THE JOURNAL OF COMPARATIVE NEUROLOGY 316~221-237 (1992)

Trigeminocerebellar Projections to the Posterior Lobe in the Cat, as Studied by Anterograde Transport of Wheat Germ Agglutinin-HorseradishPeroxidase MICHIKO IKEDA AND MATSUO MATSUSHITA Department of Anatomy, Kansai Medical University, Moriguchi, Osaka 570 (M.1.); Department of Anatomy, Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, Ibaraki 305 (M.M.), Japan

ABSTRACT Cerebellar projections of the nucleus interpolaris and oralis of the spinal trigeminal nucleus were studied in the cat by anterograde transport of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP).Injections of WGA-HRP into these nuclei labeled many mossy fiber terminals mainly ipsilaterally in the rostral folium of lobule IX (IXa or IXa + b), the simple lobule, the anterior part (sublobuleA) of the paramedian lobule and the posterior part of crus II. Labeled terminals were also seen in the anterior lobe, lobules VI and VII, the anterior part of crus I, and the paraflocculus dorsalis. Projection fields in the horizontal plane of lobules were reconstructed from a series of transverse sections through each folium of lobule IX, the paramedian lobule, and the posterior part of crus I1 on the ipsilateral side. In sublobule IXa + b, labeled terminals were distributed in five longitudinal areas extending along the apicobasal axis of the sublobule. These five areas were located in the apical two-thirds of the ipsilateral half of the sublobule. Labeled terminals were distributed in five longitudinal areas in sublobule A (the rostral part) of the paramedian lobule. In the posterior part of crus 11, four aggregations of labeled terminals were present in cross sections through a lobule. They were distributed in the apicobasal extent of the lobules. The present study indicates that the projection fields of trigerninocerebellar fibers are longitudinally arranged along the apicobasal axis of the cerebellar lobules. Key words: brainstem, spinal trigeminal nucleus, cerebellar cortex, mossy fiber

With the aid of the retrograde transport of horseradish peroxidase (HRP), cerebellar projections from the trigeminal sensory nuclei have been studied in great detail in various mammals: the cat (Faull, '77; Ikeda, '79; Somana et al., '80; Gould, '80; Jasmin and Courville, '87a), rat (Faull, '77; Watson and Switzer, '78; Kimoto et al., '78; Huerta et al., '83; Falls et al., '85; Mantle-St. John and Tracey, '87; Phelan and Falls, '911, mouse (Steindler, '771, sheep (Saigal et al., '80),and tree shrew (Patrick and Haines, '82). In the cat, the secondary trigeminocerebellar fibers originate from the nucleus interpolaris (Vi),the caudal part of the nucleus oralis (Vo), and the principal sensory nucleus (Vp) of the trigeminal nerve. They project mainly t o lobule IX, the simple lobule and its neighboring portion of crus I, the anterior part of the paramedian lobule and the posterior part of crus I1 of the hemisphere. Studies by the anterograde degeneration (Carpenter and Hanna, '61) and the anterograde labeling techniques (Courville and FaracoCantin, '78) have demonstrated that the trigeminocerebelO

1992 WILEY-LISS, INC.

lar fibers terminate as mossy fibers. No systematic study, however, has been done to reveal the projection areas in the cerebellar cortex by anterograde tracing techniques. Anatomical studies in mammals have shown that mossy fibers originating from the spinal cord and from precerebellar nuclei in the brain stem terminate in sagittal zones of the cerebellar cortex: spinocerebellar (Hazlett et al., '69; Voogd, '64, '69; Voogd et al., '69; Rossum, '69; Watson et al., '76; Matsushita, '88; Matsushita and Ikeda, '87; Matsushita and Tanami, '87; Matsushita and Yaginuma, '89; Yaginuma and Matsushita, '86, '87, '89; Xu and Grant, '901, cuneocerebellar (Voogd, '69; Voogd et al., '69; Gerrits et al., '85; Massopust et al., '85; Jasmin and Courville, '87b), reticulocerebellar (Kunzle, '75; Chan-Palay et al., '771, and

Accepted October 7,1991. Address reprint requests to Dr. Michiko Ikeda, Dept. of Anatomy, Kansai Medical University, Moriguchi, Osaka 570, Japan.

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vestibulocerebellar fibers (Epema et al., '85; Matsushita and Wang, '87). We have reconstructed the projection fields of spinocerebellar fibers (Matsushita, '88; Matsushita and Ikeda, '87; Matsushita and Tanami, '87; Matsushita and Yaginuma, '89; Yaginuma and Matsushita, '86, '87, '89) and vestibulocerebellar fibers (Matsushita and Wang, '87) in the horizontal plane of each lobule (a plane parallel to the apicobasal axis of the lobules). This procedure makes it possible to project the extent of terminal distribution upon the surface of each cerebellar lobule and to delineate the geography of functional differentiation within a cerebellar lobule. The results indicate that the spinocerebellar and the vestibulocerebellar fibers terminate in longitudinal areas extending in the apicobasal direction, and that the projection pattern is specific to the cells of origin of the fiber tracts. A similar longitudinal organization was recognized for the trigeminocerebellar projections to lobule IX, the paramedian lobule and crus I1 in the cat (Ikeda and Matsushita, '89) and in lobules VIII and JX in the pigeon (Arends and Zeigler, '89). The present study was undertaken by the anterograde wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP)technique, to systematically examine the extent of trigeminocerebellar projections in the cerebellar cortex, and the projection pattern in the horizontal plane of each lobule.

