coleman *et al.* (1997). Key: AGC – agranular (motor) cortex; GC – granular (somatosensory) cortex.
<figure><img src="image_27.png" /></figure>

<figure><img src="image_28.png" /><figcaption>**Figure 4.2: Examples of photomicrographs of motor cortex injection sites.**</figcaption></figure>

(A) The photomicrograph shows an injection of Fluoro-Ruby into the forelimb motor cortex. (B) Shows a photomicrograph of an injection of Fluoro-Emerald into the hindlimb motor cortex. These photomicrographs were taken from the same experiment (DP26). Scale bars = 500 µm.
<table><caption>**Table 4.3: Details of anterograde injection sites in the motor cortex that gave terminal labelling in BPN.**</caption><thead><tr><th>Case</th><th>Trac-er</th><th>Volume (nl)</th><th>r-c (μm)</th><th>m-l (μm)</th><th>d-v (μm)</th><th>Depth (mm)</th><th>Distance from Bregma</th><th>Injection site (mm³)</th></tr></thead><tbody><tr><td>DP13 FL</td><td>FR</td><td>300 + 100</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>DP23 FL</td><td>FR</td><td>3 x 100</td><td>300</td><td>270</td><td>175</td><td>1.7</td><td>0 mm</td><td>0.059</td></tr><tr><td>DP25 FL</td><td>FR</td><td>3 x 100</td><td>400</td><td>135</td><td>175</td><td>1.5</td><td>0.4 mm</td><td>0.039</td></tr><tr><td>DP26 FL</td><td>FE</td><td>3 x 100</td><td>100</td><td>90</td><td>210</td><td>1.6</td><td>0 mm</td><td>0.008</td></tr><tr><td>DP27 FL</td><td>FR</td><td>3 x 100</td><td>300</td><td>70</td><td>475</td><td>1.8</td><td>0.2 mm</td><td>0.042</td></tr><tr><td>DP1 HL</td><td>FR</td><td>100</td><td>300</td><td>190</td><td>265</td><td>1.6</td><td>-0.2 mm</td><td>0.063</td></tr><tr><td>DP11 HL</td><td>FR</td><td>3 x 100</td><td>300</td><td>270</td><td>115</td><td>1.2</td><td>-1.6 mm</td><td>0.039</td></tr><tr><td>DP14 HL</td><td>FR</td><td>350</td><td>300</td><td>475</td><td>165</td><td>1.7</td><td>-1 mm</td><td>0.098</td></tr><tr><td>DP24 HL</td><td>FR</td><td>3 x 100</td><td>300</td><td>105</td><td>300</td><td>1.5</td><td>-0.4 mm</td><td>0.039</td></tr><tr><td>DP25 HL</td><td>FE</td><td>3 x 100</td><td>200</td><td>70</td><td>300</td><td>1.6</td><td>-1.4 mm</td><td>0.018</td></tr><tr><td>DP26 HL</td><td>FR</td><td>3 x 100</td><td>300</td><td>50</td><td>585</td><td>1.7</td><td>-1.4 mm</td><td>0.037</td></tr><tr><td>DP27 HL</td><td>FE</td><td>3 x 100</td><td>200</td><td>155</td><td>140</td><td>1.6</td><td>-1.6 mm</td><td>0.018</td></tr></tbody></table>

The table shows a summary of the injection details for all the cases where terminal labelling was found in BPN. From left to right, the columns show case number (HL and FL refer respectively to injections into hindlimb and forelimb parts of the motor cortex); type of tracer (FE, Fluoro-Emerald, FR, Fluoro-Ruby); volume of tracer injected (nl); then details of the dimensions of the injectate rostrocaudally (r-c), mediolaterally (m-l), and dorsoventral (d-v), all in µm; the depth of the centre of the injection site from the cerebro cortical surface (mm); the distance of the injection from Bregma (mm); and the total calculated volume of injectate (mm³); –, histological data not available. The volume of the injectate was calculated by multiplying values together for volume of an ellipsoid.
<figure><img src="image_29.png" /></figure>

<figure><img src="image_30.png" /><figcaption>Figure 4.3: Examples of photomicrographs of cerebellar cortex injection sites.</figcaption></figure>

(A) The photomicrograph shows an injection of red beads into the C1 FL zone in PML. (B) Shows a photomicrograph of an injection of green beads into the C1 HL zone in CP. These photomicrographs were taken from the same experiment (DP11).
Scale bars = 500 µm.
<table><thead><tr><th>Case</th><th>Tracer</th><th>Volume (nl)</th><th>m-l (μm)</th><th>Distance to mid-line (mm)</th><th>WM interface (μm)</th><th>Injection site (mm²)</th></tr></thead><tbody><tr><td>DP1 HL</td><td>GB</td><td>50</td><td>300</td><td>3.9</td><td>455</td><td>0.107</td></tr><tr><td>DP2 FL</td><td>GB</td><td>100</td><td>300</td><td>1.9</td><td>328</td><td>0.051</td></tr><tr><td>DP2 FL</td><td>RB</td><td>100</td><td>300</td><td>2.8</td><td>419</td><td>0.099</td></tr><tr><td>DP10 FL</td><td>GB</td><td>75</td><td>300</td><td>1.7</td><td>348</td><td>0.082</td></tr><tr><td>DP11 FL</td><td>RB</td><td>50</td><td>300</td><td>2.0</td><td>188</td><td>0.030</td></tr><tr><td>DP11 HL</td><td>GB</td><td>50</td><td>200</td><td>1.6</td><td>376</td><td>0.059</td></tr><tr><td>DP12 HL</td><td>GB</td><td>75</td><td>200</td><td>1.3</td><td>684</td><td>0.107</td></tr><tr><td>DP13 FL</td><td>GB</td><td>50</td><td>300</td><td>2.0</td><td>624</td><td>0.147</td></tr><tr><td>DP14 HL</td><td>GB</td><td>75</td><td>200</td><td>1.4</td><td>328</td><td>0.052</td></tr><tr><td>DP25 HL</td><td>GB</td><td>75</td><td>200</td><td>1.2</td><td>90</td><td>0.014</td></tr><tr><td>DP26 HL</td><td>GB</td><td>50</td><td>200</td><td>1.7</td><td>353</td><td>0.055</td></tr><tr><td>DP27 FL</td><td>GB</td><td>50</td><td>200</td><td>2.1</td><td>445</td><td>0.070</td></tr></tbody></table>

**Table 4.4: Details of successful retrograde injection sites in the cerebellar cortex.**
The table shows a summary of the injection details for all the cases where cell labelling was found in BPN and in the expected area(s) of the inferior olive for the injection site. From left to right, the columns show case number (HL and FL refer respectively to injections into the forelimb C1 zone in PML or the hindlimb C1 zone in CP in the cerebellum); type of tracer (GB, green beads; RB, red beads); volume of tracer injected (nl); the mediolateral (m-l) size of the core of the injectate (μm); the distance to the anatomical midline from the middle of the core (mm); the length of the white matter (WM) interface (μm); and the injection site size (mm²). The area of the injectate for the injection site size calculation was found from multiplying the values together for the area of an ellipse (see King *et al.* [1998] for further details).
<figure><img src="image_31.png" /><figcaption>Figure 4.4: Location of retrogradely labelled cells in the inferior olive.</figcaption></figure>

