Spatial localization and projection densities of brainstem mossy fibre afferents to the forelimb C1 zone of the rat cerebellum

Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ “This is the peer reviewed version of the following article: Herrero, L., Pardoe, J., Cerminara, N.L. and Apps, R. (2012), Spatial localization and projection densities of brainstem mossy fibre afferents to the forelimb C1 zone of the rat cerebellum. European Journal of Neuroscience, 35: 539-549, which has been published in final form at https://doi.org/10.1111/j.1460-9568.2011.07977.x . This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions. This article may not be enhanced, enriched or otherwise transformed into a derivative work, without express permission from Wiley or by statutory rights under applicable legislation. Copyright notices must not be removed, obscured or modified. The article must be linked to Wiley’s version of record on Wiley Online Library and any embedding, framing or otherwise making available the article or pages thereof by third parties from platforms, services and websites other than Wiley Online Library must be prohibited." For Peer Review The relationship between medullary mossy fibre afferents and the forelimb C1 zone of the rat cerebellum. Journal: European Journal of Neuroscience Manuscript ID: Draft Manuscript Type: Research Report Date Submitted by the Author: n/a Complete List of Authors: Herrero, Luis; University of Bristol, School of Physiology and Pharmacology; University of Seville, Physiology Pardoe, Joanne; University of Bristol, School of Physiology and Pharmacology Cerminara, Nadia; University of Bristol, School of Physiology and Pharmacology Apps, Richard; University of Bristol, School of Physiology and Pharmacology Key Words: Brainstem, tract-tracing, trigeminal, cuneate, reticular formation ScholarOne, 375 Greenbrier Drive, Charlottesville, VA, 22901 1(434)964-4100 ext. 1 European Journal of Neuroscience For Peer Review 1 Journal Section: Neurosystems Associate Editor: Paul Bolam The relationship between medullary mossy fibre afferents and the forelimb C1 zone of the rat cerebellum. Luis Herrero1,2, Joanne Pardoe1,3 Nadia L Cerminara1 and Richard Apps1. 1School of Physiology and Pharmacology, Medical Sciences Building, University of Bristol, Bristol BS8 1TD, UK 2Departamento de Fisiologia, Facultad de Biologia, Universidad de Sevilla, 41012 Sevilla, Spain 3Glaxosmithkline Research and Development, Park Road, Ware, Hertfordshire, SG12 0DP, UK Number of pages: 34 Number of Figures: 5 Number of Tables: 4 Number of words in the whole manuscript: 8031 Number of words in the abstract: 232 Number of words in the introduction: 512 Running title: Mossy fibre inputs to C1 cerebellar zone. Keywords: brainstem, tract-tracing, cuneate, trigeminal, reticular formation Corresponding author: Prof. Richard Apps School of Physiology and Pharmacology Medical Sciences Building University of Bristol Bristol BS8 1TD, UK [email protected] Page 1 of 43 ScholarOne, 375 Greenbrier Drive, Charlottesville, VA, 22901 1(434)964-4100 ext. 1 European Journal of Neuroscience For Peer Review 2 ABSTRACT The present study uses a combined electrophysiological and double retrograde tracer technique in rats to examine the spatial localization and pattern of axonal branching in mossy fibres arising from three major sources in the medulla – the external cuneate nucleus, sensory trigeminal nucleus, and the reticular formation – to two rostrocaudally separated parts of the cerebellar cortex that are linked by common climbing fibre input, the forelimb-receiving parts of the C1 zone in lobulus simplex and the paramedian lobule. In each experiment a small injection of rhodamine-tagged beads was injected into one cortical region and an injection of fluorescein-tagged beads was injected into the other region. The main findings were: 1) the proportion of doublelabelled cells in each of the three precerebeller sources of mossy fibres was positively correlated to those in the inferior olive. And 2) the C1 zone in lobulus simplex was found to receive a greater density of projections from all three sources of mossy fibres than the C1 zone in the paramedian lobule. These data are consistent with the cerebellar one-map hypothesis since two rostrocaudally separated, but somatotopically corresponding, parts of the C1 zone receive common mossy fibre and climbing fibre inputs. However, the differences in projection densities also suggest a modification of the hypothesis in that the two parts of the zone differ in the extent to which they receive mossy fibre signals arising from the same precerebellar nuclei. Page 2 of 43 ScholarOne, 375 Greenbrier Drive, Charlottesville, VA, 22901 1(434)964-4100 ext. 1 European Journal of Neuroscience For Peer Review 3 INTRODUCTION Interaction between mossy fibre and climbing fibre afferents at the level of the cortex is thought to be central to cerebellar mechanisms contributing to movement coordination and motor learning processes. It is therefore important to understand their anatomical relationships (Voogd and Glickstein, 1998; Apps and