MATERIALS AND METHODS The present observations were based on the results obtained from a total of 11 successful cases in adult cats, weighing 2.0-4.0 kg. The animals were anesthetized by intraperitoneal administration of sodium pentobarbital (50 mg/kg. bw.) and placed in a stereotaxic apparatus. A glass micropipette (30-50 pm tip diameter) was filled with 2% wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP: Sigma or Toyobo), which was dissolved in a Krebs-Ringer solution containing 100 mM KCl. After the foramen magnum was opened, the pipette was stereotaxically inserted toward the spinal trigeminal nucleus (Vsp) at the angle of 45-55 degrees in the posteroanterior direction.

Abbreviations Cr. I Cr. I1 cs

D EC I0 L

LR M NIA NIP NL NM PFLd Ph PML rs

SL ST vc Vl vo

VII XI1 12N

crus I cms I1 caudal side descending vestibular nucleus external cuneate nucleus inferior olivary complex lateral vestibular nucleus lateral reticular nucleus medial vestibular nucleus anterior interpositus nucleus posterior interpositus nucleus lateral cerebellar nucleus medial cerebella nucleus paraflocculus dorsalis prepositus hypoglossi nucleus paramedian lobule rostral side simple lobule spinal tract of the trigeminal nerve nucleus caudalis of the spinal trigeminal nucleus nucleus interpolaris of the spinal trigeminal nucleus nucleus oralis of the spinal trigeminal nucleus facial nerve nucleus hypoglossal nerve nucleus hypoglossal nerve

The tracer was injected iontophoretically (2-5 pA DC for 20-30 min.) into the Vsp at planes 10.0-11.0 of Berman's atlas ('68). The pipette was left in place for at least 10 minutes after each injection. In four cases, 0.2 pl of a 30% HRP solution was injected into each of sublobules a-d of lobule IX. After 2-3 days, the animals were deeply reanesthetized and perfused transcardially with 500 ml of saline, followed by 3 liters of a fixative containing 2.5%glutaraldehyde and 0.5%paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Cerebella and brainstems, including injection sites, were removed and postfixed for 3-5 h in 2.5% glutaraldehyde buffered with 0.1 M phosphate. After washing in the buffer, they were kept in a 10% sucrose-phosphate buffer mixture for 1-2 days. Brainstems containing the injection sites were cut serially at 90 pm in the transverse plane on a freezing microtome. These sections were processed for histochemistry of HRP according to the method of Adams ('81) to evaluate the extent of diffusion of the injected WGA-HRP. Cerebella were cut serially into 100-pm-thick sagittal sections (three cases). In eight cases, each folium of lobule IX, the paramedian lobule and the posterior part of crus 11,was separated from the corpus medullare of the cerebellum, and cut serially into 100-pm-thickcross (transverse) sections in the apicobasal direction (the plane perpendicular to the longitudinal axis of the lobules). Some sections were cut at 50 pm. The rest of the cerebella, including the anterior lobe, was cut serially in the sagittal plane. The cerebellar sections were processed for HRP histochemistry according to the method of Mesulam et al. ('801, with a reaction medium containing 0.1% cobalt chloride. The sections were mounted on gelatin-chrom alum coated slides. The distribution of anterogradely labeled mossy fiber terminals in the horizontal plane of the lobules (the plane parallel to the apicobasal axis) was reconstructed from a complete series of cross sections through the lobules. Cross sections were cut in the plane vertical to the apicobasal axis of the lobules (Fig. 6A). The locations of labeled terminals in these cross sections were plotted serially on a sheet of graph paper with the aid of a drawing tube attached to the microscope and were arranged from apical to basal (Fig. 6B). The apicobasal distance was determined by the thickness of sections (100 Fm) and the magnification of plotting was adjusted to 20 times. For reconstruction, the midline in sublobule IX was determined by the constriction of the subcortical white matter in cross sections. Since there is no landmark for reconstruction in the paramedian lobule and crus 11, the midpoint between the medial and the lateral extremities of the folia of the paramedian lobule and the midpoint between the dorsal and the ventral extremities of the folia of crus I1 were used as the fixed point. Details are given in previous papers (see Fig. 5 of Yaginuma and Matsushita, '89; Fig. 3 of Matsushita, '911. The lobules of the cerebellar cortex were named according to Larsell ('53). The anterior, posterior, and copular parts of the paramedian lobule were named sublobules A, B, and C , respectively (Matsushita and Ikeda, '80; Kassel et al., '84). In addition, the anterior part of crus I1 and the anterior and the posterior folia of the posterior part of crus TI were named sublobules A, B, and C, respectively, after Kassel et al. ('84). The rostral and the caudal folia of the paramedian lobule, and the lateral and the medial folia of the posterior part of crus I1 were numbered 1 and 2, respectively (e.g., A1 and A2).When these folia divided further, each small folium was named sublobules A2a and A2b.

TRIGEMINOCEREBELLAR PROJECTIONS

A

223

B

C

D

Fig. 1. Diagrams showing the extent of diffusion of the injected WGA-HRP (hatched) in the spinal trigeminal nucleus. Transverse sections through the brainstem are arranged in caudorostral sequence (levels 1-7).

