Somatotopic nucleocortical projections to the multiple somatosensory cerebellar maps

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Neuroscience Vol. 83, No. 4, pp. 1085 1104, 1998 Copyright ~2) 1998 |BRO. Published by ElsevierScience Ltd Printed in Great Britain. All rights reserved PII: S0306-4522(97)00477-6 0306m522/98 $19.00+0.00

SOMATOTOPIC NUCLEOCORTICAL PROJECTIONS TO THE MULTIPLE SOMATOSENSORY CEREBELLAR MAPS L. P R O V I N I , * t W. M A R C O T T I , * S. M O R A R A * and A. R O S I N A * ++ *Istituto di Neuroscienze e Bioimmagini del CNR, Universita' di Milano, Milano, Italy fIstituto di Fisiologia Generale e Chimica Biologica, Universita' di Milano, Milano, Italy Abstract The cerebellum is organized in a series of parasagittal compartments: in C1-C3 and C2 compartments Purkinje cells receive climbing fibre afferents from the rostral part of the accessory olives, and project their axon to the nucleus interpositus anterior and posterior, respectively. Within these compartments electrophysiological studies have shown that the cutaneous input carried by climbing fibre afferents is topographically organized so as to design a map of peripheral body districts. The body map is replicated over the anterior lobe-pars intermedia and the paramedian lobule, and anatomical studies have indicated that the replication is partly due to the axonal branching of olivocerebellar neurons. The aim of this study was to analyse the presence of a somatotopic organization and of a branching pattern in the nucleocortical projections, in relation to the replicated body maps within C1 C3 and C2 compartments. By using double retrograde neuronal tracing we explored, in the cat, the topographic distribution of single- and double-labelled cells in the interposed nuclear subdivisions, after tracer injections into forelimb or hindlimb regions of the anterior lobe-pars intermedia, paramedian lobule and hemisphere (medial crus II). Most of the nucleocortical neurons were found in ipsilateral nucleus interpositus posterior, with smaller numbers in the ipsilateral nucleus interpositus anterior. Nucleocortical neurons projecting to forelimb- or hindlimb-related areas are completely segregated, the forelimb neurons being located laterally and the hindlimb neurons medially in the nucleus interpositus posterior. Within their respective domains both the forelimb and hindlimb populations projecting to the anterior lobe-pars intermedia are partly segregated from those projecting to the paramedian lobule, in that the two populations are slightly shifted along the dorsoventral axis of the nucleus. Although mostly different, some of the cells are common to the two forelimb populations, since they send axonal branches to the homologous areas of the anterior lobe and paramedian lobule. Contralateral fastigial or interposed nucleocortical projections are restricted to the anterior lobe-pars intermedia, and their neurons of origin are different from those that project to the ipsilateral cerebellar cortex: i.e. they are not a bilateral, but a separate contralateral component. ~) 1998 IBRO. Published by Elsevier Science Ltd. Key words': deep cerebellar nuclei, cerebellar circuitry, topography, feed-back, fluorescent tracing.

The cerebellum, a structure placed in a metasystemic position with respect to the main motor pathways, 37 plays a central role in motor, adaptive and learning processes, as indicated by ablation, functional and, more recently, regional oxidative metabolic studies in humans. 53 This structure receives peripheral and central afferences through its two main inputs, the mossy fibre (MF) and climbing fibre (CF) inputs. The one-to-one C F connection to Purkinje cell (Pc) and the fact that C F responses do not vary with either the type or intensity of the peripheral stimulus suggested that one of the significant parameters to be encoded by the C F input is the location of the stimulus on the body surface. ,tTo whom correspondence should be addressed. Abbreviations: CF, climbing fibre; dl, dorsal lamella of

principal olive; DY, Diamidino Yellow; FB, Fast Blue; IO, inferior olive; MF, mossy fibre; NF, nucleus fastigius; NIA, nucleus interpositus anterior; NIP, nucleus interpositus posterior; NL, nucleus lateralis; Pc, Purkinje cell; P1AL, pars intermedia of the anterior lobe; PML, paramedian lobule; PO, principal olive; rDAO, rostral part of the dorsal accessory olive; rMAO, rostral part of the medial accessory olive; vl, ventral lamella of principal olive.

A m o n g the concepts proposed for the organization of information relayed to the cerebellar cortex is that of a somatic representation of the body surface, summarized in the "distorted homunculus" by Snider. s2 According to the somatic organization a point-to-point representation of the body surface is relayed by C F input to the cerebellar cortex, as already suggested by the rather detailed olivocerebellar topography found in anatomical studies. 7 Contiguous parts of the body are represented contiguously, defining a complete map of the body: the resulting map is distorted since the extremities and face are represented with higher resolution than the other areas of the body. A new organizational scheme was later introduced by Voogd, 62 according to which the cat cerebellar cortex can be subdivided in a series of mediolateral compartments (A to D), 26'27 defined by the longitudinal organization of the olivocerebellar and corticonuclear pathways. 63 This anatomical scheme found support in physiological experiments in which, following electrical stimulation of the major spinoolivo-cerebellar pathways, mediolateral parasagittal

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zones (named a to d) were identified in the cerebellar cortex of the cat anterior lobe. 4° However, as already suggested by tract-tracing studies, s physiological experiments indicated, at least in some zones, a rostrocaudal segregation of forelimb-hindlimb representations, in keeping with the somatic representation concept. 7'52 More recently, studies employing natural tactile stimulation and single Pc recording of peripheral receptive fields in the intermediate cortex of the anterior lobe, revealed that CF-mediated afferences are organized in a complex mediolateral patch-like organization,45 which is reminiscent of Oscarsson's microzonal organization41 found in C1 and C3 zone. 21'22 Patches were defined by cells with common or contiguous receptive field, and contiguous patches could also represent non-contiguous parts of the body. 4'~46'51 Again, the overall organizational scheme of the intermediate cortex was found to consist of a large representation of forelimb in the caudal folia and of a minor representation of hindlimb in the more rostral folia of the anterior lobe. 44'51 The same forelimb-hindlimb segregation was observed in multiple-labellingtract-tracing studies on the spatial distribution of inferior olive (IO) axon collaterals. 9'49 Moreover, in keeping with the results of previous electrophysiological experiments,2"3 the study by Rosina and Provini49 revealed that the olivocerebellar system is topographically organized to define not only the body maps, but also, by way of axonal branching, the replication of the maps in the anterior and posterior lobes. The incidence of the axonal branching was estimated around 30% of the total neuron population projecting to any of the somatotopically homologous areas, within C1 C3 and C2 compartments.49'5° These results suggested that Pcs in the forelimb or hindlimb areas in pars intermedia of the anterior lobe (PIAL), paramedian lobule (PML) and crus II could be activated, in parallel, by the discharge of individual IO neurons and thus the information on the same peripheral receptive field could be relayed over Pcs located in different somatotopically homologous areas of a same compartment. This suggestion found experimental evidence in functional mapping studies of the cerebellar cortex during natural tactile stimulation. It was observed that the majority of the CF-mediated receptive fields encountered in the Pcs of PML were also found in the Pcs of PIAL or crus I I ] 2 That the somatotopic organization of the CF input to the cerebellar intermediate cortex is preserved in the somatotopic organization of the corticonuclear and nuclear efferent pathways from both the anterior and posterior interposed nuclei to the red nucleus and motor thalamic nuclei, has been shown by several authors. 4-6A5'23'47 In particular, in one of these studies direct evidence was given for a somatotopic alignment between the olivary projections to C1-C3 and C2 compartments and the cerebellofugal pathways from interposed nuclei to the red nucleus.47

