The organization of reticulo-olivo-cerebellar circuits in the North American opossum

Brain Research, 137 (1977) 253-266 © Elsevier/North-Holland Biomedical Press

253

T H E O R G A N I Z A T I O N OF RETICULO-OLIVO-CEREBELLAR CIRCUITS IN T H E N O R T H A M E R I C A N OPOSSUM

G. F. MARTIN, M. S. BEATTIE, H. C. HUGHES, M. LINAUTS and M. PANNETON

Department of Anatomy, The Ohio State University, College of Medicine, Columbus, Ohio 43210

(U.S.A.) (Accepted March 15th, 1977)

SUMMARY

By employing the autoradiographic method we have determined that the inferior olivary nucleus receives input from the reticular formation of the midbrain, pons and, probably, the medulla. The remarkable thing about such connections is that they are not diffuse, but targeted in large part on restricted portions of the caudal accessory nuclei. From our previous studies it is clear that some of the olivary regions receiving reticular input also receive projections from the cerebral cortex, the spinal cord and the dorsal column nuclei. In addition, the H R P technique reveals that these same olivary regions relay to parts of the spinal cerebellum, i.e. to restricted zones of the anterior lobe (present study). Taken together these observations suggest that some reticulo-olivo-cerebellar connections provide indirect routes through which cortical and spinal information gains access to the spinal cerebellum. Such circuits have also been suggested by the physiological literature. Of particular interest, however, was our finding that several reticular areas of the midbrain and pons project to that portion of the medial accessory nucleus which relays in turn to auditory-visual areas of the cerebellar vermis (declive, folium and tuber). It would appear that at least some reticulo-olivo-cerebellar circuits are significant in the organization of motor responses to visual and/or auditory stimuli.

INTRODUCTION

The inferior olivary nucleus is considered to be the major source of climbing fibers within the cerebellar cortex, as well as one origin of axons which distribute to the deep nuclei (see review by Armstrong1). The obvious significance of the olive in cerebellar function 1 and recent data attesting to the unexpected complexity of olivocerebellar circuits1-5, 9 combine to emphasize the importance of identifying all inputs to the olive as well as characterizing the targets of each in terms of connections with the cerebellum.

254 Although anatomical studies have documented the existence of olivary projections from reticular (tegmental) areas of the midbrain 7,11-15,19-21, to our knowlegde the existence of reticulo-olivary connections from more caudal areas of the brain stem has not been verified (see Armstrong 1 for a discussion). At least one autoradiographic study reports negative evidence for such connections 22, but the author recognized the possibility of technical explanations, and further, his primary purpose was to report other connections. As part of an ongoing program designed to elucidate olivary connections in the North American opossum, we have used the autoradiographic method 6 in an attempt to determine whether reticulo-olivary projections take origin within the lower brain stem and, if so, to detail their precise olivary targets. An additional objective was to reinvestigate reticulo-olivary connections from the midbrain since our earlier description of such connections was taken from material processed by degeneration methods. In each case the olivary target(s) of labeled axons have been identified as accurately as possible so that the comparable region(s) could be examined for neurons containing reaction product after injections of horseradish peroxidase into different areas of the cerebellar cortex 10. The terminology used for the opossum olive is taken from Martin et al. 12, whereas that employed for the reticular formation is from Oswaldo-Cruz and Rocha-Miranda 17. MATERIALS AND METHODS The following observations were made from the brains of 47 opossums in which [3H]leucine was injected into one or more reticular areas of the brain stem. The injections were made by means of a Hamilton syringe (No. 31 gauge needle) attached to a stereotaxically guided microdrive. Volumes of from 0.1 to 0.3/ti/injection were introduced (concentrated 50-100 #Ci/#l) over periods of 0.5-1 h and, in each case, the needle was left within the brain for about 10 min before and after the injection. After survival times of 1-10 days the animals were sacrificed by intracardiac perfusion of saline followed by one of several different fixatives. Frozen sections were mounted, coated with K o d a k NTB-2 emulsion diluted with an equal quantity of distilled water and refrigerated for approximately 4 weeks. The slides were developed (D-19 high contrast developer), stained through the emulsion, coverslipped, and examined by light and dark field optics for the 'limits' of the injection site and the presence of silver grains above background level over the olive. Clumps of grains over axonal bundles are thought to indicate labeled axons in passage, whereas grain dispersion over the olivary neuropil is interpreted as labeled axon terminals (e.g. Fig. 3B). Eighteen additional brains are available in which horseradish peroxidase (HRP) has been injected into different regions of the cerebellar cortex. Although the complete pattern of olivo-cerebellar connections is very complex and will be reported at a later date, those results which are pertinent to the present study are reported herein. The marker was delivered as described above (0.1-0.6 #1 of a 30-50 ~ solution) and after survival times of approximately 24 h the animals were anesthetized and perfused with a 1 - 3 ~ paraformaldehyde, 1 . 0 ~ gluteraldehyde solution in a 0.1 M phosphate buffer (pH = 7.4, 24 °C). The brains were removed, immersed for 6 h in the perfusate and rinsed for 24 h or more in a 0.1 M phosphate buffer (pH = 7.4) containing 30~o

