Brain Research, 95 (1975) 221-239 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

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DEMONSTRATION OF A SOMATOTOPICALLY ORGANIZED PROJECTION ONTO THE PARAMEDIAN LOBULE AND THE ANTERIOR LOBE FROM THE LATERAL RETICULAR NUCLEUS: AN EXPERIMENTAL STUDY WITH THE HORSERADISH PEROXIDASE METHOD

PER B R O D A L

Anatomical Institute, University of Oslo, Oslo (Norway)

SUMMARY

Using the retrograde axonal transport of horseradish peroxidase, the projection from the lateral reticular nucleus (NRL) to the cerebellar anterior lobe and paramedian lobule has been studied in 13 cats. Both the anterior lobe and the paramedian lobule receive a somatotopically organized projection from the NRL. The projection to the paramedian lobule is nearly exclusively ipsilateral and originates mainly in the dorsal part of NRL, while the projection to the anterior lobe is bilateral (with ipsilateral predominance) and takes origin from all parts of the NRL. The lateral part of the NRL (closely coinciding with the parvocellular nucleus) projects to the rostral part of the anterior lobe and caudal parts of the paramedian lobule (both representing the hindlimb), while the medial part of the NRL (magnocellular nucleus) projects to the caudal parts of the anterior lobe and rostral parts of the paramedian lobule (representing the forelimb), The small subtrigeminal nucleus projects onto the anterior lobe as well as the paramedian lobule, but apparently mainly to their forelimb areas.

INTRODUCTION

The lateral reticular nucleus (NRL) is one of several brain stem nuclei which relay impulses from various sources onto the cerebellum. The quantitatively most important of these are the pontine nuclei, then come the inferior olive and the reticular nuclei: the tegmental reticular nucleus and the NRL (and in addition the smaller paramedian reticular nucleus). The NRL, situated ventrolateral and a little caudal to the inferior olive, is usually subdivided into a small-celled (parvocellular) part situated laterally, and a

222 medial magnocellular part constituting the bulk of the nucleus. In addition there is a rostral small subtrigeminal part located just ventral to the spinal tract of the fifth sensory nucleus. The N R L receives afferents from various sources, the largest contingent coming from the spinal cord a,27,a2,a4. In addition, afferents come from the cerebral cortex6, 25, the red nucleusll,16, 3a,a2, the fastigial nucleus 4~, and parts of the vestibular nuclei 29. The various afferent contingents end in largely overlapping, but not identical regions of the N R L (see ref. 4). Within its spinal afferents a somatotopical organization was first demonstrated by Brodal 3 with the Glees method, and has recently been verified by Kfinzle 27, using a modified Nauta method. Fibers from lumbar levels terminate more laterally (mainly within the parvocellular part) while fibers from cervical levels terminate medially (mainly in the magnocellular part). In the projection from the N R L onto the cerebellum a somatotopical pattern has not been looked for. Only a gross pattern of topical localization was found by Brodal 2, using the modified Gudden method 1. Since anatomically there is a clear somatotopical pattern within the first link of the spino-reticulo-cerebellar pathway, one might expect this principle to be upheld in the next link, the projection from the N R L to the cerebellum. Strong support for this assumption has recently been brought forward by Clendenin et al.S, 9, who demonstrated physiologically that impulses travelling in the spino-reticulocerebellar pathway show some degree of somatotopical localization both in the N R L and the anterior lobe. The present investigation was undertaken in order to study parts of the cerebellar projection from N R L in greater detail than hitherto done. The anatomical methods available until recently are not satisfactory for this purpose. However, the method of tracing connections by way of the retrograde axonal transport of horseradish peroxidase (HRP)24,a°,al, 35 is well suited for the study of cerebellar afferent systems 4a since it allows a more precise determination of the cells of origin of a projection and usually also of the sites o f termination of fibers than does the method of retrograde degeneration. The present paper is concerned particularly with the question of a topical organization within the cerebellar projection from the NRL. It is restricted to the projection to the paramedian lobule and the anterior lobe which appear to be the parts of the cerebellum in receipt of the bulk of fibers from the N R L 2,9,41. As will be shown, a somatotopical pattern can be demonstrated within the projection from N R L onto the paramedian Iobule as well as onto the anterior lobe. Furthermore, these two cerebellar regions receive afferents from overlapping, but not quite identical parts of the NRL. M A T E R I A L A N D METHODS