Garwicz, 2005; Ito, 2005). Climbing fibres arise exclusively from the contralateral inferior olive and make direct synaptic contact with Purkinje cells whose axons project, in turn, to the cerebellar and lateral vestibular nuclei. As a function of olivocerebellar topography, the cerebellar cortex is arranged into a number of narrow longitudinally-oriented zones (designated from medial to lateral as A, X, B, C1, C2, C3 and D1, D0 and D2, e.g. Voogd and Bigare, 1980; Buisseret-Delmas and Angaut, 1993; Apps and Hawkes, 2009). Furthermore, olivocerebellar axons branch preferentially in the rostrocaudal axis; that is, a single olive cell projects to several Purkinje cells located at different rostrocaudal points in a single longitudinal zone (Armstrong et al., 1973; Apps, 2000; Sugihara et al., 2001). This anatomical organization indicates that rostrocaudally separate regions of cerebellar cortex have at least some common climbing fibre input. By contrast, mossy fibres arise from many different sources, including the spinal cord, pons and medulla. These inputs tend to terminate bilaterally within the cerebellar cortex and show a complex pattern of branching with a wide mediolateral spread, but with a tendency to form multiple, rostrocaudally-oriented stripes (e.g. Wu et al., 1999; Gravel and Hawkes, 1990; Ruigrok et al, 1995; Serapide et al., 2001; Voogd et al., 2003). Page 3 of 43 ScholarOne, 375 Greenbrier Drive, Charlottesville, VA, 22901 1(434)964-4100 ext. 1 European Journal of Neuroscience For Peer Review 4 The topographical relationship between mossy fibre and climbing fibre terminations in the cerebellar cortex has previously been studied using cholera toxin to map patterns of collateral branching (Voogd et al., 2003; Pijpers et al., 2006; Pijpers and Ruigrok, 2006); however, few double retrograde tracer studies have considered the spatial organization of cells within precerebellar sources of mossy fibres and their relationship to rostrocaudally separated parts of individual cerebellar zones. Experiments to date have focussed on the projections from the basal pontine nuclei (BPN) and lateral reticular nucleus (LRN; King et al., 1998; Herrero et al., 2002). In particular, Herrero et al., (2002) found a positive relationship between olivary and LRN inputs targeting the forelimb-related parts of the C1 zone in lobulus simplex (LS) and the paramedian lobule (PML), but no such relationship with the projection from BPN. Therefore it appears that the LRN projection is zonally organised, but the BPN projection is not. It remains unclear whether or not other sources of mossy fibres are also zonally organised. If such a relationship exists then this would support the one-map hypothesis, which postulates that the mossy fibre and climbing fibre afferent systems have a common spatial organization (Apps and Hawkes, 2009). The aim therefore of the present study was to investigate the topography of mossy fibre projections to the C1 zone in rostrocaudally separated parts of the cerebellar cortex (LS and PML), arising from three major sources of mossy fibre input in the medulla: the external cuneate nucleus (CN), the sensory trigeminal nuclei (STN), and the medullary reticular formation (RF). MATERIALS AND METHODS All experiments were carried out in accordance with the United Kingdom Animals (Scientific Procedures) Act of 1986 and were carried out on 7 male adult Page 4 of 43 ScholarOne, 375 Greenbrier Drive, Charlottesville, VA, 22901 1(434)964-4100 ext. 1 European Journal of Neuroscience For Peer Review 5 Wistar rats (255-360 g). Five of the experiments have been analyzed previously for another purpose by Pardoe and Apps (2002) and Herrero et al., (2002). All surgical procedures were carried out aseptically and were performed with surgical levels of general anaesthesia (sodium pentobarbitone injected intraperitoneally at an initial dose of 60 mg/kg, Sagatal, Rhone Merieux, Harlow, UK, maintenance doses given as required). The dorsal surface of the cerebellum was exposed in the region of the paravermal cortex in LS and the PML. The rectal temperature of the animal was monitored and kept within physiological limits using a thermostatically-controlled electric blanket. Tracer injections and histological processing Electrophysiological techniques were used to guide the tracer injections. In brief, short latency (~12 ms) field potentials were evoked in the C1 zone in both LS and PML as a result of percutaneous electrical stimulation (0.1 ms pulse) delivered to the ipsilateral forelimb. In addition, electrical stimulation (0.1 ms pulse) was delivered to the contralateral face and contralateral forelimb to distinguish C1 responses from those evoked in neighbouring zones A2 and C2, respectively (see Atkins and Apps, 1998 for further details). Retrograde tracers were injected into the centre of the C1 zone, as defined by the electrophysiological mapping, about 0.3-0.5 mm below the pial surface. The tracers were a suspension of undiluted latex microspheres tagged either with rhodamine (red beads; Lumafluor Inc., New York, NY) which was injected into the C1 zone in one