Vestibular and medullary brain stem afferents to the abducens nucleus in the cat

Brain Research, 123 (1977) 229-240

© Elsevier/North-Holland Biomedical Press, Amsterdam - Printed in The Netherlands

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VESTIBULAR A N D M E D U L L A R Y BRAIN STEM A F F E R E N T S TO T H E ABDUCENS N U C L E U S IN T H E CAT

R. J. MACIEWICZ, K. EAGEN, C. R. S. KANEKO and S. M. HIGHSTEIN Department of Neuroscience and the Rose F. Kennedy Center for Research in Mental Retardation and Human Development, Albert Einstein College of Medicine, Bronx, N.Y. 10461 (U.S.A.)

(Accepted July 6th, 1976)

SUMMARY Brain stem neurons that project to the abducens nucleus (nVI) were labeled by the technique of retrograde transport of horseradish peroxidase (HRP). Following iontophoresis of H R P into nVI a large number of labeled cells are found in the ipsilateral vestibular nuclear complex, extending from the rostral medial vestibular nucleus into the ventral lateral vestibular nucleus. A smaller number of HRP-positive cells are also found in the contralateral medial vestibular nucleus. In addition, labeled cells are localized to the contralateral dorsomedial gigantocellular tegmental field as well as the nucleus praepositus hypoglossi of both sides, evidence that these neuronal groups may also be involved in eye movement control.

INTRODUCTION The abducens nucleus (nVI) contains the motoneurons that innervate the lateral rectus muscle and as such is the final common pathway for abduction of the eye. The location of cells that project to the nVI and directly influence the activity of abducens neurons is still a matter of controversy, however. Physiological studies have shown a localized input from second order vestibular neurons onto nVI motoneurons~, 21 and interneurons 4, but anterograde degeneration studies aimed at locating the precise regions of the vestibular nuclei that project to nVI have lead to conflicting results. McMasters et al. aa have reported that, in the monkey, the superior vestibular nucleus (SV), the descending vestibular nucleus (DV), the medial vestibular nucleus (VM) and the ventral lateral vestibular nucleus (VLV) all project to nVI. In the cat, however, Tarlov 41 reported that only the SV and the rostral VM project to nVI. Gacek 12, also working in the cat, concluded that the major vestibulo-abducens pathways arise in the VM and VLV and not in the SV. The differences among these studies are probably due, in part, to: (1) the difficulty in making restricted lesions in the var-

230 ious vestibular nuclei, and (2) the fact that fibers of passage may be interrupted by such lesions 27 making the results difficult to interpret. In order to provide further information on the origin of the vestibulo-abducens pathway as well as to identify other possible brain stem afferents to the nVI we have used the retrograde transport of horseradish peroxidase (HRP)26,2s, 34 to label neurons projecting to the nVl. In addition to labeling cells in the vestibular nuclei, injections of H R P into nV1 label cells in the nucleus praepositus hypoglossi (PH) and the dorsomedial gigantocellular tegmental field (FTG) evidence that these two regions also project to nVI. An additional afferent system arising from cells in and around the oculomotor nucleus and ending in nVl is also found in all cases studied and has been reported elsewhere3L MATERIALS AND METHODS Six adult cats were anesthetized by intraperitoneal injection of Nembutal (30-40 mg/kg) and mounted in a stereotaxic frame. The bone was removed over the midline cerebellum and the posterior vermis aspirated to expose the floor of the fourth ventricle. A pipette (tip diameter = 15-70/~m) was filled with a solution of H R P (Sigma type VI) and then guided under visual control through the facial colliculus and into the vicinity of the nVl. The final placement was accomplished by maximizing the antidromic field potential recorded through the pipette evoked by stimulation of the abducens nerve within the orbit3L HRP was then iontophoresed16,30, 32 through the pipette using 2/~A, 250 msec positive pulses every 500 msec for 0.5-2.0 h. In two additional cases, 0.03/~1 of a 3 0 ~ H R P solution was pressure injected into nVI. The animals were kept anesthetized for 19-48 h following surgery and were then sacrificed by Nembutal overdose and perfused with a cold solution of 0.5-1.0 ~ paraformaldehyde and 1.25-2.5 ~ glutaraldehyde in 0.I M sodium cacodylate. The heads were stored for 12-24 hours in cold fixative. The brains were then blocked in coronal stereotaxic planes and kept in a cold, buffered, 20-30 ~o sucrose solution for 2-3 days. Frozen sections (40/~m) were cut from the brains and reacted with 3,Y-diaminobenzidine and hydrogen peroxide to localize the HRP 13. The reaction solution contained 50 mg ~ diaminobenzidine and 0.004 ~ hydrogen peroxide in a 5 ~ sucroseTris buffered solution (pH 7.6 at room temperature) 40. The sections were placed in this solution for 30-60 min at 37 °C and then rinsed in buffer and stored in 10~o formalin. Sections were mounted on glass slides and counterstained with cresyl violet or gallocyanin and then scanned using either brightfield or darkfield optics for cells containing the HRP reaction product. These sections were compared with control sections from animals in which HRP had been injected into other parts of the brain. Photographic prints were made of the brain stem sections studied and the locations of HRP-labeled cells were charted directly onto these prints. Summary line drawings such as those in Figs. 2 and 3 were then composed from representative prints. The borders of the superior, medial, lateral and descending vestibular nuclei were determined using the criteria of Brodal and Pompeiano 8, and the lateral nucleus