The figure shows the individual cases for retrogradely labelled cells for injections into forelimb-receiving C1 PML (red; n = 6) and for injections into hindlimb-receiving C1 CP (green; n = 6). Key: DL – dorsal lamella; PO – principal olive; VL – ventral lamella. Each case is denoted by its case number (DPxx) and the C1 injection site (forelimb [FL] or hindlimb [HL] receiving). Scale = 500 µm.
(A) Inferior Olive Cell Counts

<table><thead><tr><th>Case</th><th>Olive (Contra)</th><th>MAO</th><th>DAO</th><th>% Correct in Olive</th></tr></thead><tbody><tr><td>DP2 FL</td><td>3</td><td>2</td><td>1</td><td>100</td></tr><tr><td>DP2 FL</td><td>678</td><td>577</td><td>71</td><td>100</td></tr><tr><td>DP10 FL</td><td>30</td><td>4</td><td>26</td><td>100</td></tr><tr><td>DP11 FL</td><td>266</td><td>55</td><td>211</td><td>100</td></tr><tr><td>DP13 FL</td><td>75</td><td>24</td><td>51</td><td>100</td></tr><tr><td>DP27 FL</td><td>11</td><td>2</td><td>13</td><td>100</td></tr><tr><td>DP1 HL</td><td>37</td><td>6</td><td>31</td><td>83.8</td></tr><tr><td>DP11 HL</td><td>79</td><td>0</td><td>79</td><td>97.4</td></tr><tr><td>DP12 HL</td><td>119</td><td>4</td><td>115</td><td>95.5</td></tr><tr><td>DP14 HL</td><td>120</td><td>0</td><td>120</td><td>100</td></tr><tr><td>DP25 HL</td><td>1</td><td>0</td><td>1</td><td>100</td></tr><tr><td>DP26 HL</td><td>11</td><td>0</td><td>11</td><td>100</td></tr></tbody></table>

(B) Pontine Cell Counts

<table><caption>**Table 4.5: Cell counts for the successful retrograde tracer cases.**</caption><thead><tr><th>Case</th><th>BPN Ipsilateral</th><th>BPN Contra</th><th>NRTP Ipsilateral</th><th>NRTP Contra</th></tr></thead><tbody><tr><td>DP2 FL</td><td>4</td><td>103</td><td>3</td><td>3</td></tr><tr><td>DP2 FL</td><td>63</td><td>946</td><td>15</td><td>8</td></tr><tr><td>DP10 FL</td><td>137</td><td>230</td><td>13</td><td>34</td></tr><tr><td>DP11 FL</td><td>137</td><td>400</td><td>33</td><td>32</td></tr><tr><td>DP13 FL</td><td>47</td><td>286</td><td>21</td><td>22</td></tr><tr><td>DP27 FL</td><td>8</td><td>185</td><td>0</td><td>2</td></tr><tr><td>DP1 HL</td><td>0</td><td>171</td><td>0</td><td>0</td></tr><tr><td>DP11 HL</td><td>13</td><td>272</td><td>0</td><td>0</td></tr><tr><td>DP12 HL</td><td>23</td><td>823</td><td>0</td><td>0</td></tr><tr><td>DP14 HL</td><td>32</td><td>547</td><td>0</td><td>0</td></tr><tr><td>DP25 HL</td><td>2</td><td>17</td><td>0</td><td>0</td></tr><tr><td>DP26 HL</td><td>0</td><td>87</td><td>0</td><td>0</td></tr></tbody></table>

The table shows the raw cell counts for the cases where the injection in the cerebellum was in the target (as defined as having over 80% of the olivary labelling in the expected region for the injection site), where olivary data is shown in (A) and pontine in (B). The olivary projection was contralateral, whereas the pontine projection was bilateral. The pontine cell counts are broken down into contralateral side and ipsilateral side labelling from injections into the right side of the cerebellum, and show data for both BPN and NRTP. Key: BPN – basilar pontine nuclei; Contra – contralateral; DAO – dorsal accessory olive; MAO – medial accessory olive; NRTP – nucleus reticularis tegmenti pontis.
<figure><img src="image_32.png" /><figcaption>**Figure 4.5: Terminal labelling in BPN from a motor cortical injection of tracer.**
These photomicrographs show anterograde labelling from an injection of Fluoro-Ruby into the hindlimb motor cortex. (A) Low power view of BPN with heavy terminal labelling in the medial part of the right BPN, with some medially left. (B) Photomicrograph shows a high power view of the terminal labelling above. Scale bars, A = 500 µm; B = 50 µm (DP11). LFP – longitudinal fasciculus of the pons.</figcaption></figure>
A

<figure><img src="image_33.png" /></figure>

B

<figure><img src="image_34.png" /><figcaption>**Figure 4.6: Cell labelling in BPN from an injection into the cerebellum.**</figcaption></figure>

These photomicrographs show retrograde labelling from an injection of green beads into the forelimb-receiving C1 zone in PML. (A) Low power view of BPN with patches of cell labelling. (B) Photomicrograph shows a high power view of the cell labelling above. Scale bars, A = 200 µm; B = 50 µm (DP11). LFP – longitudinal fasciculus of the pons.
<figure><img src="image_35.png" /></figure>

<figure><img src="image_36.png" /></figure>

<figure><img src="image_37.png" /></figure>

<figure><img src="image_38.png" /></figure>

<figure><img src="image_39.png" /></figure>

<figure><img src="image_40.png" /><figcaption>Figure 4.7: Average and individual cases of forelimb terminal labelling in BPN.</figcaption></figure>

These figures show both sides of BPN from the ventral angle and the density of labelling for every 150 µm², where blue shading indicates light terminal labelling through to red which indicates dense terminal labelling.
<figure><img src="image_41.png" /><figcaption>Figure 4.8: Full documented case of example ‘forelimb’ BPN labelling. (DP27)</figcaption></figure>

The figure shows a full cerebro-ponto-cerebellar case for injections into the forelimb motor cortex and forelimb-receiving C1 zone in PML in the cerebellum. The cerebellum figurine demonstrates the recording site, with corresponding evoked physiological responses (3 overlaid) for both cerebral and forelimb stimulation (at 3 times threshold). Photomicrographs of the injection sites for both tracer injections are also shown (Fluoro-Ruby into the cerebral cortex; green beads in the cerebellum). The horizontal maps show labelling in the left inferior olive and bilaterally in BPN. The cell labelling in the olive was in the expected regions for the injection. The right and left BPN are shown for spread of terminal labelling and cell labelling. Scale: 500 µm for photomicrographs and horizontal maps; 2 mm cerebellar figurine.
<figure><img src="image_42.png" /><figcaption>Figure 4.9: Total extent of ‘forelimb’ BPN terminal labelling. (n = 5)</figcaption></figure>