Starting from this background, the present study was designed to verify the presence of a somatotopic organization in the nucleocortical projections in relation to the somatotopic and rostrocaudal collateral organization of CFs in C 1-C3 and C2 compartments. Indeed, the authors who first gave direct evidence of the existence of nucleo-cortical fibres in cat cerebellum, found that these projections are, at least in part, collaterals of the cerebellofugal fibres,25'54'55 a finding which was later substantiated by a lesion-electron microscopic study. 31 Moreover, electrophysiological and fluorescent double-labelling studies showed that nucleocortical fibres arise from collaterals of the nuclear efferent pathways directed to the motor thalamic nuclei. 32"42'56'57 Although it had previously been shown that specific areas of the cerebellar cortex are projected upon by topographically organized nucleocortical projections, these studies generally referred to the longitudinal topography in mediolateral compartments, 11'17As'28"3° rather than to the rostrocaudal somatotopy in multiple body maps, present in the intermediate compartments (see, however, Refs 36 and 60). Using double-fluorescent retrograde neuronal tracing (i.e. the combined use of fluorescent retrograde tracers Diamidino Yellow [DY] and Fast Blue [FB]), previously applied to the study of the olivocerebellar circuits, we explored, in the cat, the topographic distribution of labelled cells in nuclear subdivisions after tracer injections in somatotopically identified areas within C1 C3 and C2 cerebellar compartments. Our aims were to define: (i) the topographical pattern of nucleocortical projections in relation to the mediolateral sequence of the C zones; (ii) the rostrocaudal somatotopic arrangement and the degree of longitudinal collateralization in relation to the replicated somatosensory maps; (iii) the presence of any nucleocortical projections cross-linking different longitudinal compartments on the two sides of the cerebellum. EXPERIMENTAL PROCEDURES

The experiments were performed on cats. We used the technique of multiple fluorescent retrograde tracing,34 previously applied to the analysis of the olivocerebellar system,49'5° by injecting separately each of the two spectrally different retrograde fluorescent tracers, FB (trans-l-(5amidino-2-benzofuranyl)-ethylene-dihydrochloride)or DY (diamidino compound 288/26) into somatotopically-defined corticocerebellar areas of the different lobules. Two experimental paradigms were applied. In a first set of experiments (six animals), the more caudal forelimb-related folia of lobulus V-pars intermedia (forelimb-PIAL) and the more rostral (1 3b) folia of the paramedian lobule (forelimbPML), or the face and forelimb related folia of medial crus II or lobulus VI, were injected on one side, with either one or the other of the two spectrally different fluorescent tracers. In a second group of cats (four animals), the forelimb-PML or forelimb-PIAL folia were injected with one dye, while the more caudal, hindlimb-related folia of paramedian lobule (hindlimb-PML) or of lobulus IV of the anterior lobe-pars intermedia (hindlimb-PIAL) were injected with the second, always on the same cerebellar side.

Somatotopy of nucleocortical projections In a third group of animals (two cats) areas of either the forelimb-PIAL or lateral crus II were injected with one dye and the second was injected into the symmetrical areas on the contralateral side. A total of 12 young adult (two to four months) cats were used. Under Nembutal (35 mg/kg) anaesthesia, the cerebellar cortex was exposed and the tracers FB 3.0% (w/v), in distilled water, and DY 2.0% (w/v), in phosphate buffer at pH 7.2, delivered by multiple pressure microinjections through glass micropipettes (100 gm in diameter). Multiple cortical penetrations, aimed at being restricted to the superficial part of the cerebellar folia, were made at a depth of 0.7-1.0 mm, by delivering 100-150 nl at each penetration, for a total of 0.8 2.0 gl to each injected area. The injections were found to involve all the cortical layers and the subcortical white matter of the outer part of the stem of the folia. In none of the cases selected did the injected areas extend into either the cerebellar nuclei or the white matter immediately overlying them. Perfusion, histological procedures and data analysis were as previously described (see Refs 49 and 50). In brief, after an appropriate survival time for the tracers employed (10-14 days), the animals were transcardially perfused, under deep Nembutal anaesthesia, using a short rinse of saline followed by 10% cacodylate-buffered formalin (pH 7.2) for 1 h. Brainstems and cerebella were removed, postfixed for one additional hour in the same fixative at 4°C, and rinsed in 30% cacodylate-buffered sucrose (pH 7.2) at 4°C overnight. Frozen, 25 gm frontal sections were obtained from a cryostat, collected on chrome-alum-treated slides, and examined (one every three sections) under a LeitzDialux microscope equipped with a Ploem epi-illumination system, at an excitation wavelength of 360 nm. The position of retrogradely-labelled cells in the deep nuclei and the IO complex, as well as the extent of the injected areas in the cerebellar cortex, were mapped with the aid of an X- Y plotter coupled to linear transducers coaxial to the microscope stage. The incidence of double labelling was calculated by referring the number of double-marked neurons to the smaller of the two single-labelled populations of neurons, as counted in those sections where the two neuronal groups overlapped. The smaller of the two retrogradely-labelled populations was chosen since it defined the actual extent of the intermingling between the two retrogradely-labelled populations of neurons (see Ref. 49). The distribution and position of single and doublelabelled neurons in the deep cerebellar nuclei was transferred to outline maps of the structures, drawn from the counterstained sections. On the individual outline maps in the illustrations, taken at an interval of 300 ~tm through the rostrocaudal extent of the nuclei, the total number of cells from two frontal sections, 25 gm-thick each, and 150 gm apart, is reported. The position and extent of the cerebellar injected areas and of the IO labellings were transferred to a flattened dorsal view of the cerebellum (modified after Larsel135) and to an unfolded map of the IO complex, modified after Brodal. 7 RESULTS N e u r o n s retrogradely labelled by FB, DY, or by F B a n d D Y (see Fig. 1) were f o u n d in the deep cerebellar nuclei as well as in the b r a i n s t e m nuclei k n o w n to be the origin of cerebellar afferents, as previously reported. 49'5° Only those experiments in which the tracers h a d been properly injected into the cerebellar cortical areas will be considered. To this end, the t o p o g r a p h y o f single- a n d double-labelled cells in the IO complex were t a k e n as a n a posteriori control o f the actual

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location a n d extent of the injected areas (see Experimental Procedures, a n d Refs 49 a n d 50). Injections were considered as properly localized w h e n the IO cell groups retrogradely labelled by either one of the tracers were f o u n d in the rostral p a r t o f the dorsal a n d medial accessory olives ( r D A O a n d r M A O ) , related to the C 1 - C 3 a n d C2 c o m p a r t m e n t s , a n d restricted to either the forelimb or h i n d l i m b areas (within the caudal two thirds) of these IO subdivisions. 49"5° In addition, since the first group of experiments was aimed at involving somatotopically h o m o l o g o u s cerebellar areas, the injected areas were considered in register, w h e n the two IO singlelabelled p o p u l a t i o n s extensively overlapped. Moreover, in the cases considered the percentages of double-labelled n e u r o n s were f o u n d to be similar to those f o u n d in o u r previous studies on the IO axonal b r a n c h i n g to the intermediate cerebellum. 49"5°