255 sucrose. Frozen sections were incubated in a medium containing hydrogen peroxide and 3,3'-diamino benzidine tetrahydrochloride and subsequently counterstained for Nissl substance. The stained sections were examined for spread of the marker at the injection site as well as for the presence of neurons containing granules of reaction product. RESULTS

The organization of reticulo-olivary connections In the following account the results obtained from medullary injections are described first followed in order by those derived from brains in which the marker was deposited within the pons and midbrain. Results of medullary injections. Unfortunately, in many of the brains with medullary placements there is direct spread of the marker to the inferior ollve (Fig. 1D) and in others the injection is so close to the olive that it is difficult to determine whether or not extracellular spread contributed to its labeling. The cases illustrated in Fig. 1A, B, C and E, however, show little obvious spread to the olive and do contain evidence for transport of labeled protein to that nucleus. The placement illustrated in Fig. 1A includes all of the nuclei surrounding the central canal as well as the caudal dorsal column nuclei, and dispersed label is present over the caudal third of the accessory nuclei, bilaterally. Since relatively little reticular formation is included at the injection site and there is more extensive label over the olive than is present after injections, which are essentially limited to the dorsal column nuclei (same survival and exposure

Fig. 1. A-E: light field photomicrographs of injection sites (arrows) from 5 different brains after exposure times of 4 weeks and survival times of 24 h. A is from P-387, x 10; B is from P-359, x 17; C is from P-360, x 15; D is from P-395, x 15 and E is from P-451, × 13.

256

B

Fig. 2. A series of drawings through three levels of the olive (rostral A to caudal C) from P-359 (left side) and P-360 (right side). Selected sections through the injection sites are drawn in the upper right for each case and, where present, silver grains over the olive are illustrated as dots. For clarity, extraolivary labeling is not shown. times), it is tentatively concluded that non-reticular neurons a r o u n d the central canal are responsible for the observed transport. Fig. 1B and C were taken from brains in which neurons o f the d o r s a l and ventral divisions o f the m e d u l l a r y reticular f o r m a t i o n are heavily labeled, but with spillover into either the d o r s a l column nuclei a n d nuclei a r o u n d the central canal (Fig. 1B) or the spinal trigeminal and lateral reticular nuclei (Fig. 1C). The results o b t a i n e d from both o f them are plotted in Fig. 2 and it can be seen that, in spite o f the size o f the injections, terminal label is essentially limited to caudal parts o f the accessory nuclei on the ipsilateral side (Fig. 2C, o f b o t h sides). Since the olivary labeling is far m o r e extensive t h a n t h a t seen after injections which are 'restricted' to either the d o r s a l c o l u m n or trigeminal nuclei (again, same survival and exposure times), we conclude that some o f it results from t r a n s p o r t by reticular neurons. In any case, if reticulo-olivary projections arise from neurons within the injection sites o f either case they distribute to the caudal third o f the accessory nuclei. The injection shown in Fig. I E includes the caudal part o f the lateral reticular