Terminal labelling found in BPN from injections of anterograde tracer into the forelimb projecting area in the motor cortex. The figure is coded to the density of the terminal labelling (none; very light; light; medium; dense). Scale bar = 500 µm.
Caudal

<figure><img src="image_43.png" /></figure>

1

<figure><img src="image_44.png" /></figure>

2

<figure><img src="image_45.png" /></figure>

3

<figure><img src="image_46.png" /></figure>

4

<figure><img src="image_47.png" /></figure>

5

<figure><img src="image_48.png" /></figure>

6

<figure><img src="image_49.png" /></figure>

7

<figure><img src="image_50.png" /></figure>

8

<figure><img src="image_51.png" /></figure>

9

<figure><img src="image_52.png" /></figure>

10

<figure><img src="image_53.png" /></figure>

Rostral

<caption>**Figure 4.10: Total extent of ‘forelimb’ BPN cell labelling.** (n = 6)</caption>

These standard transverse maps show the total areas covered by ‘pure’ C1 FL PML injections, as defined by the olivary labelling (n = 6). The shaded areas represent dense cell labelling (scale bar = 500 µm).
<figure><img src="image_54.png" /><figcaption>Figure 4.11: Average and individual cases of hindlimb terminal labelling in BPN.</figcaption></figure>

These figures show both sides of BPN from the ventral angle and the density of labelling for every 150 µm², where blue shading indicates light terminal labelling through to red which indicates dense terminal labelling.
<figure><img src="image_55.png" /><figcaption>**Figure 4.12: Full documented case of example ‘hindlimb’ BPN labelling. (DP25)**
The figure shows a full cerebro-ponto-cerebellar case for injections into the hindlimb motor cortex and hindlimb-receiving C1 zone in CP in the cerebellum. The cerebellum figurine demonstrates the recording site, with corresponding evoked physiological responses (3 overlaid) for both cerebral and forelimb stimulation (at 3 times threshold). Photomicrographs of the injection sites for both tracer injections are also shown (Fluoro-Emerald into the cerebral cortex; green beads in the cerebellum). The horizontal maps show labelling in the left inferior olive and bilaterally in BPN. The cell labelling in the olive was in the expected regions for the injection. The right and left BPN are shown for spread of terminal labelling and cell labelling. Scale: 500 µm for photomicrographs and horizontal maps; 2 mm cerebellar figurine.</figcaption></figure>
<figure><img src="image_56.png" /><figcaption>**Figure 4.13: Total extent of ‘hindlimb’ BPN terminal labelling.** (n = 7)</figcaption></figure>

Terminal labelling found in BPN from injections of anterograde tracer into the hindlimb projecting area in the motor cortex. The figure is coded to the density of the terminal labelling (none; very light; light; medium; dense). Scale bar = 500 µm.
<figure><img src="image_57.png" /><figcaption>**Figure 4.14: Total extent of ‘hindlimb’ BPN cell labelling.** (n = 6)</figcaption></figure>

These standard transverse maps show the total areas covered by ‘pure’ C1 FL PML injections, as defined by the olivary labelling (n = 6). The shaded areas represent dense cell labelling (scale bar = 500 µm).
(A) Corresponding limb injections:

<table><thead><tr><td>Case</td><td>% Cells Compared to TL</td><td>% TL Compared to Cells</td></tr></thead><tbody><tr><td>DP13 FL</td><td>25.8</td><td>55.3</td></tr><tr><td>DP27 FL</td><td>8.8</td><td>3.5</td></tr><tr><td>DP1 HL</td><td>46.7</td><td>77.7</td></tr><tr><td>DP11 HL</td><td>56.0</td><td>55.3</td></tr><tr><td>DP14 HL</td><td>11.8</td><td>160.3</td></tr><tr><td>DP25 HL</td><td>63.5</td><td>4.8</td></tr><tr><td>DP26 HL</td><td>50.0</td><td>41.0</td></tr></tbody></table>

(B) Non-corresponding limb injections:

<table><caption>**Table 4.6: Individual overlaps in BPN in cases with both successful cerebral and cerebellar injections.**</caption><thead><tr><td>Case</td><td>% Cells Compared to TL</td><td>% TL Compared to Cells</td></tr></thead><tbody><tr><td>DP25 FL TL/HL cells</td><td>38.5</td><td>2.2</td></tr><tr><td>DP26 FL TL/HL cells</td><td>45.4</td><td>15.9</td></tr><tr><td>DP11 HL TL/FL cells</td><td>5.1</td><td>13.0</td></tr><tr><td>DP27 HL TL/FL cells</td><td>9.8</td><td>8.9</td></tr></tbody></table>

(A) The table shows the overlap in BPN of cell and terminal labelling (TL) in cases where the full cerebro-ponto-cerebellar pathway was mapped in the same animal for corresponding limb injection sites (e.g. FL cerebral and FL cerebellar). (B) The table shows the overlap in BPN of cell and terminal labelling in cases where the full cerebro-ponto-cerebellar pathway was mapped for non-corresponding injection sites (e.g. FL cerebral and HL cerebellar). There was significantly more overlap in the cases where there were corresponding injections. The percentages are expressed as the total overlap as a percentage of area of the cell labelling (left columns) or of the terminal labelling (right columns).
<table><caption>Average percentages of overlap between labelled areas in BPN from different cerebral and cerebellar injection sites</caption><thead><tr><th colspan="2"></th><th colspan="4">A</th></tr><tr><th colspan="2"></th><th>FL Cells</th><th>FL TL</th><th>HL Cells</th><th>HL TL</th></tr></thead><tbody><tr><th rowspan="4" scope="rowgroup">B</th><th scope="row">FL Cells</th><td></td><td>20.2 %</td><td>14.3 %</td><td>0.003 %</td></tr><tr><th scope="row">FL TL</th><td>23.8 %</td><td></td><td>27.5 %</td><td>25.5 %</td></tr><tr><th scope="row">HL Cells</th><td>7.8 %</td><td>12.8 %</td><td></td><td>15.3 %</td></tr><tr><th scope="row">HL TL</th><td>30.3 %</td><td>22.1 %</td><td>28.6 %</td><td></td></tr></tbody></table>

A = Labelling from injection

B = Overlapped with

<caption>**Table 4.7 Pooled overlaps in BPN cases with successful cerebral and cerebellar injections.**</caption>

The table expresses the overall pooled findings for the percentage overlaps. Columns in 'A' represent the site of injection compared to rows in 'B' to gain the overlap for all combinations of labelling. Combinations of 'forelimb' (FL TL) and 'hindlimb' (HL TL) motor cortex injections producing terminal labelling and C1 forelimb-receiving PML (FL Cells) and C1 hindlimb-receiving CP cerebellar zonal injections producing labelled cells. The percentages are therefore expressed as the total overlap as a percentage of area of the cell labelling or of the terminal labelling.
<figure><img src="image_58.png" /><figcaption>Figure 4.15: Overlap of ‘forelimb’ terminal labelling and ‘forelimb’ cell labelling in BPN.</figcaption></figure>

