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Olivocerebellar projection to the cardiovascular zone of rabbit cerebellum
240
Neuroscience Research, 12 (l 991) 240-250 © 1991 Elsevier Scientific Publishers Ireland, Ltd. 0168-0102/91/$03.50
N E U R E S 00492
Olivocerebellar projection to the cardiovascular zone of rabbit cerebellum N. Nisimaru 1, K. Okahara i and S. N a g a o 2 1Department of Physiology, Medical College of Oita, Oita and 2 Department of Physiology, Faculty of Medicine, Unit,ersity of Tokyo, Tokyo, (Japan) (Received 31 May 1991; Accepted 28 June 1991)
Key words." Cerebellum; Lobule VII; Climbing fiber; Olivo-cerebellar projection; Horseradish peroxidase; Topographical organization; Rabbit
SUMMARY Climbing fiber responses were evoked in the medial vermal cortex of lobule Vlla by stimulation of the contralateral medial accessory olive (MAO) in anesthetized, paralyzed rabbits. Effective stimulating sites were localized in a small medial part of the caudal MAO, at 0.4-1.6 m m rostral from the caudal pole of the M A O (total length of the MAO, 4.2 ram). Stimulation of this M A O area induced depression in renal sympathetic nerve activity and this depressant response disappeared after ablation of lobule VIIa. Following injections of horseradish peroxidase into the small areas of lobule Vlb, Vlc, VIIa or VIIb, retrogradely labeled cells were found in corresponding small particular regions of the MAO: lobule VIb to the most caudal part, lobule VIc to the next caudal, lobule VIIa to the most rostral within the caudal MAO, and lobule VIIb further rostrally to the intermediate MAO. There was a clear disparity between the medial halves of Iobules VI and VII projected from the medial M A O and the lateral halves from the lateral MAO. These results show that climbing fiber projections to lobules VI and Vll are topographically organized, and that the medial region of lobule VIla, related to cardiovascular function, receives climbing fibers from a localized small medial region of the caudal MAO.
INTRODUCTION
The cerebellum is involved not only in the control of motor functions but also in that of autonomic functions 3,2o. Our previous studies in anesthetized rabbits suggest that areas of the anterior and posterior lobes of the cerebellar vermis, especially the A-zone of lobule VIIa, are involved in cardiovascular control 21,22. Knowledge of the neuronal circuitry organization of this particular cerebellar area is important in understanding the mechanism of the cerebellar cardiovascular control. A subdivision of the inferior olive sends climbing afferent fibers to a narrow longitudinal zone of the cerebellar cortex 8,12. The A-zone of lobules VI and VII is projected from the caudal half of the medial accessory olive (c-MAO), part of the inferior olive, in cats 2,6,11,13,24, rabbits ~', rats 1,9,26, monkeys 7 and sheep 25. Recently, several separate cell columns in the c-MAO have been found histologically, in particular, by using cholinesterase staining in cats 16, monkeys 5, sheep 25 and opossums 17
Correspondence: N. Nisimaru, Department of Physiology, Medical College of Oita, Oita 879-55, Japan.
241 Some investigators 4.10.29 have also shown that these columns of the c-MAO project separately to A~- and A2-zones of the cerebellar vermis. Such topographical relationships between the MAO and lobule VII have not yet been revealed in rabbits. In this study, we traced olivocerebellar projections from the MAO to lobules VI and VII in rabbits by using electrophysiological techniques and horseradish peroxidase (HRP) axonal transport, and found that the projections are organized with a clear orderly pattern. MATERIALSAND METHODS Twenty-five albino rabbits (body weight, 2.1-3.9 kg) were anesthetized with a-chloralose plus urethane (30 and 400-800 mg/kg, respectively) administered intravenously and supplemented as required. Rabbits were then immobilized by intravenous injection of gallamine triethiodide and artificially respired with intermittent positive pressure. Each rabbit was mounted on a metal frame in a prone position, with the head fixed rigidly with a mouthpiece and pointed screws piercing the zygomatic arch from both the right and left sides. The posterior vermis of the cerebellum and the dorsal surface of medulla were exposed by partial craniotomy. The exposed cerebellar tissues were kept warm by applying warm paraffin oil mixed with vaseline over the surface. Rabbits were kept warm with an electric heating pad. The atlas of the rabbit cerebellum was according to Larsell 15
Electrophysiological experiments For stimulation, monopolar platinum-iridium electrodes (200 Izm diameter without insulation) or 2 M NaC1 glass microelectrodes (0.5-1 MO) were used as a cathode against a silver-wire anode electrode placed on the neck muscle. These electrodes were inserted into the inferior olive through the dorsal surface of the medulla. Stimulating electrodes were moved from the surface to about 5 mm in depth by 500-/zm steps. Electrodes were also moved mediolaterally or rostrocaudally in 500-~m steps. Single- or double-stimulation pulses (width, 0.1-0.5 ms; intensity, 5-100 ~A; interval, 3 ms) were applied through these electrodes. At the end of each experiment, the most prominent effective site was marked by passage of currents (intensity, 400/zA DC; duration, 20 s) through the wire electrode, or by iontophoretic ejection of Fast Green FCF (Sigma) from a glass microelectrode. The marked sites were later located histologically. The evoked potentials caused by stimulation of the inferior olive were recorded from the vermal cortex of lobule VII with monopolar ball-tipped silver electrodes (diameter, 600 izm) or monopolar platinum-iridium electrodes (200 /~m diameter without insulation). These electrodes were placed on the surface or inserted into the vermal cortex in lobule VII. For recording and averaging, a storage oscilloscope (Sony Tectronix 5113) and a data-processing computer (ATAC 350, Nihon Kohden Co.) were used. The surgical procedures for isolation of the left renal sympathetic nerve bundle and for recording efferent discharges from these nerves have already been described elsewhere 22. Efferent nerve discharge recorded with Ag-AgC1 wire electrodes was amplified, rectified, integrated and averaged during 20-50 successive sweeps using the data-processing computer. Arterial pressure at the abdominal aorta was recorded with a pressure gauge (MP-3, Nihon Kohden Co.) through a heparin-filled polyethylene tube inserted through the left femoral artery.