Injectons into the forelimb related areas of the pars intermedia of the anterior lobe and of the paramedian lobule To analyse the t o p o g r a p h y of the nucleocortical projections to the forelimb area o f PIAL, a n d to c o m p a r e it with the localization o f the projections to f o r e l i m b - P M L , we first m a d e r a t h e r large injections, aimed at involving the whole cortical extent of f o r e l i m b - P I A L or of f o r e l i m b - P M L , with the individual tracers. This experimental p a r a d i g m also led to the unveiling of any b r a n c h e d projections to the two forelimb areas. To this end, the g r o u p of folia of lobulus V related to the forelimb representation, i.e. folia Vb-e, or the g r o u p o f folia in P M L related to the forelimb representation, i.e. folia 1-3b, were injected with individual tracers. W h e n injecting the entire g r o u p of forelimb-related folia in these two cerebellar regions, the n e i g h b o u r i n g folia of lobulus VI or the medialmost folia of crus II (ansula) were generally involved. These last cerebellar subdivisions are also recipient of a face-forelimb r e p r e s e n t a t i o n (see Refs 49 a n d 50). A n example o f this g r o u p of experiments is case M160: the fluorochrome D Y (2.5 gl, 2.0% w/v) was injected into f o r e l i m b - P I A L a n d the spectrally different fluorescent tracer FB (2.0 ~tl, 3.0% w/v) into f o r e l i m b - P M L . A t the histological e x a m i n a t i o n the D Y injected area was f o u n d to cover the intermediolateral parts of folia Vb-c a n d VIc, with a slight involvement of Va (Fig. 2A). In the contralateral IO complex retrogradely-labelled D Y n e u r o n s were f o u n d in a r a t h e r wide area o f r M A O , covering the medial h a l f of the subdivision, over the caudal twothirds of its r o s t r o c a u d a l extent. The IO n e u r o n s t h a t project to the forelimb area of either P I A L or P M L within the C2 stripe are k n o w n to be localized in this area, 49'5° that hereinafter will be called forelimbr M A O . DY-labelled n e u r o n s were also n u m e r o u s in the medial h a l f of r D A O , the sector o f D A O where n e u r o n s projecting to the forelimb areas of the C1

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C

Fig. 1. Photomicrographs of posterior interposed (NIP) neurons, retrogradely labelled after Fast Blue (FB) injection into folia Va-c of the anterior lobe (PIAL) and Diamidino Yellow (DY) into folia 1 3a of the paramedian lobule (PML). The cytoplasms and primary dendrites are labelled by FB, nuclei by DY. Arrowheads indicate double-labelled cells. Scale bar=20 gm.

and C3 compartments are located (forelimb-rDAO). In addition, scattered DY-labelled cells were found in both the ventral lamella (vl) and dorsal lamella (dl) of the principal olive (PO), indicating that the injected area covered the entire mediolateral extent of the intermediate C compartments, involving rims of D1 and D2 stripes (see Fig. 2B). The FB injection in PML covered the more rostral folia of the lobule (folia 1-3a, Fig. 2A), and part of the medialmost folium of crus II (ansula). Retrogradely-marked FB neurons covered most of the rMAO, suggesting that also the adjacent area face-forelimb area of C2 in the ansula was involved. 48'49 Numerous FB-labelled neurons were found in the forelimb-rDAO, indicating that the forelimb area of PML was significantly involved in

C1-C3 compartments. Finally, scattered cell labelling in vl and dl of PO showed that the D1-D2 stripes had also been injected. In brief, the FB-injected area in PML involved the forelimb-related areas, as did the DY injection in PIAL, over the same compartments. As shown in Fig. 2B, the two populations of labelled cells completely overlapped in all the IO subdivisions, and double-labelled cells were found at an incidence of 24.8% in rMAO, 26.7% in rDAO, 21.4% in vl and 18.7% in dl. These figures were very similar to those reported in previous studies by Rosina and Provini. 49,5° In the deep nuclei, cells retrogradely labelled by DY were found in the ipsilateral nucleus interpositus posterior (NIP) and, to a minor extent, in the nucleus

Somatotopy of nucleocortical projections

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Fig. 2. Distribution of cerebellar nucleocortical cells retrogradely labelled after tracer injections into somatotopically homologous forelimb areas of the anterior lobe-pars intermedia (folia Vb-c and VIc) and paramedian lobule (folia 1 3a), in case MI60. (A) Dots or circles indicate position and extent of the cerebellar areas injected with FB or DY, on a schema of the unfolded cerebellum (modified after Larsel135). (B) FB- and DY-retrograde cell labelling in the 10 subnuclei are indicated on a schema of the unfolded IO complex (modified after Brodal) by continuous and dashed lines, respectively. (C) The distribution of retrogradely-labelled cells, drawn from a series of caudorostral frontal cerebellar sections, is indicated on the outlines of the nuclear subdivisions. On each outline the labellings of two non adjacent sections, over 150 jam interval, are represented. FB-labelled and DY-labelled cells are indicated (2:1) by the same symbols as in A, double-labelled neurons are indicated (1:1) by crosses. Cerebellar folia and lobuli are indicated following Larsell's nomenclature, frontal planes in B were recognized according to Berman's atlas. Abbreviations: AL, anterior lobe; c, caudal; Cr.I, crus I; Cr.II, crus II; d, dorsal; DAO, dorsal accessory olive; dl, dorsal lamella of PO; Flocc., flocculus; F.pr., fissura prima; 1, lateral; m, medial; MAO, medial accessory olive; NF, fastigial nucleus; NIA, anterior interposed nucleus; NIP, posterior interposed nucleus; NL, lateral nucleus; P.fl.d., dorsal paraflocculus; P.fl.v., ventral paraflocculus; PML, paramedian lobule; PO, principal olive; r, rostral; vl, ventral lamella of PO. interpositus a n t e r i o r (NIA), c o r r e s p o n d i n g to comp a r t m e n t s C2 and C1 C3, respectively. 63 As can be seen in Fig. 2C most of the labelled cells were f o u n d in N | P , localized in the dorsocentral part of its lateral

half a n d extending over m o s t of the c a u d o r o s t r a l extent o f the nucleus. In N I A , a DY-labelled cell p o p u l a t i o n was f o u n d caudally in the ventromedial sectors of the nucleus, while rostrally the cells were