M.IKEDA AND M. MATSUSHITA

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RESULTS ‘l’o reveal the distribution of secondary trigeminocerebellar fibers in the cerebellar cortex, we injected WGA-HRP at the middle to the rostral levels of the Vi and the caudal level of the Vo, which are known to project to the simple lobule, crus 11, and sublobule A (the anterior part) of the paramed i m lobule (Ikeda, ’79; Matsushita e t al., ’82). Prior to this, we examined the location of trigeniinocerebellar neurons projecting to lobule IX following HRP injections into lobule IX. A number of retrogradely labeled neurons were found in the dorsomedial and the ventrolateral parts of the Vi, the lateral part of the caudal Vo, and the ventral part of the Vp. Their distribution resembled that of neurons projecting to the anterior part of the paramedian lobule and overlapped the distribution of neurons projecting to the simple lobule and the posterior part of crus I1 (Ikeda, ’79; Matsushita et al., ’82).

Distribution in the sagittal plane The distribution of labeled mossy fiber terminals in the s a s t t a l plane ofthe cerebellar cortex was examined in three cases (Nos. 322, 330, and 339). In case No. 322 (Figs. lA, 2A) t,he iiijected WGA-IlRP spread from the rostral Vi to the caudal Vo (levels 5-7), whereas in case No. 330 (Fig. 1A) the injection was confined to the caudal Vi (levels 2-3). In case No. 339 (not illustrated) the injected WGA-HKP spread more extensively from the middle Vi to the caudal Vo (levels 3-6). There was no difference in the distribution of labeled mossy fiber terminals between these three cases. The projections to the cerebellar cortex were predominantly ipsilateral, with a small contingent to similar regions on the contralateral side, except for the paraflocculus dorsalis. The results on the side ipsilateral to the injection will be described below. In sections through the medial and the lateral parts of the vermis, few labeled terminals were seen in lobules I-IV and lobule VIII (Fig. 3A,B). On the other hand, many labeled terminals were seen in the apical two-thirds of lobules V-VII, particularly in sections through the lateral part of the vermis (Fig. 3B). Labeled terminals were numerous in the lateral part of sublobule IXa + b (the rostra1 folium), where they were concentrated to its apical two-thirds (Fig. 3B). No labeled terminals were seen in lobule X. In the intermediate-lateral regions of the hemisphere, many labeled terminals were seen in all folia of the lateral part of lobule VI and the simple lobule (Fig. 4A,B), and in the neighboring folium of crus I (Fig. 4C). In the paramedian lobule (Fig. 4 B ) , labeled terminals were observed only in its sublobule A (the anterior part). Labeled terminals were numerous in sublobule C of crus I1 (Fig. 4C), the neighboring folium of sublobule B of crus I1 (Fig. 4D), and the paraflocculus dorsalis (Fig. 4D). However, no labeled terminals were seen in sublobule A of crus 11, the paraflocC U ~ U Sventralis and the flocculus.

Distribution in the transverse and the horizontal plane The distribution of labeled mossy fiber terminals was examined in the transverse plane of lobule IX, the paramed i m lobule, and crus 11. A two-dimensional distribution on the rostral and the caudal sides of lobule IX (Figs. 6, 7) and the paramedian lobule (Figs. 8-10), and on the medial and the lateral sides of crus I1 (Figs. 11, 12) was reconstructed

Fig. 2. Photomicrographs showing t h e site and the cxtcnt of‘ WGA-HRP injections into the spinal trigeminal nucleus. A: nucleus oralis at lcvcl 6 (case No. 3221; B-D: nuclcus interpolaris at lcvcl 5 (B. case No. 3481, level 4 !C: case No. 3651 and level 5 !D: case No :150).X 5.5.

from a series of transverse sections through these suhlohules. The collections of labeled terminals seen in the transverse plane were named “groups” and were numbered as in previous studies (Yaginuma and Matsushita, ’86, ’87).The distributions of corresponding groups of labeled terminals, reconstructed in the horizontal plane, were named “areas” of projection. The groups and areas appearing in lobule IX, the paramedian lobule, and crus 11, were numbered independently because those appearing in different lobules could not be correlated. Lobule I X . The distribution of labeled terminals in lobule IX was analyzed in cases with injection into the Vi (levels 3-5) to the caudal Vo below level 6 (Figs. lI3,2B: case No. 348) and with injection into the dorsomedial part o f t h e Vi at levels 2-4 (Fig. 1C: case No. 353). The majority of labeled mossy fiber terminals were seen ipsilaterally in sublobule IXa (or IXa + b ) . A few labeled terminals were seen in sublobule IXb, but none were seen in sublobules IXc and IXd. I n a transverse section through sublobule IXa + b a t 2.9 mm from the apex (Figs. 5 , 6A), five groups of laheled terminals appeared on the side ipsilateral to the injection: group 1 located in the midline region, group 2 located within 0.7 mm of the midline, group 3 located between 0.8 min arid 1.1 mm lateral to the midline, group 4 located between 1.3 mm and 1.5 mm, and group 5 located between 1.7 mm and 1.8 mm. In this section, these groups were

TRIGEMINOCEREBELLAR PROJECTIONS

Fig. 3. Diagrams showing the distribution of labeled mossy fiber terminals (dots) in sagittal sections through the medial (A)and the lateral parts (B)of the vermis on the side ipsilateral to the injection into the rostra1 Vi to the caudal Vo (case No. 322). Distributions in two

225

sections are superposed in B. Figures on the upper left corner of each section denote the distance from the midline. Each dot represents one terminal. Arrowheads indicate the fissura prima.