lobule, or fluorescein-tagged latex microspheres (green beads; Lumafluor Inc., New York, NY) which was injected into the C1 zone in the other lobule (Apps and Ruigrok, 2007). Each tracer injection (a case) was delivered hydraulically via a glass micropipette attached to a 1 µl Hamilton syringe and the total volume injected was Page 5 of 43 ScholarOne, 375 Greenbrier Drive, Charlottesville, VA, 22901 1(434)964-4100 ext. 1 European Journal of Neuroscience For Peer Review 6 between 50 and 100 nl (see Table 1). Immediately after each injection, the pial surface was flushed with warm saline then covered with gelfoam. The craniotomy was sealed with dental acrylic cement before the wound was closed in layers. After recovery, each animal was carefully monitored under veterinary guidance throughout a survival period of 7 days. As a precautionary measure post-operative analgesia was maintained for 24 hours with buprenorphine (Temgesic, 0.1 mg/kg, i.m., Reckitt and Colman, Hull, UK), although none of the animals showed any signs of discomfort or distress during the survival period. At the end of the survival period, the rats were re-anaesthetised with barbiturate (200 mg/kg, i.p.) and perfusion-fixed transcardially with 500 ml of heparinised saline, followed by 1 litre of 4% paraformaldehyde then 500 ml of 10% sucrose in phosphate buffer. The cerebellum and underlying brainstem were removed and stored overnight at 4°C in 10% sucrose, phosphate buffer solution. The cerebellum and medulla oblongata were separated and a freezing microtome used to cut the medulla into 50 µm transverse sections and the cerebellum into 50 µm sagittal sections. All sections were mounted onto gelatin-coated slides and separated into two series: one for mapping, the other as a reserve. Slides were allowed to air dry and stored in the dark at 4°C without clearing or coverslipping to minimise fading of fluorescent material (Apps and Ruigrok, 2007). The sections were examined with a Leica DMRB microscope fitted with a 50W-mercury UV light source (Ploemopak) and high numerical aperture objectives (PL Fluotar). The red fluorescence was viewed with an N2.1 filter block (Dichroic mirror 580 nm, BP 515-560 nm, LP 580 nm), while the green fluorescence was viewed with an H3 filter block (Dichroic mirror 510 nm, BP 420-490 nm, LP 520 nm). Cell bodies retrogradely labelled with red and/or green beads Page 6 of 43 ScholarOne, 375 Greenbrier Drive, Charlottesville, VA, 22901 1(434)964-4100 ext. 1 European Journal of Neuroscience For Peer Review 7 were readily differentiated from background by the bright granular appearance of the fluorescent labelling in their cytoplasm. Analysis of injection sites and retrograde labelling Each injection site consisted of a core, containing a high density of fluorescent material, surrounded by an area of much lower density. A scale drawing was made of each sagittal section containing the injection site core, and the length of the injection site core at the Purkinje cell layer was measured to obtain its rostrocaudal extent in that section. All the values from each section containing the core were then added together and the total area of cortex involved in the injection site was calculated by multiplying this value by the section interval (in this case 0.1 mm). The values obtained were used to calculate projection densities (see Table 4). In addition, a standard series of 24 transverse maps (levels) was constructed (0.2 mm apart), covering the rostrocaudal extent of the medulla. Each section in one series was assigned to one of the transverse levels (typically 3 sections per level), and numbers of retrogradely labelled cells and areas occupied by cell labelling in the CN, STN and RF were calculated from these maps. However, for the purposes of illustration, only one in three levels are depicted in the Figures (i.e. the interval between the levels shown is 0.6 mm), but this includes the full rostrocaudal extent of levels in which retrogradely labelled cells were found. Because of sampling, the total number of observed labelled cells was an underestimate of the real number of labelled cells. The “raw” counts were therefore corrected, assuming that the real number of cells (Nt) is a function of the observed number of cells (No), the section thickness (50 µm), the number of series of sections (two) and particle diameter, according to the following equation (Abercrombie, 1946): Page 7 of 43 ScholarOne, 375 Greenbrier Drive, Charlottesville, VA, 22901 1(434)964-4100 ext. 1 European Journal of Neuroscience For Peer Review 14 were significantly greater than to PML by a factor ranging between 2 to 4:1 (paired ttest, two-tailed, p<0.05), with the exception of the ipsilateral STN and ipsilateral CN. Climbing fibres versus mossy fibres To determine if there was any zonal organization in the mossy fibre projection from CN, STN and RF to the C1 zone in LS and PML, for each case the percentage of overlap of the two populations of single-labelled cells in each of the medullary nuclei was compared with the percentage of overlap of the two corresponding populations of single-labelled