Olivocerebellar projections to paramedian lobule in tree shrew (Tupaia glis): a horseradish peroxidase study

Brain Research, 305 (1984) 271 - 282

271

Elsevier BRE 10171

Olivocerebellar Projections to Paramedian Lobule in Tree Shrew

(Tupaia glis):

a Horseradish Peroxidase Study R. H. WHITWORTH 1, Jr., D. E. HAINES 2 and G. W. PATRICK*

1Departments of Anatomy, Louisiana State University Medical Center, New Orleans, LA 70112-1393 and 2West Virginia University, Morgantown, WV26506-6302 (U.S,A.) (Accepted December 20th, 1983)

Key words: medulla - - cerebellum - - inferior olive - - tree shrew - - olivocerebellar projections - - horseradish peroxidase - paramedian lobule

Horseradish peroxidase (HRP) was used as a retrograde tracer to identify the distribution pattern of labeled cells in the inferior olivary nucleus (IO) of Tupaia. Crystallized HRP was implanted into dorsal (DPML) and ventral (VPML) divisions of the paramedian lobule (PML) and, following appropriate survival times, the tissues were processed using diaminobenzidine and tetramethylbenzidine as chromogens. Subsequent to implants into lateral DPML and VPML, HRP-labeling is seen in rostral subgroup a of the medial accessory olive (MAO) and in the medial part of the dorsal accessory olive (DAO~) and ventral lamellae of the principal olive (VLPO) close to their rostral poles. The lateral bend and adjacent dorsal lamella of the principal olive and rostral subgroup c of MAO also contain HRP-reactive somata following lateral DPML implants. Subsequent to implants in central DPML, labeling is seen in rostral DAO m and subgroup a of MAO. Central VPML implants result in additional clusters of labeled cells in VLPO, the lateral bend of the principal olive (PO), and subgroup c of MAO. Following implants of HRP into medial PML reactive somata are found in dorsomedial VLPO and DLPO, and clusters of labeled cells are present in caudal subgroup a of MAO and DAO m. In contrast to implants in central and lateral PML, rostral DAO m and PO are devoid of reactive neurons. These results show that olivocerebellar projections to PML of Tupaia are exclusively contralateral and topographically organized. Collectively these olivocerebellar data corroborate the existence of zones C l, C 2, C 3 and D in PML of Tupaia and show that their patterns are similar, in their essential features, to those seen in the corticonuclear pathway in this species. zones C 1, C2, C3, D1 and D 2 in P M L of cat and Maca-

INTRODUCTION

ca. Studies, particularly in cat, h a v e p r o v i d e d d a t a

W i t h the e x c e p t i o n of studies by L i n a u t s and M a r -

c o n c e r n i n g the t o p o g r a p h y and z o n a l a r r a n g e m e n t of

tin 32 and M a r t i n et al. 35 o n Didelphis no d a t a are

olivocerebellar projections

lobule

available on o l i v o c e r e b e l l a r p r o j e c t i o n s in m o r e gen-

( P M L ) (see ref. 11 for r e v i e w ) . T o p o g r a p h i c a l l y or-

eralized m a m m a l i a n forms. T h e t r e e s h r e w (Tupaia

ganized p r o j e c t i o n s f r o m the i n f e r i o r olive ( I O ) to

gl&) occupies a u n i q u e and c o n t r o v e r s a l position at

P M L h a v e b e e n r e p o r t e d using e l e c t r o p h y s i o l o g i c al 1,45 and a n a t o m i c a l tracing 2,7,8-10,12-14A9,21.30,32,5°

the p r i m a t e - n o n - p r i m a t e

techniques. A z o n a l o r g a n i z a t i o n of o l i v o c e r e b e l l a r

m a m m a l s (see ref 33). T h e p r e s e n t study will utilize

t e r m i n a t i o n s in P M L as s u g g e s t e d by e l e c t r o p h y s i o -

this m a m m a l to address the f o l l o w i n g questions: (1)

logical results/ has b e e n v e r i f i e d using a u t o r a d i o graphic21,30 and h o r s e r a d i s h p e r o x i d a s e 9,10A2,13,50

v o c e r e b e l l a r p r o j e c t i o n s to P M L t o p o g r a p h i c a l l y or-

p r o c e d u r e s . T h e s e studies indicate the p r e s e n c e of

g a n i z e d ? (3) is t h e r e a z o n a l a r r a n g e m e n t of o l i v o c e -

to p a r a m e d i a n

i n t e r f a c e and has i m p o r -

tant r e l a t i o n s h i p s with s o m e n o n - p r i m a t e e u t h e r i a n

which subdivisions of I O p r o j e c t to P M L ? (2) are oli-

* Present address: Department of Anatomy, Fort Wayne Center For Medical Education, 2101 Coliseum Blvd. East, Fort Wayne, IN 46805, U.S.A. Correspondence: R. H. Whitworth, Jr., Department of Anatomy, Louisiana State University Medical Center, 1901 Perdido Street, New Orleans, LA 70122-1393, U.S.A. 0006-8993/84/$03.00 © 1984 Elsevier Science Publishers B.V.