231 was further subdivided into dorsal and ventral parts6. The minor vestibular complex (nuclei F, X, Y and Z) are eliminated from the There is some disagreement concerning the precise borders of the nuclei (see, for example, ref. 12), and this should be considered results reported below.

cell groups of the figures for clarity. various vestibular in evaluating the

RESULTS

Injection sites The size of the injection site was defined as the limit of spread of the homogeneous HRP reaction product in the brain stem in and around the nVI. HRP readily diffuses from the site of injection and it proved impossible to limit the spread of label to the nVI with even very small (0.03 #1) pressure injections of HRP. Iontophoretic

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Fig. 1. a: photomicrograph of a section through the brain stem in a cat in which HRP has been iontophoresed into nVI. The dark HRP reaction product is concentrated immediately beneath the facial nerve in the region of nVI. b: a darkfieid micrograph from the facial nucleus. Hollow arrows indicate two neurons that are filled with granules of HRP reaction product. An axon that is homogeneously labeled with reaction product traverses the field, c: a darkfield micrograph of a cell from the ipsilateral VM that is filled with granules of HRP reaction product. The asterisk identifies an erythrocyte. d: a labeled cell from the VM contralateral to an HRP injection into nVI. Granules of reaction product extend from the cell body into the primary dendrites (arrows). The calibration bars in b, c and d are 30/zm.

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Fig. 2. Representative levels through the brain stem showing the pattern of labeled cells in individual sections following iontophoresis of HRP into nVI. The results of case AH-23 are superimposed on case AH-14. Arrowheads indicate the injection sites in nVI. The extent of spread of HRP in nVI in case AH-14 is drawn in black, while that in AH-23 is indicated by broken lines. Labeled cells in the former case are indicated by black dots and in the latter by × 's. The genu of the facial nerve and the stria acoustica are hatched for clarity. Abbreviations: CX, external cuneate nucleus; DV, descending vestibular nucleus; FTG, gigantocellular tegmental field; INT, nucleus intercalatus; PH, nucleus praepositus hypoglossi; SA, stria acoustica; SV, superior vestibular nucleus; VLD, dorsal lateral vestibular nucleus; VLV, ventral lateral vestibular nucleus; VM, medial vestibular nucleus; 6, abducens nucleus; 7, facial nerve; 10, dorsal motor nucleus of vagus; 12, hypoglossal nucleus. a p p l i c a t i o n s o f H R P yielded a much m o r e restricted area o f spread o f label at the injection site (Fig. la). The injection sites in these cases consisted o f a central region densely stained with r e a c t i o n p r o d u c t s u r r o u n d e d by a much m o r e lightly stained zone. The limits o f the injection sites shown in Figs. 2 a n d 3 represent the extent o f this h o m o g e n e o u s lightly labeled zone, a l t h o u g h the precise borders o f the injection sites were often difficult to determine. I t is d o u b t f u l whether cells projecting to the edges o f these injection sites r e t r o g r a d e l y t r a n s p o r t sufficient H R P to be labeled with reaction p r o d u c t in the present e x p e r i m e n t 9.