The figure shows the overlap from labelling produced by injections of anterograde tracer into forelimb motor cortex area and retrograde tracer into the forelimb-receiving C1 PML zone in the cerebellum. Scale bar = 500 µm.
<figure><img src="image_59.png" /><figcaption>Figure 4.16: Overlap of ‘hindlimb’ terminal labelling and ‘hindlimb’ cell labelling in BPN.</figcaption></figure>

The figure shows the overlap from labelling produced by injections of anterograde tracer into hindlimb motor cortex area and retrograde tracer into the hindlimb-receiving C1 CP zone in the cerebellum. Scale bar = 500 µm.
<figure><img src="image_60.png" /><figcaption>Figure 4.17: Overlap of ‘forelimb’ terminal labelling and ‘hindlimb’ cell labelling in BPN.</figcaption></figure>

The figure shows the overlap from labelling produced by injections of anterograde tracer into forelimb motor cortex area and retrograde tracer into the hindlimb-receiving C1 CP zone in the cerebellum. Scale bar = 500 µm.
<figure><img src="image_61.png" /><figcaption>Figure 4.18: Overlap of ‘hindlimb’ terminal labelling and ‘forelimb’ cell labelling in BPN.</figcaption></figure>

The figure shows the overlap from labelling produced by injections of anterograde tracer into hindlimb motor cortex area and retrograde tracer into the forelimb-receiving C1 PML zone in the cerebellum. Scale bar = 500 µm.
<figure><img src="image_62.png" /><figcaption>Figure 4.19: Areas of total overlap in BPN.</figcaption></figure>

The figure shows the overlap from labelling produced from all cerebral and cerebellar injections, thus the areas of total overlap. Scale bar = 500 µm.
<figure><img src="image_63.png" /><figcaption>Figure 4.20: Photomicrographs and ventral map of NRTP cell labelling.</figcaption></figure>

(A i) The photomicrograph shows an overview of mid-NRTP in the transverse plane (scale bar = 200 µm; white arrow points to a labelled cell) and (A ii), a high power photomicrograph of typical cells found (scale bar = 100 µm) in NRTP. (B) The total extent of cell labelling is shown in ventral NRTP standard maps (constructed from transverse maps and areas of labelling; scale bar = 500 µm).
(A) Peripheral Forelimb Stimulation

T

2T

3T

(B) Cerebral Forelimb Area Stimulation

(C) Peripheral Hindlimb Stimulation

T

2T

3T

(D) Cerebral Hindlimb Area Stimulation
<figure><img src="image_64.png" /><figcaption>**Figure 5.1:** Evoked climbing fibre field potentials to peripheral and central limb stimulation. Stimulation intensities of three consecutive sweeps at threshold response (T), twice threshold (2T) and three times threshold (3T). (A) Responses to ipsilateral forelimb stimulation (T=0.35V). (B) Responses to double pulse stimulation of the contralateral forelimb the motor cortex (T=0.23mA). (C) Responses to ipsilateral hindlimb stimulation (T=0.70V). (D) Responses to double pulse stimulation of the contralateral hindlimb motor cortex (T=0.26mA). Arrow indicates time of stimulation; scale bars = 0.1 mV vertical; 10 ms horizontal (case DPE23).</figcaption></figure>
<table><caption>**Table 5.1: Evoked latencies per case for climbing fibre field recordings.**</caption><thead><tr><th>Case</th><th>Ip FL PML</th><th>Co FL Ctx PML</th><th>Ip FL Ctx PML</th><th>Ip FL LS</th><th>Co FL Ctx LS</th><th>Ip FL Ctx LS</th><th>Ip HL CP</th><th>CoHL Ctx CP</th></tr></thead><tbody><tr><td>DPE2</td><td>9.93</td><td>12.41</td><td>-</td><td>-</td><td>-</td><td>-</td><td>14.33</td><td>19.00</td></tr><tr><td>DPE3</td><td>12.12</td><td>11.71</td><td>-</td><td>-</td><td>-</td><td>-</td><td>17.69</td><td>21.59</td></tr><tr><td>DPE4</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>15.88</td><td>20.10</td></tr><tr><td>DPE5</td><td>10.87</td><td>10.63</td><td>-</td><td>-</td><td>-</td><td>-</td><td>14.89</td><td>19.02</td></tr><tr><td>DPE6</td><td>11.76</td><td>11.52</td><td>-</td><td>-</td><td>-</td><td>-</td><td>17.69</td><td>17.08</td></tr><tr><td>DPE7</td><td>12.34</td><td>14.63</td><td>18.61</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>DPE8</td><td>12.81</td><td>12.91</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>DPE9</td><td>12.54</td><td>11.98</td><td>16.16</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>DPE12</td><td>-</td><td>-</td><td>-</td><td>11.74</td><td>9.37</td><td>12.49</td><td>-</td><td>-</td></tr><tr><td>DPE13</td><td>-</td><td>-</td><td>-</td><td>12.08</td><td>12.10</td><td>-</td><td>17.51</td><td>20.86</td></tr><tr><td>DPE15</td><td>13.19</td><td>13.84</td><td>-</td><td>12.46</td><td>13.49</td><td>-</td><td>-</td><td>-</td></tr><tr><td>DPE17</td><td>12.00</td><td>13.18</td><td>15.95</td><td>12.03</td><td>12.15</td><td>15.70</td><td>15.95</td><td>22.51</td></tr><tr><td>DPE18</td><td>-</td><td>-</td><td>-</td><td>11.68</td><td>10.90</td><td>-</td><td>-</td><td>-</td></tr><tr><td>DPE19</td><td>11.93</td><td>12.40</td><td>14.38</td><td>12.15</td><td>12.73</td><td>15.23</td><td>-</td><td>-</td></tr><tr><td>DPE20</td><td>12.32</td><td>14.58</td><td>13.75</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>DPE21</td><td>12.68</td><td>12.98</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>DPE22</td><td>11.73</td><td>12.78</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>DPE23</td><td>13.18</td><td>12.03</td><td>-</td><td>-</td><td>-</td><td>-</td><td>17.41</td><td>22.07</td></tr><tr><td>DPE24</td><td>12.34</td><td>13.28</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>DPE25</td><td>12.38</td><td>12.48</td><td>-</td><td>-</td><td>-</td><td>-</td><td>15.85</td><td>20.33</td></tr><tr><td>DPE26</td><td>13.05</td><td>11.34</td><td>-</td><td>-</td><td>-</td><td>-</td><td>14.68</td><td>18.61</td></tr><tr><td>DPE28</td><td>12.37</td><td>13.45</td><td>14.98</td><td>11.58</td><td>13.06</td><td>14.00</td><td>-</td><td>-</td></tr><tr><td>DPE29</td><td>-</td><td>-</td><td>-</td><td>13.27</td><td>13.83</td><td>-</td><td>18.08</td><td>22.43</td></tr><tr><td>DPE32</td><td>10.94</td><td>12.36</td><td>16.97</td><td>-</td><td>-</td><td>-</td><td>14.46</td><td>20.38</td></tr><tr><td>DP20</td><td>12.30</td><td>13.75</td><td>-</td><td>-</td><td>-</td><td>-</td><td>17.28</td><td>22.85</td></tr><tr><td>DP24</td><td>12.29</td><td>12.41</td><td>-</td><td>-</td><td>-</td><td>-</td><td>16.72</td><td>22.69</td></tr><tr><td>DP25</td><td>13.74</td><td>14.36</td><td>-</td><td>-</td><td>-</td><td>-</td><td>17.08</td><td>24.58</td></tr><tr><td>DP27</td><td>14.33</td><td>12.05</td><td>-</td><td>-</td><td>-</td><td>-</td><td>15.19</td><td>25.23</td></tr><tr><td>Average</td><td>12.31</td><td>12.74</td><td>15.83</td><td>12.12</td><td>12.20</td><td>14.35</td><td>16.29</td><td>21.21</td></tr><tr><td>n</td><td>23</td><td>23</td><td>7</td><td>8</td><td>8</td><td>4</td><td>16</td><td>16</td></tr><tr><td>SE</td><td>0.19</td><td>0.22</td><td>0.62</td><td>0.19</td><td>0.52</td><td>0.72</td><td>0.32</td><td>0.55</td></tr></tbody></table>