272 rebellar terminations within PML? (4) how does the arrangement of olivocerebellar zones compare with the zonal organization of corticonuclear projections? MATERIALSAND METHODS Twelve adult male and female tree shrews (Tupaia glis) were used. Small chips of horseradish peroxidase (HRP) were made by saturating a 5 ~1 drop of sterile saline with HRP (Sigma type VI), allowing it to air-dry for approximately 2-3 min then removing small pieces of the crystallized enzyme. After appropriate surgical exposure small pieces of this crystallized HRP were implanted into superficial folia of PML. Care was taken to orient the implants so as to minimize spread of HRP to adjacent areas of vermis or into contiguous folia of the lobule. Following sur-

vival times of 22-38 h, the animals were perfused with 0.9% saline (0.1% procaine hydrochloride added) followed immediately by a buffered solution of 1.0% paraformaldehyde/1.25% glutaraldehyde/4.25% sucrose. Brains were removed, postfixed in the same solution for 4-6 h, and washed overnight in 2 changes of wash (NaH2PO + K2HPO 4 + sucrose). Brainstems and cerebelli were cut into frozen sections at 50/~m each in either coronal or horizontal planes. Five brains were processed with the diaminobenzidine (DAB) technique; alternate sections in each were counterstained with cresyl-violet acetate. All sections from 5 additional brains were processed according to the tetramethylbenzidine (TMB) method37, 38. Alternate sections from two more brains were processed with DAB and TMB, respectively. The locations of HRP-positive cells in the

Fig. 1. Tracings of the inferior olivarycomplex (IO) and related brainstem structures from one Tupaia(case TS-86) in transverse plane from rostral (A) to caudal (L). Compare with representative photomicrographsin Fig. 2. See text for discussion. Scale = 1.0 mm.

273 inferior olive were plotted with the aid of an Olympus drawing tube. The approximate extent of the effective region of each implantation side and surrounding area of diffusion was determined according to established criteria 37,38. The morphology of Tupaia PML has been described elsewhere23, 27. RESULTS

Conformation of the inferior olive Since a detailed description of the configuration of IO in Tupaia glis is not available in the literature, the subdivisions of IO in tree shrew were identified from the Nissl-stained sections. Tree shrew IO is composed of 3 main subnuclei typical of all mammals studied to date; a principal olivary nucleus (PO), medial accessory olive (MAO) and dorsal accessory olive (DAO) (Fig. 1 A-L). The total rostrocaudal extent of IO in Tupaia is approximately 3000 pm.

Principal olivary nucleus (PO) The PO of Tupaia is bounded ventromedially by MAO, dorsally and dorsolaterally by D A O , and extends throughout the rostral one-half of the complex (Fig. 1A-F). Although appearing at mid-olivary levels as an undivided group of cells, PO is composed in its middle two-thirds of dorsal (DLPO) and ventral lamellae (VLPO) connected at their lateral extremes by a lateral bend (Figs. 1C-E and 2B). The VLPO extends dorsomedially from the ventrolateral margin of the brainstem just dorsolateral to subgroup a of MAO (Figs. 1C-E and 2B) and fuses rostrally with D A O (Figs. 1B and 2C) to form the rostral pole of the complex (Fig. 1A). A subdivision of MAO, the dorsomedial cell column (see below), is continuous with the dorsomedial extreme of VLPO at more caudal levels (Figs. 1E and 2B). The DLPO, located ventromedial to DAO, is more limited relative to VLPO in its rostrocaudal extent (Fig. 1C-E).

Dorsal accessory olive (DA O) The D A O extends throughout all but the most caudal extreme of the complex (Fig. 1A-K). Two subdivisions of D A O can be identified on cytoarchitectural bases; a group of cells located dorsomedial to DLPO, designated DAOm, and a more laterally placed cluster of somata located dorsolateral to DLPO designated D A O 1 (Figs. 1D-F and 2B). D A O m and

Fig. 2. Photomicrographsof IO and adjacent structures in Tupaia shown in transverse p!ane from caudal (A) to rostral (C). These photomicrographs correspond to specific tracings from Fig. 1 as follows:Figs. 2A to 1B; 2B to 1E; and 2C to 1L. Note typical dorsal (DLPO) and ventral (VLPO) lamellae of PO (B) and the subdivision of DAO into lateral (DAOI) and medial (DAOm) portions (B, C). The caudal pole of IO is formed by subgroup b of MAO with a minor contribution from subgroup a (A). Cresylviolet stain. Scale = 300/~m. DAO1 are frequently invaded by and/or separated from each other by intramedullary fascicles of the hypoglossal nerve (Figs. 1D, E and 2B). The D A O 1 first appears (Fig. 1K) dorsolateral to subgroup b of

274 M A O (see below) just rostral to the caudal pole of the complex. The caudal terminus of DAOm is found immediately caudal to mid-olivary levels in a position dorsolateral to subgroup a of M A O (Fig.ill). Both D A O m and D A O l extend essentially to the rostral pole of the complex (Figs. 1B and 2C) where they appear to merge.

/

Medial accessory olive (MA 0) The M A O extends throughout the caudal threefourths of Tupaia IO (Fig. 1C-L). The M A O is divisible into caudally located subgroups a, b, c and d which extend dorsomedially from the ventral border of the brainstem just above the pyramid, and a more rostrally located dorsomedial cell column (DMCC). The caudal pole of the complex is formed by subgroup b usually accompanied by a few cells of subgroup a (Figs. 1L and 2A). Subgroup b, which decreases in size more rostrally as subgroup c enlarges, terminates just caudal to mid-olivary levels (Fig. 1G and H). Subgroup a increases in size from caudal to rostral and extends throughout the caudal threefourths of the complex (Fig. 1C-L). Progressing rostrally from mid-olivary levels, subgroup a of M A O is displaced dorsomedially by the expanding ventral lamella and lateral bend of PO (Fig. 1C-G). Subgroup c of M A O begins just rostral to the caudal pole of IO in a location dorsomedial to subgroup b and lateral to the medial lemniscus (Fig. 1K). Close to its rostral terminus, subgroup c appears to be subdivided into lateral and medial parts connected by a narrow band of cells (Fig. 1G and H). Subgroup d of M A O , located dorsal to subgroup c, has a relatively limited rostrocaudal extent (Fig. 1I and J). The D M C C is located in the rostral half of IO, dorsal to subgroup a, lateral to the medial lemniscus, and medial to DAOm (Figs. 1C-E and 2B). Rostrally, D M C C is continuous ventrolaterally with V L P O (Figs. 1E and 2B) and terminates just caudal to the rostral pole of the complex (Fig. 1C).