242
Retrograde axonal transport of HRP A glass micropipette (tip diameter, 3 0 - 5 0 / z m ) was filled with 10-15% H R P (Toyobo Grade I-C) dissolved in 0.05 M T r i s - H C l buffer solution (pH 8.6) ~4. It was inserted into the vermal cortex of lobules VI and VII, the tip being placed 1-2 mm deep into the cortex. H R P was injected iontophoretically into a small region of the right lobules VI or VII with 2 - 6 p,A D C current (tip positive) for 5 - 2 0 rain. In some cases, 40% H R P solution was injected by pressure through a Hamilton syringe. Following a survival time of 24-48 h, the animals were perfused with 1% paraformaldehyde and 1.25% glutaraldehyde solution (pH 7.4). The brainstem was then cut transversely, and the cerebellum was cut parasagittally into 60-/~m-thick serial sections on a freezing microtome. The sections were incubated according to the tetramethylbenzidine (TMB) procedure r e c o m m e n d e d by Mesulam ~'~'~ and then counterstained with Nuclear Fast Red and mounted. The HRP-injected areas were mapped on the parasagittal sections and were reconstructed on an unfolded surface of the cerebellum. To compare the results obtained from 7 rabbits, the mediolateral extent of lobule VII was normalized to the average width (4.8 +_ 0.42 mm, mean_+ SD, n = 12, as shown in our companion paper 23). HRP-labeled cells in the inferior olive were examined microscopically under bright-field and dark-field illumination. Coronal 60-/xm serial sections of the brainstem obtained from one rabbit provided a standard contour of the inferior olive, in which labeled cells in each rabbit were plotted (Figs. 4 and 5). Total length of the M A O was normalized to the standard rabbit in order to compare the results obtained from 7 rabbits (standard total length, 4.2 mm). RESULTS
Climbing fiber responses on the medial vermal cortex of lobule VII Stimulation with a single pulse (current intensity 5 - 8 ~ A , duration 0.5 ms) in the right c-MAO evoked field potentials in the left medial vermal cortex of lobule V I I a (Fig. 1A). The evoked potentials consisted of a sharp positivity (duration, 2 - 3 ms) followed by 1 or 2 small negative-positive potentials (durations, 5 - 1 0 ms). The amplitude of the maximally evoked potential was 100/zV and its latency 5.2 ms (trace 3 in Fig. 1A). On averaging the measurements in 13 rabbits, the latency of the evoked potentials was 4.8 ms (0.75 ms, SD). These evoked potentials were identified as climbing fiber responses on the basis of the configuration and latency of the potentials, and the contralateral projection 10,2"7 Figure 1C summarizes the stimulating sites from which climbing fiber responses were most effectively induced in the medial vermal cortex of lobule V I I a (one symbol in each of the 13 rabbits tested). These sites are restricted to the medial part of the c-MAO including the beta-nucleus. Effect of inferior olive stimulation on renal sympathetic nerve activity Stimulation of the right medial part of the c-MAO inducing the climbing fiber response on the left medial vermal cerebellum of lobule V I I a produced a depression of the spontaneous discharges in the renal sympathetic nerve (Fig. 2A-l). In order to obtain prominent effects, two pulses paired at 3-ms intervals were applied. The latency of the depression was about 150 ms from the onset of the M A O stimulation and the depression continued for about 450 ms. In 5 rabbits, the vermal cortex of lobule VII (or both lobules VII and V I I I ) was removed by suction. In 3 of these 5 rabbits, the vermal cortex of lobule VII was totally removed and the stimulating electrode was positioned
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Fig. 1. Climbing fiber responses evoked in the medial vermal cortex of lobule VIIa by stimulation of the MAO. (A) Traces 1-4: averaged evoked potentials recorded from the left medial region of lobule VIIa under stimulation at the sites indicated in a dorsal view of the medulla in panel B. Arrows indicate the onset time of stimulation. (B) Coronal section of medulla at 1.5 mm rostral from the caudal pole of the MAO. Filled circles = sites where evoked potentials were induced with stimulating current of below 25 /zA; large filled circles = 5/~A; medium filled circles = 10/~A; small filled circles = 25/xA. zx = ineffective sites with current of 25 g A . x = obex; shaded area shows a vessel on the medullar surface, s = track of the stimulating electrode with which the evoked potential in trace 3 in panel A was recorded; N V = trigeminal nucleus; NXII = hypoglossal nucleus; d.cap = dorsal cap; D A O = dorsal accessory olive. (C) Unfolded contour of the MAO in the inferior olive summarizing the most effective stimulating sites, one symbol for each of the 13 rabbits tested (standard total length of the MAO, 4.2 mm). dm.c.c. = dorsomedial cell column; beta = beta-nucleus.
within the medial part of the c-MAO, as later confirmed histologically. After suction of lobule VII, stimulation of the medial part of the c-MAO no longer induced the depressant response in the renal sympathetic nerve activity (Fig. 2A-2). This observation implies that the depressant responses of renal sympathetic nerve activity induced by stimulation of the medial part of the c-MAO were mediated via the cerebellar vermal cortex of lobule VIIa.
Distribution of labeled cells in the inferior oliue following HRP injection into lobules VI and VII Figure 3 shows photomicrographs of the injection site of H R P and HRP-labeled cells in the c-MAO following injection of H R P into the right lobules VI and VII (R15 and R17 in Figs. 4 and 5). In Figure 4(A, B) the HRP-injected areas in rabbits R l l and R15 partly overlapped each other in lobules VIc and VIIa and together covered the right half from lobule VIb to VIIb. In these 2 cases, all HRP-labeled cells were found in the c-MAO of the contralateral side (Fig. 4C). In rabbit R15, HRP-labeled cells were found in the medial and lateral regions of the c-MAO between 0.3 and 2.7 mm from the caudal pole, and no labeled cell was found in the most caudal region of the MAO. On the other hand, in rabbit R l l , HRP-labeled cells were found in the most caudal region of the MAO (0-2.4 mm). The difference
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Fig. 2. Effect of MAO stimulation on the renal sympathetic nerve activity. (A) Integrated and averaged renal sympathetic nerve activity after stimulation of the c-MAO. A-I and A-2: before and after removal of lobules VII and VII1, respectively. Arrows show the onset time of double-pulse stimulation (width = 0.5 ms; intensity = 50 #,A; interval = 3 ms). (B) Track of the stimulating electrode in MAO illustrated similarly to Fig. 1B, but at 1.2 mm rostral from the caudal pole of MAO. (C) Line drawing of parasagittal cerebellar section through the middle of the cerebellar vermis. The ablated region in the cerebellar vermis is shaded. Other abbreviations are similar to those in Fig. 1. dc = dorsal cap.
between R15 and R l l indicates that lobule VIb is projected from the most caudal region of the M A O (0-0.3 mm), while lobule VIIb is projected from both the medial and lateral parts of the intermediate M A O (2.4-2.7 mm from the caudal pole of the MAO). The overlap of R15 and R l l indicates that lobules VIc and V I I a are projected from the medial and lateral parts of the c - M A O (0.3-2.4 m m from the caudal pole of the MAO).
