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Electrophysiological analysis of the tecto-olivo-cerebellar (lobulus simplex) projection in the rat
Brain Research, 417 (1987) 371-376 Elsevier
371
BRE 22417
Electrophysiological analysis of the tecto-olivo-cerebellar (Iobulus simplex) projection in the rat Tadashi Akaike Department of Physiology, Nagoya UniversitySchool of Medicine, Nagoya (Japan) (Accepted 5 May 1987)
Key words: Cerebellum; Superior colliculus; Medial accessory olive; Climbing fiber response; Lobulus simplex; Rat
In albino rats the deep layers of the superior colliculus were stimulated, and climbing fiber responses of Purkinje cells were explored in the medial region of the lobulus simplex. They were identified in a strip (ca. 0.7-1.0 mm wide) in the most medial region of folium b. In the tectorecipient zone of the medial accessory olive antidromically evoked potentials from the lobulus simplex were recorded laterally to those from lobule VII. Evidence is presented that climbing fibers innervating the zone in the lobulus simplex are axon collaterals of the inferior olivary neurons which project to crus II.
In mammals the olivocerebellar projection is characterized by clear longitudinal zonations of their termination in the cerebellar cortex 12A8'37'49. It was demonstrated in the cat that climbing fibers originating from inferior olivary neurons in a certain sector of the nucleus terminate in one longitudinal zone across many folia, lobules or lobes 9'13'37'5°. Inferior olivary neurons in a tectorecipient zone in the caudomedial quadrant of the medial accessory olive are so far supposed to project exclusively to the vermal region of lobule VII of Larsell in the cat and rat 3,3°'34. Recently it was reported in the rat that climbing fiber responses could be evoked in a longitudinal zone in the medial region of crus II by stimulation of the deep layers of the superior colliculus 5,6. Purkinje cells in these two areas are innervated respectively by climbing fibers originating from different inferior olivary neurons in the tectorecipient zone. In the present study climbing fiber responses were surveyed in the lobulus simplex, which were evoked by stimulation of the deep layers of the suPerior colliculus. A new zone is identified in the most medial region of the lobulus simplex (folium b). The area is supposed to be a longitudinal extention of the tecto-olivo-recipient zone in crus II. Evidence is presented that the new zone in
the lobulus simplex is innervated by axon collaterals of inferior olivary neurons projecting to the zone in crus II. Eleven albino rats were used (Wistar, male adult, 200-350 g). Experimental procedures were described elsewhere 2-6. Briefly, the animals were anesthetized with chloral hydrate (400 mg/kg, i.p.). The trachea was cannulated, but usually the animals were not, artificially respirated except when immobilized by gallamine triethiodide. The same results were obtained with or without immobilization. Body temperature was maintained by a heating pad. A burr hole was made at the midline (A 8.0, L 0.0) 4°. The caudal part of the cerebrum, cerebellum and the upper part of the spinal cord were exposed and were covered with a mixture of warmed vaseline and liquid paraffin. A bipolar electrode insulated except the tips (separated 1.0-1.5 mm) was placed through the cerebrum at the optic nerve (A 8.0, L 0.0). A monopolar electrode was placed in the deep layers of the superior colliculus (A 1.0-1.5, L 1.5-2.0). Using a current stimulus of 100-300/~A, I adjusted the depth of the tectal electrode to evoke maximum climbing fiber responses in crus II and lobule VII in the ipsilateral side 3'5,6. In 6 animals bipolar or monopolar elec-
Correspondence: T. Akaike, Department of Physiology, Nagoya University School of Medicine, Nagoya Aichi 466, Japan. 0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
372 tive potentials). By lowering the tip of the electrode for 100 or 200 # m , evoked potentials were reversed to negative field potentials having a steeply falling phase and a longer duration (ca. 7 - 1 5 ms). When the tip of the electrode was lowered a little further, amplitudes of the negative field potentials increased, but their duration decreased a little, and they had a more steeply falling phase. It suggests that at the most superficial layer there are sources of the synaptic current, and at the middle layer where the negative field potentials are recorded, there are sinks of synaptic current evoked by climbing fibers, and at the deepest layer the tip of the recording electrode picks up action currents which are generated at the dendrites of Purkinje cells. When it was lowered 50-100 # m deeper into the cerebellar cortex, it reached another layer where the negative potentials were again reversed to positive field potentials with long duration (ca. 7-15 ms: deep positive potentials). The latter were usually preceded by short negative potentials. There, unit discharges were often observed, and occasionally the glass micropipettes impinged intracellularly. It disclosed large excitatory postsynap-