cells in the inferior olive. For projections to both lobules, a highly statistically significant positive correlation was found for all three nuclei, CN (r2=0.91; p=0.0003, n=8, Fig. 4A), STN (r2=0.88; p=0.0005, n=8, Fig. 4B) and RF (r2=0.89; p=0.0004, n=8, Fig. 4C). These data imply that the degree of spatial overlap between the CN, STN and RF projections to the C1 zone in LS and PML is proportional to the involvement of common olivary inputs to rostrocaudally separated parts of the same zone. Consistent with this suggestion, the percentage of double-labelled cells in the different precerebellar sources of mossy fibres (expressed as a percentage of the total number of labelled cells) increased with the percentage of double-labelled cells in the inferior olive (r2=0.67; p=0.001; n=12; Fig. 4D). DISCUSSION The present study investigated the pattern and quantity of mossy fibre inputs to two rostrocaudally separated, but somatotopically corresponding, parts of the cerebellar cortical C1 zone in LS and PML. The major findings were: (i) mossy fibre projections from the CN, STN and RF are all zonally organized; and (ii) the projection densities from these precerebellar nuclei to the C1 zone in LS are significantly greater than those to PML by a factor of approximately 3:1. Page 14 of 43 ScholarOne, 375 Greenbrier Drive, Charlottesville, VA, 22901 1(434)964-4100 ext. 1 European Journal of Neuroscience For Peer Review 15 Comparison with previous anatomical studies Cuneate projections The present study found little or no cell labelling in the main cuneate nucleus but instead retrograde cell labelling was consistently present within its external subdivision. The latter relays skin and proprioceptive inputs from the neck and ipsilateral forelimb to the cerebellum (Campbell et al., 1974; Quy et al., 2011). A projection to the ‘forelimbreceiving’ regions of the PML and LS might therefore be expected. A bilateral projection with a heavy ipsilateral predominance is also consistent with previous anterograde (Gerrits et al., 1985; Massopust et al., 1985; Jasmin and Courville, 1987a,b; Päällysaho et al., 1991; Tolbert and Gotting, 1998; Ji and Hawkes, 1994) and retrograde tracer studies (Rinvik and Walberg, 1975; Cheek et al., 1975; Somana and Walberg, 1980; Berreta et al., 1991; Quy et al., 2011), as is our finding that labelled cells were located mainly in caudal parts of CN (Rinvik and Walberg, 1975; Gerrits et al., 1985; Quy et al., 2011). Previous anterograde mapping studies have also shown a rostrocaudally striped pattern of termination from CN to paravermal and lateral regions of cerebellar cortex (Massopust et al., 1985; Tolbert and Gotting, 1998; Gerrits et al., 1985; Jasmine and Courville, 1987a, b; Quy et al., 2011). The present study extends these previous findings by showing that CN projections are also related to climbing fibre zonal organization. Projection densities from CN have not previously been investigated, except in the double retrograde tracer study by Berretta et al., (1991). In their study, large injections were made into anterior and posterior lobes of the cerebellum in rat (presumably involving multiple zones in paravermal/lateral parts of the cortex), and the Page 15 of 43 ScholarOne, 375 Greenbrier Drive, Charlottesville, VA, 22901 1(434)964-4100 ext. 1 European Journal of Neuroscience For Peer Review 16 projection to the anterior lobe was found to be more extensive than to the posterior lobe by a factor of 2:1 This is consistent with our findings in which cells in CN projecting to LS were twice as extensive as those targeting PML. However, Beretta et al. (1991) found that 14% of all labelled cells in CN were double-labelled, whereas in our study only 3.2% of all labelled cells were double-labelled. Taken together with the findings of Beretta et al., (1991), one possible interpretation is that rat paravermal/lateral zones located in more rostral parts of the cortex receive a higher density of projections from CN than the same zones located in more caudal lobules, but that some zones have a higher proportion of axonal branching. Further study of other individual zones will be required to test this possibility. Sensory trigeminal nucleus The STN relays propioceptive, mechanical, thermal and olfactory information from craniofacial structures to various centres in the brain (Waite and Tracey, 1995). Amongst these are direct trigeminocerebellar projections. These have been studied extensively in a range of mammalian species, including rat. Similar to our results, cells projecting to the cerebellum have been reported in pars oralis and pars interpolaris of the STN (Silverman and Kruger, 1985; Yatim et al., 1996; Phelan and Falls, 1991). However, in contrast to our findings, Yatim et al., (1996), using WGA-HRP as a retrograde tracer, concluded that STN does not have a projection to the C1 zone in rat. The discrepancy may arise because their two retrograde tracer injections into the C1 zone were in lobules III and VIII which are both ‘hindlimb-receiving’ areas of the cerebellar cortex (Atkins and Apps, 1997; Jorntell et al., 2000). Yatim et