Olivocerebellar projections to paramedian lobule The paramedian lobule of Tupaia is composed of 4-5 folia; the rostral two represent the dorsal PML (DPML) while the caudal 2-3 comprise the ventral PML (VPML) z7. Three H R P implants were made in the lateral part of the most rostral folium of PML. Since the distribution of labeled somata in IO was es-

Fig. 3. Tracings of IO in Tupaiain transverse section from rostral (A) to caudal (E) showing the distribution of HRP-positive cells in IO following an implant of HRP into the lateral part of the rostral folium of PML (case H38). In this and all subsequent tracings the following conventions are followed. The distribution of large dots represents the relative concentration of HRP somata. In the upper right of tracing A is the case number (e.g. H38) and in each tracing the specific slide number (e.g. 58, 54, 48, 44, etc.) from that case from which each tracing was made is so indicated. The maximum area of spread of HRP from the implant site is indicated in the inset. Note the location of labeled neurons in subgroups a (B-D) and c (E) of MAO, dorsomedial two-thirds of VLPO (A, B) lateral parts of the lateral bend of PO (A-C), ventrolateral DLPO, and in rostral DAO (A-C). Scale = 1.0 ram. sentially identical in these experiments, case H38 (Fig. 3) is presented as representative. Following an implant in which the effective area of diffusion of H R P was confined to lateral and central parts of the rostral folium of D P M L (Fig. 3 inset), two groups of labeled cells are found within contralateral PO; a cluster restricted to the dorsomedial two-thirds of VLPO, and a group in the lateral bend of PO (Fig. 3A and B). At more caudal levels (Fig. 3C) labeled neurons are located in the lateral half of the lateral bend with a few extending into the ventrolateral extreme of adjacent D L P O . Dorsomedial D L P O , the medial half of the lateral bend, V P L O (Fig. 3C) and the caudal pole of PO (Fig. 3D) are devoid of reactive somata. Close to the rostral pole of 10, HRP-labeling occupies the entire extent of D A O m (Fig. 3A). At a slightly more caudal level two clusters of HRP-filled neurons, located in dorsomedial aspects of DAOm and D A O I, respectively, are separated by an area

275 devoid of labeled cells (Fig. 3B). Occasional HRPlabeling is seen in ventrolateral parts of D A O 1 at the same level (Fig. 3B). A small group of reactive cells is present in dorsomedial D A O m close to mid-olivary levels (Fig. 3C) while caudal to PO, D A O m and DAO1 contain no HRP-positive somata (Fig. 3D-E). The rostral half of subgroup a of M A O contains labeled somata in its dorsal and dorsomedial areas (Fig. 3B-D). Close to the rostral terminus of subgroup a HRP-positive cells are confined to the dorsal extreme (Fig. 3B) while more caudally they occupy its entire dorsomedial one-half (Fig. 3C). At the caudal pole of PO, reactive neurons in subgroup a M A O are restricted to its ventromedial aspect; the remainder of the subnucleus contains no label (Fig. 3D). The rostral part of subgroup c of M A O also contains a moderate number of HRP-positive neurons (Fig. 3E). All other subgroups of M A O , D A O and PO in the caudal half of IO are devoid of HRP-reactive somata. In order to compare the olivocerebellar projection to lateral aspects of dorsal versus ventral PML, H R P was implanted into lateral parts of the rostral two folia of VPML; the implant site slightly infringed on caudal DPML (case H32 - - Fig. 4 inset). In contrast to case

dno.

lat..

--mild.

Fig. 4. Tracings of IO in Tupaiain transverse section from rostral (A) to caudal (D) showing the distribution of reactive HRP cells in IO followingan implant of HRP into lateral parts of ventral paramedian lobule (case H32). Rostral portions of DAO (A, B) and VLPO (B) contain HRP-positive somata. Labeled ceils are also found in the medial part of subgroup a of MAO (C). Scale = 1.0 ram.

H38 (Fig. 3), fewer labeled cells are found in rostral parts of contralateral PO, M A O , and D A O (Fig. 4 A - D ) in case H32. As in the previous case, the caudal three-fourths of IO is devoid of reactive somata. Labeled cells are restricted to the medial onefourth of subgroup a of M A O close to its rostral termination (Fig. 4C). Subgroup a at more caudal levels and all other subdivisions of M A O contain no label (Fig. 4D). Reactive somata, limited in their rostrocaudal distribution, are scattered throughout VLPO close to the rostral pole of IO; the lateral bend of PO at this level and all of PO at other levels contain no HRP-positive neurons (Fig. 4B and C). At the rostral pole of IO, labeling is found exclusively in D A O (Fig. 4A), a few isolated reactive cells being located in D A O at more caudal levels (Fig. 4B). Implants of H R P were made into more central regions of PML to determine if the same subgroups of IO that project to lateral PML also send fibers to this part of cerebellar cortex. Since all implants resulted in a similar pattern of labeling, the results of two representative cases (H35 and H39) are presented. The implant in case H35 included the central area of caudal DPML with slight involvement of the rostral VPML (Fig. 5 inset - - H35). Labeled neurons located in rostral M A O are restricted to central and ventromedial parts of subgroup a just caudal to its rostral pole (H35, Figs. 5C-D and 6D). The rostral terminus (H35, Fig. 5B) and more caudal regions of subgroup a (H35, Fig. 5E) contain no HRP-labeling. In the rostral pole of IO labeled neurons are present in D A O (H35, Figs. 5A and 6E). Adjoining portions of rostral PO (H35, Fig. 5A) and all subdivisions of PO, D A O m and D A O l at more caudal levels contain no reactive somata (H35, Fig. 5B-E). Examination of the implant site placed in central regions of VPML in case H39, revealed minor involvement of the central portion of the caudal folium of DPML (H39, Fig. 5 inset). In this case a band of HRP-labeled cells extends throughout the dorsomedial one-half of VLPO at about its midpoint (H39, Figs. 5B and 6B). Another focus of reactive somata is present at the same level in the lateral aspect of the lateral bend of PO (H39, Fig. 5B). Both of these groups of reactive neurons, separated from each other by an area devoid of HRPpositive cells, extend into more rostral levels of VLPO and the lateral bend of PO, respectively (H39, Figs. 5B and 6A). The medial part of the lateral