Organization of olivary projections to lobules VI and VII When H R P injection was limited to the medial half of lobule VII, labeled cells were found in the medial part of the c-MAO (R1 in Figs. 5 and 6C). In rabbit R17, in which the injection area was limited to the medial half of lobule VIIa, HRP-labeled cells were found in the localized region of the medial part of the c-MAO (R17 in Figs. 5 and 6D). These 2 cases indicate that the medial region of lobule V I I a is projected from the more caudal region of the medial M A O than that projecting to lobule VIIb. In rabbits R1, R15 and R16 (Fig. 6B, C), it is also evident that the lateral half of lobule VII is projected from the lateral part of the c-MAO, while lobule V I I a is projected from a more caudal part of the lateral c-MAO. When H R P injection was limited to the lateral half of lobule VI, labeled cells were found in the lateral area of the most caudal region of the M A O (R21 in Fig. 6D). When the injection area was limited to the medial half of lobule VIb, labeled cells were
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Fig. 3. Inferior olive neurons retrogradely labeled by HRP injection into lobules VI and VII. (A) Bright-field photomicrograph of the injection site of lobule Vlla for R17. (B, C) Bright-field photomicrographs at a lower magnification showing HRP-labeled cells in the caudal MAO for RI7 (B) and R15 (C). (D-F) Higher magnification of labeled cells indicated by the arrows in panels B and C. Calibration bars = 500/zm in panel A, 200 p~m in panels B and C, and 50/zm in panels D-F.
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Fig. 4. Distribution of labeled cells in the inferior olive following injection of HRP into the right lobules Vl and VII. (A) Surface view of the posterior vermis (VI-VIII). The injection areas in 2 cases (RI1 and RI5) are shaded, m.l. = midline. (B) Line drawing of parasagittal cerebellar section at 1.08 mm lateral to the midline for Rll and R15. The injection areas in 2 cases are shaded similarly to panel A. (C) Line drawings of coronal sections through the left inferior olive at 300-txm intervals. Labeled cells observed in 5 successive sections (each 60/xm thick) are collectively plotted in each section. Coronal sections in which observed HRP-labeled cells are only illustrated. Numerical figures represent distances of sections from the caudal pole of the MAO. Other abbreviations are similar to those in Fig. 1. v.l.o. = ventrolateral outgrowth.
located in the medial area of the most caudal region of the M A O (R22, Fig. 6D). In these 2 cases as well as in rabbits R l l a n d R16 (Figs. 4 - 6 ) , it is clear that the medial half of lobule VI is projected from the m e d i a l area of the most caudal M A O , a n d that lobule VIc is projected from a more rostral part of the c - M A O . F i g u r e 6 s u m m a r i z e s the d i s t r i b u t i o n of H R P - i a b e l e d cells in the M A O o b t a i n e d from the 7 rabbits following the H R P injection into the posterior vermal cortex of lobules VI a n d VII. T h e s e results indicate clear topographical o r g a n i z a t i o n in the olivocerebellar projection from the M A O to lobules VI a n d V I I . C l i m b i n g fibers to lobules VIb, VIc, V I I a a n d V I I b originate from small areas of the M A O orderly aligned from the caudal to the i n t e r m e d i a t e M A O . F u r t h e r , the medial regions of lobules VI a n d VII are projected from the medial part, a n d the lateral regions of lobules VI a n d VII from the lateral part of the M A O , as shown in F i g u r e 6F.
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When H R P was injected into the medial region of lobule VII, HRP-labeled cells were also found in the lateral region of the beta-nucleus ( R l l , R15, R17 and R1 in Figs. 4-6). Furthermore, in some cases (R15 and R16 in Figs. 4 and 5), some cells were labeled in the dorsal cap (d.cap) and ventrolateral outgrowth (v.l.o.). DISCUSSION
Organization of the cardiovascular zone of rabbit cerebellum In previous studies, we have shown that, in rabbits, stimulation of the medial vermal cortex in lobule VIIa causes a depression of renal sympathetic nerve activity and a reduction of systemic blood pressure 21,22. These results indicate that the medial region of lobule VIIa plays a role in cardiovascular control. The present electrophysiological and H R P study demonstrates that these areas in rabbits receive a climbing fiber input from the medial part of the c-MAO and also that the stimulation of this part of the c-MAO causes a depression of renal sympathetic nerve activity, which disappears after ablation of the vermal cortex of lobule VII (Fig. 2). Furthermore, the latency of the depressant responses in renal sympathetic nerve discharges (150 ms) is close to that (163 + 39 ms) following stimulation of the medial region of the cerebellar lobule VII 22 These results support the view that the depressant responses on the renal sympathetic nerve activity to stimulation of the medial part of the c-MAO are mediated via Purkinje cell excitation in lobule VIIa via climbing fiber input from the medial part of the c-MAO. Our companion paper 23 shows that vagal afferent nerves project to the medial vermal cortex of lobule VIIa as climbing fiber input. It is therefore concluded that the
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Fig. 6. Summary of the olivocerebellar projections to lobules VI and VII. ( A - D ) Reconstructions of the regions in the M A O where HRP-labeled cells were found, in rabbits R l l and R15 in panel A, R1 and R16 in panel B, R1 and R15 in panel C, and R17, RZl and R22 in panel D. Contour of M A O is unfolded in the same m a n n e r as in Fig. 1C. m = medial; 1 = lateral. (E) Each injection area in lobules VI and VII of the cerebellum. Corresponding regions of the cerebellum and the M A O are marked by identical symbols. (F) Schematic illustration of the topography of the olivocerebellar projection in the M A O as revealed in the present study.
vagal afferent nerves project to lobule VIIa via the medial c-MAO, conveying information from the cardiovascular system to lobule VIIa.