233 Cells that are retrogradely labeled with appreciable amounts of H R P are characterized by the presence of dark brown granules of reaction product that fill the cell body and extend into the primary dendrites ~6,28,34. A small number of cells are also found that are homogeneously filled with HRP. In all cases, HRP-positive cells of both types were found in the facial nucleus ipsilateral to the injection site (Fig. lb). This labeling was clearly due to damage done to the facial nerve by the iontophoresis pipette, since broken axons diffusely filled with H R P reaction product were found along the path of the pipette as it passed through the genu of the facial nerve to reach the nVI immediately below. As broken axons are known to retrogradely transport H R P TM, finely tapered pipettes were used to minimize the damage done during the injection procedure. However, labeling of axons in the facial nerve and the presence of HRP-positive cells in the facial nucleus are clear evidence that at least some retrogradely labeled cells in the present experiment are due to interruption of axons in, or in this case, immediately above, the nVI. HRP-positive cells were also found bilaterally in the vestibular nuclei and the PH, as well as in the contralateral dorsomedial FTG.

Vestibular projections Fig. 2 shows the results of two cases in which H R P was iontophoresed into nVI. The injection site in case AH-14 is shown in black and the resultant labeled cells are shown as dots. Case AH-23 is drawn in dotted lines while the HRP-positive cells in this case are represented by ×'s. In both cases the H R P reaction product at the injection site appears restricted to the nVI. Within the ipsilateral vestibular nuclei labeled cells are found principally in the rostral VM (Figs. lc, 4d) adjacent to the medial border of the stria acoustica. This distribution of cells extends ventrally and laterally into the VLV. A large number of labeled neurons are found in this region of the VM and VLV in all cases examined and their presence is strong evidence that this part of the vestibular complex is the major if not exclusive origin of the ipsilateral vestibulo-abducens pathway. HRP-positive cells are only very rarely found in the other ipsilateral vestibular nuclei, although a small number of clearly labeled cells are located in the DV in all cases. Such cells seem to be randomly located in fields of cells that do not contain H R P reaction product. The scarcity and inconstant distribution of such DV cells are factors suggesting that these cells do not project to nVI; perhaps such cells have axons that are damaged during the injection procedure. In the contralateral vestibular complex HRP-positive neurons are again found in the VM (Fig. ld) in both AH-14 and AH-23, although in far fewer numbers than on the ipsilateral side. This finding is consistent with the observation from anterograde degeneration studies that the VM contributes a greater projection to the ipsilateral than the contralateral nV112,41.

Reticular projections Labeled cells are also localized within the medullary reticular formation in both cases AH-14 and AH-23 (Fig. 4a, b, c). They occur predominantly in the dorsomedial F T G at the level of the facial nucleus just caudal to nVI; at this level they lie

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Fig. 3. The distribution of labeled cells in case AH-5. The HRP iontophoresis site is noted by arrowheads and the limit of spread of HRP within nVI is indicated in black. Dots represent the location of labeled cells. For explanation of abbreviations see legend Fig. 2. ventral to the PH and just lateral to the medial longitudinal fasciculus (MLF). A few such cells are intermingled among the most lateral fibers of the MLF. Aside from this very localized grouping of HRP-positive neurons, the medullary reticular formation is devoid of labeled cells. It is interesting to note that electrophysiologica124 and anatomical experiments 15 provide evidence for a projection from the paramedian pontine reticular formation (PPRF) to nVI. Such cells are not found in cases in which the spread of H R P is restricted to nVI (cases AH-5, AH-14, AH-23). However, numerous labeled cells are found in the P P R F in cases in which a larger amount of H R P is either pressure injected or iontophoresed into nVI (cases AH-2, AH-9, AH-13), but in these cases the spread of H R P extends beyond the borders of nVI ventrally into the adjacent reticular formation and dorsally into PH. Cases in which labeled P P R F cells are