276

lat..

med.

~(

H39~

Fig. 5. Tracings of IO in Tupaiain transverse section from rostral (A) to caudal (D, E) showing the distribution of HRP-labeled cells in IO following the inplant of HRP into central regions of caudal DPML (case H35 - - left) and into rostral VPML (case H39 - - right). Following implant of HRP into dorsal paramedian lobule (case H35, A-E) labeled cells are present in rostral portions of DAO (case H35, C-D). Note the lack of labeled cells in VLPO and lateral bend of PO (case H35, E). Labeled cells are located in rostral VLPO (case H39, A-C) and c (case H39, D, of MAO. Scale = 1.0 mm.

bend, ventrolateral V L P O (H39, Fig. 5B) and D L P O (H39, Fig. 5C) contain no labeled cells. Although the rostral pole of subgroup a of M A O contains a few isolated reactive somata (H39, Fig. 5A), labeled cells are more numerous and are confined to the dorsomedial one-half of subgroup a caudally (H39, Fig. 5B). Close to the caudal pole of P O scattered labeled somata are present in v e n t r o m e d i a l subgroup a (H39, Fig. 5C), and an isolated cluster is located in the medial half of subgroup c of M A O (H39, Fig. 5D). Numerous HRP-positive somata are present in rostral D A O close to the rostral terminus of the entire complex (H39, Fig. 5A). A t slightly more caudal levels a small contingent of labeled neurons is located in the dorsomedial extreme of the rostral one-half of D A O m (H39, Figs. 5B-C and 6A). D A O 1 is devoid of reactive cells throughout its rostrocaudal extent (H39, Fig. 5 B - D ) .

A relatively large implant (case H43), which involved central and medial parts of V P M L (Fig. 7 inset) and totally avoided D P M L , revealed H R P - l a b e l ing in specific areas of M A O , D A O , and PO (Fig. 7 A - E ) . Labeled somata are present in the rostral one-third of subgroup a of M A O (Fig. 7 C - E ) . HRP-positive cells are localized in the ventromedial one-half of caudal subgroup a at mid-olivary levels (Fig. 7E) while more rostrally they are restricted to dorsolateral portions (Fig. 7C and D). No labeled cells are found in the rostral pole of subgroup a or in the adjacent D M C C (Fig. 7B). Isolated H R P - l a b e l ing is confined to the dorsomedial extremes of V L P O (Fig. 7D and E) and D L P O (Fig. 7B and D). Ventrolateral V L P O and D L P O and the contiguous lateral bend are devoid of HRP-positive neurons (Fig. 7 B - E ) . M o d e r a t e numbers of labeled somata are found in the rostral half of D A O m (Fig. 7 C - E ) . Caudally these reactive cells are scattered through-

277 Out D A O m (Fig. 7 E ) w h i l e rostrally in D A O m, the

DISCUSSION

H R P - p o s i t i v e s o m a t a are c o n f i n e d to lateral aspects of the nucleus (Fig. 7C and D ) . R o s t r a l D A O

and

P O , in c o n t r a s t to all o t h e r cases are c o n s p i c u o u s l y d e v o i d of H R P - l a b e l i n g (Fig. 7 A and B).

Conformation of Tupaia inferiorolive T h e c o n f i g u r a t i o n of the I O of Tupaia is similar

to that d e s c r i b e d and illustrated for cat 44,49, rat19.22.36, 42,

Fig. 6. High power (A, C, D) and low power (B, E) dark-field photomicrographs illustrating the presence of reactive somata in IO following implant of HRP into central regions of caudal DPML (case H35, D and E) and rostral VPML (case H39, A-C). Numerous labeled somata are present in rostral VLPO and adjacent DAO (A). At more caudal levels labeled cells occupy subgroup a of MAO in addition to VLPO and DAO (B). A cluster of labeled somata is restricted to subgroup c of MAO just caudal to mid-olivary levels (C). Subgroup a of MAO contains labeled cells following an implant of DPML (D). At a level close to the rostral pole of the complex, HRPlabeled cells are located exclusively in DAO (E). Horseradish peroxidase method. Scale = 45/~m for A, 84/~m for B-E.

278

lal

i

-.

-

-

i

. . . .