Topographical organization of olivocerebellar projection in lobules VI and VII The present study also demonstrates that each of 4 sublobules (VIb, VIc, VIIa, VIIb) of the rabbit cerebellum is respectively supplied with climbing fibers from a particular area of the MAO (Fig. 6F). Martin et al. 17 and Marani and Voogd 16 have demonstrated 3 mediolateral subdivisions of the c-MAO using the acetylcholinesterase method in the opossum and cat. Voogd et al. 28 also showed in the monkey that injection of tritiated leucine into the 'b' column of the c-MAO (intermedial column) labeled the A~-zone. Injections of tritiated leucine or lesions in both the 'a' and 'b' columns of the c-MAO in cats labeled two bundles of olivocerebellar fibers which projected to the A land A z-zones, separately 11. Injections of H R P into the rostromedial fastigial nucleus of the cat labeled Purkinje cells in the medial vermis and also in the medial column of the c-MAO. Injection to the rostrolateral part of the fastigial nucleus labeled more lateral areas in the vermis and the contralateral c-MAO 4,28. These observations are consistent with the present results that each of the medial and lateral regions of the 4 sublobules
249 (VIb, VIc, VIIa, VIIb) receives projections from climbing fibers from a particular area of the MAO (Fig. 6). The present results shown in Figure 6F are comparable to those reported in cats 8, rats 26 and monkeys 7 whereby the central portion of the c-MAO is free of labeled neurons following HRP injection into lobules VI-VIII, and lobule VII receives projections from the medial portion of the c-MAO and beta-nucleus. On the other hand, the present results, which purport that lobule VIb receives climbing fibers from the caudal pole of the c-MAO (Fig. 6), differ from the findings in rats and cats that this area of the MAO projects to lobule VIII. The present study also shows that cells vcere labeled in the d.cap or the v.l.o, following HRP injection into lobule VII in rabbits (R15, R1 and R16 in Figs. 4 and 5). This is at variance with the data in rats 26, cats 8,11,13, monkeys 7 and opossums 17 in which lobule VII receives no climbing fiber from the d.cap or the v.l.o. The discrepancy might reflect a species difference in the olivecerebellar projections among rabbits, rats and cats. ACKNOWLEDGEMENTS
We thank Prof. Kazuhiro Yamada for his constant encouragement during the course of this study, and the late Prof. Kyoji Maekawa for valuable discussion. We also thank Mrs. Kumi Goto and Miss Yumiko Oishi for their assistance. This work was supported in part by a Grant-in-Aid from the Japanese Ministry of Education, Science and Culture (No. 01570068) and a Research Grant from the Naito Foundation Natural Science Scholarship. REFERENCES 1 Apps, R., Columnar organization of the inferior olive projection to the posterior lobe of the rat cerebellum, J. Comp. Neurol., 302 (1990) 236-254. 2 Armstrong, D.M., Harvey, R.J. and Schild R.F., Topographical localization in the olivo-cerebellar projection: an electrophysiological study in the cat, J. Comp. Neurol., 154 (1974) 287-302. 3 Ban, T., Hilliard, J. and Sawyer, C.H., Autonomic and electroencephalographic responses to stimulation of the rabbit cerebellum, Anat. Rec., 136 (1960) 309. 4 Bigar6, F., De efferente Projectie van de Cerebellaire Schors van de Kat, Thesis, University of Leiden (1980). 5 Bowman, J.P. and Sladek, Jr., J.R., Morphology of the inferior olivary complex of the rhesus monkey, J. Comp. Neurol., 152 (1973) 299-316. 6 Brodal, A., Experimentelle Untersuchungen iiber die olivo-cerebell~ire Lokalisation, Z. Ges. Neurol. Psychiatr., 169 (1940) 1-153. 7 Brodal, P. and Brodal, A., The olivocerebellar projection in the monkey. Experimental studies with the method of retrograde tracing of horseradish peroxidase, J. Comp. NeuroL, 201 (1981) 375-393. 8 Brodal, A. and Kawamura, K., Olivocerebellar projection: a review, Adu. Anat. EmbryoL Cell Biol., 64 (1980) 57-72. 9 Campbell, N.C. and Armstrong, D.M., Topographical localization in the olivocerebellar projection in the rat: an autoradiographic study, Brain Res., 275 (1983) 235-249. 10 Eccles, J.C., Llinas, R. and Sasaki, K., The excitatory synaptic action of climbing fibers on the Purkinje cells of the cerebellum, J. Physiol. (Lond.), 182 (1966) 286-296. 11 Groenewegen, H.J. and Voogd, J., The parasagittal zonation within the olivocerebellar projection. I. Climbing fiber distribution in the vermis of cat cerebellum, J. Comp. Neurol., 174 (1977) 417-488. 12 Groenewegen, H.J., Voogd, J. and Freedman, S.L., The parasagittal zonation within the olivocerebellar projection. II. Climbing fiber distribution in the intermediate and hemispheric parts of cat cerebellum, J. Comp. Neurol., 183 (1979) 551-602. 13 Hoddevik, G.H., Brpdal, A. and Walberg, F., The olivocerebellar projection in the cat studied with the method of retrograde axonal transport of horseradish peroxidase. III. The projection to the vermal visual area, J. Comp. Neufol., 169 (1976) 155-170.
250 14 Katayama, S. and Nisimaru, N., Parasagittal zonal pattern of olivo-nodular projections in rabbit cerebellum, Neurosci. Res., 5 (1988) 424-438. 15 Larse!l, O. In J. Jansen (Ed.), The Comparatir'e Anatomy and Histology of the Cerebellum from Monotremes through Apes, University of Minnesota Press, Minneapolis, 1970, pp. 116-267. 16 Marani, E., Voogd, J. and Boekee, A., Acetylcholinesterase staining in subdivisions of the cat's inferior olive, J. Comp. Neurol., 174 (1977) 209-226. 17 Martin, G.F., Dom, R., King, J.S., Robards, M. and Watson C.R.R., The inferior olivary nucleus of the opossum (Didelphis marsupialins virginiana), its organization and connections, J. Comp. Neurol., 160 (1975) 507-534. 18 Mesulam, M.M., Principles of horseradish peroxidase neurochemistry and their applications for tracing neural pathways--axonal transport, enzyme histochemistry and light microscopic analysis. In M.M. Mesulam (Ed.), Tracing Neural Connections with Horseradish Peroxidase. IBRO Handbook Series, Methods in Neuroscience, Wiley, New York, (1982) pp. 1-151. 19 Mesulam, M.M., Tetramethyl benzidine for horseradish peroxidase neurohistochemistry: a noncarcinogenic blue reaction-product with superior sensitivity for visualizing neural afferents and efferents, J. Histochem. Cytochem., 26 (1978) 106-117. 20 Moruzzi, G., The cerebellar influence in the autonomic sphere. In Problems in Cerebellar Physiology, Charles C Thomas, Springfield, IL, 1950, pp. 74-96. 21 Nisimaru, N. and Yamamoto, M., Depressant action of the posterior lobe of the cerebellum upon renal sympathetic nerve activity, Brain Res., 133 (1977) 371-375. 22 Nisimaru, N., Yamamoto, M. and Shimoyama, I., Inhibitory effects of cerebellar cortical stimulation on sympathetic nerve activity in rabbits, Jpn. J. Physiol., 34 (1984) 539-551. 23 Okahara, K. and Nisimaru, N., Climbing fiber responses evoked in Iobule VII of the posterior cerebellum from a vagal afferent nerve in rabbits, Neurosci. Res., 12 (1991) in press. 24 Oscarsson, O and Sj61und, B., The ventral spino-olivocerebellar system in the cat. II. Termination zones in the cerebellar posterior lobe, Exp. Brain Res., 28 (1977) 487-503. 25 Saigal, R.P., Karamanlidis, A.N., Voogd, J., Michaloudi, H. and Mangana, O., Olivocerebellar connections in sheep studied with the retrograde transport of horseradish peroxidase, J. Comp. Neurol., 217 (1983) 440-448. 26 Sugita, S., P~i~illysaro, J, and Noda, H., Topographical organization of the olivocerebellar projection upon the posterior vermis in the rat, Neurosci. Res., 7 (1989) 87-102. 27 Szentfigothai, J. and Rajkovits, K., Uber den Ursprung der Kletterfasern des Kleinhirns, Z. Anat. Entwicklungsgesch., 121 (1959) 130-141. 28 Voogd, J., The olivocerebellar projection in the cat. Exp. Brain Res., Suppl. 6 (1982) 134-161. 29 Voogd, J. and Bigar~, F., Topographical distribution of olivary and cortico-nuclear fibres in the cerebellum: a review. In J. Courville et al. (Eds.), The Inferior Olil~ary Nucleus: Anatomy and Physiology, Raven Press, New York, 1980, pp. 207-234.