al., (1996) also made injections of anterograde tracer into pars oralis and pars interpolaris, and in both cases mossy fibre labelling was found in medial regions of LS where the C1 zone is Page 16 of 43 ScholarOne, 375 Greenbrier Drive, Charlottesville, VA, 22901 1(434)964-4100 ext. 1 European Journal of Neuroscience For Peer Review 17 located (see their Fig. 5). Terminal labelling in medial parts of PML was not found in their study, but given our finding of differences in projection density in the rostrocaudal axis this may be due to the difficulty of detecting weaker/more diffuse terminal labelling. In the rabbit, trigeminocerebellar projections have been reported to include the PML (Bukowska et al., 2003, 2006). Furthermore, Bukowska and colleagues provided evidence that the uvula, and rostral and caudal parts of PML receive independent trigeminal sensory information from neurones in separate regions of STN. This is broadly consistent with the present finding that there are partially independent trigeminal inputs to LS and PML. Medullary reticular formation The parvocellular reticular formation is thought to participate in a range of oralfacial behaviours, including eye blink reflexes (Smit et al., 2006), whisking (Hattox et al., 2002), mastication (Travers et al., 2010) and vocalization (Jurgens and Hage, 2007). Similarly, the intermediate reticular nucleus is thought to be involved in gustatory behaviour, including licking (Chen et al., 2001; Nasse et al., 2008). To our knowledge, the present study is the first to report mossy fibre projections from these regions of the RF to the cerebellar cortex. Our findings indicate there are substantial mossy fibre projections, particularly to the C1 zone in LS. Indeed, the greatest projection density to the C1 zone in LS was from the ipsilateral RF, and this exceeded the projection to PML by a factor of almost 4:1. This implies differences in function between these two rostrocaudally separated parts of the same cortical zone. Figure 5 summarizes our present findings regarding projection densities from CN, STN and RF to the C1 zone in LS and PML and also those of Herrero et al. (2002) Page 17 of 43 ScholarOne, 375 Greenbrier Drive, Charlottesville, VA, 22901 1(434)964-4100 ext. 1 European Journal of Neuroscience For Peer Review 18 regarding the projections from the BPN, nucleus reticularis tegmenti pontis (NRTP) and the LRN. In rat the largest source of mossy fibres to the C1 zone in LS is BPN, and in descending order, LRN, NRTP, RF, CN and STN (ipsilateral and contralateral projections pooled together). By comparison, the relative proportions of the projection densities to the C1 zone in PML are as follows: the largest source of mossy fibres is the LRN, followed by the BPN, CN, NRTP, STN and RF. Overall, the projection densities from all these nuclei to LS are significantly greater than to PML. Taken together these findings therefore strongly suggest that rostrocaudally separated parts of the same (C1) zone are functionally distinct. Comparison to physiological studies The current study focussed on mossy fibre projections to two ‘forelimbreceiving’ parts of the C1 zone. This somatotopical characterization is based on evidence that these regions of the C1 zone in rat, like other species, respond at short latency to sensory stimuli delivered to the ipsilateral forelimb, conveyed via the spinoolivocerebellar system (Atkins and Apps, 1997; Jorntell et al., 2000; Pardoe and Apps, 2002). Also, single-unit recording in awake behaving cats has shown that Purkinje cells in the C1 zone in the homologous region of the cat PML show a pronounced modulation in their discharge of simple spikes, time-locked to the course of the step cycle in the ipsilateral forelimb (Apps and Lidierth, 1989). This rhythmic discharge is presumably due in part to peripheral feedback from the ipsilateral limb being conveyed by the cuneocerebellar tract. It is noteworthy that this pattern of modulation is similar for lobule V of the anterior love but differences are also present (Armstrong and Edgley, 1984; Edgley and Lidierth, 1988). In particular, in lobule V the majority of Purkinje cells receive input from fairly restricted regions of the forelimb while in PML receptive Page 18 of 43 ScholarOne, 375 Greenbrier Drive, Charlottesville, VA, 22901 1(434)964-4100 ext. 1 European Journal of Neuroscience For Peer Review 19 fields tend to be more diffuse. Thus, different rostrocaudally separated parts of the same zone, while having a similar role in the control of movement may utilise different types and sources of afferent feedback (Apps and Lidierth, 1989). However the forelimb is not the only source of sensory input to the C1 zone in LS and PML. For example, other body regions also provide sensory inputs via climbing fibres, including the face/whisker area (Akaike, 1989; Jorntell el al., 2000) and the neck and thorax (Rushmer et al., 1980). Individual Purkinje cells in the C1 zone in PML can also display changes in both complex spike and simple spike activity to both face and forelimb inputs, showing convergence of sensory information from