Fig. 7. Tracings of IO in Tupaiain transverse section from rostral (A) to caudal (E) following the implant of HRP into medial portions of ventral paramedian lobule (case H43). HRP labeled cells are present in subgroup a of MAO and DAO m(C-E). Isolated reactive somata are located in DLPO (B, D) and VLPO (D-E). Note the lack of labeled cells in the rostral pole of the IO. Scale = 1.0 mm. rabbit 39, pig 6, marsupialsS, 35,34.52, prosimians20,45,53, higher primates4,9,16, 41 and man 40 (see Whitworth and Haines 53 for review). Although PO of Tupaia and other mammals consists of a D L P O and a V L P O connected by a lateral bend, Tupaia PO lacks the lamellar infoldings characteristic of monkey 4 and man 40. The DAOm of Tupaia (present study) and Galago 53 corresponds to the dm of marsupials 52, to the medial subgroup of D A O in Didelphis 34, and to the commashaped dorsomediai part of D A O in Macaca 4,9 and Saimiri 4j. The lateral subgroup of D A O in Didelphis 34, the dl of other marsupials 52 and the ventrolateral D A O of Macaca 4,9 and Saimiri41 are equivalent to D A O 1 of Galago 53 and Tupaia (present study). Subgroups of Tupaia M A O (i.e. a, b, c, d, and DMCC) appear to be homologous to those in other mammalian species 53. However, in contrast to rat 1922, cat 44,49, Galago 53, rhesus 4,9,16 and squirrel monkeys 4~, no homologue of the ventralateral outgrowth (VLO) could be identified in Tupaia (present study). Whether lateral and medial parts of subgroup c of Tupaia M A O (present study) are true subdivisions comparable to subnucleus C 1 and C of M A O in marsupials 34,s2 is unclear.

Olivocerebellar projections to Tupaia PML The occurrence and/or arrangement of zones in PML has been a point of controversy. Although Courville et al. 17 and Bishop et al. 3 denied the presence of efferent zones in PML, Dietrichs and Walberg ts, Voogd and Bigare 4s, and Haines and Patrick27 have identified 4-6 efferent zones in PML. A zonal organization of olivocerebellar fibers has also been reported for cat 1,12A3,21,30,50, opossum32,35, and macaque 9,10. Although Linauts and Martin 3z and Martin et ai. 35 did not specifically identify zones in opossum, the pattern of olivocerebellar projections they described clearly indicates that a zonal pattern, similar in its basic configuration to that seen in cat, is present in this mammal. The present data suggest the presence of olivocerebellar zones (El, C2, C 3 and D) in PML of Tupaia similar in their spatial arrangement to those reported in an earlier study of cerebellar corticonuclear fibers 27. Implants of H R P in lateral D P M L of Tupaia (present study) resulted in IO labeling similar to that reported in cat 12,13, opossum32, 35 and monkey t0. Following implant of H R P in lateral D P M L of Tupaia (present study), the labeling of cells in rostral subgroup a of M A O , D A O m and medial V L P O , subgroups of IO known to project to zones C2, C 3 and D (see ref. 11), is consistent with the spatial relationship of zones in this folium as determined in corticonuclear studies 1&27. Although occupying positions comparable to those in medial D A O of cat 12A3 and opossum 32 the labeled somata in DAOm of Tupaia were more restricted in their rostrocaudal extent. In agreement with a recent H R P study of olivocerebellar projections to rostrolateral PML of monkeytO, but in contrast to similar studies in cat 12, the present study of Tupaia revealed isolated labeled somata in DAO1. In opossum, labeled cells were found in subnuclei a and b of M A O following H R P injections of the superior folium of PML 32 while H R P injections of rostral PML in monkey I0 resulted in no labeling in M A O . The presence of labeled cells in the rostral half of M A O of cat following H R P injections into rostral PML 12,13 is also the case for Tupaia. In contrast to studies of cat, however, labeled cells were found more caudally in subgroup c of Tupaia M A O (present study). Retrograde transport of H R P from implants or in-

279 jections of rostral PML of Tupaia (present study), monkey ~0, cat ~2.~3 and opossum 32 resulted in labeling in DLPO and VLPO. Labeled cells were found in DLPO but not VLPO in monky lo and cat12 while in opossum they occupied the rostral two-thirds of DLPO and a few 'faintly positive' neurons were seen in VLPO 32. The HRP-positive somata found in VLPO of Tupaia (present study) were limited to its rostral parts and were not found in more caudal VLPO as reported in cat 13. The presence of labeled somata in the lateral bend of Tupaia PO (present study) following implants in PML has not been reported for other species. Implants of HRP into caudal DPML in Tupaia (present study) labeled somata in the rostral pole of DAO m and in rostral subgroup a of MAO; this indicates the presence of zones C 2 and C 3. Although Haines and Patrick 27 made no lesions in caudal DPML corresponding to our HRP-implant site, a lesion in central parts of rostral DPML resulted in degeneration in anterior (NIA) and posterior (NIP) interposed nuclei, an observation substantiating the presence of zones C 2 and C 3 in DPML. Examination of IO following HRP injections of central parts of rostral PML in cat revealed labeled cells in medial D A O and in rostral MAO 12,13, a point confirmed by our results on Tupaia. The HRP-implant site in lateral areas of rostral VPML of Tupaia (present study) closely approximates the location of lesions made in VPML of Tupaia in a study of its corticonuclear fibers 27. Following lesions of lateral VPML Haines and Patrick 27 reported preterminal degeneration in central NIP, lateral NIA, and in medial portions of the lateral cerebellar nucleus (NL); a projection pattern characteristic of zones C 2, C 3 and D, respectively. The presence of labeled cells in subgroup a of MAO, rostral DAOm and VLPO following implant of HRP into lateral VPML (present study) further supports this interpretation of zones in PML of Tupaia. In contrast, however, subsequent to HRP injections of lateral PML in rat TM, numerous labeled cells were found in DLPO and a few cells in cell groups b and c of caudal MAO. Injections of HRP into lateral parts of caudal PML in macaque resulted in labeling in medial DLPO and VLPO, and in medial D A O (comparable to Tupaia DAOm) indicating the presence of zones C 3, Di and D 2 in this species 10.