Neuroscience Research, 12 (l 991) 240-250 © 1991 Elsevier Scientific Publishers Ireland, Ltd. 0168-0102/91/$03.50
N E U R E S 00492
Olivocerebellar projection to the cardiovascular zone of rabbit cerebellum N. Nisimaru 1, K. Okahara i and S. N a g a o 2 1Department of Physiology, Medical College of Oita, Oita and 2 Department of Physiology, Faculty of Medicine, Unit,ersity of Tokyo, Tokyo, (Japan) (Received 31 May 1991; Accepted 28 June 1991)
Key words." Cerebellum; Lobule VII; Climbing fiber; Olivo-cerebellar projection; Horseradish peroxidase; Topographical organization; Rabbit
SUMMARY Climbing fiber responses were evoked in the medial vermal cortex of lobule Vlla by stimulation of the contralateral medial accessory olive (MAO) in anesthetized, paralyzed rabbits. Effective stimulating sites were localized in a small medial part of the caudal MAO, at 0.4-1.6 m m rostral from the caudal pole of the M A O (total length of the MAO, 4.2 ram). Stimulation of this M A O area induced depression in renal sympathetic nerve activity and this depressant response disappeared after ablation of lobule VIIa. Following injections of horseradish peroxidase into the small areas of lobule Vlb, Vlc, VIIa or VIIb, retrogradely labeled cells were found in corresponding small particular regions of the MAO: lobule VIb to the most caudal part, lobule VIc to the next caudal, lobule VIIa to the most rostral within the caudal MAO, and lobule VIIb further rostrally to the intermediate MAO. There was a clear disparity between the medial halves of Iobules VI and VII projected from the medial M A O and the lateral halves from the lateral MAO. These results show that climbing fiber projections to lobules VI and Vll are topographically organized, and that the medial region of lobule VIla, related to cardiovascular function, receives climbing fibers from a localized small medial region of the caudal MAO.
INTRODUCTION
The cerebellum is involved not only in the control of motor functions but also in that of autonomic functions 3,2o. Our previous studies in anesthetized rabbits suggest that areas of the anterior and posterior lobes of the cerebellar vermis, especially the A-zone of lobule VIIa, are involved in cardiovascular control 21,22. Knowledge of the neuronal circuitry organization of this particular cerebellar area is important in understanding the mechanism of the cerebellar cardiovascular control. A subdivision of the inferior olive sends climbing afferent fibers to a narrow longitudinal zone of the cerebellar cortex 8,12. The A-zone of lobules VI and VII is projected from the caudal half of the medial accessory olive (c-MAO), part of the inferior olive, in cats 2,6,11,13,24, rabbits ~', rats 1,9,26, monkeys 7 and sheep 25. Recently, several separate cell columns in the c-MAO have been found histologically, in particular, by using cholinesterase staining in cats 16, monkeys 5, sheep 25 and opossums 17
Correspondence: N. Nisimaru, Department of Physiology, Medical College of Oita, Oita 879-55, Japan.
241 Some investigators 4.10.29 have also shown that these columns of the c-MAO project separately to A~- and A2-zones of the cerebellar vermis. Such topographical relationships between the MAO and lobule VII have not yet been revealed in rabbits. In this study, we traced olivocerebellar projections from the MAO to lobules VI and VII in rabbits by using electrophysiological techniques and horseradish peroxidase (HRP) axonal transport, and found that the projections are organized with a clear orderly pattern. MATERIALSAND METHODS Twenty-five albino rabbits (body weight, 2.1-3.9 kg) were anesthetized with a-chloralose plus urethane (30 and 400-800 mg/kg, respectively) administered intravenously and supplemented as required. Rabbits were then immobilized by intravenous injection of gallamine triethiodide and artificially respired with intermittent positive pressure. Each rabbit was mounted on a metal frame in a prone position, with the head fixed rigidly with a mouthpiece and pointed screws piercing the zygomatic arch from both the right and left sides. The posterior vermis of the cerebellum and the dorsal surface of medulla were exposed by partial craniotomy. The exposed cerebellar tissues were kept warm by applying warm paraffin oil mixed with vaseline over the surface. Rabbits were kept warm with an electric heating pad. The atlas of the rabbit cerebellum was according to Larsell 15
Electrophysiological experiments For stimulation, monopolar platinum-iridium electrodes (200 Izm diameter without insulation) or 2 M NaC1 glass microelectrodes (0.5-1 MO) were used as a cathode against a silver-wire anode electrode placed on the neck muscle. These electrodes were inserted into the inferior olive through the dorsal surface of the medulla. Stimulating electrodes were moved from the surface to about 5 mm in depth by 500-/zm steps. Electrodes were also moved mediolaterally or rostrocaudally in 500-~m steps. Single- or double-stimulation pulses (width, 0.1-0.5 ms; intensity, 5-100 ~A; interval, 3 ms) were applied through these electrodes. At the end of each experiment, the most prominent effective site was marked by passage of currents (intensity, 400/zA DC; duration, 20 s) through the wire electrode, or by iontophoretic ejection of Fast Green FCF (Sigma) from a glass microelectrode. The marked sites were later located histologically. The evoked potentials caused by stimulation of the inferior olive were recorded from the vermal cortex of lobule VII with monopolar ball-tipped silver electrodes (diameter, 600 izm) or monopolar platinum-iridium electrodes (200 /~m diameter without insulation). These electrodes were placed on the surface or inserted into the vermal cortex in lobule VII. For recording and averaging, a storage oscilloscope (Sony Tectronix 5113) and a data-processing computer (ATAC 350, Nihon Kohden Co.) were used. The surgical procedures for isolation of the left renal sympathetic nerve bundle and for recording efferent discharges from these nerves have already been described elsewhere 22. Efferent nerve discharge recorded with Ag-AgC1 wire electrodes was amplified, rectified, integrated and averaged during 20-50 successive sweeps using the data-processing computer. Arterial pressure at the abdominal aorta was recorded with a pressure gauge (MP-3, Nihon Kohden Co.) through a heparin-filled polyethylene tube inserted through the left femoral artery.