From a larger material of adult cats and some kittens in which injections of HRP had been made in various parts of the cerebellum, altogether 13 cats were selected for the present study. Parts of the same material have been used for studies of the olivocerebellar 5, the cuneocerebellar as and pontocerebellar projections a9. For a detailed description and discussion of the method of retrograde axonal transport

223

Fig. 1. A: photomicrograph showing several labeled cells in the magnoceUular part of the NRL 3 days following an injection of horseradish peroxidase into the ipsilateral anterior lobe in cat B.St.L. 629. × 350. B: photomicrograph showing a labeled cell in the magnocellular part of the NRL 1 day following an injection of the ipsilateral paramedian lobule in cat B.St.L. 636. × 1400.

of H R P used in the present material, the reader is referred to Walberg et all. 4~. In most instances 0.5 #l of a 50 % H R P solution was injected into the cerebellar cortex. The animals were killed after 1-3 days, and were then perfused under Nembutal anesthesia through the heart with saline followed by a buffered mixture of 0.4 % formaldehyde and 1.25 % glutaraldehyde. Following 1 day's further fixation the brain stem and cerebellum were cut at a freezing microtome at 50 #m. The brain stem was cut transversally, the cerebellum sagittally. The sections were collected in groups of 5 consecutive ones, and two sections from each group were mounted. Both of these sections were transferred to a solution containing 0.95% 3,3'-diaminobenzidine tetrahydrochloride in Tris-HC1 buffer (pH 7.6) for 5 min at r o o m temperature. They were then incubated for a further 15 min at r o o m temperature in a similar solution but with the addition of 66 #l of 30% hydrogen peroxide per 100 ml. One section was weakly counterstained with cresyl violet, the other left unstained. The sections from the cerebellum were drawn under a projection apparatus and examined microscopically in order to determine the extent and localization of the staining of the cerebellum. The sections containing the N R L were drawn by use of an electronic panthograph, and every labeled cell was plotted in the drawings.

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Coudol Fig. 2. The distribution of labeled cells in the ipsilateral N R L following injection of the paramedian lobule (cat B.St.L. 634). Above: a drawing of the left paramedian lobule with the extent of staining seen after 3 days. The black area indicates the total extent of staining, regardless of whether the staining is present in the cortex or the white matter. White circle indicates site of injection. Only within parts of the black area is there unequivocal staining of the granular layer. This is the case also in the following figures. Below: drawings of transverse sections through the brain stem, passing through the ipsilateral lateral reticular nucleus. Labeled cells are indicated with dots. Every labeled cell observed in the represented sections has been marked. Note that labeled cells are restricted to the dorsal part of the NRL, but are found throughout its rostrocaudal extent. Labeled cells are also seen in the subtrigeminal part, not included in the drawings.

225 RESULTS

One to three days following the injection of H R P into the paramedian lobule or the anterior lobe, labeled cells can be observed in the NRL. The labeling consists of brownish granules within the perikarya and proximal dendrites (Fig. 1A and B). The size of the granules varies considerably within the same cell and from one cell to another, and in some instances they can only be identified with certainty with the oil immersion lense. The appearance of peroxidase labeled cells in the N R L does not differ from what has been described in other regions of the central nervous system ~°22,26,30,35,37,39.

In the following, cases demonstrating the organization of the projection from the N R L onto the paramedian lobule and the anterior lobe will be described and illustrated. The paramedian lobule The projection from N R L onto the paramedian lobule is practically entirely ipsilateral, as is evident from the cases to be described below. In none of the cases more than a relatively small part of the paramedian lobule has been injected. However, by adding the labeled areas in N R L found in cases with injections which together cover most parts of the paramedian lobule, it appears that the paramedian lobule receives afferents predominantly from the dorsal half of the N R L (see Fig. 2). Below, some cases with staining of parts o f the paramedian lobule will be described. In cat B.St.L. 634 (3 kg, injected 0.5/,1 of a 5 0 ~ H R P solution, killed after 3 days, Fig. 2), the middle part of the left paramedian lobule has been incompletely stained. The 2 rostralmost and the 4 caudalmost folia are free from staining (Fig. 2). In the ipsilateral N R L labeled cells are found scattered, often in small groups, mainly in the dorsal half throughout the rostrocaudal extent of the nucleus, and within both the magno- and parvocellular parts. There are also labeled cells in the subtrigeminal part of the nucleus (not included in the diagram of Fig. 2). Only a few labeled cells are found in the contralateral NRL. Corresponding observations are made in two other animals (not illustrated). In one cat, B.St.L. 636 (3 kg, 0.5 #1 o f a 50~o H R P solution, killed after 1 day), there is partial staining of the rostral 5 folia of the paramedian lobule, somewhat more extensive than in B.St.L. 634 (Fig. 2), in the other, cat B.St.L. 647 (1.8 kg, 0.25 #1 of a 100 ~ H R P solution, killed after 2 days), the staining is restricted to the middle 3 folia of the paramedian lobule. Even if the amount of enzyme solution injected is the same, the number of labeled cells in cat B.St.L. 636 is significantly higher than in cat B.St.L. 634, apparently corresponding to the larger cerebellar area stained. In the former case up to 70 labeled cells are found in the N R L in one section, in the latter not more than 40. Such a correlation between the number of labeled cells in the N R L and the size of the stained cerebellar area, even if the amount of enzyme solution injected is the same, is generally found throughout this study. When cases with small stained areas restricted to the rostral- and caudalmost parts of the paramedian lobule are studied, a topical principle within the projection

