afferent and efferent connections of the oculomotor cerebellar vermis in the macaque monkey

THE JOURNAL OF COMPARATIVE NEUROLOGY 265~224-241(1987)

Afferent and Efferent Connections of the Oculomotor Cerebellar Vermis in the

Macaque Monkey

JINZO YAMADA AND HIROHARU NODA Visual Science Department, School o f Optometry, Indiana University,

Bloomington, Indiana 47405

ABSTRACT Saccadic eye movements were evoked with weak currents applied to a

circumscribed vermal area. The area was confined to lobule VII in the majority of the monkeys and coincided with the distribution of saccade- related neural activity. We defined this area as the oculomotor vermis and studied its anatomical connections with wheat germ-agglutinin conjugated horseradish peroxidase (WGA/HRP) and HRP.

When injected HRP was confined to the oculomotor vermis, most labeled Purkinje axons terminated ipsilaterally in an ellipsoidal region in the mediocaudal aspect of the fastigial nucleus. Retrogradely labeled cells were found in two relatively circumscribed regions in the fastigial nucleus: one group was in the lateral half of the ellipsoidal terminal region and the other group was in a spherical region near the lateral margin of the nucleus.

Following the injection of HRP into the oculomotor vermis, the largest population of retrogradely labeled neurons was found in the nucleus reticu- lark tegmenti pontis. Labeled cells were located only in the medial and dorsolateral portions of the nucleus. The cell aggregates in the dorsoIatera1 portion merged with densely labeled cells of the processus tegmentosus lateralis. The second largest population of labeled cells was found in the pontine nuclei. Approximately 28% of the labeled pontine cells aggregated in the paramedian pontine nucleus, whereas the other labeled pontine cells were widely distributed in the dorsal part of the pontine peduncular nucleus and the dorsolateral pontine nucleus. Labeled cells were scattered also in the pontine raphe, the paramedian pontine reticular formation, and the interfascicular nucleus at the rostra1 level of the hypoglossal nucleus. Fewer labeled cells were discovered in the vestibular nuclear complex and the prepositus hypoglossi. In the inferior olivary nucleus, labeled cells were located in the subnucleus b of the medial accessory nucleus.

Key words: fastigial nucleus, HRP study, eye movements, nucleus rcticularis tegmenti pontis, pontine nuclei

Previous experiments have shown that saccadic eye movements (saccades) are evoked by stimulation of the posterior cerebellar vermis (Ron and Robinson, '73; Keller et al., '83; McElligott and Keller, '84). In these studies, except for five stimulus tracks made in lobule VII by Ron and Robinson ('731, microstimulation was applied primarily t o vermal lobules V and VI. It was commonly observed that currents necessary to evoke saccades from lobules V and VI always exceeded 100 PA, unless they were applied during the saccades (Keller et al., '83) or applied to the deep white

matter near the fastigial nucleus (McElligott and Keller, '84). Recently, however, we discovered that saccades could be evoked with much smaller currents from a circum-

Accepted June 3, 1987. Address reprint requests to Dr. Hiroharu Noda, School of Optorn-

etry, Indiana University, 800 E. Atwater, Bloomington, IN 47405. J. Yamada is now at the Department of Anatomy, Nippon Medi-

cal School, Sendagi, Bunkyo-ku, Tokyo, Japan.

0 1987 ALAN R. LISS, INC.

CONNECTIONS OF THE OCULOMOTOR VERMIS 225

scribed region located more posteriorly than the area pre- viously described. Neurophysiological experiments conducted on twelve macaque monkeys revealed that the ocu- Iomotor vermis, characterized by a low threshold (typically < 10 /LA) and by a presence of vigorous saccade-related background activity, was confined to lobule VII in seven monkeys. This region extended into lobule VIc in the remaining five monkeys. The anterior border of the region in lobule VI varied from monkey to monkey. However, it always ended in the folia (lobule VIc) that constitute the anterior wall of the posterior superior fissure and never included the folia (lobule VIa, b) that make up the posterior wall of the primary fissure (Fujikado and Noda, '87; Noda and Fujikado, '87a). The low threshold region coincided with the portion of the posterior vermis from which saccade- related Purkinje-cell activity was recorded in monkeys (Kase et al., '80).

By demonstrating that the presence of intact Purkinje cells was essential for evoking saccades, we have shown that the saccades elicited by microstimulation of the oculomotor vermis were caused by orthodromic impulses con- veyed through the Purkinje axons. When the Purkinje cells were destroyed by topical injection of kainic acid, the ver- ma1 microstimulation no longer evoked saccades despite the fact that afferent fibers were intact as evidenced by the presence of saccade-related mossy fiber activity. The ab- sence of evoked saccades during stimulation of the white matter indicated that antidromic activation of the afferent fibers was not the mechanism subserving the vermal oculomotor responses (Noda and Fujikado, '87b). We have also shown that a transient excitation of the Purkinje cells by the microstimulation caused a n inhibition in the fastigial neurons and that y-aminobutyric acid (GABG) was respon- sible for the synaptic mechanism. When a small dose of bicuculline, a potential blocker of GABA, was injected in the fastigial nucleus, the saccades evoked by vermal stimulations were suppressed for several hours (Noda et al., '85). These neurophysiological data suggested that the impulses from the cerebellar vermis were transmitted to the brainstem oculomotor circuitries by way of the fastigial nucleus.

According to Clarke and Horsley ('05), cerebellar cortico- fugal fibers end in the nearest nuclear mass beneath their region of origin. This notion is supported by Jansen and Brodal ('40, '42) who concluded that in rabbits, cats, and monkeys each of three paired longitudinal zones of cortex projected ipsilaterally to correspondingly situated cerebellar nuclei. Thus, a bilateral vermal zone projects to the fastigial nucleus; a paravermal or intermediate zone projects to the nucleus interpositus; and a lateral zone projects to the dentate nucleus. In a recent study on the monkey, Tolbert et al. ('78) found anterogradely labeled Purkinje axons in the fastigial nucleus after an HRP injection in the midline cerebellar cortex of lobule VI and VII.

With regard to the afferent fibers to the cerebellar vermis, a number of studies of the pontocerebellar projection have been performed in cats by injecting HRP in the posterior lobule (Shinnar et al., '73; Batini et al., '78; Hoddevik, '78; Gould, '80) or by injecting tritiated amino acid into various parts of the pons (Kawamura and Hashikawa, '81). These experimental data indicate precise but complex patterns of convergence and divergence in the projections from various parts of the brainstem to different lobules of the vermis.