trodes were placed in the tecto-olivo-recipient zones of lobule VII, crus II and/or the lobulus simplex. For electrical stimulation single rectangular pulses (0.2 ms in duration, 50-500 # A of current intensity) were derived through isolation units and delivered at a rate of 0.5-1.0 Hz. For recording glass micropipettes filled with 2 N NaC1 solution (usually saturated with Fast green) and having an electric resistance of 3 - 5 Mr2 were used. They were connected to a neutralized preamplifier and then to an A T A C 250 (a storage type of oscilloscope, Nihonkohden). Responses were photographed and analyzed after experiments. Recording and stimulating sites were marked by ejecting dye or by making a small lesion with electric current. These were identified histologically after experiments. Recording electrodes were lowered perpendicularly in the cerebellum. When a tip of the recording electrode touched the cerebellar surface in the medial region of folium b in the lobulus simplex (Fig. 1), positive field potentials were obtained (latency; 9-11 ms), which had steeply rising and falling phases, and a relatively short duration (ca. 3 - 5 ms: surface posi-
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2 4. J Fig. 1. Laminar analysis of climbing fiber responses of Purkinje cells in the cerebellar cortex of an albino rat. They were evoked by stimulation of the deep layers of the superior colliculus with single pulses (200 #A, 0.2 ms), and were recorded in the medial region of folium b of the lobulus simplex by a perpendicular penetration of a recording electrode (vertical line). Evoked responses (1-24) were reco~ed every 50 #m in depth. Twenty responses were superposed. The recording sites were numbered, and were indicated by short horizontal bars in a camera lucida drawing of a parasagittal section of the cerebellum. Purkinje cell somas were indicated by dots. Inset: a camera lucida drawing of a parasagittal section of the cerebellum and the lower brainstem, in which the recording area was indicated by a square. Calibration: 2 mV, 50 ms (see text for further explanation).
373 tic potentials, often superposed by a few action potentials, which were characteristic of climbing fiber responses of Purkinje cells 2,3,5,2i. Histological examination revealed that the first reversal points were within the molecular layer, and the second reversal points were between the molecular and the granular layers of the cerebellar cortex. The profile of the evoked potentials across the cortical layers of the cerebellum is characteristic of climbing fiber responses of Purkinje cells in the rat. The laminar analysis of climbing fiber responses has been described in detail in the cat 22, and it is essentially the same as in the rat. Climbing fiber responses evoked by the tectal stimulation, which had the laminar profile described above, were surveyed in the lobulus simplex, mainly in its medial half. They were obtained in the most medial region of folium b, often adjacent to and lateral to the paravermal vein. The responsive area in the cerebellar surface extended longitudinally; usually the caudal region was wider (ca. 0.7-1.0 mm) than the rostral region (ca. 0.5-0.7 mm). Furthermore, these potentials were surveyed and identified deeply in the fissure of folium b. The responsive area seems to extend longitudinally throughout the cerebellar cortex not only in the cerebellar surface but also in the fissure in the medial region of folium b of the lobulus simplex. The amplitudes of the evoked potentials were almost of the same size within the zone, and they decreased steeply at the boundary and disappeared outside the zone. Electrical stimulation of the optic nerve was of no effect. Stimulation of the face region, specifically the vibrissae area in the contralateral side with subcutaneously placed metal electrodes, sometimes evoked climbing fiber responses in the area, but with a much longer latency. Under deep anesthesia they fluctuated or were not evoked. In 6 animals the inferior olivary nucleus was surveyed to record field potentials which were evoked either orthodromically by tectal stimulation, or antidromically by stimulation of the cerebellar cortices within the tecto-olivo-recipient zone of lobule VII, crus II or the lobulus simplex. The tectorecipient zone was, as described in previous papers 4'6 immediately rostral to a caudal, visually responsive area. It extended from the midline to 0.4-0.5 mm laterally, 0.4-0.5 mm long rostrocaudally and 0.1-0.2 m m thick in depth. Histologically it corresponded to subnucleus c in the caudomedial quadrant of the medial