242
Retrograde axonal transport of HRP A glass micropipette (tip diameter, 3 0 - 5 0 / z m ) was filled with 10-15% H R P (Toyobo Grade I-C) dissolved in 0.05 M T r i s - H C l buffer solution (pH 8.6) ~4. It was inserted into the vermal cortex of lobules VI and VII, the tip being placed 1-2 mm deep into the cortex. H R P was injected iontophoretically into a small region of the right lobules VI or VII with 2 - 6 p,A D C current (tip positive) for 5 - 2 0 rain. In some cases, 40% H R P solution was injected by pressure through a Hamilton syringe. Following a survival time of 24-48 h, the animals were perfused with 1% paraformaldehyde and 1.25% glutaraldehyde solution (pH 7.4). The brainstem was then cut transversely, and the cerebellum was cut parasagittally into 60-/~m-thick serial sections on a freezing microtome. The sections were incubated according to the tetramethylbenzidine (TMB) procedure r e c o m m e n d e d by Mesulam ~'~'~ and then counterstained with Nuclear Fast Red and mounted. The HRP-injected areas were mapped on the parasagittal sections and were reconstructed on an unfolded surface of the cerebellum. To compare the results obtained from 7 rabbits, the mediolateral extent of lobule VII was normalized to the average width (4.8 +_ 0.42 mm, mean_+ SD, n = 12, as shown in our companion paper 23). HRP-labeled cells in the inferior olive were examined microscopically under bright-field and dark-field illumination. Coronal 60-/xm serial sections of the brainstem obtained from one rabbit provided a standard contour of the inferior olive, in which labeled cells in each rabbit were plotted (Figs. 4 and 5). Total length of the M A O was normalized to the standard rabbit in order to compare the results obtained from 7 rabbits (standard total length, 4.2 mm). RESULTS
Climbing fiber responses on the medial vermal cortex of lobule VII Stimulation with a single pulse (current intensity 5 - 8 ~ A , duration 0.5 ms) in the right c-MAO evoked field potentials in the left medial vermal cortex of lobule V I I a (Fig. 1A). The evoked potentials consisted of a sharp positivity (duration, 2 - 3 ms) followed by 1 or 2 small negative-positive potentials (durations, 5 - 1 0 ms). The amplitude of the maximally evoked potential was 100/zV and its latency 5.2 ms (trace 3 in Fig. 1A). On averaging the measurements in 13 rabbits, the latency of the evoked potentials was 4.8 ms (0.75 ms, SD). These evoked potentials were identified as climbing fiber responses on the basis of the configuration and latency of the potentials, and the contralateral projection 10,2"7 Figure 1C summarizes the stimulating sites from which climbing fiber responses were most effectively induced in the medial vermal cortex of lobule V I I a (one symbol in each of the 13 rabbits tested). These sites are restricted to the medial part of the c-MAO including the beta-nucleus. Effect of inferior olive stimulation on renal sympathetic nerve activity Stimulation of the right medial part of the c-MAO inducing the climbing fiber response on the left medial vermal cerebellum of lobule V I I a produced a depression of the spontaneous discharges in the renal sympathetic nerve (Fig. 2A-l). In order to obtain prominent effects, two pulses paired at 3-ms intervals were applied. The latency of the depression was about 150 ms from the onset of the M A O stimulation and the depression continued for about 450 ms. In 5 rabbits, the vermal cortex of lobule VII (or both lobules VII and V I I I ) was removed by suction. In 3 of these 5 rabbits, the vermal cortex of lobule VII was totally removed and the stimulating electrode was positioned
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Fig. 1. Climbing fiber responses evoked in the medial vermal cortex of lobule VIIa by stimulation of the MAO. (A) Traces 1-4: averaged evoked potentials recorded from the left medial region of lobule VIIa under stimulation at the sites indicated in a dorsal view of the medulla in panel B. Arrows indicate the onset time of stimulation. (B) Coronal section of medulla at 1.5 mm rostral from the caudal pole of the MAO. Filled circles = sites where evoked potentials were induced with stimulating current of below 25 /zA; large filled circles = 5/~A; medium filled circles = 10/~A; small filled circles = 25/xA. zx = ineffective sites with current of 25 g A . x = obex; shaded area shows a vessel on the medullar surface, s = track of the stimulating electrode with which the evoked potential in trace 3 in panel A was recorded; N V = trigeminal nucleus; NXII = hypoglossal nucleus; d.cap = dorsal cap; D A O = dorsal accessory olive. (C) Unfolded contour of the MAO in the inferior olive summarizing the most effective stimulating sites, one symbol for each of the 13 rabbits tested (standard total length of the MAO, 4.2 mm). dm.c.c. = dorsomedial cell column; beta = beta-nucleus.
within the medial part of the c-MAO, as later confirmed histologically. After suction of lobule VII, stimulation of the medial part of the c-MAO no longer induced the depressant response in the renal sympathetic nerve activity (Fig. 2A-2). This observation implies that the depressant responses of renal sympathetic nerve activity induced by stimulation of the medial part of the c-MAO were mediated via the cerebellar vermal cortex of lobule VIIa.
Distribution of labeled cells in the inferior oliue following HRP injection into lobules VI and VII Figure 3 shows photomicrographs of the injection site of H R P and HRP-labeled cells in the c-MAO following injection of H R P into the right lobules VI and VII (R15 and R17 in Figs. 4 and 5). In Figure 4(A, B) the HRP-injected areas in rabbits R l l and R15 partly overlapped each other in lobules VIc and VIIa and together covered the right half from lobule VIb to VIIb. In these 2 cases, all HRP-labeled cells were found in the c-MAO of the contralateral side (Fig. 4C). In rabbit R15, HRP-labeled cells were found in the medial and lateral regions of the c-MAO between 0.3 and 2.7 mm from the caudal pole, and no labeled cell was found in the most caudal region of the MAO. On the other hand, in rabbit R l l , HRP-labeled cells were found in the most caudal region of the MAO (0-2.4 mm). The difference
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Fig. 2. Effect of MAO stimulation on the renal sympathetic nerve activity. (A) Integrated and averaged renal sympathetic nerve activity after stimulation of the c-MAO. A-I and A-2: before and after removal of lobules VII and VII1, respectively. Arrows show the onset time of double-pulse stimulation (width = 0.5 ms; intensity = 50 #,A; interval = 3 ms). (B) Track of the stimulating electrode in MAO illustrated similarly to Fig. 1B, but at 1.2 mm rostral from the caudal pole of MAO. (C) Line drawing of parasagittal cerebellar section through the middle of the cerebellar vermis. The ablated region in the cerebellar vermis is shaded. Other abbreviations are similar to those in Fig. 1. dc = dorsal cap.
between R15 and R l l indicates that lobule VIb is projected from the most caudal region of the M A O (0-0.3 mm), while lobule VIIb is projected from both the medial and lateral parts of the intermediate M A O (2.4-2.7 mm from the caudal pole of the MAO). The overlap of R15 and R l l indicates that lobules VIc and V I I a are projected from the medial and lateral parts of the c - M A O (0.3-2.4 m m from the caudal pole of the MAO).