226

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M. IKEDA AND M. MATSUSHITA (No.322)

C

v

Cr. I

-

8.0 1

PML

B (No.330)

SL

5.9

D 11.

Cr.11-B

Fig. 4. Diagrams showing the distribution of labeled mossy fiber terminals (dots! in sagittal sections through the intermediate-lateral regions of the hemisphere (A-D) on the side ipsilateral to the injection. A case No. 322 with injection into the rostral Vi to the caudal Vo; B-D:

case No. 330 with injection into the caudal Vi. Figures on the upper left corner of each section denote the distance from the midline. Each dot represents one terminal. Arrowhead indicates the fissura prima.

Fig 5. Photomicrograph shou-ingthe distribution of labeled termi-

labcled termin;ilh wrrrspondir1g to those

in

Fig 6A are seen i n t h e

nals 111 a c m h s stvtiirn through suhlohiilr IXa at 3.75 m m from the apex !cast So 3531.on t h r side ipsilateral to the injection. Groups 2 5 of

,qanule cell layer on the rostral side !rs).Arrows indicate the midline.

relatively well-demarcated. However, the labeled terminals were diffusely or sparsely distributed, depending on sections, in the corresponding locations. This made the outlines of projection areas less distinct in reconstruction (Fig. 6 8 ) .Nevertheless, the corresponding groups could be traced in a series of' transverse sections. Reconstruction of the distribution on the rostral side shows that five longtudinal areas extend in the apical two-thirds of sublobule IXa + b (Fig. 6Bi: area 1 extending along the midline, area 2 extending from 1.0 mni to 5.3 mm from the apex, area 3 extending from 1.4 inn1 to 5.5 mm, area 4 extending from 1.4 min to 4 . 5 mm, and area 5 Extending from 1.8 mm to 5.5 mm. Labeled terminals were sparse in area 4 at the depth of3.4 to 4.6 mm. Areas 2 and 3 curved medially at the depth of3.5 mm to 5.0 mm from the apex, as the width of the sublobule narrowed. In transverse sections, the distribution of labeled terminals on the caudal side of sublobule IXa + b was compared with that on the rostral side described above. However, identification of the five groups was not always possible because the labeled terminals were sparse and were not closely grouped (Fig. 6Aj. In reconstruction (Fig. 7J, the labeled terminals appeared t o he distributed in thc longitudinal direction and to form five less-demarcated and discontinuous ;ireus. No clear-cut pattern of distribution could be recognized on the contralateral side (Figs. 6H, 7 ) . Pararirrdian lo6ult. The distribution of labeled terminals in the paramedian lobule was examined in cases with injections a t middle levels (levels 3-5) of the Vi (Figs. l C , 2C: casc No. 365) and with deposits only in the dorsomedial part of the Vi at levels 2-4 (Fig. 1C: case No. 3533. Labeled terminals were seen predominantly in the rostral sublobule A, except for a siiiall number in the adjacent folium of sublobule U. No labeled terminals were seen in the other folia of sublobule B or in sublobule C. Lnbclcd terminals were distributed more sparsely and grouped Iws densely than in suhlohule IXa + b. However, they appeared to gather in five places when traced in a

series of transverse sections, a n d could he placed tentatively into groups of five. In a transverse section through sublobule A2a a t 0.9 mm from the apex (Fig. 8A: case No. 365), groups 1 and 2 were located in the medial half of sublobule A2a; group 3 was located near the midline region and; groups 4 and 5 were located in the lateral half of the sublobule. Although these five groups were not clearly demarcated, they appeared to be arranged along the apicobasal axis of the lobules (Fig. XB,, as seen on the caudal side of sublobule A2a. Similar areas appeared to exist on the rostral side, but they were not clear-cut and discontinuous (Fig. 8 C ) . On the other hand, labeled terminals were more numerous and gathered a t five sites in a transverse section through sublobule A2b a t 0.9 mm from the apex (Fig. 9A). In reconstruction, they were distributed in five areas extending longtudinally on the rostral side of sublobule A2b (Fig. 913). On the caudal side, labeled terminals were densely distributed (Fig. 9C), so that the projection areas could not be distinguished from each other. The distribution in sublobule A1 was examined in a different case (Fig. 10: case No. 3531. In a transverse section a t 1.1 mm from the apex (Fig. 10Aj, five small groups o f labeled terminals were arranged from medial to lateral, in locations similar to those in sublobule A2a (cf. Figs. 8A, 9A): groups 1 and 2 in the medial half, and groups %5 in the lateral half. In reconstruction, the five areas were identified, based on the density and the distribution (the distance from the midline) of labeled terminals. On the rostral side (Fig. lOB), labeled terminals were concentrated in the apical half of the sublobule, and appeared t o be distributed mainly in two ill-defined areas corresponding to areas 4 and 5 of sublobule A2a (Figs. 8B, 9B). On the caudal side of sublobule A1 (Fig. 10A,C), only group 4 could be followed in a series of transverse sections. Cnis I I . Projections to the posterior part of crus I1 were examined in a case with injection mainly into the rostral half of the Vi (levels 3-51, involving part of the spinal tract of the trigerninal nerve (Figs. I D , 2D: case No. 350). Since

Bar

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TRIGEMINOCEREBELLAR PROJECTIONS (No. 348) IXa+b ( c a u d a l s i d e )