accessory olive at the rostrocaudal level of the middle one-third of nucleus ft. Antidromic field potentials evoked by stimulation of lobule V I I were recorded in the most medial region (from the midline to 0.2-0.3 mm laterally) in the tectorecipient zone, while those evoked by stimulation of crus II were recorded in the lateral region (from 0.2-0.3 m m to 0.4-0.5 m m laterally) in the zone of the inferior olive. Antidromic field potentials, evoked by stimulation of the medial region of folium b of the lobulus simplex were recorded in the lateral region of the tectorecipient zone. The area for the lobulus simplex was included in and almost overlapped with the area for crus II. However, amplitudes of antidromically evoked potentials by stimulation of the lobulus simplex were smaller or larger than those evoked by crus II stimulation at certain sites within the tectorecipient zone. In 3 animals I investigated whether or not these climbing fiber responses of Purkinje cells in the lobulus simplex were evoked by axon collaterals of the inferior olivary neurons projecting to the lobule VII and/or crus II (Fig. 2). When the tip of a recording electrode was in the upper portion of the molecular layer in the lobulus simplex, it recorded surface positive potentials in response to the tectal stimulation (latency; 9-11 ms); the same form of field potentials was obtained with a little shorter latency (ca. 8 - 1 0
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3 4Fig. 2. Climbing fiber responses of Purkinje cells evoked by axonal reflex in the lobulus simplex of a rat cerebellum. Experimental procedures were drawn in the dorsal view of the cerebellum. The cerebellar cortices within the tecto-olivo-recipient zones (blackened areas) of crus II (Cr II), or lobule VII (VII) were stimulated with bipolar electrodes (150 #A, 0.2 ms). The evoked responses (1-3) were recorded at 3 different depths of a penetration of the recording electrode in the cerebellar cortex in the zone of folium b of the lobulus simplex (LS b). When the electrode picked up the surface positive (1), negative (2) or deep positive potentials (3) in response to tectal stimulation (SC: 200 #A, 0.2 ms), the same response types were obtained by crus II stimulation at the same depths. Twenty responses were superposed. Calibration: 1 mV, 25 ms.
374 ms) by stimulation of the cerebellar cortex within the tecto-olivo-recipient zone of crus II. When the tip of the electrode was moved deeper, and it recorded negative field potentials or deep positive potentials in response to tectal stimulation, the same forms of the field potentials were evoked by crus II stimulation. The reverse relation was also true; that is, climbing fiber responses were evoked in the tecto-olivo-recipient zone of crus II in response to stimulation of the zone in the lobulus simplex. Intracellular recordings also confirmed typical climbing fiber responses of Purkinje cells in crus II by stimulation of the lobulus simplex. Stimulation of lobule VII was of no effect in the present study. These findings suggest that at least some climbing fibers innervating Purkinje cells in the most medial region of folium b of the lobulus simplex are axon collaterals of inferior olivary neurons projecting to the tecto-olivo-recipient zone of crus II. The last question concerns the extent to which climbing fibers innervating the lobulus simplex (folium b) are axon collaterals. Climbing fiber responses were surveyed every 100 ktm across the tecto-olivorecipient zone of the lobulus simplex while the cerebellar cortex within the zone of crus II was stimulated. They were always recorded in any sector in the zone of the cerebellar surface of the lobulus simplex. Although I have not investigated systematically the responses in the cerebellar cortex deep in the fissure of folium b, they seemed to be evoked. However, the reverse relation was not always true: stimulation of the lobulus simplex evoked climbing fiber responses in the zone of crus II, except for its medial portion. This is reasonable since the area of the zone in crus II seems to be wider than that in the lobulus simplex. These findings suggest that a large part of climbing fibers innervating Purkinje cells in the tecto-olivo-recipient zone of the lobulus simplex are axon collaterals. Anatomical investigations by Wiklund et al. 5°, using the anterograde axonal transport techniques, provided evidence in the rat that there were axonal branches of inferior olivary neurons; one projects to the lobulus simplex, the other to crus II. The possibility of current spread from the tectal stimulating electrode to the visual and other afferent pathways and/or various mesencephalo-olivary pathways may be eliminated because the intensity of stimulus current (50-100 ktA) was low enough to evoke climbing fiber responses in the lobulus simplex, and