The information about the pathways in primates avail- able from retrograde degeneration experiments (Sunder- land, '40; Lafleur et al., '74) is scanty. In a systematic HRP study of the pontocerebellar projection in monkeys, the injection was placed in the middle of lobule VIILA, but the staining involved lobule VII as well (Brodal, '79). It has been found that lobules VII-VIIIA receive afferents mainly from two distinct cell groups in the pontine nuclei, one located dorsomedially and the other dorsolaterally. Frank- furter et al. ('77) injected HELP into lobule VII of the squirrel monkey and found that the subnucleus b of the medial accessory nucleus was the only source of olivary afferent to lobule VII. Moreover, two other precerebellar relay nuclei, the dorsolateral pontine nucleus (DL) and the nucleus reticularis tegmenti pontis (NRTP), were also found to project substantially upon lobule VII.

In the present study, we tried to provide the anatomical basis for: (1) pathways for the impulses from the oculomotor vermis, and (2) sources of the afferent fibres that convey visual and oculomotor information necessary for eye movement control by the cerebellum.

MATERIALS AND METHODS Afferent and efferent connections of lobule VII of the

cerebellar vermis in monkeys were studied by using the technique of retro- and anterograde transport of WGA/HRP and HRP. These studies were conducted in three adolescent pigtailed monkeys (Macaca nerneslrina), 4-5 kg in body weight. Data from three other monkeys, which were used primarily for neurophysiological experiments, are also included. The neuroanatomical tracer was injected in alert monkeys after identifying the oculomotor vermis neuro- physiologically.

Surgical preparations Following an initial training period, each monkey was

intubated under Ketalar sedation and was deeply anesthetized with a gas mixture of N20-Oa plus a varying amount of methoxyflurane. A search coil for measuring eye position was implanted on one eye, by using the method described by Judge et al. ('80). A stainless steel chamber was implanted over the occipital lobe for later insertion of microelectrodes and injection cannulae. The chamber was placed in a trephined hole; its outer circumference reached the superior nuchal line posteriorly. When a hydraulic micro- drive was mounted, the microelectrode inclined 5 posteriorly from the stereotaxic vertical and the tip was directed to a point 15-18 mm posterior to the Horsley-Frankfurt stereotaxic zero. Two transverse tubes were placed on the skull and embedded in dental arcylic cement for stabilizing the head during the experiment.

Mapping procedures Mapping by single-unit recording of the posterior vermis

was carried out by testing throughout the depth of the track. Tungsten microelectrodes with resistance 0.5-2 MQ at 200 Hz and insulated with isonel 31 were introduced into the cerebellum through the chamber. Extracellular action potentials were led through an FET preamplifier (Dagan 2400) with a band pass of 35-10K Hz and were displayed on an oscilloscope. By recording neuronal activity, we identified the cerebellar layers. The characteristic discharge patterns of various vermal neurons during eye movements (Kase et al., '80) helped to identify the position of an elec-

226

trode during the experiment. Complex spikes from Purkinje cell activity characterized the Purkinje cell layer. The presence of complex spikes in a relatively quiet background activity was a sign that the tip of the microelectrode was in the molecular layer. The distinction between the granular layer and the white matter was difficult unless granular cell activity was identified. Both structures were characterized by vigorous saccade-related background activity.

Mapping of the thresholds for evoking saccadic eye movements by microstimulation was carried out by using the same electrode and switching between the amplifier and stimulator channels of the Dagan 2400 preamplifier. By means of repetitive pulses (0.2 ms negative pulses at 600 Hz) for 20 ms, the minimum current necessary for evoking at least seven saccades out of ten stimulations was determined. The current was applied via a homemade constant current isolation unit. Over a period of 2-3 weeks, approximately 20-30 electrode penetrations were made before the tracer injection.

WGA/HRP and HRP injection After the mapping of thresholds for evoking saccades was

completed, tracks for HRP injection were selected in each monkey. Prior to the injection, the depth of the lowest threshold for evoking saccades was confirmed by remap- ping the selected track with a tungsten microelectrode. The electrode was guided by a 22-gauge cannula down to 2-3 mm below the tentorium; this guide tube was retained in the brain during the entire injection procedure. When the lowest threshold site was confirmed, the microelectrode was replaced with a 33-gauge cannula for HRP injection. Be- cause the length and diameter of the injection cannula were identical to those of the microelectrode, it was possible to place the tip of the cannula within 300 pm of the previous location of the microelectrode. A mixture containing 2% WGALHRP and 5% HRP in a 0.1 M phosphate buffer containing 0.2 M NaCl solution was injected in the selected vermal site. In monkey Jo (see Fig. ZA), two tracks in the right half of lobule VII were selected and each site was injected hydraulically with 0.5 ~1 of the mixture. In monkey Rh (see Fig. 2B), 1 p1 of the mixture was injected in one right vermal track. The neuroanatomical tracer was injected with a 10-pl Hamilton microsyringe driven by a Pe- tipump (Model 1150, Harvard apparatus) in several aliquots during a 20-30-minute period. Both monkeys survived 72 hours. In monkey An (see Fig. 2C), the mixture was injected electrophoretically through a micropipette made of 450 pm (OD.) tubing. The inner diameter at the tip was 10-15 pm. The tracer was injected into two vermal sites selected from each side after identifying their function neurophysiologi- cally. With the same micropipette, the saccade-related background activity was recorded and saccades were evoked with current less than 5 pA. For the WGMHFtP and HRP injection, 5 pA DC was applied for seven seconds at 14 second-intervals for a total period of 10 minutes with a High Voltage Precision Current Source (Model CS-3, Midgard Electronics). This monkey survived 48 hours.

Histological processing After 72 and 48 hours, respectively, the monkeys were

deeply anesthetized with N ~ 0 - 0 ~ plus the maximum evap- oration rate of methoxyflurane and were perfused transcar- dially by fluid delivered under controlled pressure through a cannula inserted into the ascending aorta. Four liters of physiological saline solution were introduced, followed by

J. YAMADA AND H. NODA

three liters of fixative solution containing 1.25% glutaral- dehyde and 1% paraformaldehyde in a 0.1 M phosphate buffer (pW 7.4). Following perfusion, the brains were ex- posed, blocked in the stereotaxic plane, and removed into a 0.1 M phosphate buffer with 30% sucrose. After the excess tissue was trimmed, the blocked brains were placed in a sucrose buffer and held in a refrigerator overnight. The following day they were sectioned into 80-pm parasagittal sections on a freezing microtome and collected in compart- mentalized trays. The serial sections of the brains were segregated into three groups, putting every third section in a group. The first group was treated with TMB (Mesulam, '78). The second group was treated with TMB and counter- stained with neutral red. The third group was incubated with DAB (Graham and Karnovsky, '66) and stained with cresyl violet.