The table documents the average response latency per case for combinations of stimulation and recording sites. All values in ms. At the bottom of the table, the average response latency overall for each stimulation/recording site is given and the number of experiments (n) with the standard error (SE). Key: Co – contralateral; CP – copula pyramidis; Ctx – motor cortex; FL – forelimb; HL – hindlimb; LS – lobulus simplex; Ip – ipsilateral; PML – paramedian lobule; -, data not collected.
<table><caption>Table 5.2: Significant differences between onset latencies comparing stimulation and recording sites.</caption><thead><tr><th></th><th>Ip PML</th><th>Co FL Ctx PML</th><th>Ip FL Ctx PML</th><th>Ip FL LS</th><th>Co FL Ctx LS</th><th>Ip FL Ctx LS</th><th>Ip HL CP</th><th>Co HL Ctx CP</th></tr></thead><tbody><tr><td>Ip FL PML</td><td></td><td>ns</td><td>p&lt;0.001</td><td>ns</td><td>ns</td><td>ns</td><td>p&lt;0.001</td><td>p&lt;0.001</td></tr><tr><td>Co FL Ctx PML</td><td>ns</td><td></td><td>p&lt;0.001</td><td>ns</td><td>ns</td><td>ns</td><td>p&lt;0.001</td><td>p&lt;0.001</td></tr><tr><td>Ip FL Ctx PML</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td></td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>ns</td><td>ns</td><td>p&lt;0.001</td></tr><tr><td>Ip FL LS</td><td>ns</td><td>ns</td><td>p&lt;0.001</td><td></td><td>ns</td><td>ns</td><td>p&lt;0.001</td><td>p&lt;0.001</td></tr><tr><td>Co FL Ctx LS</td><td>ns</td><td>ns</td><td>p&lt;0.001</td><td>ns</td><td></td><td>ns</td><td>p&lt;0.001</td><td>p&lt;0.001</td></tr><tr><td>Ip FL Ctx LS</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td></td><td>ns</td><td>p&lt;0.001</td></tr><tr><td>Ip HL CP</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>ns</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>ns</td><td></td><td>p&lt;0.001</td></tr><tr><td>Co HL Ctx CP</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td></td></tr></tbody></table>

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

B

C

D

E

<figure><img src="image_65.png" /><figcaption>**Figure 5.2:** Paired pulse tests for climbing fibre responses to repeated stimulation sites. (Case DPE8)</figcaption></figure>

Olivary neurones responding to both types of stimulation as shown by conditioning tests. Varied paired pulse tests showing the refractory period of climbing fibre responses. Initial climbing fibre response from the first stimulation (0s) then second stimulation at (A) 20ms, (B) 40ms, (C) 60ms, (D) 80ms, (E) 100 ms (see stimulation artefact) for both peripheral and cerebral sites. Sets of three consecutive responses. Corresponding graphs below, showing the appearance and increase in response with increased inter-stimulus interval. Scale bars, 0.025s horizontal; 0.15mV vertical.
A

B

C

D

E

<figure><img src="image_66.png" /><figcaption>**Figure 5.3: Conditioning tests for climbing fibre responses to different stimulation sites.** (Case DPE8)</figcaption></figure>

This demonstrates olivary neurones responding to both types of stimulation as shown by conditioning tests. (A – E) Varied paired pulse tests show the refractory period of climbing fibre responses. Initial climbing fibre response from the first stimulation (0 s) then second stimulation at (A) 20 ms, (B) 40 ms, (C) 60 ms, (D) 80 ms, (E) 100 ms (see stimulation artefact). Sets of three consecutive responses, scale bars = 0.025 s horizontal; 0.15 mV vertical.
<figure><img src="image_67.png" /><figcaption>**Figure 5.4: Convergent responses from stimulation at threshold.**</figcaption></figure>

Threshold stimulation recorded in PML, LS and CP, column (A) peripheral threshold, (B) cerebral threshold and (C) simultaneous peripheral and cerebral at threshold. (see stimulation artefact). Sets of three consecutive responses, scale bars = 0.025 s horizontal; 0.15 mV vertical (Case DPE8 PML and DPE29 for LS and CP recordings).
**Figure 5.5: Labelled fibres in pyramidal tract under inferior olive.**

The figure displays the consistent lack of terminal labelling in the olive from anterograde tracer injections into the motor cortex. This photomicrograph shows short, labelled axons in the pyramidal tract in case DP11 from an injection of Fluoro-Ruby into the hindlimb area of the motor cortex (scale bar = 200 µm). Some cells labelled from an injection of red beads into the forelimb-receiving C1 zone in PML in the cerebellum can also be seen, which are located in the expected part of DAO that projects to that zone.
<figure><img src="image_68.png" /><figcaption>Figure 5.6: Photomicrograph and camera lucida drawing of labelling in the matrix area. (DP11)</figcaption></figure>

The high power photomicrograph shows terminal labelling in the matrix labelled with Fluoro-Ruby (scale bar = 50 µm) from an injection into the hindlimb motor cortex. The camera lucida drawing displays the path of the labelled fibres along the pyramidal tract (small black lines) and the location of the terminal labelling (square blue box). This plane of section was approximately 2 mm caudal to the inferior olive and 16.5 mm caudal to bregma (scale bar = 1 mm).
<figure><img src="image_69.png" /><figcaption>Figure 5.7: Double anterograde labelling in the caudal brainstem. (DP25)</figcaption></figure>

Anatomical and physiological documentation of a full double tracer experiment. (A) The dorsal view of the cerebral cortex shows the sites of stimulation (red – forelimb; green – hindlimb) where the corresponding climbing fibre responses were recorded in the cerebellum. Terminal labelling was found in the matrix from an injection of Fluoro-Ruby into the forelimb motor cortex (injection site in B and labelling in red in D) and an injection of Fluoro-Emerald into the hindlimb motor cortex (injection site in C and labelling in green in E). The axonal labelling is shown in C and D as coloured lines, where axons could be traced crossing the midline to the matrix. Calibration (A): 5 ms, 0.1 mV. Scale bars: A, 2 mm, B-F, 0.5 mm.
<figure><img src="image_70.png" /><figcaption>Figure 5.8: Overview of pooled and individual cases giving terminal labelling in the matrix.</figcaption></figure>