because all mesencephalo-olivary pathways except the tecto-olivary projection, were described exclusively to terminate in the ipsilateral side 3,5,14,48. Furthermore, the tectorecipient zone in the inferior olive, identified in the present study, was almost the same area as that described by Hess 3°, and by Swenson and Castro 48, using anterograde axonal transport techniques in the rat. Shambes et al. 35'45'46reported that there are extensive tactile projections to the granule layers of the rat cerebellum, specifically in crus I, crus II, paramedian lobule, lobulus simplex (folium b) and the uvula (folia 9a) of the posterior vermis. When the tecto-olivorecipient zone of the lobulus simplex in the present study is referred to their Fig. 1 (ref. 45), it is composed of 3 regions where the granule cells have receptive fields in the contralateral and ipsilateral lower lip, and the ipsilateral vibrissae, respectively. The zone in crus II is also composed of 3 regions where the granule cells have receptive fields in the contralateral, bilateral and ipsilateral perioral areas, respectively46. Purkinje cells in the medial region of crus II are described to innervate neurons in the dorsolateral protuberance of the cerebellar medial nucleus (Fdlp) 1°. It is probable that Purkinje cells in the zone of the lobulus simplex also project to the Fdlp 26. In a previous paper s, I discussed differences in modalities of their afferents such as mossy fibers between the zone of lobule VII and that of crus II, and speculated a sequence of the information processing starting from Purkinje cells in those zones. It may also apply to the zone of the lobulus simplex. Recently Redgrave et al. 44 disclosed that the deep layers of the superior colliculus in rats can be divided into two laminae which contain different neuronal groups, respectively. That is, in the upper layer (stratum album intermediale according to his nomenclature) are located most neurons of origin of the contralateral predorsal bundle, which include neurons of origin of the tecto-olivary projection 33. He suggested that most of the tectal cells of origin of the predorsal bundle also project rostrally to targets in the ipsilateral caudal diencephalon. This is consistent with the electrophysiological findings of Chevalier and Deniau 19. Anatomical and electrophysiological investigations 1'8'17'20'23'24'27'28'32'38'39'41'42indicate that neurons in the deep layers of the superior colliculus, which include neurons of origin of the tecto-olivary
375 projection, have multimodal sensory properties. That is, they are responsive to somatosensory, visual and/or auditory inputs. They have many axonal branches: one projects rostrally to targets in the ipsilateral side, which are supposed to be the center of attention 19'36'43'44, and the other caudally to the precerebellar nucleus in the tegmental reticular nucleus ~5,43, to the p r e o c u l o m o t o r region in the bulbar reticular formation 29,43, and to the medial accessory olive in the contralateral side 3'6'3°'33'4s. In the bulbar reticular formation they may have many axonal branches 29. Thus, in b o t h afferent and efferent aspects these neurons m a y have integrative functions. The presumptive integrative p r o p e r t i e s of neurons of origin of the tecto-olivary p r o j e c t i o n clearly contrast with the visual pretecto-olivary projection. The latter has definite properties of the receptive field, such as direction and velocity of m o v e m e n t of the receptive fieldt6,47. 1 Abrahams, V.C. and Rose, P.K., Projections of extraocular, neck muscle and retinal afferents to superior colliculus in the cat: their connections to cells of origin of tectospinal tract, J. Neurophysiol., 38 (1975) 10-18. 2 Akaike, T., Lobular distribution of visual climbing fiber responses in the cerebellum, Brain Research, 327 (1985) 359-361. 3 Akaike, T., Electrophysiological analysis of the tecto-olivocerebellar (lobule VII) projection in the rat, Brain Research, 340 (1985) 369-372. 4 Akaike, T., Spatial distribution of evoked potentials in the inferior olivary nucleus by stimulation of the visual afferents in the rat, Brain Research, 368 (1986) 183-187. 5 Akaike, T., Electrophysiological analysis of the tecto-olivo-cerebellar (crus II) projection in the rat, Brain Research, 378 (1986) 186-190. 6 Akaike, T., Differential localization of inferior olivary neurons projecting to the tecto-olivo-recipient zone of lobule VII or crus II in the rat cerebellum, Brain Research, 386 (1986) 400-404. 7 Anderson, G., Demonstration of a cuneate relay in a cortico-olivo-cerebellar pathway in the cat, Neurosci. Lett., 46 (1984) 47-52. 8 Antonetty, C.M. and Webster, K.E., The organization of the spino-tectal projection. An experimental study in rat, J. Comp. Neurol., 163 (1975) 449-466. 9 Armstrong, D.M., Harvey, R.J. and Schild, R.F., The spatial organization of climbing fibre branching in the cat cerebellum, Exp. Brain Res., 18 (1973) 40-58. 10 Armstrong, D.M. and Schild, R.F., An investigation of the cerebellar corticonuclear projections in the rat using an autoradiographic tracing method. II. Projections from the hemispheres, Brain Research, 141 (1978) 235-249. 11 Berkley, K.J. and Worden, I.G., Projection to the inferior olive of the cat. I. Comparisons of the input from the dorsal column nuclei, the lateral cervical nucleus, the spino-olivary pathways, the cerebral cortex and the cerebellum, J.