A substantial part of the study involved an electrophys- iological identification of the function of the injection sites and a parallel examination of Nissl-stained sections from an additional three monkeys. In the final week of experiments on these three monkeys, the recording and stimulation were conducted with stainless-steel microelectrodes (made of Elgiloy orthodontic wires) to identify electrode tracks histologically. A 3V cathodal potential was applied through the electrode for 2-3 seconds. The brains were perfused with a physiological saline solution, followed by 10% formalin and aqueous K4Fe(CN)6. Parasagittal sections (60-pm thick) were made for two monkeys, and frontal sections (60-pm thick) were made for the remaining monkey; all sections were stained with cresyl violet. When iron reacted with K4Fe(CN),, it was found as a characteristic blue reaction spot of approximately 150-pm diameter against the Nissl-stained background.

Analysis of data Each section was examined with both low and high mag-

nification lenses using bright-field illumination. In addition, selected slides were also examined with dark-field illumination. The location of cell bodies labeled with HRP was carefully mapped onto large drawings with the aid of a drawing tube at magnifications of 100-25OX. Only cell bodies with a complete nucleus were mapped by focusing the microscope at different levels of the 80-pm-thick section. The number of labeled neurons was then evaluated by counting cells on the camera lucida drawings made from TMB reacted sections. Labeled neurons were classified into three groups: large, intermediate, and small. The appear- ance of a cell was, however, strongly influenced by configu- ration of the dendrites. Because we did not measure the size in every cell, the classification was somewhat arbitrary and did not necessarily reflect the actual sizes of cell bodies. Examples of large neurons are seen in the paramedian pontine reticular formation (PPRF) and in the processus tegmentosus lateralis (PL) (see Fig. 9D,E). The long and short axes of the cell body were measured for randomly selected neurons from high magnification drawings. The long and short axes of large cells were 35.5 f 9.3 and 18.6 5.0 pm, respectively. We decided to count the majority of multipolar neurons in the medial portion of the NRTP as intermediate-size cells (see Fig. 9A). The long and short axes were 23.1 k 6.0 and 13.6 k 3.9 pm, respectively. The great majority of pontine neurons (see Fig. 9C, GI and of inferior olivary neurons (see Fig. 9F) were considered as small cells. The long and short axes of small cells were 17.7 3.4 and 9.2 & 2.1 pm, respectively. Because the cells


classification was based mainly on the first impression of the cell body, an error rate of approximately 10% was noted when the cells were recounted.

The boundary of each nucleus in the brainstem was carefully marked onto the drawings of reference parasagittal sections made from every third section, which was stained with cresyl violet after incubation with DAB, The area of each nucleus was measured by tracing the boundary marked on the camera lucida drawings, using a Zeiss Video- plan image analysis system.

RESULTS Injection sites

Being a thin, complexly folded structure, the cerebellar cortex proved to be a difficult target when we attempted to confine an HRP injection to the cortical layers or to fill a lobule completely with HRP. In this study, therefore, the center of the injection was placed in the f h i t e matter of lobule VII, identified electrophysiologically by low thresholds for evoking saccades and by characteristic saccade- related background activity. Figure 1 shows an injection site found in two consecutive sections, one treated with the DAB and one with the TMB method. It is clear that differences in the histochemical procedure influenced the appar- ent size of the injection site. In the TMB section, dense deposits of reaction product are seen within the granular layer. The diffusion in this layer is more important than that in the white matter because the HRP uptake is more active at the axonal terminals than along the myelinated

BC: DBC: DL: DN: DPy: FN: ICP IP: LL: L t MAO: MCP ML: MLF: MRF: NRTP n.VI: PD: PL: PMPN: PPRF: Pre: Pr.F. FT: Py: Ra: Rt: S?T: VeI VeL: VeM VeSpr: VeSu: VI: VII:

Ahbreuiations

brachium conjunctivum decussation of brachium conjunctivum dorsolateral nucleus of pontine &ray dentate nucleus decussation of pyramidal tract fastigial nucleus inferior cerebellar peduncle interposed nuclei lateral lemniscus left side medial accessory nucleus of inferior olive medial cerebellar peduncle medial lemniscus medial longitudinal fasciculus midbrain reticular formation nucleus reticularis tegmenti pontis rootlets of abducens nerve dorsal part of pontine peduncular nucleus processus tegmentosus lateralis paramedian pontine nucleus paramedian pontine reticular formation nucleus prepositus hypoglossi primary fissure principal sensory nucleus of trigeminal netve pyramidal tract pontine raphe right side spinal tract nucleus of trigeminal nerve inferior vestibular nucleus lateral vestibular nucleus medial vestibular nucleus supravestibular nucleus superior vestibular nucleus lobule VI of cerebellar vermis lobule VII of cerebellar vermis

Fig. 1. Comparison of tha HRP injection site in the oculomotor vermis of monkey Jo as revealed in two conaecutive sections, each processed with a DAB and TMB method.

228 J. YAMADA AM, H. NODA

00 prn

An

I t I60 p m u 5 mm

Fig. 2. Locations of injection sites in lobule VII in monkeys Jo (A), Rh (s), and An (C). The injection sites are indicated with solid black and the extent of diffusion. on tho basis of DAB-reacted sections, is shaded. A bilateral injection was made in An. Unilateral large injection was made in Rh in which the diffusion invaded the paravermal zone on the right side. The diffusion covered both sides but was confined to the vermis in Jo and An.


fibers. Nevertheless, the center of the injection in both DAB and TMB sections was in the white matter of the down- stream from folia VIIAb, VIIAc, VIIB of Larsell’s nomencla- ture (’53). HRP diffusion also extended into folium VIIAa.

The injections into the oculomotor vermis of the three monkeys are summarized in Figure 2. The midsagittal plane was determined by identifying the middle section between the bilateral fastigial nuclei. In all three monkeys, the identified midsagittal section coincided with that identified by the characteristic brainstem midsagittal structure in which fibers ran dorsolaterally and numerous blood ves- sels perforated in the ventrodorsal direction. Prior to section, the borders between the vermis and the hemispheres had been marked with India ink, which served as an indi- cation of the paravermal zone.

The two injections in Jo (Fig. 2A) were confined to lobule VII of the right vermis: one was in folium VIIBa, 1.3 mm right; the other was in the white matter of lobule VII, 0.6 mm right. HRP diffusion from the two injection sites par- tially overlapped and formed a horizontally elongated ellipsoid. It extended from approximately l mm left to the right boundary of the vermis. The injection in Rh (Fig. ZB) was large but still confined within lobule VII, centering at 1 mm right. HRP diffusion extended from 0.6 mm left to the right paravermal zone. The injections in An (Fig. 2C) were located in sections 1.6 mrn left and 0.6 mm right. In both injections, HRP diffusion was confined within 0.4 mm of the injection sites. The vermes of Jo and An were straight and the right and left halves were symmetrical. The posterior vermis of monkey Rh was slightly curved and shifted to the left.