B

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Fig. 3. A-D: light field photomicrographs of sections through the injectionsites of P-403 (A) and P-446 (C). B is a dark field photomicrograph of the caudal medial accessory nucleus showing terminal label (arrow) over the dorsolateral part of subnucleus 'c' after the injection shown in A. D illustrates the lighter labeling (arrow) over the same part of subnucleus 'c' after the smaller placement shown in C. The insert in C is a low-power, dark field photomicrograph of the section from which the illustration in D was taken. The arrow indicates the site of terminal label in each section. A and C are × 11 and in B the bar equals 0.5 ram. D is taken atthe same magnification as B. The exposure time for all sections was 4 weeks and the survival time was 10 days. nucleus, a n d the a u t o r a d i o g r a m s suggest that labeled protein was transported to the caudal third of the medial accessory nucleus o n the same side. Suggestion of a n additional projection to the lateral part of the dorsal accessory nucleus at more rostral levels was also noted. A n o t h e r case (not illustrated) was subjected to an injection of the rostral part of the lateral reticular nucleus and adjacent regions, b u t showed no evidence for t r a n s p o r t to the caudal olive. It does c o n t a i n some evidence for a projection to rostral regions of the olive, although the proximity of the injection site to the labeled part of the olive makes it difficult to rule out extracellular spread. It should be noted that our material provides n o i n f o r m a t i o n relative to the possible olivary target(s) of n e u r o n s in the p a r a m e d i a n medulla, since all injections of that region show some spread to the olive itself (e.g. Fig. I D). Results from pontine injections. The results obtained from p o n t i n e injections are

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Fig. 4. A - F : A s h o w s the injection site f r o m P-404 a n d B illustrates the axonal labeling present in the rostral medulla o f the ipsilateral side. C a n d D are light a n d dark field p h o t o m i c r o g r a p h s of the caudal medial accessory nucleus on the side of the injection. T h e arrow points to the s a m e vessels in each figure a n d in D terminal label can be seen mainly in the area referred to herein as subnucleus 'b' (C). E a n d F are higher power views of C a n d D, respectively, a n d in each the s a m e vessels are indicated (arrows) as in C a n d D. The exposure time for all sections was 4 weeks a n d the survival time was 24 h. T h e bar in D is 0.5 ram.

259

~

Fig. 5. A series of drawings through three levels of the olive (rostral A to caudal C) from P-403 (left side) and P-404 (right side). A selected section is drawn through the injection site in each case (upper right) and terminal label where present is illustrated as dots. For clarity, labeling outside the olive is not shown.

more definitive since direct spread to the olive is no problem. The injection shown in Fig. 3A is intentionally large and includes the nucleus gigantocellularis and the nucleus gigantocellularis, pars ventralis, bilaterally, as well as the nuclei raphe magnus and pallidus. As might be expected from the size of the placement, silver grains are abundant and clumped over axons on both sides of the paramedian medulla. In the olive, however, labeling is sparse and essentially limited to the region corresponding to the dorsolateral part of subnucleus 'c' (compare Fig. 3B and the left side of Fig. 5 with Fig. 9A). Although the olivary label is bilateral, it is more extensive on the side indicated by the arrow in Fig. 3A. When the injection is smaller and more restricted to one side (e.g. Fig. 3C), axonal labeling is still bilateral in the paramedian medulla. Silver grains are fewer in number over the olive, but they are located over precisely the same regions of the olive described above (compare Fig. 3B and D). Labeling of axonal bundles which distribute to more caudal targets can be seen by referring to the insert of Fig. 3C as well as Fig. 3D.

U

-

260

-

x

Fig. 6. A-D: a series of light field photomicrographs through the injection sites (arrows) of P-444 (A), P-445 (B), P-399 (C), P-385 (C, insert) and P-422 (D). A and B are • 13, C and its insert are × 11 and D is × 15. A 4-week exposure time was used for all sections and the animals were sacrificed 10 days after the injection. Two of the brains with more lateral and rostral injections of the pontine reticular formation (Fig. 4A) also show evidence for a projection to the caudal olive, but to a slightly different region. The section shown in Fig. 4A was taken from a case in which neurons are heavily labeled deep to the abducens nucleus and the genu of the facial nerve. Although axonal bundles within the paramedian medulla are covered with silver grains (Fig. 4B), olivary label is limited to the caudal thrid o f the medial accessory olive where it mainly overlies the region referred to in our previous description as subnucleus ' b ' (Fig. 4C-F). The results from the placement shown in Fig. 4A are plotted on the right side o f Fig. 5. When comparing Fig. 4D with Fig. 3B and D it should be remembered that the label shown in Fig. 4D was obtained after a 24 h survival, whereas that in Fig. 3B and D was derived from cases which survived for 10 days. Grain dispersion is present over the dorsolateral part of subnucleus 'c' in several brains with injections centered within rostral portions of the pontine reticular formation. The placement shown in Fig. 6A resulted in heavy labeling of reticular neurons medial and rostral to the m o t o r trigeminal nucleus as well as labeling o f axons in the paramedian medulla, mainly on the same side. The results from that injection are plotted in Fig. 7 (left side) where it can be seen that the label extends over the cap o f Kooy, as well as over the dorsolateral part of subnucleus 'c', and that it is lighter than