The superior colliculus is now focused as an output stage of the basal ganglia through the striatonigral, and nigrotectal projections 25'31'5t. The ipsilateral nigrotectal projections in rats were identified in the full extent of the stratum album intermediale 25. Therefore it is most p r o b a b l e that neurons of origin of the predorsal bundle are targets of the nigrotectal fibers in rats. Thus, the p e r i p h e r a l sensory and central motor information converged in and g e n e r a t e d in the cerebral cortex is t r a n s f o r m e d and t r a n s p o r t e d to the cerebellum as climbing fiber inputs, not only via cortico-olivary, cortico-tectal and other cortieo-preolivary nuclear projections 7'11'24'4t'48'52, but also via the cerebral cortex-striatum-substantia nigra-superior colliculus-inferior olive. To u n d e r s t a n d the function of climbing fiber afferents to the cerebellum it is important to clarify physiological properties of neurons identified as origin of the tecto-olivary projections.
Comp. Neurol., 180 (1978) 237-252. 12 Beyerl, B.D., Borgers, L.F., Swearingen, N. and Sidman, R.L., Parasagittal organization of the olivocerebellar projection in the mouse, J. Comp. Neurol., 209 (1982) 339-346. 13 Brodal, A. and Kawamura, K., Olivocerebellar projection: a review, Adv. Anat. Embryol. Cell Biol., 64 (1980) 1-140. 14 Brown, J.T., Chan-Palay, V. and Palay, S.L., A study of afferent input to the inferior olivary complex in the rat by retrograde and anterograde axonal transport of horseradish peroxidase, J. Comp. Neurol., 176 (1977) 1-22. 15 Burne, R.A., Azizi, S.A., Mihailoff, G.A. and Woodward, D.J., The tectopontine projection in the rat with comments on visual pathways to the basilar pons, J. Cornp. Neurol., 202 (1981) 287-307. 16 Cazin, L., Precht, W. and Lannou, J., Firing characteristics of neurons mediating optokinetic responses to rat's vestibular neurons, Pfliigers Arch. Ges. Physiol., 386 (1980) 221-230. 17 Chalupa, L.M. and Rhoades, R.W., Responses of visual, somatosensory, and auditory neurons in the golden hamster's superior colliculus, J. Physiol., 270 (1977) 595-626. 18 Chan-Palay, L.M., Palay, S.M., Brown, S.L. and Van Itallie, C., Sagittal organization of olivocerebellar and reticulocerebellar projections: autoradiographic studies with 35Smethionine, Exp. Brain Res., 30 (1977) 561-576. 19 Chevalier, G. and Deniau, J.M., Spatio-temporal organization of a branched tecto-spinal/tecto-diencephalicneuronal system, Neuroscience, 12 (1984) 429-439. 20 Dr~iger, U.C. and Hubel, D.H., Responses to visual stimulation and relationship between visual, auditory and somatosensory inputs in mouse superior colliculus, J. Neurophysiol., 38 (1975) 690-713. 21 Eccles, J.C., Llinas, R. and Sasaki, K., The excitatory synaptic action of climbing fibres on the Purkinje cells of the cerebellum, J. Physiol. (London), 182 (1966) 268-296. 22 Eccles, J.C., Provini, L., Strata, P. and T~iborikov~i,H.,
376 Analysis of electrical potentials evoked in the cerebellar anterior lobe by stimulation of hindlimb and forelimb nerves, Exp. Brain R es. , 6 (1968) 171-194. 23 Finlay, B.L., Schneps, S.E., Wilson, K.G. and Schneider, G.E., Topography of visual and somatosensory projections to the superior colliculus of the golden hamster, Brain Research, 142 (1978) 223-235. 24 Fujikado, T., Fukuda, Y. and Iwama, K., Two pathways from the facial skin to the superior colliculus in the rat, Brain Research, 212 (1981) 131-135. 25 Gerfen, C.R., Staines, W.A., Arbuthnott, G.W. and Fibiger, H.C., Crossed connections of the substantia nigra in the rat, J. Comp. Neurol., 207 (1982) 283-303. 26 Goodman, D.C., Hallett, R.E. and Welch, R.B., Patterns of localization in the cerebellar cortico-nuclear projections of the albino rat, J. Comp. Neurol., 121 (1963) 51-67. 