Anterograde labeling of terminal fields in the fastigial nucleus

Injection of the oculomotor vermis with WGlVHRP and HRP resulted in terminal fields located ipsilaterally in the

mediocaudal aspect of the fastigial nucleus. The labeIing of the terminal fields observed in section 800 l m right in J o is shown in Figure 3. The labeled Purkinje axons that arose in lobule VII descended mainly in the ventral half of the white matter and terminated in an ellipsoidal region of the fastigial nucleus. The ellipsoidal area containing labeled terminal fields started from the dorsocaudal end and wedged ventrorostrally toward the bottom of the nucleus. The major axis of the terminal area was directed to the fastigium of the fourth ventricle.

A small number of anterogradely labeled terminal fields was scattered outside the ellipsoidal region as well. Fine clusters of plexuses could be found in the region vental to the ellipsoidal region, in the vicinity of the fastigium, and in an area anterior to the spherical region. Although a few labeled fibers could be traced to the y group and the lateral vestibular nucleus, no labeled terminal fields could be seen in these nuclei. The labeled fibers were so few that we were not able to determine their destination.

Retrograde labeling of fastigial neurons An important and consistent finding was that the distri-

bution of retrogradely labeled fastigial neurons did not coincide with that of anterogradely labeled terminals and preterminals of Purkinje axons. Retrogradely labeled fastigial neurons were confined almost exclusively either in the lateral part of the ellipsoidal region or in the spherical region. The medial portion of the ellipsoidal region, where the highest density of labeled terminals was discovered, was always free of retrogradely labeled fastigial neurons (Fig. 4). In contrast with the massive HRP-labeled terminals of Purkinje axons, the number o f retrogradely labeled fastigial neurons was surprisingly small. The retrogradely labeled cells were intermingled with the labeled terminals in the lateral half of the ellipsoidal region where labeled synaptic terminals were less numerous than in the medial

Fig. 3. Distribution of anterogradely labeled terminal fields in the right fastigial nucleus following WGAMRP and HRP injection into lobule VII (indicated by an arrow). Pr.F., primary fissure.

230 J. YAMADA AND H. NODA

Fig. 4. Camera lucida drawing of the ellipsoidal region of the mediocaudal fastigial nucleus where dense aggregates of labeled terminal fields were found. Note that there was no retrogradely labeled fastigial neuron in this region.

half. The labeled cells were of various sizes, but large cells were more prevalent in the caudodorsal part of the region (Fig. 5A).

In addition to the ellipsoidal terminal region, there was a cluster of retrogradely labeled fastigial neurons at the lateral border of the nucleus. These neurons were found in a spherical region that was relatively well isolated from the other parts of the fastigial nucleus. When the HRP diffusion was confined to the vermis, there was no anterograde labeling in the spherical region (Fig. 5B).

In parasagittal Nissl-stained sections (monkey Nn), the identification of the cell group that occupied the ellipsoidal region was relatively simple (Fig. 6Ca). Large cells dominated the dorsal part and small cells characterized the vental part. In frontal Nissl-stained sections (monkey Sd, Fig. 6A,B), the dorsal part of the ellipsoidal terminal area was characterized by dorsoventrally oriented fusiform cells (Fig. 6Aa). It i s likely, therefore, that the axons of Purkinje cells terminate in large flattened multipolar cells in the dorsal part and in small and medium-size cells in the vental part of the region. The spherical region was clearly sepa- rated by fibers in both parasagittal and frontal sections and contained small to large cells (Fig. 6A, B, Db). There were many multiform large cells, but the majority of labeled cells were small (Fig. 5B). It is important to note that anterogradely labeled terminal fields appeared in the spherical region only when HRP diffusion invaded the paravermal zone.

Projections to and from the paravermal zone When HRP diffusion included the paravermal zone (mon-

key Rh), the distribution of labeled terminals was slightly different,. Labeled terminals increased in the medial part of the fastigial nucleus. Labeled terminals also appeared in the spherical region. However, the number of retrogradely labeled cells did not increase significantly in either the ellipsoidal or the spherical regions. Numerous terminal fields appeared outside these regions, particularly in the

ventral portion of the fastigial nucleus. Labeled fibers increased in the white matter lateral to the fastigial nucleus and terminated in the marginal zones of the anterior and posterior interposed nuclei. The cores of these nuclei were, however, free of terminal fields. Labeled fibers were further traced t o the vestibular nuclear complex. Some labeled fibers were found also in the basal interstitial nucleus &an- ger, '85), but there were no labeled terminal fields. Only a few retrogradely labeled cells were discovered in this nucleus. There were some retrogradely labeled cells in the dentate nucleus (DN). However, there were neither labeled axons nor terminals in this nucleus. Labeled axons that penetrated the y group reached all four vestibular nuclei. Although they were not numerous, labeled terminals were discovered in all vestibular nuclei. Retrogradely labeled cells in the vestibular nuclei were of intermediate size, but were significantly less numerous when compared with the other brainstem nuclei.

Retrograde labeling of brainstem neurons The distribution of retrogradely labeled cells in the brain-

stem was consistent in the two monkeys (Jo and An) in which the HRP diffusion was confined to the oculomotor vermis. Except for an additional reciprocal connection of the paravermal zone with the other deep cerebellar nuclei and the vestibular complex, the results of the third monkey (Rh) were in agreement with those of Jo and An. Unless otherwise specified, the following descriptions are based on the findings from monkey Jo.

A striking feature of the brainstem projection to the oculomotor vermis was that a large portion of the afferent fibers arose from the paramedian brainstem structure. Ap- proximately 55% of retrogradely labeled cells were found within 1 mm of the midline; the other aproximate half was widely distributed laterally to 1 mm of the midline.

Figure 7 shows a portion of a camera lucida drawing made from the left 160-pm parasagittal section (TMB reacted) of monkey Jo. Dense aggregates of labeled cells were


/ I m m

B \

\ \

/-------- \

\

Lt 1920 p m

Fig. 5. A. Camera lucida drawing of the lnteral part of the ellipsoidal terminal zone where retrogradely labeled fastigial neurons were found. B. Camera lucidn drawing of a cluster of retrogradely labeled fastigial neurons; there were no labeled terminals in this region.


'- I .

Lt 4 3 2 I 0 (mm)

-2 -3 - 4 -5 -6 (mm)

Lt 4 3 2 I 0 ( m m )

D

. .. , .. . .. 1 . L -. . . . :

. . 1. ' .

I .