(A) The extent of all the labelling in the matrix from injections into the motor cortex is shown, where blue indicates locations where terminal labelling from both fore- and hindlimb cortical injections was found and green indicates terminal labelling from hindlimb motor cortical injections only, which extended slightly further rostral and caudal than the forelimb labelling. Distance from Bregma is shown on the right of the diagram. Scale bar = 0.5 mm. (B) The extent of terminal labelling is shown diagrammatically for individual cases (n=10) for hindlimb (green) and forelimb (red) injections. The extent of labelling is shown to scale, in graphical format for its rostrocaudal location in the matrix. The cases are identified by their case number and injection site (see Table 4.2 for motor cortical injection site details).
<figure><img src="image_71.png" /><figcaption>Figure 5.9: Photomicrograph of an example injection site made to inactivate the matrix area. (DPE28)</figcaption></figure>

The photomicrograph shows an injection of GABA/KA mixture (dyed with pontamine sky blue) into the matrix area (marked by the white arrow) to inactivate the descending signals from the motor cortex in the location where the anterograde labelling was found in the anatomical experiments. Scale bar = 500 µm.
<table><caption>**Table 5.3: Significant differences and percentage changes in responses from injections into the matrix area.**</caption><thead><tr><th rowspan="3">Case</th><th colspan="10">Statistical Significant Difference to Control</th></tr><tr><th colspan="4">Peripherally Evoked Responses</th><th rowspan="2">5 mins</th><th colspan="4">Cerebrally Evoked Responses</th></tr><tr><th>5 mins</th><th>10 mins</th><th>15 mins</th><th>20 mins</th><th>5 mins</th><th>10 mins</th><th>15 mins</th><th>20 mins</th></tr></thead><tbody><tr><td>DPE22 PML</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>ns</td><td>p&lt;0.001</td><td>ns</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>ns</td><td>p&lt;0.001</td></tr><tr><td>% Change</td><td>132.2</td><td>130.0</td><td>99.8</td><td>140.5</td><td>100.6</td><td>48.6</td><td>72.9</td><td>81.6</td><td></td></tr><tr><td>DPE24 PML</td><td>ns</td><td>ns</td><td>p&lt;0.05</td><td>-</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>ns</td><td>-</td><td></td></tr><tr><td>% Change</td><td>83.6</td><td>91.0</td><td>120.4</td><td>-</td><td>25.2</td><td>75.2</td><td>104.3</td><td>-</td><td></td></tr><tr><td>DPE26 PML</td><td>p&lt;0.001</td><td>ns</td><td>p&lt;0.05</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td></td></tr><tr><td>% Change</td><td>76.4</td><td>88.3</td><td>86.4</td><td>74.4</td><td>34.0</td><td>31.6</td><td>34.1</td><td>36.2</td><td></td></tr><tr><td>DPE25 CP</td><td>ns</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td></td></tr><tr><td>% Change</td><td>108.4</td><td>120.3</td><td>123.7</td><td>129.7</td><td>22.6</td><td>25.5</td><td>32.4</td><td>69.5</td><td></td></tr><tr><td>DPE26 CP</td><td>ns</td><td>ns</td><td>ns</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td></td></tr><tr><td>% Change</td><td>100.7</td><td>107.3</td><td>107.6</td><td>112.7</td><td>21.2</td><td>19.2</td><td>21.5</td><td>27.7</td><td></td></tr><tr><td>DPE28 LS</td><td>p&lt;0.001</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>p&lt;0.05</td><td>ns</td><td>p&lt;0.001</td><td></td></tr><tr><td>% Change</td><td>128.7</td><td>111.6</td><td>102.6</td><td>114.0</td><td>128.8</td><td>63.5</td><td>102.3</td><td>57.1</td><td></td></tr><tr><td>DPE28 PML</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>ns</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td></td></tr><tr><td>% Change</td><td>127.4</td><td>142.1</td><td>124.9</td><td>140.8</td><td>140.5</td><td>86.9</td><td>40.0</td><td>40.3</td><td></td></tr><tr><td>DPE32 PML</td><td>p&lt;0.05</td><td>ns</td><td>p&lt;0.05</td><td>ns</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td></td></tr><tr><td>% Change</td><td>85.0</td><td>85.2</td><td>84.7</td><td>86.3</td><td>61.6</td><td>65.0</td><td>64.8</td><td>79.0</td><td></td></tr><tr><td>DPE29 CP</td><td>ns</td><td>ns</td><td>p&lt;0.001</td><td>-</td><td>ns</td><td>ns</td><td>ns</td><td>-</td><td></td></tr><tr><td>% Change</td><td>92.1</td><td>94.3</td><td>88.2</td><td>-</td><td>108.6</td><td>110.3</td><td>94.7</td><td>-</td><td></td></tr><tr><td>DPE32 CP</td><td>ns</td><td>ns</td><td>ns</td><td>p&lt;0.05</td><td>ns</td><td>p&lt;0.05</td><td>p&lt;0.001</td><td>ns</td><td></td></tr><tr><td>% Change</td><td>95.0</td><td>101.5</td><td>100.0</td><td>111.1</td><td>92.4</td><td>77.5</td><td>67.4</td><td>85.6</td><td></td></tr></tbody></table>

The table shows a summary of individual cases and injections into the putative relay, 2 mm caudal to obex. The responses are to peripheral and cerebral stimulation and values are taken at 5, 10, 15 and 20 minutes, compared to pre-drug baseline. Shaded boxes indicate significant differences from Dunnett's multiple comparison post test in conjunction with repeated measures ANOVA; yellow = decrease and green = increase, to control. C1 zone recording site is also indicated (CP – hindlimb receiving copula pyramidis; LS – forelimb receiving lobulus simplex; PML – forelimb receiving paramedian lobule). DPE22, 23, 24, 25 and 26 were injected with local anaesthetic, DPE28, 29 and 32 were injected with GABA/KA.
<figure><img src="image_72.png" /></figure>

<figure><img src="image_73.png" /></figure>
Responses in CP to Peripheral Hindlimb Stimulation

Responses in PML to Cerebral Forelimb Stimulation

<figure><img src="image_74.png" /></figure>

Responses in CP to Cerebral Hindlimb Stimulation

<figure><img src="image_75.png" /></figure>

**Figure 5.10: Bar graphs of binned raw cerebellar responses to peripheral and cerebral stimulation from an injection of LA into the matrix.**

The graphs show the bins of 30 raw climbing fibre responses per stimulation/recording site in mV x ms, over time measured in minutes. The responses to cerebral stimulation decrease by 2.5 minutes post-injection of LA and recover after approximately 30 minutes, where the peripherally evoked responses remain essentially unchanged. The forelimb and hindlimb responses were recorded simultaneously (DPE26).
<figure><img src="image_76.png" /><figcaption>Figure 5.11: Selective decrease in climbing fibre responses to cerebral stimulation from an injection of GABA/KA into the matrix. (DPE28)</figcaption></figure>