Following implants of HRP into central regions of VPML (present study) labeling was found in subgroups a and c of MAO, rostral D A O m rostromedial VLPO, and parts of the lateral bend of PO. Following lesions of the same relative region of Tupaia VPML 27 axonal debris is restricted to NIP with sparse amounts of degeneration in adjacent areas of NIA and 'possibly' NL. Both studies therefore support the concept that zones C 2 and C 3 are present in central VPML. Subsequent to HRP injections into central and caudal PML in monkey labeling was seen in DLPO, caudal VLPO, D A O and subgroup a of MAO lo indicating involvement of zones C 2, C l and/or C 3 and D. Labeled cells in subgroup a of monkey which project to central VPML 9 are located more caudally than those in subgroup a of Tupaia (present study) or the corresponding part of MAO of cat 12A3. In contrast to results of HRP studies in most other species, a focus of labeled cells was present in the lateral aspect of the lateral bend and no cells in DLPO were labeled after implant of central VPML of Tupaia (present study). In agreement with studies in rat ~4 but in contrast with studies in catt2.13 and monkey 10 implants of HRP in central VPML in Tupaia (present study) also resulted in labeling in subgroup c of MAO. In HRP studies of olivocerebellar projections to monkey PML, labeled cells were present in subgroup c (nucleus fl) of MAO only when the injection site involved adjacent portions of uvula lo. In Tupaia (present study) the implant site in case H39 was confined to PML with no invasion of adjacent uvula, therefore labeled cells in subgroup c of Tupaia MAO apparently represent a true projection to VPML. Brodal and Brodal 9 reported labeled cells in the lateral lamella (lateral bend of other authors) following an injection of PML which extended into the caudalmost folium of crus II and part of the dentate nucleus. In the present study of Tupaia, cells in the lateral bend were labeled following HRP implants limited to lateral VPML. The presence of HRP-labeling in subgroup a of MAO and DAOm, subsequent to implants of medial parts of rostral VPML (present study), corroborates evidence from a study of corticonuclear projections in Tupaia in which degeneration resulting from lesions in this area was distributed to caudomedial NIA and medial NIP representing zones C1 and C 2, respectively27. Zone C 3 appears to be absent in medial

280 V P M L of Tupaia as evidenced by the lack of H R P labeling in rostral D A O m (present study) and the lack of preterminal degeneration in dorsolateral N I A following lesion of medial V P M L 27. Subsequent to H R P injections of medial P M L of rat, the occurrence of reactive ceils in rostral D A O and rostral M A O also suggests the presence of zones C1 and C2 in medial PML in this species 19. A disparity of opinion exists in the literature concerning the presence of a zone B in PML. Fibers from caudolateral areas of contralateral D A O terminating in zone B of lobules I - V I I and in medial regions of V P M L have been r e p o r t e d 8,12,21,3°,47 suggesting the presence of zone B in medial PML. A l t h o u g h lesions of zone B of lobules I - V I I p r o d u c e degeneration in the ipsilateral vestibular complex2*-26,28,29, anterograde 27.51 and r e t r o g r a d e 48 studies of efferents of medial P M L revealed projections primarily into medial N I A . Corticonuclear projections from medial P M L to N I A and not to N M or the vestibular complex 27 and the lack of retrogradely labeled Purkinje cells in medial P M L following H R P injections into vestibular nucleiC5, suggest the most medial zone of P M L should be designated the C 1 zone. L a b e l e d cells in rostromedial D A O (present study) support the existence of a C1 zone in medial V P M L but the lack of labeled somata in caudolateral D A O suggests that in tree shrew, zone B is not r e p r e s e n t e d in medial VPML. It must be acknowledged, however, that neither H R P ABBREVIATIONS a b c d dao dao I daom dlpo

subgroup a of mao subgroup b of mao subgroup c of mao subgroup d of mao dorsal accessory olive dorsal accessory olive (lateralpart) dorsal accessory olive (medial part) dorsal lamella of principal olive

injections into P M L (present study) nor lesions of P M L 27, involved the medial extremes of VPML. The possibility that zone B is r e p r e s e n t e d in caudomedial V P M L in Tupaia (or in some other mammals) remains problematic and will require further investigation. The present data have shown a topographically organized projection to P M L from specific stlbgroups of the contralateral IO in Tupaia. In addition, the distribution patterns of labeled cells in IO following injections which collectively included most of P M L indicates a zonal a r r a n g e m e n t of olivocerebellar terminals in both D P M L and V P M L of Tupaia. The pattern of zones described here based on the distribution of olivocerebellar input is similar in its essential features to that previously described 27 in a study of cerebellar corticonuclear fibers in this species. ACKNOWLEDGEMENTS This research was s u p p o r t e d by U S P H S G r a n t NS 11327 from N I N C D S to D . E . H . The authors express their sincere appreciation to Ms. A n n e Carr, Mr. Jeff A t l e m u s and Mr. Garbis Kermian who provided histologic and photographic assistance. Ms. A n g e l a Pepin and Ms. Augustine C h a p m a n typed earlier versions of this manuscript and Ms. Sandra S. W a r n e r and Ms. D o n n a M. Borland typed the final version.

dmcc lr mao ml po py vlo vlpo XII

dorsomedial cell column lateral reticular nucleus medial accessory olive medial lemniscus principal olive pyramid ventrolateral outgrowth ventrallamella of principal olive facicles of hypoglossal nerve