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Fig. 3. Four cases showing a somatotopical pattern within the projection from the lateral reticular nucleus onto the paramedian lobule. Above: the extent of cortical staining subsequent to injections of various parts of the paramedian lobule is shown (see legend to Fig. 2). Below: the outlines of the NRL as appearing in transverse sections. Labeled cells are indicated by dots. There is a clear mediolateral difference in the location of labeled cells following injections of the rostral part of the paramedian lobule ('forelimb' area, the two cases to the left) and injections of the caudal part of the paramedian lobule ('hi.ndlimb' area, the two cases to the right). The 'forelimb' part of the paramedian lobule receives its afferents mainly from the dorsal part of the magnocellular nucleus, while the 'hindlimb' part of the paramedian lobule is connected with the dorsal part of the parvocellular nucleus. The subtrigeminal part is not included, but contains labeled cells in cat B.St.L.619. from the dorsal part o f the N R L onto the p a r a m e d i a n lobule becomes evident, as the cases illustrated in Fig. 3 show. In cat B.St.L. 619 (2.1 kg, 0.5/zl o f a 5 0 ~ H R P solution, killed after 3 days, Fig. 3), the staining is restricted to parts o f the 3 rostralmost folia o f the left param e d i a n lobule. I n the ipsilateral N R L labeled cells are f o u n d in the dorsal twothirds o f the nucleus except that the dorsolateral part of the nucleus is practically

227 free. There is labeling of many cells in the subtrigeminal division (not illustrated). The sparing of the dorsolateral part of N R L following injections of the rostral part of the paramedian lobule is more clearly shown in another case, B.St.L. 650 (750 g, 0.05/zl of a 75 ~ HRP solution, killed after 1 day) where the staining of the cortex is more restricted than in B.St.L. 619, but similarly located (Fig. 3). The number of labeled cells is much smaller than in the preceding case and they are restricted to the medial or intermediate part of the dorsal half of the NRL. The lateralmost part is completely free from labeled cells. Closely corresponding observations are made in two further cases (B.St.L. 662 and 663, not illustrated) with injections confined to the rostralmost folia of the paramedian lobule. The stained cerebellar areas are very small in both, and the number of labeled cells in the N R L correspondingly low. When the staining of the cortex is restricted to the caudalmost part of the paramedian lobule, the labeled cells are confined mainly to the lateralmost regions of the dorsal part of NRL. In cat B.St.L. 653 (2.3 kg, 0.4 #I o f a 50~o HRP solution, killed after 3 days, Fig. 3), the staining of the cortex is restricted to minor parts of the two folia next to the caudalmost one. In the N R L only a few labeled cells are seen in each section, and all are confined to the lateralmost part (see Fig. 3). In cat B.St.L. 656 (1.6 kg, 0.4 #1 of a 5 0 ~ HRP solution, killed after 2 days, Fig. 3) with an injection very similar to that in B.St.L. 653, a closely corresponding distribution of labeled cells is present, except that there are some more labeled cells. Comments. The cases described above (4 of them illustrated in Fig. 3) show clearly that when the staining is restricted to the rostral and caudal parts of the paramedian lobule, respectively, the location of the labeled cells in the N R L is clearly different. The lateral part of the N R L (corresponding closely to the parvocellular division) sends fibers mainly to the caudalmost part of the paramedian lobule, while the rostral part of the paramedian lobule receives afferents mainly from more medial parts of the NRL. In all cases described above with injections of the paramedian lobule, the labeled cells are confined to approximately the dorsal half of the NRL. In some cases the percentage of labeled cells in the dorsal half probably exceeds 50 ~ in some sections. The number of labeled cells occurring seems to be related to the extent of the staining of the cerebellar cortex more than to the amount of enzyme injected. The anterior lobe