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few and scattered. Ipsilateral labelling was also found in nucleus lateralis (NL), along the medial border of the dorsal sectors of the nucleus. Also FB-labelled neurons were found ipsilaterally in the lateral half of NIP, like the DY-labelled neurons. However, the FB population was shifted slightly ventrally covering only the central two-thirds of the lateral half of the nucleus. Very few FBmarked cells were found in NIA, and these were localized to the ventrocaudal sectors of the nucleus, overlapping the DY-labelled cells. Similarly, FB neurons were found along the medial border of NL, ipsilaterally localized like the DY cells. Thus, there was an extensive intermingling of the DY- or FB-labelled populations in all the deep cerebellar nuclei. In particular, in NIP, doublelabelled cells were found irregularly distributed within the celatral region of the overlap (see Fig. 2C), where they represented 22.5% of the smaller of the two labelled populations. In N I A few labelled neurons were seen. Furthermore, very few doublelabelled neurons were found in NL, and these were restricted to the caudomedial sectors of the nucleus where the cell populations tended to cluster. Thus, nucleocortical neurons sending axon collaterals to the forelimb areas of PIAL and PML were found to be significantly present only in the intermediate C2 compartment. In addition to these ipsilateral nucleocortical neurons, which are reciprocal in terms of C compartments, 18"24'3°'54 few ipsilateral non-reciprocal DYlabelled neurons were seen in the nucleus fastigius (NF), in its more rostral portion. Several DY neurons were also found contralaterally, in the rostral part of NF, laterodorsally located, and in the rostroventral sectors of NIP. No FB-labelled cells were found either in the ipsilateral N F or in the contralateral NIP and NF. Thus, whereas ipsilateral interposed projections to PML mirrored the PIAL ones, fastigial or contralateral nucleocortical neurons projecting to the forelimb-PML were absent. To further identify the topographic organization of nucleocortical projections in relation to the forelimb areas of the intermediate cerebellum, more restricted injections were made, like in the two cats M167 and M 162 that are representative examples and described in the following in some detail. In cat M167 the DY injections (1.3 gl) were limited to the intermediate parts of folia Vb-d and the FB injections (1.5 gl) to folia 2 and 3a of PML (see Fig. 3A). In the IO complex, the two labelled populations showed a slight rostrocaudal shift in that the DY, forelimb-PIAL projecting cells covered slightly more caudal areas in rMAO and rDAO than the FB ones projecting to forelimb-PML (see Fig. 3B), as described in previous studies. 49 However, both labellings were limited to the forelimb areas of rDAO and rMAO, corresponding to compartments C1-C3 and C2, respectively. Double-labelled neurons were present in wide areas of both accessory olives, show-

ing percentages (18.9% in rMAO and 21.0% in rDAO) similar to the figures reported in previous studies. In the PO practically no DY labelling was seen, while scattered FB-labelled neurons were found in both lamellae, indicating a marginal involvement of D1-D2 compartments of PML. In summary, the two injected areas resulted in somatotopic and compartmental registers. Large populations of neurons were seen ipsilaterally in NIP, localized to the lateral half of the nucleus (see Fig. 3C). Within this sector, DY neurons occupied the dorsal region, whereas the FB population was shifted ventrally to cover also the more central region of the sector. The two nucleocortical populations also showed a rostrocaudal shift, in that FB neurons were found at the very caudal pole of NIP, left free by the DY neurons. This last population extended rostrally to cover the rostral third of the nucleus, where FB cells were rarely found. In the central part of the lateral sector of the nucleus the two populations overlapped, resulting in nucleocortical neurons double-labelled by FB and DY i.e. sending axonal collaterals to both PIAL- and PML-forelimb areas. The incidence of such neurons (10.5%) was, however, lower than that found in experiment M 160. In contrast to the sustained and specific topographic nuclear labellings found in NIP, very few neurons labelled by either tracer were found in NIA. Neurons singly labelled by the FB tracer were also found in the nucleus lateralis, due to the involvement of the D1 D2 stripes at the PML injection site, as indicated by the neuronal labelling in both lamellae of the PO. In addition to these ipsilateral interposed projections, scattered DY-cells, labelled by the PIAL injection, were found bilaterally in the NF, while virtually no FB-labelled neurons were seen either in ipsilateral N F or contralateral N F and NIP (Fig. 3C). Thus, the two populations of neurons projecting to either forelimb-PIAL or forelimb-PML showed a specific localization in the posterior interposed nucleus, where they together occupied the dorsolateral sectors throughout the rostrocaudal extent of the nucleus. Inside this area the two populations showed a dorsoventral and rostrocaudal shift, the forelimb-PIAL projecting neurons being dorsally and rostrally located with respect to the forelimb-PML projecting cells. Following the same experimental paradigm, cat M162 (Fig. 4) had DY (2.0 ~tl) injected into folia Vb-c, with slight involvement of Va, and FB (1.4 gl) into folia 1, 2 and 3a of PML (Fig. 4A). In the IO, both DY and FB retrograde labellings covered the forelimb-related areas of both rMAO and rDAO, but the FB-labelled neurons, although numerous, showed a more restricted localization (Fig. 4B). However, the double-labelled neurons showed percentages (18.8% in rMAO and 33.1% in rDAO) comparable to those found in our previous studies on olivocerebellar

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axonal branching. 49'5° Thus, the two injected areas involved C2 a n d C1 C3 zones, a n d were in somatotopic register. A l t h o u g h there was n o involvement of the A or B zones, as attested by the absence of retrograde labellings in the caudal accessory olives, the scattered D Y a n d FB retrograde labelling in the lamellae of the P O indicated t h a t b o t h the P I A L a n d

P M L injections also slightly involved the D 1 - D 2 zones. Retrogradely-labelled nuclear cells could be traced ipsilaterally to the injected areas, localized in the posterior interposed nucleus (Fig. 4C). There, D Y nuclear neurons, projecting to the forelimb region of P I A L , were f o u n d distributed over the dorsal sectors

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Fig. 4. Distribution of cerebellar nucleocortical cells retrogradely labelled after tracer injections into somatotopically homologous forelimb areas of the anterior lobe-pars intermedia (folia Vb~z) and paramedian lobule (folia 1 3a), in case M162. (A) Dots or circles indicate position and extent of the cerebellar areas injected with FB or DY, on a schema of the unfolded cerebellum (modified after Larsel135). (B) FB- and DY-retrograde cell labelling in the IO subnuclei are indicated on a schema of the unfolded IO complex (modified after Brodal) by continuous and dashed lines, respectively. (C) The distribution of retrogradely-labelled cells, drawn from a series of caudorostral frontal cerebellar sections is indicated on the outlines of the nuclear subdivisions. On each outline the labellings of two non-adjacent sections, over 150 [am interval, are represented. FB-labelled and DY-labelled cells are indicated (1:1) by the same symbols as in A, double-labelled neurons are indicated (1:1) by crosses. of the lateral half of the nucleus, whereas the FB neurons, projecting to the forelimb region of PML, were also located laterally, but slightly shifted to a more central position along the dorsoventral axis, as in the previously described case. The two neuronal groups were also partly shifted along the anteroposterior axis. Thus, the two populations of neurons projecting to either the forelimb region of P M L or to the forelimb-PIAL only partly overlapped along both

the dorsoventral and rostrocaudal axes of NIP. In the central sectors, where the two populations intermingled, neurons labelled by both tracers were seen, with an incidence around 12.0%. In contrast to the specific topography of the nucleocortical projections found in the posterior interposed nucleus, very few labelled neurons were found in the anterior interposed, in spite of the massive involvement of the C1-C3 compartments. In addition to these ipsilateral