261

,~u ~

~

.~-~.

P-445

Fig. 7. A series of drawings through three levels of the olive (rostral A to caudal C) from P-444 (left) and P-445 (right). A selected section through the injection site is drawn on the upper right for each case and, where present, silver grains over the olive are illustrated as dots. For clarity, labeling outside the olive is not shown. that present after more caudal placements. The placement shown in Fig. 6B also resulted in olivary labeling (Fig. 7, right side), but spread of the marker makes it impossible to be sure that the grains over the olive reflect transport by reticular neurons. It is interesting, however, that the terminal site is limited to the medial extreme of subnucleus 'b'. Fig. 6C (including the insert) and 6D illustrate the injection site in three cases which contain no evidence for a projection to the olive. Results from midbrain injections. Injections of [aH]leucine into certain dorsolateral reticular (tegmental) and deep tectal areas of the midbrain result in light label over caudal portions of the medial accessory nuclei of both sides. The injection of P488 of our collection (Fig. 8A) produced heavy label over neurons within the deep superior colliculus and the underlying interstitial tegmental (reticular) area. On the side opposite the placement there is light terminal label over the same dorsolateral part of subnucleus 'c' which receives input from the pontine reticular formation (Fig. 8B).

262

Fig. 8. A D: A and C show light field views of sections through the injection sites (arrows) of P-488 (A) and P-467 (C) of our midbrain collection. B shows light terminal label (arrow) over part of subnucleus 'c' on the side opposite the injection in A. D illustrates the heavy labeling over the principal nucleus on the side ipsilateral to the right side of the injection shown in C. A is x 10 and C is × 13. The exposure time is 4 weeks for each section and in both cases the postoperative survival time is 10 days.

Equally light label is present over the ventromedial part of subnucleus 'c' on the same side (the beta nucleus of the feline olive 12) as well as over restricted portions of the ipsilateral dorsal accessory nucleus. In cases with somewhat more ventral and lateral tegmental injections, the ipsilateral labeling of the medial and dorsal accessory nuclei is often seen without the contralateral component. The light labeling produced by tectotegmental placements (Fig. 8B) can be contrasted with that present over the ipsilateral principal olive after injections in and around the red nucleus (Fig. 8C and D). The postoperative survival and autoradiographic exposure times are the same in both cases. It should be noted that not all injections within the midbrain reticular formation provided evidence for a projection to the olive as described above. Midbrain-olivary projections are very complex and will be the subject of a subsequent communication.

Cerebellar projections from those portions of the olive receiving reticular inputs Brains are available with injections of H R P into all major regions of the cerebellum except the flocculus and the nodule. From such cases it is clear that the reticular targets of the medial accessory olive contain reactive neurons when the

263

Fig. 9. A-C : A is a photomicrograph of a Nissl stained section through that part of the olive receiving most of the projections described in this communication. The arrow indicates the dorsolateral part of subnucleus 'c'. B is a sagittal section through the cerebellar vermis which shows a HRP injection (arrow) of the tuber. C illustrates, in dark field, the HRP-labeled neurons (arrow) seen after the foliumtuber injection shown in transverse section in the insert (arrow). The bar in A and C indicates 0.5 mm and B is × 9.5.