The cases to be described in the following will show that the anterior lobe receives afferents from practically the entire NRL. The projection is bilateral with an ipsilateral predominance. Like the projection to the paramedian lobule, the total area of origin in the N R L of fibers to the vermal and intermediate parts of the anterior lobe has to be inferred by adding the findings made in several cases with restricted stained areas. The lateral parts of the anterior lobe have not been systematically explored. In cat B.St.L. 629 (2.2 kg, 0.5 #l of a 50 ~ HRP solution, killed after 3 days, Figs. 1A and 4), the staining is relatively extensive, but restricted to the vermal and

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Fig. 4. The projection from the lateral reticular nucleus onto lobule V of the anterior lobe (cat B.St. L.629). The staining of the cortex (el legend to Fig. 2) involves both vermis and the intermediate zone of lobule V on the left side. To the left are drawings of 4 representative transverse sections through the ipsilateral NRL. Labeled cells are marked with heavy dots, unlabeled cells with light dots. Note that labeled cells are present in the ventral as well as in the dorsal part of the NRL (of. Fig. 3) and that the normal cells are concentrated far laterally, in a region that coincides closely with the parvocellular part (which projects onto rostral parts of the anterior lobe, cf. Fig. 5). The subtrigeminal part of NRL, not included in the drawing, contains labeled cells as well. Note furthermore that most of the cells in the magnocellular nucleus are labeled, especially in the three lower sections (2-9). i n t e r m e d i a t e p a r t s o f l o b u l e V, m a i n l y o n the left side, b u t e x t e n d i n g slightly o v e r the midline. In the N R L on b o t h sides l a b e l e d cells are f o u n d p r a c t i c a l l y all o v e r t h e nucleus, e x c e p t t h a t t h e r e is a c o n s p i c u o u s s p a r i n g laterally, c o r r e s p o n d i n g to t h e p a r v o c e l l u l a r p a r t o f N R L . T h e n u m b e r o f labeled cells d e c r e a s e s in the r o s t r a l d i r e c t i o n , b u t t h e r e is a m p l e l a b e l i n g a l s o in the s u b t r i g e m i n a l d i v i s i o n ( n o t illustrated). I n this

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Caudal Fig. 5. Three cases demonstrating the somatotopical organization within the projection from the N R L onto the anterior lobe. Diagrams according to the same principle as in Fig. 2. Note that the more rostrally in the anterior lobe the injection is situated, the more laterally in the N R L labeled cells occur. The labeled cells are found more ventrally than after injections of the paramedian lobule (cf. Fig. 3). For the subtrigeminal part (not included in the drawings) see text.

230 case all normal as well as labeled cells were plotted in 4 representative sections, illustrated in Fig. 4. Labeled cells are marked with heavy dots, normal ones with light dots. The number of cells has been counted in each section of the left NRL, and in some sections more than 80 ~ of the total number of cells in the magnocellular part are labeled. The normal cells are located mainly far laterally. The distribution on the two sides is entirely symmetrical, but the number of labeled cells on the ipsilateral side is roughly twice that on the contralateral side. This case indicates that the lobule V receives afferents from most of the magnocellular division of NRL, but very few from the parvocellular part. This is substantiated in another case, cat B.St.L. 654 (1 kg, 0.075 ,ul o f a 5 0 ~ HRP solution, killed after 1 day, Fig. 5), with an injection of lobule V where the stained area is relatively small and situated mainly in the vermal part (Fig. 5). The area occupied by labeled cells in the N R L is smaller in this case than in the former and is restricted to the medial part of the nucleus. The labeled cells are found in equal number on the two sides, and symmetrically located. When the injection is made in lobule IV, the labeled cells in NRL are found somewhat more laterally than following injections of Iobule V, as appears from cat B.St.L. 623 (2.3 kg, 0.5/~1 o f a 5 0 ~ HRP solution, killed after 3 days, Fig. 5). The injection was made paravermally in the left lobule IV and the stained area extends from the vermis to the lateral part of the lobule. In the N R L labeled cells are found on both sides, slightly more numerous on the ipsilateral side. The distribution is very similar on the two sides. From Fig. 5 it is seen that the labeled cells mainly are located more laterally and ventrally following an injection of lobule 1V than in the cases described above with injections of lobule V. There appears thus to be a tendency for rostral parts of the anterior lobe to receive afferents from cells located laterally in the N R L and for caudal parts of the anterior lobe to receive afferents from more medial NRL cells. This is substantiated by the findings in the following case, eat B.St.L. 665 (3 kg, 0.5 #1 o f a 5 0 ~ HRP solution, killed after 1 day, Fig. 5). An injection oflobule I1 was made stereotaxically from the dorsal aspect of the cerebellum. The bulk of the staining is found in lobule I and lobule II (Fig. 5), but in addition there is a small area of slight staining also in the rostralmost part of the left fastigial nucleus. In the NRL labeled cells are found in about equal numbers on both sides and the distribution is entirely symmetrical, Labeled cells are almost exclusively found in the parvocellular division of NRL, although the dorsalmost part of it is spared. A few labeled cells are also present in the subtrigeminal part (not illustrated). It is highly unlikely that the very moderate staining in the fastigial nucleus can be responsible for the heavy labeling found in the NRL (see Discussion). Thus is seems permissible to conclude that Iobules I and II project mainly to the lateralmost zone of the NRL. Comments. Even if the cases with injections of the anterior lobe are relatively few, the results are clear enough to warrant some conclusions. Thus the anterior lobe receives afferents from most of the NRL, including most of the parvocellular, the magnocellular and the subtrigeminal subdivisions. From a comparison of cases with small, well restricted injections, a clear somatotopical arrangement within the