Organization of olivary projections to lobules VI and VII When H R P injection was limited to the medial half of lobule VII, labeled cells were found in the medial part of the c-MAO (R1 in Figs. 5 and 6C). In rabbit R17, in which the injection area was limited to the medial half of lobule VIIa, HRP-labeled cells were found in the localized region of the medial part of the c-MAO (R17 in Figs. 5 and 6D). These 2 cases indicate that the medial region of lobule V I I a is projected from the more caudal region of the medial M A O than that projecting to lobule VIIb. In rabbits R1, R15 and R16 (Fig. 6B, C), it is also evident that the lateral half of lobule VII is projected from the lateral part of the c-MAO, while lobule V I I a is projected from a more caudal part of the lateral c-MAO. When H R P injection was limited to the lateral half of lobule VI, labeled cells were found in the lateral area of the most caudal region of the M A O (R21 in Fig. 6D). When the injection area was limited to the medial half of lobule VIb, labeled cells were
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Fig. 3. Inferior olive neurons retrogradely labeled by HRP injection into lobules VI and VII. (A) Bright-field photomicrograph of the injection site of lobule Vlla for R17. (B, C) Bright-field photomicrographs at a lower magnification showing HRP-labeled cells in the caudal MAO for RI7 (B) and R15 (C). (D-F) Higher magnification of labeled cells indicated by the arrows in panels B and C. Calibration bars = 500/zm in panel A, 200 p~m in panels B and C, and 50/zm in panels D-F.
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Fig. 4. Distribution of labeled cells in the inferior olive following injection of HRP into the right lobules Vl and VII. (A) Surface view of the posterior vermis (VI-VIII). The injection areas in 2 cases (RI1 and RI5) are shaded, m.l. = midline. (B) Line drawing of parasagittal cerebellar section at 1.08 mm lateral to the midline for Rll and R15. The injection areas in 2 cases are shaded similarly to panel A. (C) Line drawings of coronal sections through the left inferior olive at 300-txm intervals. Labeled cells observed in 5 successive sections (each 60/xm thick) are collectively plotted in each section. Coronal sections in which observed HRP-labeled cells are only illustrated. Numerical figures represent distances of sections from the caudal pole of the MAO. Other abbreviations are similar to those in Fig. 1. v.l.o. = ventrolateral outgrowth.
located in the medial area of the most caudal region of the M A O (R22, Fig. 6D). In these 2 cases as well as in rabbits R l l a n d R16 (Figs. 4 - 6 ) , it is clear that the medial half of lobule VI is projected from the m e d i a l area of the most caudal M A O , a n d that lobule VIc is projected from a more rostral part of the c - M A O . F i g u r e 6 s u m m a r i z e s the d i s t r i b u t i o n of H R P - i a b e l e d cells in the M A O o b t a i n e d from the 7 rabbits following the H R P injection into the posterior vermal cortex of lobules VI a n d VII. T h e s e results indicate clear topographical o r g a n i z a t i o n in the olivocerebellar projection from the M A O to lobules VI a n d V I I . C l i m b i n g fibers to lobules VIb, VIc, V I I a a n d V I I b originate from small areas of the M A O orderly aligned from the caudal to the i n t e r m e d i a t e M A O . F u r t h e r , the medial regions of lobules VI a n d VII are projected from the medial part, a n d the lateral regions of lobules VI a n d VII from the lateral part of the M A O , as shown in F i g u r e 6F.
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When H R P was injected into the medial region of lobule VII, HRP-labeled cells were also found in the lateral region of the beta-nucleus ( R l l , R15, R17 and R1 in Figs. 4-6). Furthermore, in some cases (R15 and R16 in Figs. 4 and 5), some cells were labeled in the dorsal cap (d.cap) and ventrolateral outgrowth (v.l.o.). DISCUSSION
Organization of the cardiovascular zone of rabbit cerebellum In previous studies, we have shown that, in rabbits, stimulation of the medial vermal cortex in lobule VIIa causes a depression of renal sympathetic nerve activity and a reduction of systemic blood pressure 21,22. These results indicate that the medial region of lobule VIIa plays a role in cardiovascular control. The present electrophysiological and H R P study demonstrates that these areas in rabbits receive a climbing fiber input from the medial part of the c-MAO and also that the stimulation of this part of the c-MAO causes a depression of renal sympathetic nerve activity, which disappears after ablation of the vermal cortex of lobule VII (Fig. 2). Furthermore, the latency of the depressant responses in renal sympathetic nerve discharges (150 ms) is close to that (163 + 39 ms) following stimulation of the medial region of the cerebellar lobule VII 22 These results support the view that the depressant responses on the renal sympathetic nerve activity to stimulation of the medial part of the c-MAO are mediated via Purkinje cell excitation in lobule VIIa via climbing fiber input from the medial part of the c-MAO. Our companion paper 23 shows that vagal afferent nerves project to the medial vermal cortex of lobule VIIa as climbing fiber input. It is therefore concluded that the
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Fig. 6. Summary of the olivocerebellar projections to lobules VI and VII. ( A - D ) Reconstructions of the regions in the M A O where HRP-labeled cells were found, in rabbits R l l and R15 in panel A, R1 and R16 in panel B, R1 and R15 in panel C, and R17, RZl and R22 in panel D. Contour of M A O is unfolded in the same m a n n e r as in Fig. 1C. m = medial; 1 = lateral. (E) Each injection area in lobules VI and VII of the cerebellum. Corresponding regions of the cerebellum and the M A O are marked by identical symbols. (F) Schematic illustration of the topography of the olivocerebellar projection in the M A O as revealed in the present study.
vagal afferent nerves project to lobule VIIa via the medial c-MAO, conveying information from the cardiovascular system to lobule VIIa.