enzyme is deposited into either the anterior lobe or some portion o f the cerebellar vermis. Caudal subnucleus 'a' (Fig. 9A) is labeled when H R P is injected into the medial anterior lobe, after large injections which include parts o f the folium and tuber and after injections limited to the uvula. Subnucleus ' b ' (Fig. 9A) contains reactive neurons after large injections of the anterior lobe and after some injections o f the vermis. O f particular interest, however, is the observation that the dorsolateral part of subnucleus 'c' (Fig. 9A) is backfilled only when the injection is made into the declive, folium or tuber or when the marker spreads into those regions (Fig. 9B and C). DISCUSSION Because o f the tendency for the marker to spread, our autoradiographic material does not provide definitive evidence for reticulo-olivary projections from the medulla. Supportive evidence for these projections is available, however. After H R P injections which are limited to the feline olive, labeled neurons are present within reticular areas comparable to those covered by the medullary placements illustrated in this study (G. A. Bishop, R. A. M c C r e a and S. T. Kitai, personal communication). Our results

264 suggest that the caudal third of the accessory nuclei is a major target of such connections. Although it is clear that reticulo-olivary projections take origin from several areas of the pons and midbrain they are limited and distribute to restricted portions of the accessory nuclei. Some of the reticular neurons located rostral and lateral to the nucleus gigantocellularis project to subnucleus 'b' and 'a' of the medial accessory olive, but those within the nucleus gigantocellularis and/or the nucleus gigantocellularis, pars ventralis, project to that part of the medial accessory nucleus referred to as the dorsolateral part of subnucleus 'c '12. An additional projection to subnucleus 'c' and the adjacent cap of Kooy takes origin from more rostral portions of the pontine reticular formation. Most of the above connections are predominantly ipsilateral. Connections from the interstitial tegmental (reticular) nucleus of the midbrain and/or the deep tectum also pick out the dorsolateral part of subnucleus 'c', but on the opposite side (see Martin et al.a2). In spite of the fact that the reticular formation is often thought of as 'diffuse' its projections to the inferior olive are remarkably restricted. Since beginning this study we have re-examined a number of brains which have been subjected to lesions of the reticular formation and subsequently processed by the Fink-Heimer technique. Although lesions comparable in position to the placements shown in Figs. 3C, 4A and 6A produce terminal degeneration in those portions of the medial accessory nucleus which are labeled in the autoradiographic experiments, degeneration is extensive in other olivary regions as well. For example, a lesion in the area injected in Fig. 6A interrupts axons of midbrain origin producing extensive degeneration within the principal nucleus of the ipsilateral olive as well as throughout most of the medial accessory complex. Such results are a further testimony to the power of the autoradiographic method to unravel the efferent connections of regions traversed by axons from distant sources. Unfortunately, however, all techniques have limitations and two of the serious drawbacks of the autoradiographic method are the tendency for the marker to spread when injected and the difficulty one encounters in drawing 'boundaries' around injection sites. Extracellular spread is a particular problem in the reticular formation, especially since it often takes fairly large injections to produce olivary labeling at the concentrations we employed. Although a detailed account of the opossum olive has been given previously 12, two points deserve emphasis so as to avoid problems of atlas semantics. First, it is possible that the dorsolateral part of subnucleus 'c' in the opossum medial accessory olive is comparable to the most dorsomedial portion of subnucleus 'b' in the cat and monkey (see Martin et al. 12 for a discussion). Secondly, it seems clear that the ventromedial bend of subnucleus 'c' in the opossum and primate is the same as the region referred to as the beta nucleus in the cat. Afferent connections to the olive become most meaningful when interpreted in light of the cerebellar projections of their olivary targets. Since the physiological literature suggests that reticulo-olivary connections provide indirect routes through which spinal information reaches the cerebellum 1,16, it is worth noting that certain