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Fig. 6. Schematic representation of the somatotopical pattern within the projection from the NRL onto the paramedian lobule and the anterior lobe. To the right, a diagram of the cerebellum. To the left, 4 representative transverse sections through the left NRL. The anterior lobe (vertical and oblique hatchings) receives afferents from more ventral parts of the NRL than does the paramedian lobule (filled and open circles), although there is extensive overlap. Furthermore, the 'hindlimb' areas within the anterior lobe (vertical hatchings) and the paramedian lobule (open circles) are connected mainly with the lateral (parvocellular) part of the NRL, while the cerebellar 'forelimb' areas (oblique hatching and filled circles respectively) mainly receive afferents from the medial or magnocellular part of the NRL. The projection from the subtrigeminal part onto the anterior lobe and paramedian lobule is not illustrated.

t e r m i n a t i o n o f fibers f r o m the N R L emerges: fibers to lobule V originate in medial parts o f N R L , while fibers to lobules IV a n d II come f r o m successively more lateral parts o f N R L (Figs. 4 a n d 5). Fig. 6 shows a d i a g r a m m a t i c s u m m a r y o f the present results. It should be em-

232 phasized that the representation is schematic, and the sharpness of localization enhanced for the sake of clarity. DISCUSSION

In the present investigation the use of the H R P method has revealed some principles of organization in the cerebellar projection from the N R L which have so far not been shown anatomically. Although the tracing of pathways in the central nervous system by way of retrograde axonal transport of H R P is a quite new neuroanatomical tool, its usefulness has been well documented (refs. 30, 31, 35, 37, 43 and several others). Only a few points with regard to the method will, therefore, be discussed here. When labeled cells are found in the NRL following injection of H R P in the cerebellar cortex, it can safely be concluded that the injected part of cerebellum receives afferents from the labeled cells (provided that the extension of cerebellar staining due to the injection has been carefully mapped). However, for several reasons it is at present impossible to decide to what extent a stained area of cortex indicates uptake of HRP in all axon terminals within it. It follows that the evaluation of the 'effectiveness' (with regard to uptake) of the injected area will be uncertain. Consequently comparisons of quantities of projections must be made with great care. The present study is, therefore, mainly restricted to the topographical aspects of the reticulocerebellar projection. Even if it can be said with certainty that labeled cells project onto the injected cerebellar area, one can not without qualifications conclude that unlabeled ceils do not project to the same injected area. There are several reasons for this. It may be virtually impossible in the light microscope to decide that an apparently unlabeled cell is in fact free of peroxidase granules, since the size of the granules in the perikarya varies from 65 #m to almost 1 #m (in the chick optic system3°). Cells with a modest labeling may, therefore, escape recognition in the light microscope. Furthermore, it is possible that not all NRL axon terminals within an injected area take up the enzyme, and will transport H R P in the retrograde direction, particularly since Kfinzle and Cu6nod 28 found that small and large cells in the N R L of the cat behave differently with regard to uptake of radioactive amino acids. Even if, in the present study, ample HRP labeling was observed of small as well as of large cells, there may nevertheless exist differences between N R L cells with regard to uptake of HRP. Due to these difficulties with regard to the interpretation of results based on H R P injected material, conclusions from single experiments and from negative findings may be hazardous. However, in the present study the conclusions are based on several experiments with corresponding positive findings and the pattern of organization observed can therefore scarcely be due to individual variations or artifacts.