-2 -3 - 4 -5 -6 ( m m )

Fig. 6. Camera lucida drawings of fastigial neurons in Nissl-stained sections. A and R. Frontal sections through the fastigial nucleus of monkey Sd, at the levels indicated in C. C and I). Parasagittal stactions of monkey Nn, at the levels indicated in A. a, ellipsoidal region. b, spherical rcgion.

located in the nucleus reticularis tegmenti pontis (NRTP), paramedian pontine nucleus (PMPN), pontine raphe ma), and paramedian pontine reticular formation (PPRF). The photomicrographs of Fig. SA,B,C,D were taken from four portions of this section and indicated with rectangles and corresponding letters in Figure 7.

Figure 8 shows the distribution of retrogradely labeled cells in three other parasagittal sections of the same monkey. The photomicrographs of Figure SE,F,G,H were taken from the portions in TMB-reacted sections that are marked with small rectangles and labeled with corresponding letters.

Figure 10 compiles the number of retrogradely labeled cells in each paramedian structure. Figure 10A shows the

HRP diffusion areas measured from every third section (DAB-reacted) within 2 mm of the midline. As seen in Figure 1, the injection site did not appear homogeneous in the cerebellar cortex, and the region containing dense de- position of reaction product expanded along the granular layer (as typically seen in the TMB section). Because of numerous HRP-labeled axons, the portion of the white matter that continues from the injection center appeared darker than the other portions. In addition, although the white matter occupies a large portion of the injection area, HRP uptake may be less active than that in the granular layer. Therefore, the precise determination of effective injection site was extremely difficult. As an estimate of the volume of the injection site, we measured only the densely and

233 CONNECTIONS OF THE OCULOMOTOR VERMIS

Fig. 7. Camera lucida drawing of a region of the paramcdinn brainstem of monkey Jo showing the boundaries and labeled cells in the nucleus reticularis tegmenti pontis (NRTP), paramedian pontine nucleus (PMPN), pontine raphe ma), and paramedian pontine reticular formation (PPRF). The photomicrographs of Pig. SA,B,C,D were taken from the four portions that are depicted in rectangles and indicated with corresponding letters.

homogenously stained area in the DAB sections. Estimated in this way, the area extended from 1.5 mm left to 2.0 mm right; the largest diffusion covered 4.3 mm2 of lobule VII in a parasagittal section through the 0.3 mm right plane.

The number ofretrogradely labeled cells for each nucleus was counted from the camera lucida mappings, an example of which is shown in Figure 7. The boundaries of the nuclei were determined from the Nissl-stained (DAB-reacted) sections. Sections without counterstaining (TMB reacted) were also useful in identifying the direction of background fibers, such as those running in ventrodorsal direction in the pontine raphe. All labeled neurons with a complete nucleus found at different levels of focus in the 80-pm thick sections are superimposed in the drawing. As seen in Figure 9, when a microscope is focused on a labeled cell, many other labeled cells were not in the focus, indicating that the cells

were sca.ttered at different depths in the 80-pm thick section. It was necessary, therefore, to examine every cell by focusing with a microscope on the cell body level. There were differences in the cell counts between the two successive sections each treated with TMB and DAB. As we found that DAB sections were not suitable for the quantitative evaluation, even with the aid of dark-field illumination, the labeled cells were counted only from the TMB sections. The cell count for the DAB section was supplemented by that from the next TMB section. As every third section was reacted with DAB, the total number for each structure (N) in the histogram represents 1.5 times the number of cells in the TMB sections. The retrogradely labeled cells were classified into large (filled columns), intermediate (stippled columns), and small (open columns) cell groups, as described in Methods.


5 mm

Fig. 8. Distribution of retrogradely labeled cells in three other sections of the brainstem of monkey Jo, indicating the locations of the labeled cells in the photomicrographs of Fig. SE,F,G,H. Open arrow indicates the injection site in lobule VII.

Fig. 9. Photomicrograph# of labeled cells in the nucleus reticularis tegmenti pontis (A), pontine raphe (B), paramedian pontine nucleus (C), paramedian pontine reticular formation Cn), processus tegmentosua lateralis (E), medial accessory olive (F), dorsolateral pontine nucleus (G), and prepositus hypoglossi (H). Scale marker 100 pm.


multiplying by 1.5. The numbers of labeled cells in the remaining brainstem structures were relatively few and are summarized in Table 1. Here again, the numbers rep- resent 1.5 times the cells counted from TMB sections.

NRTP

236

5

"E : & I F

O Q

400 8 v)

300 m E

200 E

100 2 Q

1200 .c

D

Among the brainstem nuclei, the largest population of retrogradely labeled cells was found in the NRTP, following HRP injection into the oculomotor vermis. In the medial portion of the nucleus, multipolar intermediate-size cells with thick dentrites were intricately distributed, reflecting

M A 0 ( r o s t r o l ) N = 270

I

I

n i 300

1200 I 1 I00

lT I n 1200 b

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N=2269

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Fig. 10. Distribution of retrogradely labeled cells in the paramedian structures in the brainstem. A. Area of HRP diffusion in the oculomotor verrnis, evaluated from DAB-reacted parasagittal sections, using a Zdss Video-plan image analysis system. R-E. Numbers of retrogradely labeled cells in each 80-pm thick TMB-reacted section. Filled columns, large cells. Stippled columns, intermediate cells. Open columns, small cells. See Meth- ods for the diameters of the longer and shorter axes. Total numbers (N) were evaluated by counting the cells in two consecutive TMB-reacted sections and multiplied by 1.5.

As compared with the paramedian structures, labeled cells in the other parts of the brainstem were widely spread. The histograms in Figure 11 show the numbers of retrogradely labeled cells in the brainstem structures outside the paramedial 1 rnm region. Each column represents the number of cells in 240-pm-thick tissue obtained by counting the cells in two successive TMB parasagittal sections and

Proc Tegrnent Lat N = 3988

E

0 d 400 (\J

0 *

5 ~r n Dorsolat Pont Nucl - 1 9

a,

0 200 :

0 5 F r Peduncular N ~ C I ( rostrai) N-1837 1 =

100 & n

100

n " 3mm Lt Middle ' 3 m m R t

Fig. 11. Distribution of retrogradely labeled cells in various nuclei outside the paramedian structures. Each column represents the number of cells for each nucleus counted from two consecutive TMB-reacted, 80-prn thick sections and multiplied by 1.5. Filled columns, large cells. Stippled columns, intermediate cells. Open columns, small cells.