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Basa I Fig. 7. Diagram showing the distribution of labeled terminals (dots) on the caudal side of sublobule IXa + b (case No. 348). Side ipsilateral to the injection is to the right. Five projection areas extend apicobasally.

the folia of crus I1 curved in the anterior direction, the plane of section was not precisely perpendicular to the longitudinal axis of the folia. Sublobule C of crus I1 consisted of two folia named sublobules C2 and C1 (Fig. 1l:insets). Both sublobules C2 and C1 were further divided into the medial and the lateral parts, respectively, by a

Fig. 6 . Diagrams showing the distribution of labeled terminals (dots) in sublobule IXa + b (case No. 348). Inset shows the plane of section in A. A Distribution in a cross section at 2.9 mm from the apex. Broken line indicates the border between the granule cell layer and the molecular layer. B: Reconstruction of the distribution on the rostral (rs) and the caudal sides (cs) of sublobule 1% + b. Side ipsilateral to the injection is to the right. For reconstruction, sublobule IXa b was cut serially in the plane perpendicular to the apicobasal axis of the sublobule. Then, the location of labeled terminals on the rostral side of the sublobule was plotted serially in the apicobasal sequence. Numbers 1-5 are groups of labeled terminals (A) and projection areas (B). Horizontal and vertical scales (mm) indicate the mediolateral distance from the midline of the sublobule (vertical line through zero) and the apicobasal distance (zero at the apex of the sublobule), respectively, in Figs. 6-10.

+

shallow groove on their apical surfaces. They were named the C2-medial and the C2-lateral (Fig. 11A), and the C1-medial and the C1-lateral (Fig. 12A). The midpoint between the dorsal and the ventral extremities of sublobules was regarded as the midline of the folia of crus 11. In a transverse section at 0.5 mm from the apex of the C2-medial (Fig. llA), four groups of labeled terminals were identified on its medial side: groups 1and 2 located between 0.5 mm and 1.0 mm and within 0.5 mm, respectively, in the dorsal half, and groups 3 and 4 located within 0.5 mm and between 0.5 mm and 1.0 mm, respectively, in the ventral half. These four groups were seen in many transverse sections, but they could not be followed as well-demarcated longitudinal areas (Fig. 11B). Corresponding aggregations of labeled terminals could be seen only in the basal part of the lobule. On the lateral side of the C2-lateral, labeled terminals were sparsely distributed over its horizontal plane (not illustrated). In transverse sections through sublobule C1, labeled terminals were not clearly aggregated (Fig. 12A). On the medial side of the C1-medial, labeled terminals were diffusely distributed (not illustrated), whereas they were

230

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Fig. 8. Diagrams showing the distribution of labeled terminals in sublobule A2a of the paramedian lobule (case No. 365).Inset: Plane of section in A. A: Distribution in transverse sections at 0.8 mm from the apex of sublobule A1 and at 0.9 mm from the apex of sublobule A2a. B,C: Reconstruction of the distribution on the caudal (B) and the

rostral sides (C) of sublobule A2a. Numbers 1-5 are groups of labeled terminals (A) and projection areas (B and C). The midpoint between the medial and the lateral extremities of the sublobule was chosen as the fixed point for reconstruction.

densely distributed in the entire apicobasal extent on the lateral side of the C1-lateral (Fig. 12B). On the medial side of sublobule B2 (Fig. 12C), labeled terminals were concentrated in its basal half, whereas on the lateral side of sublobule B2 they were sparse (not shown). No labeled terminals were found in sublobule A.

was suggested that trigeminocerebellar projections are primarily ipsilateral and that they extend over the simple lobule, the neighboring portion of crus I, the anterior part of the paramedian lobule, the posterior part of crus I1 (Ikeda, '79; Somana et al., '801, and the rostral folia of lobule IX (Somana et al., '80). This was confirmed by anterograde tracing from the Vi and the Vo with radioactive amino acids (Jasmin and Courville, '87a) and with WGAHRP in the present study. Projections from the Vi and the Vo have also been reported to the vermal region, although there is some discrepancy. It is agreed that projections to the lateral part of lobules V-VII are abundant. However, projections to lobules I-IV and VIII were few, as suggested by the previous retrograde HRP study (Ikeda, '791, whereas Jasmin and Courville ('87a) found relatively dense projections to lobules 11-IV, in keeping with the findings of

DISCUSSION The extent of the projection fields Studies with the retrograde HRP technique showed that, in the cat, trigeminocerebellar projection neurons are located in the Vi, the caudal part of the Vo, and the ventral division of the Vp (Ikeda, '79; Somana et al., '80; Gould, '80; Matsushita et al., '82). Based on the relationship between the injection sites and the retrograde labeling pattern, it

231

TRICEMINOCEREBELLAR PROJECTIONS

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A

C

(~0.365)

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Lateral

Fig. 9. Diagrams showing the distribution of labeled terminals (dots) in sublobule A2b of the paramedian lobule (case No. 365). A Distribution in a transverse section at 0.9 mm from the apex. B,C: Reconstruction of the distribution on the rostral (B) and the caudal

sides (C) of sublobule A2b. Numbers 1-5 are groups of labeled terminals (A) and projection areas (B). They were identified by comparison with those seen in sublobule A2a (Fig. 8A-C).