Somatotopy of nucleocortical projections interposed projections, scattered neurons, labelled by DY, were found in N F both ipsi- and contralaterally, and in the contralateral NIP. Fastigial or contralateral neurons labelled by FB, projecting to forelimb-PML, were not observed in this case. To detect whether the nucleocortical projections to other forelimb-related areas within the intermediate compartments show a similar topography, cortical areas in lobulus VI and medial crus lI, recipient of CF-mediated face-forelimb representations, were explored. It is known that these cerebellar cortical areas receive olivary afferents from neurons in rMAO and rDAO, located rostrally but partly intermingling the neuronal groups projecting to forelimb-HAL and forelimb-PML49 and that medial crus II and lobulus VI share, with forelimb-HAL and forelimb-PML, axon collaterals from common olivary neurons (Ref. 49 and unpublished observations). In one of these experiments, cat M161, the fluorochrome DY (1.5 ~tl) was injected in the intermediolateral part of lobule VI and FB (1.8~tl) was deposited in the medial tblia of crus II. At the histological examination, DY staining was found to involve not only lobule VI (folia VIa,b,c), but also the adjacent folium Vc, from the paravermal vein to the lateral border of the folia (Fig. 5A). It was evident from the IO cell labelling, localized over the whole forelimb area of both rMAO and rDAO, that the DY-injected area covered the forelimb area over stripes C2 and C1-C3. An irregular cell labelling in vl and dl of the PO indicated a partial involvement of stripes D1 D2 (Fig. 5B). The FB staining was found to cover the cortex of the three medialmost folia of crus II. Labelling in contralateral IO was restricted to the rostral pole of rMAO and to a stripe of cells along the medial border of rDAO, indicating the involvement of the ansula48 in both C2 and C1-C3 compartments. PO lamellae were also diffusely labelled. Thus, the overlap between the two FB- and DY-labelled populations was confined to the small forelimb areas defined by FB cells in rMAO, rDAO, and PO (Fig. 5B). Within these areas double-labelled cells were found, with an incidence of 18.0°/o, 29.4%, 13.0% and 11.8% in rMAO, rDAO, dl and vl, respectively. In the deep cerebellar nuclei, DY-labelled neurons were found in the dorsal two-thirds of the lateral part of NIP, i.e. in the areas of forelimb-HAL and forelimb-PML projecting neurons (Fig. 5C). Also FB-labelled cells were found in lateral sectors of NIP, but shifted caudoventrally with respect to the DY cells. Thus, the neurons feeding back to the forelimb area of lobulus VI intermingled with the nucleocortical cells projecting to the medial crus II only in a very small area of NIP, where a few neurons branched to both areas (eight cells, 14.8% of the minor population). DY cells were in this case consistently found in NIA, localized to its rostromedial sectors, as well as in NF, bilaterally. In contrast, virtually no FB cells were seen either in NIA or in

1093

NF, or contralaterally. A few DY and FB cells were found also in NL, in the medial region of the caudal half of the nucleus, due to the involvement of D1-D2 stripes by both tracers, as indicated by the neuronal labelling in both lamellae of the PO.

Injections into the forelimb- or hindlimb-related areas o f the pars intermedia o f the anterior lobe or o f the paramedian lobule or ol" medial Crus H (ansula) Further evidence for a somatotopic segregation in the ipsilateral component of the nucleocortical projections to the intermediate cerebellum is given by the results of experiments where the two fluorescent tracers were injected separately into the forelimb or hindlimb areas of either the same lobule (HAL) or different lobules (HAL and PML), on one side. In one of these experiments (cat M 166, Fig. 6) the fluorochrome FB (1.0 btl) was injected into the hindlimb area of H A L (lobule IVa-b), while the other tracer (DY, 1.1 btl) was deposited into the forelimb area of PIAL (lobule Vb-c, with slight involvement of Va; see Fig. 6A). In fact, the injected cerebellar areas were found to be limited to the C1-C3 and C2 compartments, as indicated by the FB and DY retrograde labellings, which were both restricted to rDAO and rMAO. The injected areas were also somatotopically centred, in that FB labelling was restricted to the lateral half of either rDAO or rMAO, known as hindlimb areas, 49 while DY labelling was limited to the forelimb areas of both accessory olives. The two injection sites showed no overlap, as shown by the FB and DY retrograde labellings which were fairly well segregated, except for a very modest rim of overlap where very few double-labelled cells, projecting to both hindlimb and forelimb areas, were seen (Fig. 6B). In the deep nuclei (see Fig. 6C), the hindlimbrelated FB population was localized to the dorsal sector of the medial half of NIP, left free by the forelimb-related DY neuronal groups, that occupied the forelimb (dorsolateral) area of the nucleus, as in the forelimb cases described above (see Figs 2 5). In NIA, a very small population of labelled neurons, mostly DY-labelled, localized in the dorsocaudal sectors of the nucleus was seen. A few neurons were found in NF bilaterally, whereas neurons in contralateral NIP were practically absent. No doublelabelled cells were found in these experiments, indicating that the nucleocortical projections from NIP to the hindlimb- or forelimb-related areas of H A L are indeed segregated. Similar results were obtained in another experiment, cat M165, in which tracer injections were made separately into the forelimb area of PIAL and the hindlimb area of PML. Following FB injection (1.4 btl) into the forelimb-HAL (folia Vb-c) and DY injection (1.6 lal) into the hindlimb-PML (caudal folia 4a,b and 5), retrogradely-labelled FB or DY neurons were found in completely segregated domains of the

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Fig. 5. Distribution of cerebellar nucleocortical cells retrogradely labelled after tracer injections into somatotopically homologous face-forelimb areas of the anterior lobe-pars intermedia (folia Vlc a and Vc) and medialmost folia of crus II, in case M161. (A) Dots or circles indicate position and extent of the cerebellar areas injected with FB or DY, on a schema of the unfolded cerebellum (modified after Larsel135). (B) FB- and DY-retrograde cell labelling in the IO subnuclei are indicated on a schema of the unfolded IO complex (modified after Brodal) by continuous and dashed lines, respectively. (C) The distribution of retrogradely-labelled cells, drawn from a series of caudorostral frontal cerebellar sections is indicated on the outlines of the nuclear subdivisions. On each outline the labellings of two non-adjacent sections, over 150 lain interval, are represented. FB-labelled and DY-labelled cells are indicated (l:l) by the same symbols as in A, double-labelled neurons (1:1) are indicated by crosses.

contralateral IO complex (Fig. 7A,B). FB labelling covered the forelimb areas of both accessory olives, and the rostral regions of the PO lamellae, whereas DY-labelled populations covered the caudal portions of the hindlimb domain of both accessory olives, and small caudal areas of PO lamellae. Thus, as a whole the injected areas were perfectly centred in the forelimb or hindlimb areas of the injected lobules and

were found to cover C 1 - C 3 and C2 zones and limited parts of the D 1 - D 2 stripes. In the deep nuclei, most of the FB neurons, projecting to forelimb-PIAL, were found localized to the dorsolateral (forelimb) sectors of ipsilateral N I P (Fig. 7C), as in the cases previously described (see Figs 2-6). Also most of the DY-labelled cells were found in the ipsilateral NIP, located medially to the

Somatotopy of nucleocortical projections

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v /~<~,, (_. Fig. 6. Distribution of cerebellar nucleocortical cells retrogradely labelled after tracer injections into forelimb-related (folia Vb~z) areas and hindlimb-related (folia IV a-b) areas of the anterior lobe-pars intermedia, in case M166. (A) Dots or circles indicate position and extent of the cerebellar areas injected with FB or DY, on a schema of the unfolded cerebellum (modified after Larsell3S). (B) FB- and DY-retrograde cell labelling in the IO subnuclei are indicated on a schema of the unfolded 10 complex (modified after Brodal) by continuous and dashed lines, respectively. (C) The distribution of retrogradelylabelled cells, drawn from a series of caudorostral frontal cerebellar sections is indicated on the outlines of the nuclear subdivisions. On each outline the labellings of two non-adjacent sections, over 150 pm interval, are represented. FB-labelled and DY-labelled cells are indicated (1:1) by the same symbols as in A, double-labelled neurons (1:1) are indicated by crosses. cell p o p u l a t i o n projecting to f o r e l i m b - P M L , in the dorsocentral sectors of the medial h a l f of the nucleus (see Fig. 7C). As it can be seen, the n e u r o n p o p u l a t i o n projecting to h i n d l i m b - P M L showed the same dorsomedial localization as the n e u r o n p o p u l a t i o n projecting to h i n d l i m b - P I A L , but was slightly shifted to a more ventral position within the medial half of N I P (cf. Fig. 6C). Very few FB or DY n e u r o n s were seen in NIA, as in the

previously described cases, a n d these were localized to the caudal regions of the nucleus. In ipsilateral NL, small but consistent groups of n e u r o n s retrogradely labelled by F B or D Y were f o u n d in the ventral regions along the caudorostral extent of the nucleus. Scanty if any N F or contralateral n e u r o n s were seen. F r o m these last a n d previously described cases, it can thus safely be concluded t h a t nucleocortical