The origin and termination of fibers from NRL to the cerebellum In the present study all parts of the NRL have been found to project onto the cerebellum in agreement with the findings made in a study with the modified Gudden

233 method by Brodal 2. However, the subtrigeminal part appears to have a more widespread cerebellar projection than hitherto assumed. The anterior lobe and paramedian lobule are found to receive afferents from the subtrigeminal part of NRL, while Brodal ~ concluded that the subtrigeminal part projects onto the uvula and flocculus. However, he describes moderate retrograde changes in the subtrigeminal part also in some cases with extensive damage to the vermis as well as the hemispheres. There may therefore not be any discrepancy between his results and those presented here. Thus, if the projection from the subtrigeminal part onto the anterior lobe and paramedian lobule occurs by way of collaterals of axons passing to the flocculonodular lobe, only widespread destruction of the cerebellar cortex would presumably be able to produce clear-cut retrograde changes in this part of the NRL. It is characteristic that following injections of the cerebellum, labeled cells are usually found scattered over relatively large parts of the NRL. Even if the injection is very small, and regardless of its exact site within the anterior lobe or paramedian lobule, labeled cells are found practically throughout the whole rostrocaudal extent of the NRL, while there are differences with regard to their distribution in the dorsoventral and mediolateral directions, dependent on which cerebellar area is injected. Thus, a rostrocaudally oriented column of cells in the NRL projects onto a relatively small cerebellar area, for example one or two folia of the paramedian lobule. This arrangement is somewhat similar to that present within the pontocerebellar projection 19. Longitudinal columns in the pontine nuclei project onto small areas of the cerebellum. It should be stressed, however, that even if the pontocerebellar and the reticulocerebellar pathways both are somatotopically organized and the cells projecting onto the cerebellum are arranged into columns, they differ in important respects. The pontine columns are slender and sharply circumscribed, and each small cerebellar area as a rule receives afferents from several pontine columns 19. In the NRL, on the other hand, only one column appears to project to a restricted cerebellar area and the NRL columns are wide and without sharp borders. Thus, afferents from large parts of the NRL converge onto one small cerebellar area. On the other hand, a very high percentage of labeled cells was found in the NRL even after rather small injections (see Fig. 4). This suggests that each axon from the NRL may branch extensively within the cerebellum. This has indeed recently been shown electrophysiologically to be the case by Clendenin et al. s,l°. They found that single NRL cells could be fired antidromically from up to 5 discrete cerebellar spots, often situated far apart, for example on both sides. A number of cells could further be activated both from the anterior lobe and the paramedian lobule. The NRL projection onto the anterior lobe appears from the present study to be bilateral with ipsilateral preponderance, while the projection to the paramedian lobule is practically entirely ipsilateral. This is in agreement with the physiological observations of Clendenin et alp using antidromic activation of NRL cells. They further found that stimulation of the paramedian lobule and the intermediate part of the anterior lobe evoked antidromic potentials ipsilaterally in the dorsal part of the NRL, while cells in ventral portion of NRL were activated bilaterally from the vermis and the intermediate part of the anterior lobe, but not from the paramedian lobule 9. Con-

234 cerning the main points there is good agreement between the present anatomical results and these physiological data. Thus, I find that the paramedian lobule receives afferents mainly from the dorsal part o f the NRL, while the anterior lobe in addition is supplied from the ventral part. Since all the present injections of the anterior lobe involve to some extent both vermal and intermediate parts, it can unfortunately not be decided whether there are differences between these two zones with regard to afferents from the NRL. As stressed above, the present material is not suited for quantitative estimates of projection. However, in some cases which correspond with regard to survival time, amount of enzyme injected and extension of cortical staining, the number of labeled ceils in the ipsilateral N R L is somewhat higher following injections of the anterior lobe than after injections of the paramedian lobule (compare Figs. 3, 4 and 5). However, the difference does not seem to be as great as reported by Clendenin et a12, who were able to activate antidromically relatively few N R L cells from the paramedian lobule. Further they found very few N R L cells antidromically activated from the cerebellar hemispheres. This is in general agreement with the observations of Voogd 41 using the Nauta method, and of Brodal z who found considerably less retrograde changes in the N R L following destructions of the hemispheres than after lesions of the intermediate and vermal parts of the cerebellum. In one case in the present material with a relatively large injection of crus I a moderate number of H R P labeled ceils were present particularly in the dorsal part of the rostral half of the ipsilateral NRL, but the number of labeled cells appeared to be much lower than in cases with comparable injections in the anterior lobe or the paramedian lobule (unpublished observations).