different body parts (Wise et al., 2010). Mossy fibre inputs tend to form multiple, rostrocaudally-oriented stripes (Wu et al., 1999; Gravel and Hawkes, 1990; Ruigrok et al., 1995; Serapide et al., 2001; Voogd et al., 2003). However, the somatotopical map in the cerebellar cortex established by detailed electrophysiological investigation of these mossy fibre inputs does not seem to consist of stripes, but rather, a mosaic of multiple representations (patches) of individual body parts to form a ‘fractured somatotopy’ (e.g. Shambes et al., 1978). To reconcile the two descriptions, it has been recently proposed that Purkinje cell stripes (zones) could be subdivided into smaller units that correspond to small patches (Apps and Hawkes, 2009). Different sources of mossy fibres displaying different projection densities to the C1 zone in LS and PML might then reflect the presence of different patches within the rostrocaudal extent of an individual zone. Such an arrangement may be involved in integrating different patterns of sensory input conveyed by the climbing Page 19 of 43 ScholarOne, 375 Greenbrier Drive, Charlottesville, VA, 22901 1(434)964-4100 ext. 1 European Journal of Neuroscience For Peer Review 20 fibre and mossy fibre systems to coordinate skilled movements which involve paw-facemouth interactions such as feeding and grooming (Rushmer et al., 1980). Mossy fibres versus climbing fibre inputs: zones and the one-map hypothesis The present findings extend our previous study (Herrero et al., 2002) by demonstrating that in addition to the LRN, other sources of mossy fibres to the C1 zone in LS and PML, the CN, STN and RF, also show a positive relationship between the degree of branching in the olivocerebellar axons. However, no such relationship could be found between olivocerebellar and pontocerebellar projections (Herrero et al., 2002). As 9 of the cases used in the current analysis were used in our earlier study, it is unlikely that the apparent difference between the BPN and other medullary sources of mossy fibres is due to a limitation of methods used. Rather, the difference may reflect the fact that CN, STN and RF (like the inferior olive and LRN) receive substantial somatosensory inputs, while BPN receives mainly descending inputs (Apps and Trott, 1997, Herrero et al., 2002). Following tracer injections into the C1 zone in LS and PML, the number of double-labelled cells in the three medullary sources of mossy fibres as well as the inferior olive varied substantially between experiments. A possible explanation for this is the fact that some, if not all, cerebellar zones can be further divided into microzones (Oscarsson, 1979, Garwicz et al., 1998). Each injection site may have occupied different combinations of microzones within the rostrocaudally separated parts of the C1 zone in LS and PML. Microzones are defined by their common climbing fibre input (Oscarsson, 1978; Garwicz et al., 1998). Therefore it follows that in double-tracer experiments a high proportion of double-labelled cells in the inferior olive would indicate that similar Page 20 of 43 ScholarOne, 375 Greenbrier Drive, Charlottesville, VA, 22901 1(434)964-4100 ext. 1 European Journal of Neuroscience For Peer Review 21 microzones have been targeted by the two tracer injections. In the present study, the percentage of double-labelled cells in the different medullary nuclei increased with the percentage of double-labelled cells in the inferior olive. There was also a strong positive correlation in the percentage of overlap of the territories occupied by the two populations of labelled cells in the inferior olive with the percentage of overlap between the two populations of labelled cells in the three sources of mossy fibres. Taken together these findings therefore support the one-map hypothesis, which predicts that each microzone within a particular longitudinal zone receives common mossy fibre and climbing fibre inputs (Apps and Hawkes, 2009). However, the present study also shows there are systematic differences in mossy fibre projection densities to the C1 zone in LS compared to PML, contrasting with similar climbing fibre projection densities from the inferior olive to the same zone (Pardoe and Apps, 2002). This suggests a refinement of the one-map hypothesis, because if the Purkinje cells are the scaffold during development that guide the distribution of climbing fibre and mossy fibres to form longitudinal zones/microzones (Apps and Hawkes, 2009), then rostrocaudal differences in projection densities imply that the scaffold accommodates a variable synaptic weight of mossy fibre inputs at different points along the length of the same zone/microzone. Acknowledgements We thank Ms Rachel Bissett and Ms Clare Everard for the preparation of histological material. This work was supported by the Medical Research Council, the Wellcome Trust, and the Spanish Ministerio de Innovación y Ciencia. Abbreviations Page 21 of 43 ScholarOne, 375 Greenbrier Drive, Charlottesville, VA, 22901 1(434)964-4100 ext. 1 European Journal of Neuroscience For Peer Review 22 BPN, basilar pontine nuclei; CN, external