REFERENCES 1 Armstrong, D. M., Harvey, R. J. and Schild, R. F., Topographical localization in olivocerebellar projection: an electrophysiological study of the cat, J. comp. Neurol., 154 (1974) 287-302. 2 Beyerl, B. D., Borges, L. F., Swearingen, B. and Sidman, R. L., Parasagittal organization of the olivocerebellar projections in the mouse, J. comp. Neurol., 209 (1981) 339-346. 3 Bishop, G. A., McCrea, R. A., Lighthas, J. W. and Kitai, S. T., An HRP and autoradiographic study of the projec-

tion from the cerebellar cortex to the nucleus interpositus anterior and nucleus interpositus posterior of the cat, J. comp. Neurol., 185 (1979) 735-756. 4 Bowman, J. P. and Sladek, J. R., Morphology of the inferior olivary complex of the Rhesus monkey (Macaca mulatta), J. comp. NeuroL, 152 (1973) 229-316. 5 Bowman, M. H. and King, J. S., The conformation, cytology and synaptology of the opossum inferior olivary nucleus, J. comp. Neurol., 148 (1973) 491-524. 6 Breazile, J. E., The cytoarchitecture of the brain stem of the domestic pig, J. comp. Neurol., 129 (1967) 169-188.

281 7 Brodal, A., Experimentelle Untersuchungen fiber die olivo-cerebellare Lokalization, Z. Ges. Neurol. Psychiat., 169 (1940) 1-153. 8 Brodal, A., Olivocerebellocortical projection in the cat as determined with the method of retrograde transport of horseradish peroxidase. 2. Topographical pattern in relation to the longitudinal subdivision of the cerebellum. In J. Courville, C. deMontigny, and Y. Lamarre (Eds.), The Inferior Olivary Nucleus. Anatomy and Physiology, Raven Press, New York, 1980, pp. 187-205. 9 Brodal, P. and Brodal, A., The olivocerebellar projection in the monkey. Experimental studies with the method of retrograde tracing of horseradish peroxidase, J. comp. Neurol., 201 (1981) 375-393. 10 Brodal, P. and Brodal, A., Further observations on the olivocerebellar projection in the monkey, Exp. Brain Res., 45 (1982) 71-83. 11 Brodal, A. and Kawamura, K., Olivocerebellar projection: a review, Advanc. Anat. Emb. Cell Biol., 64 (1980) 1-140. 12 Brodal, A. and Walberg, F., The olivocerebellar projection in the cat studied with the method of retrograde axonal transport of horseradish peroxidase. VI. The projections onto longitudinal zones of the paramedian lobule, J. comp. Neurol., 176 (1977) 281-294. 13 Brodal, A., Walberg, F. and Hoddevik, G. H., Olivocerebellar projection in the cat studied with the method of retrograde axonal transport of horseradish peroxidase. I. The projection to the paramedian lobule, J. comp. Neurol., 164 (1975) 449-470. 14 Brown, P. A., The inferior olivary connections to the cerebellum in the rat studied by retrograde axonal transport of horseradish peroxidase. Brain Res. Bull., 5 (1980) 267-275. 15 Courville, J. and Faraco-Cantin, F., Cerebellar corticonuclear projection demonstrated by the horseradish peroxidase method, Soc. Neurosci. Abstr., 2 (1976) 108. 16 Courville, J. and Otabe, S., The rubro-olivary projection in the Macaque: an experimental study with silver impregnation methods, J. comp. Neurol., 158 (1974) 479-494. 17 Courville, J., Diakiw, N. and Brodal, A., Cerebellar corticonuclear projection in the cat. The paramedian lobule. An experimental study with silver methods, Brain Research, 50 (1973) 25-45. 18 Dietricks, E. and Walberg, F., The cerebellar corticonuclear and nucleocortical projections in the cat as studied with anterograde and retrograde transport of horseradish peroxidase. I. The paramedian lobule, Anat. Embryol., 158 (1979) 13-39. 19 Furber, S. E. and Watson, C. R. R., Organization of the olivocerebellar projection in the rat. Brain Behav. EvoL, 22 (1983) 132-152. 20 Gerhard, L. and Olszewski, J. Medulla oblongata and pons, Primatologia, 2 (1969) 141-143. 21 Groenewegen, H. J., Voogd, J. and Freedman, S. L., The parasagittal zonation within the olivocerebellar projection. II. Climbing fiber distribution in the intermediate and hemispheric parts of the cat cerebellum, J. comp. Neurol., 183 (1979) 551-602. 22 Gwyn, D. G., Nicholson, G. P. and Flumerfelt, B. A., The inferior olivary nucleus of the rat: A light and electron microscopic study, J. comp. Neurol., 174 (1977) 489-520. 23 Haines, D. E., The cerebellum of Galago and Tupaia. I. Corpus cerebelli and flocculonodular lobe, Brain Behav. Evol., 2 (1969) 377-414. 24 Haines, D. E. Cerebellar cortical efferents of the posterior