retrograde labeling (Somana et al., '80). No projections were observed to lobule X, paraflocculus ventralis, and flocculus. From the present and previous findings, it can be concluded that secondary trigeminocerebellar fibers project primarily to the rostral folia of lobule IX, sublobule A (the anterior part) of the paramedian lobule, sublobule C (the posterior part) of crus 11,and the dorsal paraflocculus. They also project to lobules V-VII, the simple lobule and the anterior part of crus I (Fig. 3). However, the projections to the rostral lobules of the anterior lobe are far less significant than those to the above regions.

attempt has been made in the cat as to the trigeminocerebellar projections. The identified terminal fields can also be utilized as a map for electrophysiological exploration. The injection of the tracer was confined to the Vi or the Vo without diffusion to the adjacent reticular formation and did not cover the whole rostrocaudal extent of the Vi and the Vo, which give rise to trigeminocerebellar projections. Therefore, the projection patterns composed of loose aggregation of terminals may be due to the extent of the injections. Figure 13 illustrates a fundamental pattern of trigeminocerebellar projections observed in the present study. In the transverse plane of the most rostral folium of lobule IX, labeled mossy fiber terminals derived from the Vsp were aggregated in five groups (Figs. 6A, 13). These groups were continuous along the apicobasal axis of the sublobule and formed five longitudinal areas in the horizontal plane (Figs. 6B, 7). Since lobule IX is composed of zones A1 (or A1 + A2),A3 and C2 (Voogd and BigarB, '80; Gerrits et al., '851, the five longitudinal areas are considered to be

The projection pattern The primary objective of the present study was to reconstruct the distribution of mossy fiber terminals in the horizontal plane of each cerebellar lobule. This procedure is necessary to determine exactly the terminal fields of trigeminocerebellar fibers in a given lobule and the degree of overlap with various d e r e n t s , as discussed below. No such

232

M. IKEDA AND M. MATSUSHITA

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(B)and the caudal sides (C) of sublobule A l . Numbers 1-5 are groups of labeled terminals (A) and projection areas (B,C). They were identified by comparison with those seen in sublobule M a (Fig.SA-C).

contained in these zones. However, no correlation could be made between each of the projection areas and Voogd's cortical zones because there is no landmark for comparison. Based on the location and the extent of the cortical zones illustrated by Voogd and Bigare ('80) and Gerrits et al. ('85),areas 1and 2 are considered to be included in zone A1 (or zones A1 + A2)and area 5 in zone C2. It is, however, not

certain whether these areas are confined within the respective zones or extend into zone A3, and in which cortical zones areas 3 and 4 are contained. The distribution was bilateral, but predominantly ipsilateral. Retrograde and anterograde experiments with WGA-HRP in the pigeon (Arends and Zeigler, '89) showed that secondary trigeminocerebellar fibers originate only from subnucleus oralis of

233

TRIGEMINOCEREBELLAR PROJECTIONS

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Fig. 11. Diagrams showing the distribution of labeled terminals in the posterior part of crus I1 (case No. 350). Upper inset shows the sublobules of crus I1 and lower inset, the plane of cross section through sublobule C2 (A) and the medial side of sublobule C2 (B). A Distribution in cross-sections at 0.5 mm from the apex of the medial side (C2-Med) and at 1.6 mrn from the apex of the lateral side (C2-Lat) of

sublobule C2. B: Reconstruction of the distribution on the medial side of sublobule C2. Numbers 1-4 are groups of labeled terminals (A) and projection areas (B). Horizontal and vertical scales (mrnl indicate the dorsoventral distance from the midline in the sublobule (vertical line through zero) and the apicobasal distance (zero at the apex of the sublobule),respectively.

the spinal nucleus and that they terminate on the ventral side of lobule VIII and on the dorsal side of sublobule IXa. The projection areas reconstructed on the horizontal surface of these lobules were arranged longitudinally along the apicobasal axis of the lobules. The projections were dense in the medial region (zone A), flanked laterally by longitudinal areas.

Sublobule A (the anterior part) of the paramedian lobule was composed of two to three folia (sublobules A l , A2a and A2b). In the transverse plane, the labeled terminals tended to gather in five tentative sites across the sublobules (Figs. 8-10, 13). The terminals in these sites are distributed along the apicobasal axis. Projections were most abundant in sublobule A2b. and less abundant in sublobules A2a and

234

M. IKEDA AND M. MATSUSHITA

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Basal Dorsal Fig. 12. Diagrams showing the distribution of labeled terminals in the posterior part of crus I1 (case No. 350). Inset shows the plane of cross section through sublobule C1 (A) and the lateral side of sublobule C 1 (B) and the medial side of sublobule B2 (C). A: Distribution in a

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cross-section at 1.8mm from the apexof sublobule C1. B,C:Reconstruction of the distribution on the lateral side of sublobule C1 (B) and on the medial side of sublobule B2 (0.

235

TRIGEMINOCEREBELLAR PROJECTIONS Paramedian I

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Fig. 13. Summary diagrams of the main projection fields of the trigeminocerebellar fibers in the posterior lobe. The trigeminocerebellar fibers arising from the nuclei interpolaris and oralis project to sublobule IXa + b, sublobule A (consisting of sublobules Al, A2a, and A2b) of the paramedian lobule and sublobules B and C of the posterior part of crus 11. Spinocerebellar fibers project mainly to lobule VIII and sublobules B and C of the paramedian lobule. The trigeminocerebellar

projections overlap the spinocerebellar projections in sublobule A of the paramedian lobule (not shown). In the transverse plane, terminals of trigeminocerebellar fibers are aggregated in five groups in sublobule 1% + b and sublobules A2a and A2b of the paramedian lobule and in four groups in sublobule C2 of crus 11. These groups of terminals can be followed in the apicobasal extent of these sublobules and form projection areas.