cuneate nucleus; DAO, dorsal accessory olive; ION, inferior olivary nucleus; iRT, intermediate reticular nucleus; LRN, lateral reticular nucleus; LS, lobulus simplex; NRTP, nucleus reticularis tegmenti pontis; MAO, medial accessory olive; pRF, parvocellular reticular nucleus; PML, paramedian lobule; RF, medullary reticular formation, STN, sensory trigeminal nuclei; STNc, caudal trigeminal sensory nucleus; STNi, intermediate trigeminal sensory nucleus ipsilateral; STNo, oral trigeminal sensory nucleus; Page 22 of 43 ScholarOne, 375 Greenbrier Drive, Charlottesville, VA, 22901 1(434)964-4100 ext. 1 European Journal of Neuroscience For Peer Review 23 References Abercrombie, M. (1946) Estimation of nuclear populations from microtome sections. Anat. Rec., 94, 239-247 Akaike, T. 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Res., 25, 267283 Page 31 of 43 ScholarOne, 375 Greenbrier Drive, Charlottesville, VA, 22901 1(434)964-4100 ext. 1 European Journal of Neuroscience For Peer Review 32 FIGURE LEGENDS Table 1 Summary of injection site details. Case number indicates the animal involved; R and G refer to red and green tracer material, respectively. Abbreviations: MAO, medial accessory olive; DAO, dorsal accessory olive. Table 2 Summary of retrograde labelled cell counts in the different nuclei studied. Double-labelled cell counts expressed as a percentage of smaller cell population. 1Cell counts are the totals for both sides of the brain; x refers to no retrograde transport. Abbreviations: CN, external cuneate nucleus; STN, sensory trigeminal nucleus; RF, medullary reticular formation. Table 3 Relative distribution of retrogradely labelled neurones between different nuclei providing mossy fibres to the injection sites. Ipsilateral (ipsi) and contralateral (contra) indicate laterality relative to the injection site. Table 4 Projection densities calculated for the different medullary nuclei. RDL2R and RDL3R were excluded because in these cases it was not possible to reliably calculate a site size due to difficulty in measuring the length of the Purkinje cell layer within the injection site core. Figure 1. Location of the injection sites. (A) Schematic map of a sagittal section of the cerebellum at the level of the paravermis to show the location of the injections sites in the lobulus simplex (LS) and paramedian lobule (PML) for an example experiment. The drawing also shows the microelectrodes to record (rec) cerebellar field potentials evoked by ipsilateral forelimb stimulation to guide the tracer injections (inj). The Page 32 of 43 ScholarOne, 375 Greenbrier Drive, Charlottesville, VA, 22901 1(434)964-4100 ext. 1 European Journal of Neuroscience For Peer Review 33 dashed boxes represent the areas shown in B, C. (B, C) Field potentials recorded in LS and PML following electrical stimulation of the ipsilateral forelimb (st). Each trace is an average of four sweeps. (D, E) Photomicrographs showing the injection sites in the LS and PML. (F) Photomicrograph showing example of a retrogradely labelled cell in the medullary reticular formation. Figure 2. Distribution of retrogradely labelled cells in the external cuneate nucleus, medullary reticular formation and trigeminal sensory nucleus in a representative experiment (RDL7). Cell labelling plotted on five equally spaced transverse levels of the medulla (AP levels -11.3 to -13.7) after injection of green tracer into the C1 zone in LS and red tracer into PML. Panel LS +PML shows the overlapping regions between the two single-labelled populations which correspond with areas in which doublelabelled cells were observed. Arrows indicate the distribution of labelled cells in the external cuneate nucleus. Interval between levels is 0.6 mm. Abbreviations: ION, inferior olivary nucleus; iRF, intermediate reticular nucleus; LRN, lateral reticular nucleus; pRF, parvocellular reticular nucleus; STNc, caudal trigeminal sensory nucleus; STNi, intermediate trigeminal sensory nucleus ipsilateral; STNo, oral trigeminal sensory nucleus; (ipsi) and contralateral (contra) indicate laterality relative to the injection site. Figure 3. Pooled data from all single and double-tracer experiments. From left to right each plot shows the total area containing retrogradely labelled cells after single-tracer injections into the C1 zone in LS and single-tracer injections into PML, and the overlapping areas of the two single-labelled cell populations in double-tracer experiments (LS + PML). The regions containing labelled cells from at least three Page 33 of 43 ScholarOne, 375 Greenbrier Drive, Charlottesville, VA, 22901 1(434)964-4100 ext. 1 European Journal of Neuroscience For Peer Review 34 animals is represented by black shading. Interval between levels is 0.6 mm. Abbreviations as in Figure 2. Figure 4. Relationship between divergence from inferior olive and from medullary nuclei to the C1 zone in lobulus simplex and the paramedian lobule. Area of overlap in the external cuneate nucleus (A), trigeminal sensory nucleus (B) and medullary reticular formation (C) between the two single-labelled populations (expressed as a percentage