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Fig. 4. a: a low power photomicrograph of a coronal brain stem section just caudal to nVI. The rectangle locates an HRP-labeled cell in the dorsomedial FTG contralateral to an HRP injection into nVI. b: higher magnification of the rectangle shown in a. HRP reaction product diffusely fills the cell body and extends for some distance into the dendrites of this FTG neuron, c: a brightfield micrograph of another FTG neuron at higher magnification. In this case the HRP reaction product is granular and is largely restricted to the cell body and initial dendrites, d: an HRP-positive cell from the VM ipsilateral to an HRP injection into nVI. The size, density and perikaryal distribution of HRP-positive granules are similar in c and d. The asterisk identifies an erythrocyte. The calibration bar for c and d is 20/~m.

f o u n d are therefore n o t illustrated, because o f the possibility t h a t such cells actually p r o j e c t t o nuclear g r o u p s a d j a c e n t to, r a t h e r t h a n within, nVI.

Perihypoglossalprojections T h e nucleus p r a e p o s i t u s hypoglossi (PH), the nucleus intercalatus, a n d the nucleus o f R o l l e r are c o m m o n l y g r o u p e d t o g e t h e r descriptively as the perihypoglossal nuclei 7. I n b o t h cases A H - 1 4 a n d A H - 2 3 , H R P - p o s i t i v e cells are f o u n d bilaterally in the P H (Fig. 5). W i t h i n this nucleus, l a b e l e d cells are i n t e r m i n g l e d with u n l a b e l e d cells a n d occur with a p p r o x i m a t e l y equal frequency on b o t h sides. Such cells are often l o c a t e d in the ventral p a r t o f the nucleus b o r d e r i n g on the reticular f o r m a t i o n . I n case A H - 2 3 (Fig. 2), this d i s t r i b u t i o n o f H R P - p o s i t i v e cells continues c a u d a l l y into the c o n t r a l a t e r a l nucleus intercalatus, b u t in all a n i m a l s studied the nucleus o f R o l l e r is d e v o i d o f l a b e l e d cells.

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Fig. 5. Brightfield micrographs of two PH cells filled with HRP-positive granules. The arrows in a identify axons homogeneously labeled with HRP reaction product. The calibration bar for a and b is 20/~m. Fig. 3 shows the distribution of HRP-positive cells in case AH-5. The spread of label is restricted to nVI and the distribution of labeled cells is similar to that in cases AH-14 and AH-23 shown in Fig. 2. This case serves to summarize our findings, as again in AH-5 the rostral VM and ipsilateral VLV contain a large number of labeled cells. A smaller number of labeled cells are found in the contralateral VM and, in this case, a few labeled cells are found in the contralateral VLV as well. Within the medulla, HRP-positive cells are bilaterally localized to the PH. The contralateral dorsomedial F T G also contains labeled cells. In this case, a few labeled cells are located in the ipsilateral F T G as well. DISCUSSION Two major sources of error attend the retrograde transport method used in the present experiment. First, not all of the cells terminating in the nVI are necessarily clearly labeled34, 39, so that certain afferents to nVI may not have been identified in this study. Second, axons broken during the injection procedure can retrogradely transport H R P even though they do not terminate at the injection site11A 8. Micropipettes were used to iontophorese H R P in the present experiment in an attempt to minimize the amount of tissue damage done during the injection procedure. However, the finding of labeled axons in the facial nerve along the pipette path as well as the presence of HRP-positive cells in the facial nucleus clearly show that at least some labeled cells in the present experiment are due to H R P uptake by injured axons. Despite these limitations, the retrograde transport method has proved an invaluable aid in defining candidates for the cells of origin of a variety of pathways. Its use in the present study provides strong evidence that vestibular, reticular and perihypoglossal cells project to the nVI.

Vestibular projections Our demonstration that the VM projects to both the ipsilateral and contralateral nVI, while the VLV projects at least to the ipsilateral nVl, is most consistent

237 with the conclusions of the anterograde degeneration studies of Gacek 12. Physiological studies in the rabbit 21 and the cat 5 also support this view, as microstimulation of the VM generates monosynaptic EPSPs in contralateral nVI motoneurons and monosynaptic IPSPs in ipsilateral motoneurons. Stimulation of SV has no direct effect on nVI cellsS, 2~, a finding that is consistent with our inability to demonstrate a direct pathway from SV to nVI in the present experiment. By contrast, the trochlear nucleus and all four of the subdivisions of the oculomotor nucleus controlling extraocular muscles receive a direct inhibitory input from the ipsilateral SV 19. The nVI seems, therefore, to be the extraordinary case, as inhibition is apparently mediated by the ipsilateral VM rather than the SV z°.