A l . Five longitudinal areas were identified in sublobule A2a (Fig. 8B,C) and on the rostral side of sublobule A2b (Fig. 9B). Areas 2 4 were considered to be the main projection areas in the sublobules of the paramedian lobule. In the most rostral folium, sublobule A1 (Fig. 101, labeled terminals were also arranged longitudinally in the apical half of the apicobasal extent (Fig. 10B), as seen in sublobule A2a. Area 4 contained most abundant terminals in sublobule A1 (Fig. 10B,C). Similar longitudinal areas appeared to exist on the caudal side of sublobule A2b, but they could not be distinguished (Fig. 9C). The anterior part of the paramedian lobule consists of zones C1, C2, C3, and D, which are arranged from medial to lateral (Groenewegen et al., '79; Kawamura and Hashikawa, '79). Since the width of these zones is not known, it was not possible to determine the exact correlation between these cortical zones and the projection areas of the present study. Comparison with the illustrations by Groenewegen et al. ('79) and Kawamura and Hashikawa ('79) suggests that, as for sublobule A2, area 1,located in the medialmost part of the sublobule, lies in zone C1; area 5, located in the lateralmost part, lies in zone D. However, it is not clear whether groups or areas 2-4 occupying the middle part of the sublobule are situated in zone C2, extending medially to zone C1, and laterally to zone D. In cross sectional plane of the posterior part of crus 11, labeled terminals were diffusely distributed, but they appeared to be collected in four sites on the medial side of the most medial folium of crus 11, sublobule C2 (Fig. 11B, 13). These collections were continuous in the apicobasal extent,

but the longitudinal areas were not clearly separated. Crus I1 contains all cortical zones, zones C1, C2, C3, D1, and D2 (Groenewegen et al., '79; Kawamura and Hashikawa, '79; Voogd and Bigare, '80). The relationship between four terminal groups and the cortical zones remains unknown.

Comparison of the terminal field of cerebellar afferents Available data are limited about the projection pattern of other cerebellar afferents to the lobules where the trigeminocerebellar fibers terminate. Jasmin and Courville ('87a) noted that cuneo-cerebellar afferents, labeled with tritiated amino acids from the external cuneate nucleus and the main cuneate nucleus, converge with afferents from the Vsp in the rostral folium of lobule IX. Since the cuneocerebellar fibers terminate in the medial half of lobule IX, corresponding to zone A1 or zones A1 + A2 (Gerrits et al., '851, the terminal fields appear t o overlap, in part, those of trigeminocerebellar fibers. Secondary vestibulocerebellar fibers were also shown to terminate in lobule IX in the monkey (Carleton and Carpenter, '831, cat (Magras and Voogd, '85; Sat0 et al., '891, and rabbit (Epema et al., '85; Thunnissen et al., '89). Since these fibers terminate mainly in sublobule IXd in the rabbit (Thunnissen et al., '89) or in the ventral lobule IX in the cat (Sato et al., '89), there may be little overlap of termination between vestibulo- and trigeminocerebellar fibers. On the other hand, the cerebellar projections from the pontine nuclei proper and the nucleus reticularis tegmenti pontis are extensive in the

236

M. IKEDA AND M. MATSUSHITA

dorsal lobule IX, the rostral folium (sublobule A) of the paramedian lobule, and the posterior part of crus I1 (Kawamura and Hashikawa, '81). Although the projection pattern has not been studied, the extensiveness of projections to each of the above areas and the mediolateral extent of projections in lobule IX (Sato et al., '89) suggest the possibility of the overlap between pontocerebellar and trigeminocerebellar projections. The spinocerebellar system is composed of different tracts (Matsushita et al., '79). The projection fields of spinocerebellar fibers have been analyzed in great detail by means of the anterograde tracing technique (Matsushita, '88; Matsushita and Ikeda, '87; Matsushita and Tanami, '87; Matsushita and Yaginuma, '89; Yaginuma and Matsushita, '86, '87, '89). Although the projection areas are different, depending on the tracts (or cells of origin), the spinocerebellar system projects mainly to the anterior lobe, lobules VI and VIII, and sublobules B and C of the paramedian lobule (Fig. 13). Only small projections were present to lobule IX, the medial part of the simple lobule, sublobule C (the posterior part) of crus 11,and sublobule A of the paramedian lobule, which are the primary projection areas of trigeminocerebellar fibers. Thus, in the cat, the major projection areas of spinocerebellar fibers seem to be separate from those of trigeminocerebellar fibers (Fig. 13). Only a small number of fibers from the central cervical nucleus projected to lobule IX and the rostral part of sublobule A of the paramedian lobule (Matsushita and Tanami, '87). A significant number of the dorsal spinocerebellar tract fibers from the thoracic cord project to sublobule A of the paramedian lobule (Yaginuma and Matsushita, '87). Since the distribution of spinocerebellar fibers in lobule IX and the paramedian lobule was not reconstructed, the relationship with the four longitudinal areas of trigeminocerebellar fibers is not clear. Functional considerations. Following light tactile stimulation of the orofacial region in the cat, multi-unit activity was recorded from the granule cell layer in the paramedian lobule and crus I1 (Kassel et al., '841, corresponding to the trigeminocerebellar projections demonstrated electrophysiologically in the rat (Woolston et al., '81).Micromapping of the receptive field in these lobules (cat: Kassel et al., '84; rat: Shambes et al.,'78a; Bower and Woolston, '831, lobule IX (rat: Joseph et al., '78), and the simple lobule (rat: Shambes et al., '78b), showed that a particular small facial area can be represented in separate patches. These patches of receptive fields form a mosaic-like disjunctive representation of the body surface. Since the recording was made from the apical surface of the paramedian lobule and crus I1 (Kassel et al., '84), they can not readily be correlated with the longitudinal projection areas shown in the present study. The longitudinal areas apparently represent the overall projection of the Vsp, and may be constituted of small modality-specific receptive fields (patches) identifiable by natural stimulation of a discrete body area.