of the total area occupied by both LS and PML projections) plotted as a function of the area of overlap between the two single-labelled populations in the olive (expressed as a percentage of the total area). (D) Incidence of double-labelled cells in the medullary nuclei as a function of the proportion of double-labelled cells in the inferior olive (all expressed as percentages of the corresponding total labelled cell count). Figure 5. Diagram summarising differences in density of brainstem mossy fibre projections to the C1 zone in lobulus simplex and the paramedian lobule. Abbreviations as in Figure 2. BPN, basilar pontine nuclei; NRTP, nucleus reticulus tegmenti pontis. Includes data from Hererro et al (2002). Page 34 of 43 ScholarOne, 375 Greenbrier Drive, Charlottesville, VA, 22901 1(434)964-4100 ext. 1 European Journal of Neuroscience For Peer Review Figure 1. Location of the injection sites. (A) Schematic map of a sagittal section of the cerebellum at the level of the paravermis to show the location of the injections sites in the lobulus simplex (LS) and paramedian lobule (PML) for an example experiment. The drawing also shows the microelectrodes to record (rec) cerebellar field potentials evoked by ipsilateral forelimb stimulation to guide the tracer injections (inj). The dashed boxes represent the areas shown in B, C. (B, C) Field potentials recorded in LS and PML following electrical stimulation of the ipsilateral forelimb (st). Each trace is an average of four sweeps. (D, E) Photomicrographs showing the injection sites in the LS and PML. (F) Photomicrograph showing example of a retrogradely labelled cell in the medullary reticular formation. 145x132mm (300 x 300 DPI) Page 35 of 43 ScholarOne, 375 Greenbrier Drive, Charlottesville, VA, 22901 1(434)964-4100 ext. 1 European Journal of Neuroscience For Peer Review Figure 2. Distribution of retrogradely labelled cells in the external cuneate nucleus, medullary reticular formation and trigeminal sensory nucleus in a representative experiment (RDL7). Cell labelling plotted on five equally spaced transverse levels of the medulla (AP levels -11.3 to -13.7) after injection of green tracer into the C1 zone in LS and red tracer into PML. Panel LS +PML shows the overlapping regions between the two singlelabelled populations which correspond with areas in which double-labelled cells were observed. Arrows indicate the distribution of labelled cells in the external cuneate nucleus. Interval between levels is 0.6 mm. Abbreviations: ION, inferior olivary nucleus; iRF, intermediate reticular nucleus; LRN, lateral reticular nucleus; pRF, parvocellular reticular nucleus; STNc, caudal trigeminal sensory nucleus; STNi, intermediate trigeminal sensory nucleus ipsilateral; STNo, oral trigeminal sensory nucleus; (ipsi) and contralateral (contra) indicate laterality relative to the injection site. 195x212mm (300 x 300 DPI) Page 36 of 43 ScholarOne, 375 Greenbrier Drive, Charlottesville, VA, 22901 1(434)964-4100 ext. 1 European Journal of Neuroscience For Peer Review Figure 3. Pooled data from all single and double-tracer experiments. From left to right each plot shows the total area containing retrogradely labelled cells after single-tracer injections into the C1 zone in LS and single-tracer injections into PML, and the overlapping areas of the two singlelabelled cell populations in doubletracer experiments (LS + PML). The regions containing labelled cells from at least three animals is represented by black shading. Interval between levels is 0.6 mm. Abbreviations as in Figure 2. 193x230mm (300 x 300 DPI) Page 37 of 43 ScholarOne, 375 Greenbrier Drive, Charlottesville, VA, 22901 1(434)964-4100 ext. 1 European Journal of Neuroscience For Peer Review Figure 4. Relationship between divergence from inferior olive and from medullary nuclei to the C1 zone in lobulus simplex and the paramedian lobule. Area of overlap in the external cuneate nucleus (A), trigeminal sensory nucleus (B) and medullary reticular formation (C) between the two singlelabelled populations (expressed as a percentage of the total area occupied by both LS and PML projections) plotted as a function of the area of overlap between the two singlelabelled populations in the olive (expressed as a percentage of the total area). (D) Incidence of double-labelled cells in the medullary nuclei as a function of the proportion of double-labelled cells in the inferior olive (all expressed as percentages of the corresponding total labelled cell count). 307x783mm (300 x 300 DPI) Page 38 of 43 ScholarOne, 375 Greenbrier Drive, Charlottesville, VA, 22901 1(434)964-4100 ext. 1 European Journal of Neuroscience For Peer Review Figure 5. Diagram summarising differences in density of brainstem mossy fibre projections to the C1 zone in lobulus simplex and the paramedian lobule. Abbreviations as in Figure 2. BPN, basilar pontine nuclei; NRTP, nucleus reticulus tegmenti pontis. Includes data from Hererro et al., (2002). 146x228mm (300 x 300 DPI) Page 39 of 43 ScholarOne, 375 Greenbrier Drive, Charlottesville, VA, 22901 1(434)964-4100 ext. 1 European Journal of Neuroscience