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partly overlapped since the PML-related population is consistently shifted to a more ventral position in respect to the P I A L population. In contrast the overlap of the two labelled neuronal groups in the IO was always almost complete. Although not directly demonstrated, since no hindlimb-PIAL and hindlimb-PML experiments were made, a similar degree of overlap and possibly of axonal branching, together with a partial segregation of the two

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Fig. 8. Distribution of cerebellar nucleocortical cells retrogradely labelled after symmetrical tracer injections into the cerebellar forelimb areas of the anterior lobe-pars intermedia (folia Vb~z) on the two sides, in case M163. (A) Dots or circles indicate position and extent of the cerebellar areas injected with FB or DY, on a schema of the unfolded cerebellum (modified after Larsel135). (B) FB- and DY-retrograde cell labelling in the IO subnuclei are indicated on a schema of the unfolded 10 complex (modified after Brodal) by continuous and dashed lines, respectively. The two opposite IO labellings are represented on a single schema of the unfolded IO. (C) The distribution of retrogradely-labelled cells, drawn from a series of caudorostral frontal cerebellar sections is indicated on the outlines of the nuclear subdivisions. On each outline the labellings of two non-adjacent sections, over 150 gm interval, are represented. FB-labelled and DY-labelled cells are indicated (1:1) by the same symbols as in A.

nucleocortical populations, is also likely to apply to the h i n d l i m b areas of P I A L a n d P M L .

Bilateral injections into symmetrical areas of the intermediate cerebellum In case M163 (Fig. 8), the f l u o r o c h r o m e FB (1.9 ~tl) was injected into the intermediate part o f folia V b ~

o n the left side, while D Y (1.7 ~tl) was injected into the symmetrical area of lobulus V on the right side. F r o m the analysis of the cerebellar injected areas as well as of the respective contralateral IO complexes, it was clear t h a t b o t h injected cortical areas were limited to the forelimb areas of c o m p a r t m e n t s C1 C3 a n d C2. In fact in this case b o t h the FB a n d D Y retrograde labellings were restricted to the forelimb

L. Provini et al.

1098

regions of rMAO and rDAO, without any involvement of other olivary subdivisions and, therefore, of any other cortical stripe (Fig. 8A,B). Nuclear cells retrogradely labelled by FB were found localized to the dorsal sector of the lateral half of NIP, as already observed after forelimb-PIAL injections in the above reported cases (see Figs 2- 7). In N I A only few scattered cells were seen, in the dorsocentral portion of the nucleus, and very few in the adjacent NL, N F s or contralateral NIP. Neurons retrogradely labelled by DY had a nearly symmetrical localization in the ipsilateral NIP, while hardly any labelled neurons were seen in the ipsilateral N I A or NL (Fig. 8C). Very few DY-labelled cells were found in N F s or contralateral NIP. In the limited regions of NIP and N F where the two labelled neuronal groups were to some extent intermingled, no double-labelled neurons were seen. In another case M164 (Fig. 9), bilateral injections were made into the lateralmost folia of crus II of the hemisphere. The fluorochrome FB (3.0 p-l) was injected on the left, while DY (1.8 gl) was injected into the symmetrical area on the right side. From the analysis of the cerebellar-injected areas as well as of the respective contralateral IO complexes, it appeared that both injected cortical areas were limited to C2 and D1-D2 compartments. Both the FB and DY retrograde labellings were restricted to the rMAO and to the lamellae of the PO, without any involvement of rDAO, and, therefore, of stripes C1-C3 (Fig. 9A,B), in keeping with the results of a previous study on the olivary projections to the cat ansiform lobule, which showed that no C1 or C3 zone are found in the lateralmost folia of crus II (see Ref. 48). Nuclear cells retrogradely labelled by FB were found localized to the ventralmost sector of the posterior half of NIP, partly overlapping the nuclear cells projecting to medial crus II and partly further shifted to the ventromedial corner of the nucleus left free by the hindlimb-PML projecting cells. FB retrogradely-labelled cells were also numerous in NL, localized to the dorsal sectors of the nucleus along the posterior two-thirds of its extent. In N I A no ipsilateral or contralateral cell labelling was seen. In addition no retrogradely-labelled cells were found in the N F on both sides, nor contralaterally in NIP or NL, in agreement with the results reported above on the lack of fastigial and symmetrical contralateral projections to the posterior lobe cortex. Neurons retrogradely labelled by DY were also numerous, but restricted to the ipsilateral NIP and NL where they showed a nearly symmetrical localization in respect to the FB ones. DISCUSSION

The technique of multiple fluorescent tracing applied to the analysis of cerebellar circuits has been extensively discussed in previous papers. 49'5° How-

ever, a few comments on its application to the present investigation are needed. As has already been reported by other authors, 57 we too observed that significant numbers of nuclear neurons can be retrogradely labelled only after injections of relatively large volumes of tracer solution (1.0-1.5 p-l), i.e. involving a relatively large volume of cerebellar cortex. This is most likely due to the fact that only part of the terminal axonal field of nucleocortical cells was involved by cerebellar cortical injections, since nucleocortical axons are mostly collaterals of nuclear fibres directed to extracerebellar targets. 54 The labelled nuclear neurons were generally multipolar in shape, less frequently fusiform (see Fig. 1), and of medium to large size (15-30 lain in diameter). 33"3s'54 No attempt was made to relate the morphology of labelled nuclear neurons to their still debated neurochemical identity (see Refs 13, 31, 33 and 58). Labelled neurons were generally found scattered within a population of unlabelled cells. This topological distribution sometimes made it difficult to define both the area specifically occupied by a population of labelled cells, and the areas of overlap between the two labelled populations. Using double-retrograde neuronal tracing (i.e. the combined use of fluorescent retrograde tracers DY and FB) previously applied to olivocerebellar circuit studies we explored, in the cat, the topographic distribution of labelled cells in the nuclear subdivisions, after tracer injections into different cerebellar areas in relation to: (i) the topographical pattern of nucleocortical projections in relation to the mediolateral sequence of the C zones; (ii) the rostrocaudal somatotopic arrangement and the degree of longitudinal collateralization in relation to the replicated somatosensory maps; (iii) the presence of any nucleocortical projections cross-linking different longitudinal compartments on the two sides of the cerebellum. Three main points emerged from this analysis. First, the topographic organization of the nucleocortical projections in terms of mediolateral compartments was here confirmed. Second, there was evidence of a somatotopic arrangement of these projections, in register with the olivocerebellar somatotopy within compartments. Third, nucleocortical axon collaterals were found to link somatotopically homologous cerebellar areas within C longitudinal compartments, whereas no nucleocortical collaterals were found to link the two sides of the cerebellar cortex.