TABLE. 1. Number of Retrogradely Labeled Cells in Monkey Jo

Size of cells Side Large Medium Small Total

Spherical regian R. (L.) 11 (15) 3 (15) 27 (21) 41 (51) FN Ellipsoid region R. (I,.) 12 (35) 5 (18) 23 (48) 40 (101)

IP R. (L.) 8 (0) 8 (0) 0 (0) 16 (0) DN R. (L.) 6 (0) 39 (0) 18 (0) 63 (0) VeSu R. (L.) 15 (5) 24 (47) 21 (24) 60 (76) VeL R. (z.) 12 (6) 18 (11) 2 (61 32 (23) VeI R. (L.) 9 (12) 54 (60) 33 (45) 96 (1 17) VeM R. (L.) 11 Pa, 29 (12) 68 (42) 108 (56)

Y group R. (z.) 3 (0) 23 (0) 25 (0) 51 (0) Pre. dorsal R. (L.) 3 (5) 0 (0) 140 (150) 143 (155)

ventral R. (L.) 23 (38) 15 (0) 117 (75) 155 (103) MRF R. (L.) 0 (21) 0 (8) 0 (0) 0 (29)

VeSpr R. (z.1 2 (0) 3 (0) 5 (0) 10 (0)


the cytoarchitecture of the nucleus (Fig. 9A). The labeled cells appeared throughout the rostrocaudal extent in the medial portion of the nucleus (Fig. 7), whereas they were localized exclusively in the dorsal part when the lateral portion of the NRTP was examined. Compared with the density of cells in the Nissl-stained sections (every third, DAB-reacted section), approximately 45% of the intermediate-size cells and 28% of the small cells were found to be retrogradely labeled. In contrast with the dense aggregates of labeled cells in the medial, dorsal, and dorsolateral as- pects of the NRTP, the central and ventral parts of the nucleus were almost free of labeled cells, indicating that numerous unlabeled neurons in this region may project to other portions of the cerebellum. In the lateral part, labeled cells in the dorsal NRTP merged with densely labeled cells of the processus tegmentosus lateralis (PL). Next to the NRTP, this region contained the second largest number of retrogradely labeled neurons as a single structure (Fig. 1lCj. The boundary between this region and the NRTP was difficult to distinguish and the separation of the cells into the NRTP (Fig. 10B) and the PL (Fig. 11C) may be somewhat arbitrary. Large and small labeled cells were intermingled in the NRTP (Fig. 9A), but large cells were found only in the medial part of the PL (Fig. 9Ej; the majority of cells in the lateral part were small (Fig. 11C). In the NRTP, large cells were aggregated in the dorsal portion, whereas small cells characterized the ventral portion (Fig. 9Aj. The small NRTP cells were almost identical to labeled neurons in the paramedian pontine nucleus (PMPN). Labeled cells were found in the PMPN and in the peduncular nucleus (PD), but boundaries among these cell groups were also difficult to distinguish.

Pontine nuclei The second largest population of retrogradely labeled

neurons was found in the pontine nuclei. Similar to the distribution observed in the NRTP, dense aggregates of labeled neurons were found in the paramedian region. At levels of the rostral two-thirds of the pons, approximately 28% of the labled PMPN cells were within 500 pm of the midline (Fig. 1OD). The majority of the labeled pontine cells were widely distributed in the dorsal portion of the peduncular nucleus (PD) a t the rostral and middle levels of the pons. Another group of labeled pontine neurons was found in the dorsolateral nucleus (DL) at 4-5 mm lateral from the midline (Fig. 11D). The labeled neurons in the PMPN had small cell bodies (Fig. 9Cj, which characterized the majority of pontine cells in the Nissl-stained sections. The labeled cells of the PD were similar to those of the PMPN and the boundary between these cell groups was not clear.

Pontine raphe (Ra) Labeled cells appeared in this midline structure mainly

in the caudal half of the area between the oculomotor and abducens nuclei. The boundaries between the NRTP and the Ra and between this structure and the PPRF were obscure, but we identified the raphe by the ventrodorsal direction of fibers in parasagittal sections (Fig. 9C,D). Var- ious sizes of labeled cells were widely distributed throughout the raphe (Fig. 7).

PPRF Relatively large multipolar cells were labeled in the

PPRF, in the caudal half of the area between the oculomotor and abducens nuclei. The labeled cells were found only

in parasagittal sections medial to the abducens nucleus. The cells were more numerous in a region near the medial longitudinal fasticulus (MLF). In the more lateral sections, labeled cells appeared near the anterior edge of the rootlets of the abducens nerve and near a region anterior to the abducens nucleus. Some labeled cells were discovered also in the anterior part of the nucleus. Some labeled PPRF neurons continued to a subgroup of the supragenu nucleus. Large cells dominated small labeled cells in numbers and both groups of cells were intermingled throughout the extent of PPRF (Fig. 10E). At the rostral level of the hypoglossal nucleus, relatively large numbers of small labeled cells were interspersed among the axon bundles of the MLF in the interfascicular nucleus.

Nucleus prepositus hypoglossi Labeled cells were found in this nucleus after HRP injec-

tion into the oculomotor vermis as well. Most labeled cells were small and lightly stained (Fig. 9H). There were two major groups of labeled cells: one group was found in the ventral and the other group in the caudodorsal portion of the nucleus. The ventral labeled cells were distributed from the anterior end of the hypoglossal nucleus to the posterior end of the abducens nucleus. Some labeled cells were large in the latter group. The caudal part of the nucleus contained more labeled cells than the anterior part. Labeled cells in the latter merged in neurons of the supragenu nucleus.

Inferior accessory olivary nuclei Despite the fact that the HRP diffusion was only slightly

more extensive on the right side of the vermis (see Fig. IOA), the distribution of labeled cells differed between the right and left accessory inferior olivary nuclei. On the right side, the labeled cells were located exclusively in the subnucleus b. On the left side, the distribution of labeled cells extended anteriorly and the total area was almost twice as large as the subnucleus b. No labeled cells were found in the dorsal cap of either side.

DISCUSSION The present experiment demonstrated that there are re-

ciprocal connections between the oculomotor vermis (lobule VII) and a specific area of the fastigial nucleus by injecting a small amount of WGA/HRP and HRP mixture in physiologically identified vermal sites of alert monkeys. However, it was also shown that the fastigial area with reciprocal connections did not correspond to the area of the heavy distribution of anterogradely labeled terminals and preterminals of Purkinje axons from the oculomotor vermis. A cluster of retrogradely labeled cells in a spherical region that was clearly circumscribed by fiber bundles was discovered near the lateral border of the fastigial nucleus. This region was almost free of labeled axon terminals when HRP diffusion was confined to the oculomotor vermis. These results directly confirm a previous argument that the cerebellar corticonuclear and nucleocortical projection are not organized in a strict reciprocal fashion (Tolbert et al., '78; Tolbert and Bantli, '79). As compared with the enormous projection within the ellipsoidal region in the mediocaudal portion of the fastigial nucleus, labeled fibers observed in the same nucleus outside the ellipsoidal region and in the interposed nuclei were sparse and, therefore, they did not appear to be the mainstream of the projection.