Responses to peripheral (left column) and cerebral (right column) stimulation before drug (control - top waveforms), during inactivation (10 mins post-drug - middle waveforms) and recovery (35 mins post-drug - bottom waveforms). The responses show that injections of the GABA and KA mixture selectively inactivate only the cerebrally evoked responses and not the peripherally evoked ones. Calibration = 0.1 mV, 2ms.
<figure><img src="image_77.png" /><figcaption>Figure 5.12: Effects of injecting local anaesthetic into matrix on climbing fibre field responses.</figcaption></figure>

These graphs show the effect of the local anaesthetic injected into the matrix area, recording climbing fibre response from different limb-receiving areas of the C1 zone of the posterior cerebellum. The bottom right graph demonstrates a case where dual recordings were made in the cerebellar cortex (forelimb and hindlimb C1).
<figure><img src="image_78.png" /></figure>

<figure><img src="image_79.png" /></figure>

<figure><img src="image_80.png" /></figure>

— Peripheral Forelimb LS

— Cerebral Forelimb LS

— Peripheral Forelimb PML

— Cerebral Forelimb PML

— Peripheral Hindlimb CP

— Cerebral Hindlimb CP

<caption>Figure 5.13: Effects of injecting GABA/KA into the matrix on climbing fibre field responses.</caption>

These graphs show the effect of the GABA/KA solution injected into the matrix area, recording climbing fibre response from different limb-receiving areas of the C1 zone. The top and bottom graphs are taken from cases where dual recordings were made in the cerebellar cortex.
<figure><img src="image_81.png" /><figcaption>Figure 5.14: Pooled cases to demonstrate the overall effect of inactivating the matrix on climbing fibre field responses.</figcaption></figure>

The graph shows the pooled cases (with standard error bars) for all the cases where either local anaesthetic or the GABA/KA mixture was injected into the matrix and responses were recorded from limb-receiving areas in the C1 zone in the cerebellar cortex. This graph demonstrates the overall effect that within a few minutes after injection of the inactivating agent, there is a clear selective block of cerebrally evoked responses, with a full recovery of response by 40 minutes post-injection.
<table><caption>**Table 5.4: Significant differences and percentage changes in recordings at injection sites rostral to the labelling in the matrix area.**</caption><thead><tr><th rowspan="3">Case</th><th colspan="10">Statistical Significant Difference to Control</th></tr><tr><th colspan="4">Peripherally Evoked Responses</th><th colspan="4">Cerebrally Evoked Responses</th></tr><tr><th>5 mins</th><th>10 mins</th><th>15 mins</th><th>20 mins</th><th>5 mins</th><th>10 mins</th><th>15 mins</th><th>20 mins</th></tr></thead><tbody><tr><td colspan="9"><strong>0.5 mm Caudal to Obex</strong></td></tr><tr><td>DPE22 PML</td><td>ns</td><td>ns</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>ns</td><td>ns</td><td>p&lt;0.001</td><td>p&lt;0.001</td></tr><tr><td>% Change</td><td>99.8</td><td>104.3</td><td>80.5</td><td>66.0</td><td>100.0</td><td>111.2</td><td>72.9</td><td>73.8</td></tr><tr><td>DPE23 PML</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>ns</td><td>ns</td><td>ns</td></tr><tr><td>% Change</td><td>84.0</td><td>68.5</td><td>54.3</td><td>29.7</td><td>135.0</td><td>107.8</td><td>88.0</td><td>85.8</td></tr><tr><td>DPE24 PML</td><td>ns</td><td>ns</td><td>ns</td><td>p&lt;0.001</td><td>p&lt;0.05</td><td>ns</td><td>ns</td><td>p&lt;0.05</td></tr><tr><td>% Change</td><td>103.0</td><td>101.2</td><td>92.9</td><td>67.2</td><td>85.6</td><td>89.1</td><td>91.8</td><td>86.6</td></tr><tr><td>DPE23 CP</td><td>ns</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>p&lt;0.001</td></tr><tr><td>% Change</td><td>101.1</td><td>70.0</td><td>12.9</td><td>13.6</td><td>68.7</td><td>37.7</td><td>27.2</td><td>26.6</td></tr><tr><td>DPE28 LS</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>ns</td><td>ns</td><td>p&lt;0.001</td><td>p&lt;0.001</td></tr><tr><td>% Change</td><td>115.7</td><td>132.2</td><td>137.2</td><td>154.0</td><td>96.0</td><td>99.6</td><td>125.7</td><td>142.7</td></tr><tr><td>DPE28 PML</td><td>p&lt;0.05</td><td>p&lt;0.05</td><td>ns</td><td>p&lt;0.001</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td></tr><tr><td>% Change</td><td>85.1</td><td>81.9</td><td>91.1</td><td>81.5</td><td>96.5</td><td>88.3</td><td>105.4</td><td>103.8</td></tr><tr><td colspan="9"><strong>1 mm Caudal to Obex</strong></td></tr><tr><td>DPE23 PML</td><td>ns</td><td>ns</td><td>p&lt;0.05</td><td>-</td><td>ns</td><td>ns</td><td>ns</td><td>-</td></tr><tr><td>% Change</td><td>94.7</td><td>111.4</td><td>119.4</td><td>-</td><td>96.6</td><td>97.7</td><td>89.6</td><td>-</td></tr><tr><td>DPE24 PML</td><td>ns</td><td>ns</td><td>ns</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>p&lt;0.001</td></tr><tr><td>% Change</td><td>85.6</td><td>103.8</td><td>95.4</td><td>76.4</td><td>59.8</td><td>58.3</td><td>49.6</td><td>57.9</td></tr><tr><td>DPE25 PML</td><td>p&lt;0.001</td><td>ns</td><td>ns</td><td>p&lt;0.001</td><td>ns</td><td>ns</td><td>p&lt;0.001</td><td>p&lt;0.001</td></tr><tr><td>% Change</td><td>60.8</td><td>91.5</td><td>122.3</td><td>133.0</td><td>98.9</td><td>139.5</td><td>208.3</td><td>266.5</td></tr><tr><td>DPE26 PML</td><td>ns</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>p&lt;0.05</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>ns</td><td>ns</td></tr><tr><td>% Change</td><td>116.7</td><td>140.3</td><td>137.5</td><td>123.3</td><td>130.4</td><td>149.1</td><td>116.9</td><td>86.1</td></tr><tr><td>DPE26 CP</td><td>ns</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td></tr><tr><td>% Change</td><td>128.0</td><td>171.5</td><td>174.7</td><td>31.0</td><td>88.2</td><td>105.8</td><td>114.5</td><td>116.6</td></tr><tr><td colspan="9"><strong>1.5 mm Caudal to Obex</strong></td></tr><tr><td>DPE23 PML</td><td>p&lt;0.05</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>-</td><td>ns</td><td>p&lt;0.05</td><td>ns</td><td>-</td></tr><tr><td>% Change</td><td>86.3</td><td>74.1</td><td>75.6</td><td>-</td><td>95.0</td><td>126.2</td><td>113.2</td><td>-</td></tr><tr><td>DPE23 CP</td><td>ns</td><td>ns</td><td>p&lt;0.001</td><td>-</td><td>ns</td><td>ns</td><td>ns</td><td>-</td></tr><tr><td>% Change</td><td>98.7</td><td>83.1</td><td>68.0</td><td>-</td><td>109.6</td><td>105.4</td><td>93.2</td><td>-</td></tr><tr><td>DPE28 LS</td><td>ns</td><td>ns</td><td>ns</td><td>p&lt;0.001</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td></tr><tr><td>% Change</td><td>95.2</td><td>92.3</td><td>96.3</td><td>60.3</td><td>89.7</td><td>94.3</td><td>93.6</td><td>92.3</td></tr><tr><td>DPE28 PML</td><td>ns</td><td>ns</td><td>ns</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>ns</td></tr><tr><td>% Change</td><td>100.9</td><td>101.3</td><td>101.6</td><td>62.6</td><td>125.7</td><td>124.2</td><td>140.3</td><td>97.1</td></tr></tbody></table>