Nucleus praepositus hypoglossi Injections of HRP into nVI label cells in both the ipsilateral and contralateral PH evidence that the PH may play a role in the generation of eye movements. Prior anatomical studies support this view, demonstrating projections from PH to the oculomotor nucleus 17 and to the cerebellar cortex 7,4z. Afferent inputs to PH arise in the flocculus 1 and fastigial nucleus 43, the interstial nucleus of CajaP ° (or the tegmentum immediately rostral to it31), the cerebral cortex ~8, and, perhaps, the PPRF 15. Physiological studies z indicate that PH cells probably receive an input from secondary vestibular cells as well. Baker et al. 3 have shown in addition that the discharge of many PH neurons is correlated with and often precedes eye movements, including eye movements in the horizontal plane. In light of the direct connections between PH and the eye muscle motor nuclei, this finding strongly implicates the PH in eye movement control.

Reticular formation HRP-positive cells are found in the PPRF only after large injections of HRP into nVI; injections restricted to nVI fail to label any neurons in this region. It is possible that the very limited quantities of HRP iontophoresed into nVI at the small injection sites are insufficient to clearly label neurons in the PPRF. This view is consistent with the findings of Leger et al. 29, who reported that brain stem neurons projecting to the dorsal lateral geniculate nucleus could only be demonstrated with injections of at least 0.2/tl of a 33 ~ solution of HRP into the lateral geniculate. This is a much larger amount of HRP than that injected in any of the experiments of the present study. As large injections of HRP inevitably label much more than the nVI, it may be impossible to determine with the retrograde transport technique whether PPRF cells terminate within the nVI or in adjacent nuclei. Even with the smallest HRP injections into nVI, however, labeled cells are found in the dorsomedial F T G just caudal to nVI. Graybiel and Hartwieg 17 have described cells in a similar location that project to the oculomotor nucleus. Hoddevik et al. 22 have shown that this region also projects to all four of the major vestibular nuclei, and the vestibular nuclei in return project back upon the FTG 27. In addition, afferents to this part of the reticular formation arise from the fastigial nucleus 44 and the deep layers of the superior colliculus 25. Several lines of investigation have pro-

238 vided evidence that the superior colliculus is involved in eye movement control 37, although cells in the superior colliculus do not appear to project directly to nV135,~6. The dorsomedial F T G may represent one site where information from the vestibular system interfaces with that from the superior colliculus before being relayed to the eye muscle nuclei. Stimulation of the superior colliculus evokes disynaptlc EPSPs in contralateral nVl motoneurons 14,35 and the pathway for this effect appears to involve the descending, crossed, tecto-reticular projection that travels in the predorsal bundle~4, 25. The interneurons of this disynaptic excitatory pathway are, therefore, most likely located contralateral to the superior colliculus and ipsilateral to the nVl involved in the circuit ~a. As the dorsomedial FTG neurons labeled in the present study are predominantly contralateral to the nVI they innervate, it is unlikely that they are the cells involved in this disynaptic excitatory tecto-abducens pathway. In conclusion, we have been able to demonstrate projections to the nVI that appear to arise in the VM and VLV, as well as the PH and dorsomedial FTG. The cells we have labeled in the VM and VLV are likely candidates for the origin of the direct vestibulo-abducens pathway. Cells in the PH and FTG, on the other hand, may represent sites where vestibular information is combined with and modified by other neuronal systems before being relayed to nVI. However, the specific contributions to horizontal eye movements of the afferents to nVI identified in these experiments remain to be elucidated. ACKNOWLEDGEMENTS This work was supported by N I H Grants ! R01 EY01670, 1 K04 EY00003, 5 P01 07512, 5T5 GM 1674 and 5 R01 11431. Marie Buschke is thanked for her continuing technical advice.

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