J. YAMADA AND H. NODA 238

It is well accepted that in the cat the nucleocortical projection from the individual deep cerebellar nuclei is principally organized into three rostrocaudally oriented, longitudial zones (Walberg and Jansen, '64; Courville and Diakiw, '76; Gould and Graybiel, '76; Tolbert et al., '76; Tolbert et al., '78). The dentate nucleus projects to the hemispheric zone, the anterior and posterior interposed nuclei project to the paravermal-medial hemispheric zone, and the fastigial nucleus projects to the vermal cortical zone (Tolbert et al., '78). According to Tolbert et al. ('78, however, in the primate the nucleocortical projection to these cortical zones arises principally from the dentate nucleus with only secondary input to the paravermis and vermis arising from the interposed and fastigial nucleus, respectively. The present findings have shown that the primary 'source of the nucleocortical projection to the oculomotor vermis resides in two distinct cell groups: one located in the lateral part of the ellipsoidal region in the mediocaudal aspect of the fastigial nucleus and the other group located in the spherical region at the lateral margin of the nucleus. Only a small number of retrogradely labeled cells were discovered in the dentate nucleus (DN). When the HRP diffusion invaded the paravermal zone, however, retrogradely labeled cells also appeared in the interposed nuclei (IP). It is likely that such a fine sagittal zone organization as seen in cats (Tolbert et al., '78; Gould, '79) exists also in the nucleocortical relationship of macaques, How- ever, as compared with the total number of labeled neurons in the brainstem, the retrogradely labeled cells in the fastigial nucleus were so few that it is doubtful that such a precise feedback loop as proposed for cats between the cerebellar cortex and deep cerebellar nuclei (Gould, '79) exists in macaques.

Retrogradely labeled brainstem neurons A striking aspect of the present findings is the distribu-

tion of retrogradely labeled neurons that resembled the results noted after HRP injection into the flocculus (Langer et al., '85). There were, however, several differences in the nuclei that provide the major afferents to these functionally distinct oculomotor cerebellar cortices. When HRP was injected into the flocculus of monkeys, the greatest numbers of labeled neurons were in the vestibular complex and the nucleus prepositus hypoglossi (Langer et al., '85). On the other hand, when HRP was injected into the oculomotor vermis, relatively fewer retrogradely labeled vestibular neurons were scattered in all four of the vestibular nuclei. There were no dense clusters of labeled cells in any part of the vestibular complex and the density of labeled neurons was markedly less as compared with other nuclei in the brainstem. In the nucleus prepositus hypoglossi, lightly labeled small cells were scattered only in the ventral and caudodorsal portions. This observation was also in contrast with the results of the flocculus from which the majority of the neurons in all the subdivisions of the nucleus were labeled (Langer et al., '85).

NRTP Following the injection of HRP into the oculomotor ver-

mis, the largest number of labeled neurons was distributed in the NRTP. This agrees with the previous studies of axonal transport techniques in cats (Hoddevik et al., '77; Batini et al., '78; Kawamura and Hashikawa, '81; Gould, '80). The pattern of the distribution of labeled cells was generally consistent with the observation of Langer et al.

('85) following HRP injection into the flocculus of macaques, but differences were noted. First, a large injection of HRP was not necessary to produce large fractions of labeled cells from the vermis. Second, the majority of neurons labeled from the vermis were small, although large- and intermediate-size cells characterized the dorsal part of the NRTP (Fig. 9A). Finally, multipolar neurons were discovered in the present study throughout the extent of NRTP following vermal injection, whereas these neurons were located only in the caudal part of the NRTP following floccular injection (Langer et al., '85). On the other hand, an important simi- larity was that the central regions of the NRTP that lay dorsal to the "hook" formed by each medial lemniscus were devoid of labeled neurons in both floccular and vermal injections. Langer et al, ('85) have described a group of small and intermediate neurons of the lateral margin of NRTP as it extends dorsally to the body of the medial lemniscus. This cell group was also labeled in the present study and has been located in the processus tegmentosus lateralis.

Pontine nuclei A second major source of input to the oculomotor vermis

was the pontine nuclei. Intermediate-size multipolar neurons were labeled in the dorsal midline slightly ventral to the NRTP after floccular injection (Langer et al., '85). In contrast, the main body of the corresponding area was occupied by small labeled neurons in the present study. Small neurons in a thin peripheral lamina adjacent to the fibers of the contralateral brachium pontis were irregularly distributed with labeled cells in both floccular and vermal cases. Another cell group of small and intermediate-size neurons was labeled in the dorsolateral nucleus (DL) in the present study. In addition, we observed two groups of small labeled cells at the rostral and middle pontine levels, respectively. These cell groups formed two transverse zones extending from the midline to the lateral margin of the peduncular nucleus (PD).

Haphe and PPRF Langer et al. ('85) have observed a rather extensive la-

beled neuron distribution that extends from the rostral medulla through the pons after floccular injection. Neurons in various nuclei, such as the nucleus raphe obscurus, para- medianus dorsalis, pararaphales, reticulasis paramedi- anus, reticularis paragigantocellularis dorsalis, and these medullary interfascicular nuclei, have been labeled from the flocculus (Langer et al., '85). Except for the interfascicular nuclei, there was no notable labeling of cells in these midline and paramedian structures following HRP injection into the vermis. In the present study, a significant number of labeled cells were discovered in both the pontine raphe and the FPRF in the area rostral to the abducens nucleus. The labeled area extended rostrally in the pons midway between the oculomotor and abducens nuclei in the present study.

Inferior olivary complex Large injections into the flocculus labeled nearly every

neuron in the dorsal cap of Kooy (Langer et al., '85). When HRP was injected into the vermis, labeled neurons appeared mainly in caudal subnucleus b of Bowman and Sla- dek ('73). There were no labeled neurons in either the dorsal cap of Kooy or the subnucleus c, which corresponds to the nucleus p of the cat. These findings are in agreement with

CONNECTIONS OF THE OCULOMOTOR VEKMIS

those of Frankfurter et al. ('77) in the squirrel monkey and of Hoddevik et al. ('76) in the cat.

239

of visual input. In both monkeys and cats, the visual cortical projections are centered in the rostral half of the nons.

Functional considerations A possible function of the oculomotor vermis is suggested

by the convergence of input signals necessary to orient a target in a spatial frame of reference. Evoked-potential and single-unit studies have shown visual (Freeman, '70; Buch- tel et al., '72; Donaldson and Hawthorne, '79), extraocular proprioceptive (Fuchs and Kornhuber, '69; Wolfe, '71; Baker et al., '72; Berthoz and Llinas, '74; Schwarz and Tomlinson, '771, and vestibular inputs (Precht et al., '77) to the vermal lobules VII and to neighboring lobules in the cat. Single- unit recordings in alert monkeys have shown that this vermal region receives mossy fibers whose discharges are modulated during saccadic eye movements (Kase et al., '80). Mossy fiber units responding to movements of visual stim- uli are also recorded from the same vermal region in monkeys (Kase et al., '79; Suzuki et al., '81).