Mediolateral topography of nucleocortical projections With regard to the first point, our results indicate that the ipsilateral component of the nucleocortical projections is organized in a sequence that parallels the mediolateral sequence of the olivo-corticonuclear compartments, as indicated by the olivary labelling. Even if we did not individually inject the C1, C2 and C3 zones, and thus direct evidence of the

Somatotopy of nucleocortical projections

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were only f o u n d in N I P a n d NIA, a n d no labelled cells were f o u n d in NL. Conversely, in the other cases in which the injected areas involved C2, C 1 - C 3 a n d D 1 - D 2 zones, there was retrograde labelling in all the c o r r e s p o n d i n g deep nuclei, i.e. NIP, N I A a n d N L respectively. Finally, in case M 164 (Fig. 9) in which two spectrally different tracers were injected o n b o t h sides into the lateralmost folia of crus II, where only C2 (and D1 D2) zone is present, w i t h o u t any

1100

L. Provini et al.

involvement of C1-C3 zones (see Ref. 48), retrogradely-labelled neurons were found in NIP (and NL) whereas no labelling was seen in NIA. Thus, it can be concluded that here reported nucleocortical projections are reciprocally organized (i.e. they are directed toward the cortical zones from which arise the corticonuclear projections they receive), in particular nucleocortical projections directed to C2 zone stem from NIP and those to C1-C3 from NIA. Within the mediolateral sequence of the C zones the incidence of the nucleocortical neurons was strikingly different, pointing to a different weight of the feedback circuit among the different compartments of the intermediate cerebellum. Thus, whereas retrogradely-labelled neurons were always very numerous in NIP, that corresponds to the cortical stripe C2, very few nucleocortical neurons were generally found in NIA, corresponding to C1 C3 compartments (see Figs 2, 4, 6 and 8). An uneven distribution of the nucleo-cortical neurons between the two interposed nuclei had already been observed in studies in which the injections involved the mediolateral sequence of the intermediate compartments (in the cat, ~7,18,19,24,56 in the rat, 1o,61 in the monkey,55 in the bushbaby29). More recently, failure to retrogradely label nucleocortical neurons in N I A has been reported, in a study on the cat cerebellum where the injections were restricted to the individual C1, C2 or C3 cortical zones. 6° In our experiments, where the C1 and C3 zones were simultaneously injected, as indicated by the olivary labelling, a few labelled cells were usually found in NIA. Our findings on N I A labelling following tracer injections that simultaneously involved CI and C3 were interpreted by us in a way that reconcile both our positive findings and Trott's negative findings. It is well known that the amount of retrogradelytransported tracer, and therefore the neuronal labelling, increases when the axonal terminal field of a neuron is more extensively injected, the hypothesis is here advanced that N I A neurons may, at least in part, send axonal branches both to C1 and C3 zones. This suggestion is made in analogy with the evidence that rDAO neurons send, at least in part, axonal branches to C1 and C3 zones of PIAL. 2° Our conclusion is, therefore, that N I A labelling can only be obtained when both C1 and C3 zones are involved at the injection site. The variability in the density of nucleocortical projections found in N I A through our cases, is a finding which may be attributed to a different involvement of C1 versus C3 zone in the different cases, if one assumes that also in the cat the C1 zone is the main recipient of nucleocortical projections from NIA, as already reported for the rat. 1°'~1 Despite the key role of an interaction between the nuclear and the cerebellar cortical neurons, it is only recently that studies have attempted to systematically relate nucleocortical projections to specific subdivi-

sions of the cerebellar cortex. 11'17--19'6° However, such studies have mostly dealt with the nucleocortical organization in terms of olivo-cortico-nuclear compartments, stressing the concept of a reciprocity between projections from the nuclear neurons to a given cortical stripe and the distribution of the Purkinje cell axons by which the nuclear neurons are i m p i n g e d upon. 11'17 19,24,30,54,59,60 In our experiments we consistently found, in addition to the above described ipsilateral interposed projections, which are reciprocal to the injected zones, minor ipsi- and contralateral non-reciprocal projections stemming from the fastigial nucleus on both sides, as already described in previous reports.11,17- |9,24,30,54,59,60 In particular, the present findings are consistent with earlier observations in that a few cells were labelled contralaterally in NIP after tracer injections in PIAL. 11'3° On the other side, our results show that after tracer injections into the posterior lobe cortex of PML or crus II contralaterally-labelled cells were very seldom seen (see Figs 2-5, 7 and 9), indicating that only the anterior lobe is recipient of symmetrical contralateral projections. In addition, no retrogradely-labelled cells were seen in N F either ipsi- or contralaterally after tracer injections into PML or the hemisphere, indicating that the intermediate and lateral compartments of the cat posterior lobe are not recipient of fastigial projections, in contrast with previous observations in the cat. 18'~9

Somatotopy of nucleocortical projections The second and main point emerging from this study is that the ipsilateral interposed nucleocortical projections to the intermediate cerebellum are somatotopically organized. In brief, the nucleocortical neurons projecting to the forelimb areas of PIAL, PML or crus lI are localized to the lateral half of the ipsilateral NIP, while neurons in the medial half of NIP are related to hindlimb representations. Our observations that the interposed nucleocortical neurons projecting to the ipsilateral forelimb (or hindlimb) areas are topographically organized, and segregated from the neurons projecting to the hindlimb (or forelimb) areas are, to the best of our knowledge, the first direct evidence of a somatotopic organization in the nucleocortical projections. This result mainly applies to NIP, related to C2 compartments, where the density of the projecting neurons was consistently high. In NIA, related to C1-C3 compartments, the topography of the projections could not be precisely defined, due to the paucity of the retrograde labelling. However, the majority of the retrogradely-labelled cells observed in N I A seem to follow the somatotopic organization described for NIA efferents to the red n u c l e u s 15"23"47 o r N I A corticonuclear afferents.16 The clearcut somatotopic pattern found in the nucleocortical projections from NIP related to the C2