The somatotopical organization of the reticulocerebellar projection The present study shows that fibers from the lateral part of the N R L (receiving afferents from lumbar levels3, 27) terminate within the caudal part of the paramedian lobule and the rostral part of the anterior lobe, respectively. Thus, the somatotopical pattern within the spinal pathway to the N R L is upheld in the further projection to the cerebellum, since rostral parts of the anterior lobe and caudal parts of the paramedian lobule both represent the hindlimb with respect to other afferent as well as efferent connections (see refs. 17, 36). Medial parts of the NRL, receiving afferents from cervical levels of the spinal cord 27,34, send fibers to the parts of the anterior lobe and paramedian lobule representing the forelimb, namely lobule V and the rostral part of the paramedian lobule. The evidence presented under Results with regard to the somatotopical pattern within the projection to the paramedian lobule hardly needs further comments, since several cases with very similar injections yielded completely corresponding results (see Fig. 3). However, with regard to the anterior lobe there is only one case with a small injection of lobules I and II and there is in this case some spread of staining to the rostral end of the fastigial nucleus. Since there is physiological evidence for a projection from the N R L to the fastigial nucleus TM ~5 it may be argued that the labeling found in the N R L in this experiment is due to uptake o f fibers terminating in the fastigial nucleus. However, it is very unlikely

235 that the slight staining of the rostral tip of the fastigial nucleus is alone responsible for the heavy labeling in the lateral part of the NRL (Fig. 5). Most probably the much more intense staining of the lobules I and II in this case is mainly or exclusively responsible for the labeling in NRL. The conclusion made above that the rostral part of the anterior lobe receives fibers from the extreme lateral part of the NRL (Fig. 5) seems, therefore, warranted. Furthermore, a projection from the NRL to the fastigial nucleus may quite likely take origin from the same areas of the NRL as do the fibers to lobules I and II, particularly since efferents from these lobules terminate rostrally in the fastigial nucleus44. The present experiments with injections restricted to lobules IV and V, respectively (Figs. 4 and 5), further support the conclusion that rostral parts of the anterior lobe are connected with lateral parts of the NRL, while caudal parts of the anterior lobe receive fibers from more medial parts of the NRL. Physiologically Clendenin et al. 1° found a pattern of termination in the lobules IV and V corresponding closely to what is presented here (compare their Figs. 7 and 8 with my Fig. 5). They did not explore more rostral parts of the anterior lobe than lobule IV, but suggested that few fibers from NRL supply the lobules I, II and III. This was based on their observation that relatively few NRL cells could be antidromically activated from the rostralmost part of lobule IV, and that Voogd 41 found few or no degenerated fibers in the lobules rostral to lobule IV after electrolytic lesions of the NRL. However, in Voogd's one relevant case (his Fig. 3) the lesion of NRL involves only its medial part. Thus the absence of degeneration in the rostral part of the anterior lobe in his experiment is best explained on the basis of the present findings that this part is supplied from the lateralmost part of NRL (Figs. 4 and 5). A somatotopical pattern within the projection onto the paramedian lobule is not described by Clendenin et al. 1°, presumably due to the relatively low number of NRL cells found by them to be antidromically activated from the paramedian lobule, and because the dorsolateral arm of the parvocellular division shown here to project to the caudal part of the paramedian lobule, was not explored at all. Also more ventrally in the parvocellular division relatively few cells were studied by Clendenin et al. 8-1o, and those studied were all located medially in the parvocellular part. This may explain why they found no clear-cut difference between the distribution within the cerebellum of fibers from the parvo- and magnocellular division of the NRL respectively, while in the present study where many labeled cells were located far laterally in the parvocellular part clearcut differences are found (see above and Figs. 3, 4 and 5). The subtrigeminal part of the NRL contains labeled cells in most of the present cases (see Results), but the impression is gained that the projection from the subtrigeminal part to the cerebellar forelimb areas is heavier than that to the hindlimb areas. In a case with a large injection of lobules VI and VII (parts of face area), the number of labeled cells in the subtrigeminal part is relatively high, compared to the number of cells occurring in other parts of the NRL (unpublished observations). It is, therefore, particularly interesting that Darian-Smith and Phillips 13 found many NRL cells, apparently situated in the subtrigeminal part, which were antidromically activated from lobules V and VI, and orthodromically activated with short latency