lobe vermis in a prosimian primate (Galago) and the tree shrew (Tupaia), J. comp. Neurol., 163 (1975) 21-40. 25 Haines, D. E., Cerebellar corticovestibular fibers of the posterior lobe in a prosimian primate, the lesser bushbaby (Galago senegalensis), J. comp. Neurol., 160 (1975) 363-398. 26 Haines, D. E. Cerebellar corticonuclear and corticovestibular fibers of the anterior lobe vermis in a prosimian primate (Galago senegalensis), J. comp. Neurol., 170 (1976) 67-96. 27 Haines, D. E. and Patrick, G. W., Cerebellar corticonuclear fibers of the paramedian lobule of tree shrew (Tupaia glis) with comments on zones. J. comp. Neurol., 210 (1981) 99-119. 28 Haines, D. E. and Rubertone, J. A., Cerebellar corticonuclear fibers of the dorsal culminate lobule (anterior lobe - lobule V) in a prosimian primate, Galago senegalensis, J. comp. Neurol., 186 (1979) 321-342. 29 Haines, D. E., Patrick, G. W., and Satrulee, P., Organization of cerebellar corticonuclear fiber systems. In S. L. Palay and V. Chan-Palay (Eds.), The Cerebellum -- New Vistas, Springer-Verlag, New York, 1982. 30 Kawamura, K. and Hashikawa, T., Olivocerebellar projections in the cat studied by means of anterograde axonal transport of labeled amino acids as tracers, Neuroscience, 4 (1979) 1615-1635. 31 Kooy, F. H., The inferior olive in vertebrates, Folia Neurobiol., 10 (1917) 205-369. 32 Linauts, M. and Martin, G. F., The organization of olivocerebellar projections in the opossum, Didelphis virginiana, as revealed by the retrograde transport of horseradish peroxidase, J. comp. Neurol., 179 (1978) 355-382. 33 Luckett, W. P. (Ed.), Comparative Biology and Evolutionary Relationships of Tree Shrews, Plenum Press, New York, 1980, pp. 3-312. 34 Martin, G. F., Dom, R., King, J. S., Robards, M. and Watson, C. R. R. The inferior olivary nucleus of the opossum (Didelphis marsupialis virginiana), its organization and connections, J. comp. Neurol., 160 (1975) 507-534. 35 Martin, G. F., Bishop, G. A., Culberson, J. L., King, J. S., Laxson, C., Linauts, M. and Panneton, M., Spino-olivocerebellar circuits in the North American Opossum with notes on their development. In: S. L. Palay and V. Chan-Palay (Eds.), The Cerebellum -- New Vistas, Springer-Verlag, Berlin, 1982, pp. 162-188. 36 McGrane, M. K., Woodward, D. J., Eriksson, M. A., Burne, R. A. and Saint-Cyr, J. A., The inferior olivary complex in the rat: gross nuclear organization and topography of olivocerebellar projections, Soc. Neurosci. Abstr., 3 (1977) 59. 37 Mesulam, M.-M., Tetramethyl benzidine for horseradish peroxidase neurohistochemistry: a non-carcinogenic blue reaction-product with superior sensitivity for visualizing neural afferents and efferents, J. Histochern. Cytochem., 26 (1978) 106--117. 38 Mesulam, M.-M., Principles of horseradish peroxidase neurohistochemistry and their applications for tracing neural pathways - - axonal transport, enzyme histochemistry and light microscopic analysis. In M.-M. Mesulam (Ed.), Tracing Neural Connections with Horseradish Peroxidase, John Wiley, New York, 1982, pp. 1-151. 39 Mizuno, N., An experimental study of the spino-olivary fibers in the rabbit and the cat, J. comp. Neurol., 127 (1966) 267-291.

282 40 Olszewski, J. and Baxter, D., Cytoarchitecture o f the Human Brainstem, J. B. Lippincott, Philadelphia, 1954. 41 Rutherford, J. G. and Gwyn, D. G., A light and electron microscopic study of the inferior olivary nucleus of the squirrel monkey, Saimiri sciureus, J. comp. Neurol., 189 (1980) 127-155. 42 Shild, R. F., On the inferior olive of the albino rat, J. comp. Neurol., 140 (1970) 255-260. 43 Schroeder, D. M. and Jane, J. A., Projections of dorsal column nuclei and spinal cord to brain stem and thalamus in tree shrew, Tupaia glis, J. comp. Neurol., 142 (1971) 309-350. 44 Taber, E., The cytoarchitecture of the hrainstem of the cat, J. comp. Neurol., 116 (1961)27-69. 45 Tilney, F. The brain stem of Tarsius. A critical comparison with other primates, J. comp. Neurol., 43 (1927) 371-432. 46 Van Gilder, J. C. and O'Leary, J. L., Topical projection of the olivocerebellar system in the cat: an electrophysiological study, J. comp. Neurol., 140 (1970) 69-80. 47 Voogd, J. The olivocerebellar projection in the cat. In S. L. Palay and V. Chan-Palay (Eds.), The Cerebellum - - New Vistas, Springer-Verlag, New York, 1982.

48 Voogd, J. and Bigare, F., Topographical distribution of olivary and corticonuclear fibers in the cerebellum. A review. In J. Courville, C. deMontigny and Y. Lamarre (Eds.), The Inferior Olivary Nucleus. Anatomy and Physiology, Raven Press, New York, 1980, pp. 207-234. 49 Walberg, F., Descending connections to the inferior olive. An experimental study in the cat, J. comp. Neurol., 104 (1956) 77-173. 50 Walberg, F. and Brodal, A., The longitudinal zonal pattern in the paramedian lobule of the cat's cerebellum. An analysis based on a correlation of recent HRP data with results of studies with other methods, J. comp. Neurol., 187 (1979) 581-588. 51 Walberg, F. and Jansen, J., Cerebellar corticonuclear projection studied experimentally with silver impregnation methods, J. Hirnforsch., 6 (1964) 338-354. 52 Watson, C. R. R. and Herron, P., The inferior olivary complex of marsupials, J. comp. Neurol., 176 (1977) 527-537. 53 Whitworth, R. H. and Haines, D. E., The inferior olive of a prosimian primate Galago senegalensis. I: Conformation and spino-olivary projections, J. comp. Neurol., 219 (1983) 215-227.