ACKNOWLEDGMENTS We thank Fumio Yamashita for his technical assistance in the histological work and Tetsuji Yamamoto and MasaAki Teranishi for their photographic assistance. This work was supported in part by a grant to M. Matsushita for University of Tsukuba Project Research.

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237 the Cerebellum. An Experimental Investigation of the Anterior Lobe, the Simple Lobule and the Caudal Vermis in the Rabbit. Assen: Van Gorcum, pp. 1-169. Saigal, R.P., A.N. Karamanlidis, J. Voogd, 0. Mangana, and H. Michaloudi (1980) Secondary trigeminocerebellar projections in sheep studied with the horseradish peroxidase tracing method. J. Comp. Neurol. 189:537553. Sato, Y., K.-I. Kanda, K. Ikarashi, and T. Kawasaki (1989) Differential mossy fiber projections to the dorsal and ventral uvula in the cat. J. Comp. Neurol. 279.149-164. Shambes, G.M., J.M. Gibson, and W. Welker (1978a) Fractured somatotopy in granule cell tactile areas of rat cerebellar hemispheres revealed by micromapping. Brain Behav. Evol. 15:94-140. Shambes, G.M., D.H. Beerman, and W. Welker (1978b) Multiple tactile areas in cerebellar cortex: Another patchy cutaneous projection to granule cell columns in rats. Brain Res. 157:123-128. Somana, R., N. Kotchabhakdi, and F. Walberg (1980) Cerebellar af€erents from the trigeminal sensory nuclei in the cat. Exp. Brain Res. 3857-64. Steindler, D.A. (1977) Trigemino-cerebellar projections in normal and reeler mutant mice. Neurosci. Lett. 6:293-300. Thunnissen, I.E.,A.H. Epema, and N.M. Gerrits (1989) Secondary vestibulocerebellar mossy fiber projection to the caudal vermis in the rabbit. J. Comp. Neurol. 290t262-277. Voogd, J. (1964) The Cerebellum of the Cat. Structure and Fibre Connexions. Assen: Van Gorcum, pp. 1-215. Voogd, J. (1969) The importance of fiber connections in the comparative anatomy of the mammalian cerebellum. In R. Lhnas (ed): Neurobiology of Cerebellar Evolution and Development. Chicago: American Medical Association Education and Research Foundation, pp. 493-514. Voogd, J., and F. Bigare (1980) Topographical distribution of olivary and cortico nuclear fibers in the cerehellum: A review. In J. Courville, C. de Montigny, and Y. Lamarre (eds): The Inferior Olivary Nucleus: Anatomy and Physiology. New York: Raven Press, pp. 207-234. Voogd, J., G. Broere, and J. van Rossum (1969) The rnedio-lateral distribution of the spinocerebellar projection in the anterior lobe and the simple lobule in the cat and a comparison with some other afferent fibre systems. Psychiat. Neurol. Neurochir. 72: 137-151. Watson, C.R.R., and R.C. Switzer, 111 (1978) Trigeminal projections to cerebellar tactile areas in the rat-origin mainly from n. interpolaris and n. principalis. Neurosci. Lett. 10:77-82. Watson, C.R.R., A. Broomhead, and M.-C. Holst (1976) Spinocerebellar tracts in the brush-tailed possum, Trichosurus uulpecula. Brain. Behav. Evol. 13:142-153. Woolston, D.C., J. Kassel, and J.M. Gibson (1981) Trigeminocerebellar mossy fiber branching to granule cell layer patches in the rat cerebellum. Brain Res. 209:255-269. Xu, Q . ,and G. Grant (1990) The projection of spinocerebellar neurons from the sacrococcygeal region of'the spinal cord in the cat. An experimental study using anterograde transport of WGA-HRP and degeneration. Arch. Ital. Biol. 128:209-228. Yaginuma, H., and M. Matsushita (1986) Spinocerebellar projection fields in the horizontal plane of lobules of the cerebellar anterior lobe in the c a t An anterograde wheat germ agglutinin-horseradish peroxidase study. Brain Res. 365:345-349. Yaginuma, H., and M. Matsushita (1987) Spinocerebellar projections from the thoracic cord in the cat, as studied by anterograde transport of wheat germ agglutinin-horseradish peroxidase. J. Comp. Neurol. 258:1-27. Yaginuma, H., and M. Matsushita (1989) Spinocerebellar projections from the upper lumbar segments in the cat, as studied by anterograde transport of wheat germ agglutinin-horseradish peroxidase. J. Comp. Neurol. 281:298-319.