265 r e t i c u l a r a r e a s o f t h e p o n s , a n d p r o b a b l y t h e m e d u l l a , p r o j e c t to o l i v a r y sites w h i c h receive d i r e c t i n p u t s f r o m t h e spinal c o r d a n d d o r s a l c o l u m n nuclei 12 a n d w h i c h , in t u r n , relay to spinal a r e a s o f t h e c e r e b e l l u m ( s u b n u c l e i ' a ' a n d ' b ' o f the m e d i a l a c c e s s o r y nuclei). R e t i c u l o - o l i v a r y circuits h a v e also b e e n i m p l i c a t e d as r o u t e s t h r o u g h w h i c h c o r t i c a l i n f o r m a t i o n g a i n s access t o t h e c e r e b e l l u m a n d it is o f i n t e r e s t t h a t r e t i c u l a r a r e a s o f the p o n s a n d p r o b a b l y the m e d u l l a d i s t r i b u t e to t h a t p a r t o f the m e d i a l a c c e s s o r y olive ( s u b n u c l e u s ' b ' ) w h i c h also receives c o r t i c a l i n p u t l L A n u n e x p e c t e d finding, h o w e v e r , was t h a t several r e g i o n s o f the p o n t i n e r e t i c u l a r f o r m a t i o n , as well as t e c t o t e g m e n t a l a r e a s o f t h e m i d b r a i n , p r o j e c t to t h e d o r s o l a t e r a l p a r t o f s u b n u c l e u s ' c ' , a r e g i o n w h i c h receives little if a n y spinal, d o r s a l c o l u m n o r c o r t i c a l i n p u t lz a n d w h i c h is u n l a b e l e d a f t e r H R P i n j e c t i o n s o f the spinal c e r e b e l l u m ( a n t e r i o r lobe, p a r a m e d i a n l o b u l e s a n d p y r a m i s ) . A s i l l u s t r a t e d in F i g . 9C, h o w e v e r , the d o r s o l a t e r a l p a r t o f s u b n u c l e u s ' c ' c o n t a i n s r e a c t i v e n e u r o n s s u b s e q u e n t to H R P i n j e c t i o n s o f the declive, f o l i u m o r t u b e r (see H o d d e v i k et al. 9 f o r g e n e r a l l y c o m p a r a ble results in the cat). It is i n t e r e s t i n g t h a t t h e l a t t e r a r e a s o f the c e r e b e l l u m are r e s p o n s i v e to a u d i t o r y a n d visual stimuli 18 a n d t h a t t h e i r e x c i t a t i o n elicits t u r n i n g o f the h e a d t o w a r d s t h e side o f s t i m u l a t i o n s . T h e s e a n a t o m i c a l a n d f u n c t i o n a l d a t a suggest t h a t at least s o m e r e t i c u l o - o l i v o - c e r e b e l l a r circuits are i n v o l v e d in the m o d u l a tion of such movements. ABBREVIATIONS a b bp c caud CcD CcS Cn cr Cu CuL d De Fac g GM gr Hg Lg LR Nd oi OS ped pr PrC

= subnucleus 'a' of the medial accessory olive = subnucleus'b'ofthemedialaccessory olive = brachium pontis = subnucleus'c' ofthe medial accessory olive = caudal = dorsal cochlear nucleus = superior central nucleus = culmen of cerebellum = restiform body = cuneate nucleus ~ lateral (accessory) cuneate nucleus -- dorsal accessory nucleus of the inferior olive = declive of cerebellum ~ facial nucleus = genu of facial nerve = medial geniculate nuclei = nucleus gracilis = hypoglossal nucleus -- lingula of cerebellum = lateral reticular nucleus = nodulus of cerebellum = inferior olivary nucleus = superior olivary nucleus - cerebral peduncle = principal nucleus of the inferior olive = preculmen of cerebellum

pr.d.

= dorsal lamella of the principal olivary nucleus PrTc = caudal nucleus of the pretectal complex pr.v. = ventral lamella of the principal olivary nucleus PyC = pyramis of cerebellum pyr = pyramidal tract Ra - raphe nuclei RD -- dorsal nucleus of the medullary reticular formation RGc = gigantocellular reticular nucleus RGcv -- ventral part of gigantocellular reticular nucleus RN = red nucleus ROST = rostral RP = pontine reticular nucleus RPc = parvocellular reticular nucleus RTg = reticulotegmental nucleus RV = ventral nucleus of the medullary reticular formation rVm -- sensory root of the trigeminal nerve sp = spinal trigeminal nucleus trMo = motor trigeminal nucleus trs = spinal trigeminal tract Tz = trapezoid nucleus Uv = uvula of cerebellum VstL = lateralvestibular nucleus VstM -- medial vestibular nucleus

266 A C K N O W L E D G MENTS This investigation was supported by USPHS Grants NS-08798 and NS-07410. The

authors

Malinda Amspaugh

wish

to t h a n k

Mrs.

Nann

Patterson

f o r t e c h n i c a l help, M s .

for typing the manuscript and Mr. Gabriel Palkuti for photo-

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