Topographical organization of olivocerebellar projection in lobules VI and VII The present study also demonstrates that each of 4 sublobules (VIb, VIc, VIIa, VIIb) of the rabbit cerebellum is respectively supplied with climbing fibers from a particular area of the MAO (Fig. 6F). Martin et al. 17 and Marani and Voogd 16 have demonstrated 3 mediolateral subdivisions of the c-MAO using the acetylcholinesterase method in the opossum and cat. Voogd et al. 28 also showed in the monkey that injection of tritiated leucine into the 'b' column of the c-MAO (intermedial column) labeled the A~-zone. Injections of tritiated leucine or lesions in both the 'a' and 'b' columns of the c-MAO in cats labeled two bundles of olivocerebellar fibers which projected to the A land A z-zones, separately 11. Injections of H R P into the rostromedial fastigial nucleus of the cat labeled Purkinje cells in the medial vermis and also in the medial column of the c-MAO. Injection to the rostrolateral part of the fastigial nucleus labeled more lateral areas in the vermis and the contralateral c-MAO 4,28. These observations are consistent with the present results that each of the medial and lateral regions of the 4 sublobules
249 (VIb, VIc, VIIa, VIIb) receives projections from climbing fibers from a particular area of the MAO (Fig. 6). The present results shown in Figure 6F are comparable to those reported in cats 8, rats 26 and monkeys 7 whereby the central portion of the c-MAO is free of labeled neurons following HRP injection into lobules VI-VIII, and lobule VII receives projections from the medial portion of the c-MAO and beta-nucleus. On the other hand, the present results, which purport that lobule VIb receives climbing fibers from the caudal pole of the c-MAO (Fig. 6), differ from the findings in rats and cats that this area of the MAO projects to lobule VIII. The present study also shows that cells vcere labeled in the d.cap or the v.l.o, following HRP injection into lobule VII in rabbits (R15, R1 and R16 in Figs. 4 and 5). This is at variance with the data in rats 26, cats 8,11,13, monkeys 7 and opossums 17 in which lobule VII receives no climbing fiber from the d.cap or the v.l.o. The discrepancy might reflect a species difference in the olivecerebellar projections among rabbits, rats and cats. ACKNOWLEDGEMENTS
We thank Prof. Kazuhiro Yamada for his constant encouragement during the course of this study, and the late Prof. Kyoji Maekawa for valuable discussion. We also thank Mrs. Kumi Goto and Miss Yumiko Oishi for their assistance. This work was supported in part by a Grant-in-Aid from the Japanese Ministry of Education, Science and Culture (No. 01570068) and a Research Grant from the Naito Foundation Natural Science Scholarship. REFERENCES 1 Apps, R., Columnar organization of the inferior olive projection to the posterior lobe of the rat cerebellum, J. Comp. Neurol., 302 (1990) 236-254. 2 Armstrong, D.M., Harvey, R.J. and Schild R.F., Topographical localization in the olivo-cerebellar projection: an electrophysiological study in the cat, J. Comp. Neurol., 154 (1974) 287-302. 3 Ban, T., Hilliard, J. and Sawyer, C.H., Autonomic and electroencephalographic responses to stimulation of the rabbit cerebellum, Anat. Rec., 136 (1960) 309. 4 Bigar6, F., De efferente Projectie van de Cerebellaire Schors van de Kat, Thesis, University of Leiden (1980). 5 Bowman, J.P. and Sladek, Jr., J.R., Morphology of the inferior olivary complex of the rhesus monkey, J. Comp. Neurol., 152 (1973) 299-316. 6 Brodal, A., Experimentelle Untersuchungen iiber die olivo-cerebell~ire Lokalisation, Z. Ges. Neurol. Psychiatr., 169 (1940) 1-153. 7 Brodal, P. and Brodal, A., The olivocerebellar projection in the monkey. Experimental studies with the method of retrograde tracing of horseradish peroxidase, J. Comp. NeuroL, 201 (1981) 375-393. 8 Brodal, A. and Kawamura, K., Olivocerebellar projection: a review, Adu. Anat. EmbryoL Cell Biol., 64 (1980) 57-72. 9 Campbell, N.C. and Armstrong, D.M., Topographical localization in the olivocerebellar projection in the rat: an autoradiographic study, Brain Res., 275 (1983) 235-249. 10 Eccles, J.C., Llinas, R. and Sasaki, K., The excitatory synaptic action of climbing fibers on the Purkinje cells of the cerebellum, J. Physiol. (Lond.), 182 (1966) 286-296. 11 Groenewegen, H.J. and Voogd, J., The parasagittal zonation within the olivocerebellar projection. I. Climbing fiber distribution in the vermis of cat cerebellum, J. Comp. Neurol., 174 (1977) 417-488. 12 Groenewegen, H.J., Voogd, J. and Freedman, S.L., The parasagittal zonation within the olivocerebellar projection. II. Climbing fiber distribution in the intermediate and hemispheric parts of cat cerebellum, J. Comp. Neurol., 183 (1979) 551-602. 13 Hoddevik, G.H., Brpdal, A. and Walberg, F., The olivocerebellar projection in the cat studied with the method of retrograde axonal transport of horseradish peroxidase. III. The projection to the vermal visual area, J. Comp. Neufol., 169 (1976) 155-170.
250 14 Katayama, S. and Nisimaru, N., Parasagittal zonal pattern of olivo-nodular projections in rabbit cerebellum, Neurosci. Res., 5 (1988) 424-438. 15 Larse!l, O. In J. Jansen (Ed.), The Comparatir'e Anatomy and Histology of the Cerebellum from Monotremes through Apes, University of Minnesota Press, Minneapolis, 1970, pp. 116-267. 16 Marani, E., Voogd, J. and Boekee, A., Acetylcholinesterase staining in subdivisions of the cat's inferior olive, J. Comp. Neurol., 174 (1977) 209-226. 17 Martin, G.F., Dom, R., King, J.S., Robards, M. and Watson C.R.R., The inferior olivary nucleus of the opossum (Didelphis marsupialins virginiana), its organization and connections, J. Comp. Neurol., 160 (1975) 507-534. 18 Mesulam, M.M., Principles of horseradish peroxidase neurochemistry and their applications for tracing neural pathways--axonal transport, enzyme histochemistry and light microscopic analysis. In M.M. Mesulam (Ed.), Tracing Neural Connections with Horseradish Peroxidase. IBRO Handbook Series, Methods in Neuroscience, Wiley, New York, (1982) pp. 1-151. 19 Mesulam, M.M., Tetramethyl benzidine for horseradish peroxidase neurohistochemistry: a noncarcinogenic blue reaction-product with superior sensitivity for visualizing neural afferents and efferents, J. Histochem. Cytochem., 26 (1978) 106-117. 20 Moruzzi, G., The cerebellar influence in the autonomic sphere. In Problems in Cerebellar Physiology, Charles C Thomas, Springfield, IL, 1950, pp. 74-96. 21 Nisimaru, N. and Yamamoto, M., Depressant action of the posterior lobe of the cerebellum upon renal sympathetic nerve activity, Brain Res., 133 (1977) 371-375. 22 Nisimaru, N., Yamamoto, M. and Shimoyama, I., Inhibitory effects of cerebellar cortical stimulation on sympathetic nerve activity in rabbits, Jpn. J. Physiol., 34 (1984) 539-551. 23 Okahara, K. and Nisimaru, N., Climbing fiber responses evoked in Iobule VII of the posterior cerebellum from a vagal afferent nerve in rabbits, Neurosci. Res., 12 (1991) in press. 24 Oscarsson, O and Sj61und, B., The ventral spino-olivocerebellar system in the cat. II. Termination zones in the cerebellar posterior lobe, Exp. Brain Res., 28 (1977) 487-503. 25 Saigal, R.P., Karamanlidis, A.N., Voogd, J., Michaloudi, H. and Mangana, O., Olivocerebellar connections in sheep studied with the retrograde transport of horseradish peroxidase, J. Comp. Neurol., 217 (1983) 440-448. 26 Sugita, S., P~i~illysaro, J, and Noda, H., Topographical organization of the olivocerebellar projection upon the posterior vermis in the rat, Neurosci. Res., 7 (1989) 87-102. 27 Szentfigothai, J. and Rajkovits, K., Uber den Ursprung der Kletterfasern des Kleinhirns, Z. Anat. Entwicklungsgesch., 121 (1959) 130-141. 28 Voogd, J., The olivocerebellar projection in the cat. Exp. Brain Res., Suppl. 6 (1982) 134-161. 29 Voogd, J. and Bigar~, F., Topographical distribution of olivary and cortico-nuclear fibres in the cerebellum: a review. In J. Courville et al. (Eds.), The Inferior Olil~ary Nucleus: Anatomy and Physiology, Raven Press, New York, 1980, pp. 207-234.