Somatotopy of nucleocortical projections compartment needs further comment. Though a clear forelimb-hindlimb subdivision is recognized for the C1 and C3 compartments, a gross type of forelimbhindlimb segregation is recognized in the C2 zone. 1.43.44.51 Such a forelimb-hindlimb segregation was not recognized in the C2 zone in studies based on the electrical stimulation of spino-olivo-cerebellar pathways. 4° It must be noted that these studies employed high-intensity electrical stimulation of the peripheral nerves after selective lesioning of spinal cord tracts, in decerebrate animals: they may, therefore, have isolated pathways and systems other than those activated during more natural conditions.45 Indeed, field-potential analyses of the cerebellar cortex following activation of somatotopically-defined areas of the sensori-motor cortex ~43 or electrical stimulation of the peripheral nerves43 in intact animals, recognized a fairly clear separation of the hindlimb and forelimb representations over the entire mediolateral extent of the intermediate cortex, a finding later substantiated by the results of studies which employed natural stimulation of the peripheral body receptors and single-unit recording from Purkinje cells. 44'5L In addition to the somatotopic arrangement, the two populations of ipsilateral interposed nucleocortical neurons projecting to the forelimb areas of PIAL or PML partly overlapped but were also partly segregated each other, in contrast with the olivocerebellar input which is much more extensively overlapped. This is particularly evident in NIP where the nucleocortical cells feeding-back the individual forelimb areas of PIAL, PML and medial crus II only partly overlap each other, and show a progressive shift along the dorsoventral axis of the nucleus. Whether or not this partial segregation could to be attributed to an incomplete involvement of the individual forelimb- or hindlimb-related areas at the injection site has been considered. Indeed, in those cases, as the first experimental case presented (MI60, Fig. 2), in which the forelimb area of PIAL and the forelimb area of PML were completely injected, it was common to find a higher degree of overlap between the two labelled populations. However, in such cases it was common to involve also the adjacent forelimb areas lying in the neighbouring lobules (i.e. lobulus VI for the forelimb-PIAL and medial crus II for the forelimb-PML injections). Thus, part of the retrogradely-labelled neurons had to be attributed to nucleocortical projections to the forelimb areas additionally involved by the injection. Therefore, only those cases in which the injected areas were restricted to the forelimb (or hindlimb) areas of PIAL or PML were here considered (i.e. M167, M162, M161, Figs 3-5). The analysis of retrograde cell labelling in the IO complex was further taken to compare the degree of overlap between labelled olivary populations and nucleocortical ones. It was possible to verify, that, independently of the extent of the individual forelimb or hindlimb injected area,

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whereas in the IO complex the overlap of the related populations was always almost complete, the nucleocortical neurons projecting to forelimb- or hindlimbP1AL were partly segregated from those projecting to forelimb- or hindlimb-PML, and showed a shift both along the dorsoventral and rostrocaudal axis of the nucleus. This segregation underlines a difference in the projection patterns to the cerebellar cortex between the olivary afferents and the nucleocortical feedback system to somatotopically homologous areas, within C2 compartment.

Somatotopy of nucleocortical branching The third point emerging from our study is that individual nucleocortical cells may project to forelimb-related ipsilateral cerebellar areas by way of branched axon collaterals. Direct evidence was here given for pairs of forelimb-related areas in PIAL, PML and medial crus II. Although such evidence for the hindlimb-related areas is not given here, the comparative analysis of the localization of the hindlimb-PIAL and hindlimb-PML related populations (see Figs 6 and 7) suggests that a similar degree of branching is also likely to occur between the hindlimb areas. Thus, the branching pattern of the nucleocortical feedback system is somatotopically organized as the collateral organization of the olivocerebellar afterents. 49"5° However, the degree of branching found in the nucleocortical projections is significantly lower than that found in the olivocerebellar system (see cases M 160, M 167, M 162 and M 161), due to the fact that in contrast with the olivocerebellar input the nucleocortical neuronal groups projecting to the individual forelimb areas in PIAL, PML or crus lI only show a partial overlap in NIP (see Figs 2 5). Early studies already showed that the nucleocortical fibres branch en route to the cerebellar cortex, 3~'56 but it was Haines' study 28 on a prosimian primate that gave evidence of a branching of individual nuclear neurons to areas of the cerebellar cortex as far apart as the anterior lobe-pars intermedia and paramedian lobule. Our results, while supporting these observations, give a direct functional implication to this interlobar branching, by showing that the nucleocortical fibre system is in somatotopic register with the olivary input also with its interlobar component. This finding implies the existence of a feed-back loop between NIP (and N1A) nucleocortical projections and the olivocerebellar input to somatotopically homologous areas within C compartments. With regard to the collateral organization of nucleocortical projections, a question that remained unanswered in the former studies is whether or not the ipsilateral and contralateral symmetrical projections could, at least in part, be sustained by divergent axon collaterals of individual nuclear neurons. In the present study we found that the contralateral projections from NIP are, in the cat, limited to the anterior

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lobe. In the experiments where somatotopically homologous areas of forelimb-PIAL (Fig. 8) or lateral Crus II (Fig. 9) were injected on both sides with spectrally different tracers, no double-labelled neurons were found. Thus, the existence of a transcommissural nucleocortical branching linking the two sides of the anterior lobe-pars intermedia, or the lateral hemisphere, can be ruled out for the cat. Relation o f nucleocortical projections to cerebellofugal pathways

The observation that the nucleocortical projections to the C zones are topographically organized, and that their topographic pattern is well related to the functional somatotopic organization of the cerebellar cortex in roughly organized forelimb and hindlimb areas, was not an unexpected finding. In fact, as already mentioned in the introduction, the nucleocortical pathways stem, at least in part, as axon collaterals of deep cerebellar nuclear neurons whose axons are directed to the red nucleus and/or the thalamus. These nucleofugal efferent pathways have been repeatedly reported to be somatotopically organized into rough forelimb or hindlimb domains,4 6,15,23.47 and therefore, it could have been inferred that the nucleocortical neurons are organized, at least in part, in a similar way. Particularly evident is the somatotopic organization described in a study on the NIA and NIP efferents to the red n u c l e u s . 47 Indeed, when the pattern of localization of our forelimb- or hindlimbrelated nucleocortical projecting neuronal groups in NIP were compared with the topographic organization of interposito-rubral projecting n e u r o n s , 47 it was evident that they overlapped fairly well. In addition, preliminary double-labelling experiments, in which ventralis anterior-ventralis lateralis (VAVL) thalamic nuclei and the intermediate cerebellar cortex were injected with two spectrally different fluorescent tracers (Rosina and Provini, unpublished observations), further indicate that also in the cat most of the intermediate nucleocortical fibres are collaterals of NIP efferent pathways, as previously reported by Payne in the rat. 42 These observations

indicate that the nucleocortical pathways feed back to the cortical neurons by which the nuclear output is regulated, following a pattern which is not only reciprocally but also somatotopically organized. Since the nucleocortical projections from the deep cerebellar nuclei provide a route to the output stage of the cerebellum to influence neural processing within the cortical stage, knowledge of how they are organized bears relevant functional implications in terms of input-output cerebellar relations and, ultimately, on the transformation processes that take place in the cerebellum. Therefore, the finding that the neuronal groups projecting to individual forelimb or hindlimb corticocerebellar areas are partly segregated within somatotopically homologous areas of the nucleus, raises two relevant questions. The first question is to what extent the corticonuclear projections in the posterior interpositus nucleus mirror the nucleocortical ones. The second question is how can these partly segregated somatotopically homologous nucleocortical neurons, which are at least in part nucleofugal neurons, be reconciled with the presence of a single output map in the cerebellar posterior interpositus n u c l e u s . 6't5'47 Reports in the literature point to the presence of a multiple representation of individual body segments in the rat nucleus lateralis 14 and in the monkey dentate and posterior interpositus efferents, through the red nucleus and thalamus, to the cerebral cortex. 39 The here reported partial segregation of NIP nucleocortical populations related to individual forelimb areas in PIAL, PML and crus II, and their shifted localization within the forelimb domain of the nucleus, leads us to hypothesize that both the corticonuclear and the interposed nuclear outputs may be organized in partly shifted domains as already shown for the rat nucleus lateralis ~4 and the monkey dentate and posterior interpositus outputs. 39

Acknowledgements--Our thanks go to Ms Laura Zambusi for her excellent histological assistance and to Ms Barbara Carey for revising the English. This work was partially supported by EC Contract no. SC1-CT91-0639to L.P. and A.R.W.M. was recipient of CNR fellowship no. 201.04.1720.04.04.

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