236 from the face. Thus, the subtrigeminal part of the N R L may constitute a relay station for impulses from the face traveling to the face area of the anterior lobe. The N R L as a precerebellar relay nucleus

As mentioned in the Introduction, the N R L receives afferents from several sources. The quantitatively most important one, taking origin from various levels of the spinal cord, has been considered above in connection with the discussion of a somatotopical principle within the spino-reticulo-cerebellar pathway. Suffice it here to mention that practically all parts of the magnocellular and parvocellular divisions receive spinal fibers27,~2,~4, and that the small subtrigeminal part, lacking spinal afferents, probably has a corresponding peripheral input from the trigeminal nerve (see above). Concerning the other afferent systems to the N R L it is characteristic that although they overlap extensively, they terminate in somewhat different parts of the nucleus (see ref. 4). The functional significance of this partial separate termination is not clear, but the anatomical data suggest that many N R L cells, particularly in the rostral part of the magnocellular division, may be influenced from several afferent sources. Physiological studies indicate that this is the case for at least some N R L cells. Thus, Kitai et aL 23 have recently shown convergence on single N R L cells of impulses (presumably monosynaptic activation) from the red nucleus, the nucleus interpositus, the cerebral peduncle and the spinal cord. Unfortunately, they do not indicate the exact site in the N R L of the cells studied. More often, convergence from the periphery or the spinal cord and one other source has been reported. Thus, Crichlow and Kennedy 12 found convergence of impulses with short latency from the spinal cord and the cerebral motor cortex on some N R L cells. Ghelarducci et al.lS observed convergence o f vestibular and somatic sensory peripheral impulses on single cells in large parts of the magnocellular division of the NRL. On the other hand, Bruckmoser et al. 7 failed to find convergence of monosynaptic activation from the red nucleus and the fastigial nucleus on single N R L cells, although polysynaptically transmitted convergence was often observed. Taken together, the physiological data referred to above indicate that there is a convergence of impulses from often several sources on many N R L cells. On the other hand, some N R L cells are apparently monosynaptically contacted only by some afferent systems, but not by others. This is in general agreement with the topographical distribution in the N R L of the various afferent systems. With regard to the transmission of information from the N R L to the cerebellum, the high degree of convergence and divergence within this projection should be recalled (see above). Thus, a small part of the cerebellum receives afferents from large parts of the NRL, and a small part of the N R L sends fibers to large parts of the cerebellum. It seems likely, therefore, that even if information from various afferent sources to some degree is separated within the NRL, there may not be a corresponding separation within the cerebellum. Detailed anatomical and physiological studies are needed to elucidate this problem further. As mentioned above a somatotopical pattern exists within the spinal projection

237 onto the N R L , and the present study shows that the somatotopical organization is upheld in the next link, from the N R L to the cerebellum. It would therefore be of interest to know whether the other afferent systems to the N R L are somatotopically organized as well, particularly since all of the cell groups projecting onto the N R L are somatotopically organized with regard to other connections. The cerebrocortical afferents to the N R L take origin mainly from the anterior sigmoid gyrus ('motor cortex') in the cat, and terminate in most of the magnocellular part ('forelimb'), but not in the parvocellular 6 part ('hindlimb'). N o somatotopical pattern was found by P. Brodal et al. 6 within this projection when lesions were placed in different parts of the anterior sigmoid gyrus. However, according to some recent data (see ref. 40) the m o t o r hind limb area in the cat is completely buried in the depth o f the cruciate sulcus. This region was not explored in the study of P. Brodal et al. ~ and it can therefore not be excluded that a somatotopical pattern exists within the cortical projection to the N R L . The projection from the red nucleus terminates dorsolaterally in the rostral part of the N R L , including the subtrigeminal portionll,16,z2, 42. Although looked for, a somatotopical pattern was not observed11, 4z, but as stressed by Courville 11, this is not conclusive due to the difficulties in obtaining discrete lesions of the red nucleus, and the tortuous form of the terminal area in the N R L . Although the terminal areas in the N R L of fibers from the fastigial nucleus 45 and the lateral vestibular nucleus z9 have been mapped in some detail, a somatotopical pattern within their projection onto the N R L has apparently not been specifically looked for. However, in light of the somatotopical principle within the projection from the N R L to the cerebellum, the detailed organization of the various supraspinal pathways to the N R L deserves further study.

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