The present anatomical study has shown that the oculomotor vermis receives afferent fibers from the brainstem structures in which neurons discharge with saccades. A considerable number of fibers were shown to arise from the NRTP. The same region of the NRTP in monkeys contains neurons that discharge during saccadic eye movements (Crandall and Keller, '85). The mossy fibers arising from the NRTP region, therefore, could transmit eye movement signals to the oculomotor vermis. They were directionally selective and discharged long before (20-30 msec) the onset of a saccade. These responses during saccades are characteristic of a group of mossy fibers (long-lead units) that constitute only a small population of burst mossy fiber units in the oculomotor vermis of the monkey (Kase et al., '80). There is another group of burst mossy fibers that discharges with every saccade (pan-directional) immedi- ately before the onset of a saccade (0-5 msec), and the discharge is closely related to saccade metrics (Kase et al., '80). If there are no such neurons in the NRTP, the origin of pan-directional mossy fibers must be sought in the other brainstem nuclei.

Large retrogradely labeled neurons were widely distributed in the PPRF, an area considered as the final common pathway for all rapid eye movements (Bender and Shanzer, '84; Luschei and Fuchs, '72; Keller, '74; Henn and Cohen, '76). It is generally accepted that the unilateral, medum- lead burst units provide the input that generates a high frequency burst of activity in oculomotor neurons during saccades. However, these units also show directional selec- tivity primarily in the horizontal direction.

The present study has shown that the oculomotor vermis receives mossy fibers from a region in the NRTP. The same region has been shown to project to the flocculus in the monkey (Langer et al., '85). The NRTP in rabbits serves as the main pathway of the visually evoked mossy fiber responses in the flocculus (Maekawa and Takeda, '75; Mae- kawa et al., '81). It is likely, therefore, that visual signals are transmitted to the oculomotor vermis via this region of the NRTP, which receives input signals primarily from the pretectum. The portion of the NRTP that receives fibers from the superior colliculus is at least close to the region projecting onto the oculomotor vermis and flocculus (Hod- devik, '78).

It is important to note that the pontine areas projecting onto the oculomotor vermis coincide with the terminal fields

1 ~ ~~

The- visual cortico-ponto-cerebellar pathway have been described in some detail in cats. The visual cortex (Brodal, '72a,b; Clickstein et a1.,'72; Sanides et al., '78; Gibson et al., '78; Albus et al., '811, the superior colliculus (Kawamura and Brodal, '731, and the ventral lateral geniculate nucleus (Graybiel, '74) project to circumscribed and largely non- overlapping regions of the pontine nuclei. The pontine terminal regions, however, appear to differ between the cat and the monkey. In cats, the visual cortex projects to a region just medial and ventral to the pyramidal tracts in the rostral half of the pons. In monkeys, the projection is to the dorsolateral peduncular nucleus, the dorsolateral nucleus, and the lateral nucleus (Brodal, '78; Glickstein et al., '80). The dorsolateral nucleus is the projection field of fibers from the superior colliculus in the cat (Altman and Carpen- ter, '61; Mizuno et al., '73; Graybiel and Hartwieg, '74; Mower et al., '801, and there is evidence (Harting, '77) that dorsolateral pons also receives an input from the superior colliculus in the monkey. However, fibers arising from the superior colliculus seem to terminate more caudally in the pontine nuclei than do those from the cerebral cortex (Hart- ing, '77). Pontine neurons within the terminal fields of the cortical and collicular fibers are exclusively visual. They are powerfully excited by moving visual stimulation and most are tuned for the direction speed of movement.

The present experiment has shown that the mainstream of the pathway for oculomotor command signals is via the fastigial nucleus. When the diffusion of HRP was confined to the oculomotor vermis, most Purkinje axons terminated ipsilaterally in the ellipsoidal region, which is located in the mediocaudal aspect of the fastigial nucleus. Some labeled fibers could be traced farther to the y group and the lateral vestibular nucleus, but these fibers were scanty and did not show any impressive terminal fields in brainstem nuclei. It is unlikely, therefore, that these fibers could con- tribute to the transmission of oculomotor control signals. On the other hand, the presence of the abundant terminal fields aggregated in the ellipsoidal region suggests that the major output signals of the oculomotor vermis are transmitted through this region of the fastigial nucleus.

With regard to the efferent fibers of the fastigial nucleus, it is generally accepted that: (1) fibers of the uncinate fasciculus arise predominantly from caudal parts of the nucleus and cross within the cerebellum, and (2) uncrossed fastigial efferent fibers arise predominantly from rostral parts of the nucleus and emerge from the cerebellum via the juxtarestiform body (Rasmussen, '33; Jansen and Jan- sen, '55; Cohen et al., '58; Carpenter et al., '58; Carpenter et al., '59; Flood and Jansen, '61; Walberg et al., '62; Voogd, '64; Angaut and Bowsher, '70). Although the separation of the rostral and caudal parts of the fastigial nucleus in relation to the crossed and uncrossed efferent pathways has been argued by Batton et al. ('77) on the basis of axoplasmic transport studies using 3H amino acid, fibers in the uncinate fasciculus appear to terminate in the brainstem nuclei that project back to the oculomotor vermis. For example, Asanuma et al. ('83) observed terminal labeling in the primate NRTP following an injection of tritiated amino acids in the fastigial nucleus (although the injection was not confined to the mediocaudal part of the fastigial nucleus). Interestingly, the labeled fastigial terminals within the NRTP occurred in zones that are different from those la-

240

beled following dentate or interposed nucleus injections. Clustered silver grains were not seen in the central zone but occurred in the dorsomedial region and in the processus tegmentosus lateralis (Asanuma et al., '83). Retrogradely labeled cells were found in the latter portions following HRP injections in the oculomotor vermis in the present study and in the flocculus of monkeys (Langer et al., '85). The central zone of the NRTP was free of HRP labeled cells in both studies. The present finding that a considerable number of fibers arise from these NRTP regions suggests the significance of the oculomotor vermis in a feedback system for oculomotor control consisting of a loop of anatomical pathways from the NRTP to the oculomotor vermis, from this cerebellar cortex to the fastigial nucleus, and back to the NRTP,

ACKNOWLELIGMENTS We are grateful to Drs. C.R.S. Kaneko and T.P. Langer

for their helpful criticism and correction of the manuscript. We thank also Mr. Jaque Kubley for photographic assis- tance. This work was supported by the National Institutes of Health Grant EY04063.


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afferent and efferent connections of the oculomotor cerebellar vermis in the macaque monkey

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