The table shows a summary of individual cases and injections at sites 0.5 mm, 1 mm and 1.5 mm caudal to obex (putative relay at 2 mm caudal to obex). The responses are to peripheral and cerebral stimulation and values are taken at 5, 10, 15 and 20 minutes, compared to pre-drug baseline. Shaded boxes indicate significant differences from Dunnett's multiple comparison post test in conjunction with repeated measures ANOVA; yellow = decrease and green = increase, to baseline. C1 zone recording site is also indicated (CP – hindlimb receiving copula pyramidis; LS – forelimb receiving lobulus simplex; PML – forelimb receiving paramedian lobule). DPE22, 23, 24, 25 and 26 were injected with local anaesthetic, DPE28, 29 and 32 were injected with GABA/KA.
<table><caption>**Table 5.5: Significant differences and percentage changes in recordings at injection sites caudal to the labelling in the matrix relay area.**</caption><thead><tr><th rowspan="3">Case</th><th colspan="10">Statistical Significant Difference to Control</th></tr><tr><th colspan="4">Peripherally Evoked Responses</th><th colspan="4">Cerebrally Evoked Responses</th></tr><tr><th>5 mins</th><th>10 mins</th><th>15 mins</th><th>20 mins</th><th>5 mins</th><th>10 mins</th><th>15 mins</th><th>20 mins</th></tr></thead><tbody><tr><td colspan="9"><strong>2.5 mm Caudal to Obex</strong></td></tr><tr><td>DPE23 PML</td><td>ns</td><td>ns</td><td>p&lt;0.001</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td></tr><tr><td>% Change</td><td>109.8</td><td>113.9</td><td>137.2</td><td>105.3</td><td>103.4</td><td>106.8</td><td>98.9</td><td>84.9</td></tr><tr><td>DPE23 CP</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>p&lt;0.001</td><td>p&lt;0.001</td></tr><tr><td>% Change</td><td>99.1</td><td>97.8</td><td>96.4</td><td>87.1</td><td>88.6</td><td>84.7</td><td>77.5</td><td>66.6</td></tr><tr><td colspan="9"><strong>3 mm Caudal to Obex</strong></td></tr><tr><td>DPE24 PML</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>p&lt;0.001</td></tr><tr><td>% Change</td><td>28.8</td><td>28.7</td><td>23.8</td><td>25.0</td><td>40.7</td><td>42.6</td><td>39.0</td><td>63.5</td></tr><tr><td>DPE28 LS</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>ns</td><td>p&lt;0.001</td><td>ns</td><td>ns</td></tr><tr><td>% Change</td><td>40.7</td><td>42.6</td><td>39.0</td><td>63.5</td><td>90.9</td><td>80.9</td><td>95.0</td><td>93.7</td></tr><tr><td>DPE28 PML</td><td>ns</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>ns</td><td>ns</td><td>p&lt;0.001</td><td>p&lt;0.001</td></tr><tr><td>% Change</td><td>121.8</td><td>146.3</td><td>152.9</td><td>162.6</td><td>153.4</td><td>157.2</td><td>196.0</td><td>221.3</td></tr><tr><td colspan="9"><strong>3.5 mm Caudal to Obex</strong></td></tr><tr><td>DPE22 PML</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>p&lt;0.001</td></tr><tr><td>% Change</td><td>51.6</td><td>39.6</td><td>52.3</td><td>48.4</td><td>73.3</td><td>77.3</td><td>65.0</td><td>61.4</td></tr><tr><td colspan="9"><strong>4 mm Caudal to Obex</strong></td></tr><tr><td>DPE26 PML</td><td>p&lt;0.05</td><td>p&lt;0.001</td><td>p&lt;0.05</td><td>p&lt;0.001</td><td>ns</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>p&lt;0.001</td></tr><tr><td>% Change</td><td>84.1</td><td>51.9</td><td>82.5</td><td>39.2</td><td>102.9</td><td>50.3</td><td>34.4</td><td>56.2</td></tr><tr><td>DPE25 CP</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>p&lt;0.05</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>p&lt;0.001</td></tr><tr><td>% Change</td><td>35.3</td><td>44.5</td><td>58.8</td><td>109.9</td><td>16.3</td><td>28.5</td><td>24.1</td><td>82.7</td></tr><tr><td>DPE26 CP</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>p&lt;0.001</td><td>ns</td><td>p&lt;0.05</td><td>p&lt;0.001</td><td>ns</td></tr><tr><td>% Change</td><td>34.4</td><td>27.4</td><td>26.8</td><td>20.9</td><td>68.6</td><td>66.4</td><td>62.8</td><td>69.0</td></tr></tbody></table>

The table shows a summary of individual cases and injections at sites 2.5 mm, 3 mm, 3.5 mm and 4 mm caudal to obex (putative relay at 2 mm caudal to obex). The responses are to peripheral and cerebral stimulation and values are taken at 5, 10, 15 and 20 minutes, compared to pre-drug baseline. Shaded boxes indicate significant differences from Dunnett's multiple comparison post test in conjunction with repeated measures ANOVA; yellow = decrease and green = increase, to control. C1 zone recording site is also indicated (CP – hindlimb receiving copula pyramidis; LS – forelimb receiving lobulus simplex; PML – forelimb receiving paramedian lobule). DPE22, 23, 24, 25 and 26 were injected with local anaesthetic, DPE28, 29 and 32 were injected with GABA/KA.
<figure><img src="image_82.png" /><figcaption>Figure 5.15: Potential relay sites for bilateral forelimb motor cortical projection to cerebellar forelimb-receiving areas via inferior olive.</figcaption></figure>

From the results, the cortical forelimb pathway seemed to be conveyed along different paths, depending upon whether contralateral or ipsilateral to the C1 recording site. Taking into account the physiological and anatomical data gained, the above suggested pathway would be feasible to interpret the findings.
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