27 Gordon, B.G., Receptive fields in deep layers of cat superior colliculus, J. Neurophysiol., 36 (1973) 157-178. 28 Graham, J., Pearson, H.E., Berman, N. and Murphy, E.H., Laminar organization of superior colliculus in the rabbit: a study of receptive-field properties of single units, J. Neurophysiol., 45 (1981) 915-932. 29 Grantyn, A. and Grantyn, R., Axonal patterns and sites of termination of cat superior colliculus neurons projecting in the tecto-bulbo-spinal tract, Exp. Brain Res., 46 (1982) 243-256. 30 Hess, D.T., The tecto-olivo-cerebellar pathway in the rat, Brain Research, 250 (1982) 143-148. 31 Hikosaka, O. and Wurtz, R.H., Visual and oculomotor functions of monkey substantia nigra to superior colliculus, J. Neurophysiol., 49 (1983) 1285-1301. 32 Huerta, M.F., Frankfurter, A. and Hatting, J.K., Studies of the principal sensory and spinal trigeminal nuclei of the rat: projection to the superior colliculus, inferior olive and cerebellum, J. Comp. Neurol., 220 (1983) 147-167. 33 Huerta, M.F. and Harting, J.K., The mammalian superior colliculus: studies of its morphology and connections. In H. Vaneges (Ed.), Comparative Neurology of the Optic Tecturn, Plenum, New York, 1984, pp. 687-793. 34 Jeneskog, T., Zonal termination of the tecto-olivocerebellar pathway in the cat, Exp. Brain Res., 49 (1983) 353-362. 35 Joseph, J.W., Shambes, G.M. and Welker, W., Tactile projections to granule cells in caudal vermis of the rat's cerebellum, Brain Behav. Evol., 15 (1978) 141-149. 36 Kaufman, E.F.S. and Rosenquist, A.C., Efferent projections of the thalamic intralaminar nuclei in the cat, Brain Research, 335 (1985) 257-280. 37 Kawamura, K. and Hashikawa, T., Olivocerebellar projections in the cat studied by means of anterograde axonal transport of labelled amino acids as tracers, Neuroscience, 4 (1979) 1615-1633. 38 Killackey, H.P. and Erzulumlu, R.S., Trigeminal projections to the superior colliculus of the rat, J. Comp. Neurol.,
201 (1981) 221-242. 39 King, A.J. and Palmer, A.R., Integration of visual and auditory information in bimodal neurones in the guinea-pig superior colliculus, Exp. Brain Res., 60 (1985) 492-500. 40 K6nig, J.F.R. and Klippel, R.A., The Rat Brain: A Stereotaxic Atlas of the Forebrain and Lower Parts of the Brain Stem, Williams and Wilkins, Baltimore, 1968. 41 Matsuyama, T. and Kawamura, S., Cytoarchitectonic coincidence with the discontinuous connectional pattern in the deep layers of the superior colliculus in the rat, Neurosci. Res., 2 (1985) 335-348. 42 Meredith, M.A. and Stein, B.E., Visual, auditory, and somatosensory convergence on cells in superior colliculus results in multisensory integration, J. Neurophysiol., 56 (1986) 640-662. 43 Otani, K., Kimura, M. and Tanaka, K., The laminarly differentiated proiections of the superior colliculus in the rat, Neurosci. Lett., Suppl. 6 (1981) $113. 44 Redgrave, P., Odekuule, A. and Dean, P., Tectal cells of origin of predorsal bundle in rat: location and segregation from ipsilateral descending pathways, Exp. Brain Res., 63 (1986) 279-293. 45 Shambes, G.M., Berman, D.H. and Welker, W., Multiple tactile areas in cerebellar cortex: another patchy cutaneous projection to granule cell columns in rats, Brain Research, 157 (1978) 123-128. 46 Shambes, G.M., Gibson, M. and Welker, W., Fractured somatotopy in granule cell tactile areas of rat cerebellar hemispheres revealed by micromapping, Brain Behav. EvoL, 15 (1978) 94-140. 47 Simpson, J.T. and Alley, K.E., Visual climbing fiber input to rabbit vestibulo-cerebellum: a source of direction-specific information, Brain Research, 82 (1914) 302-308. 48 Swenson, R.S. and Castro, A.J., The afferent connections of the inferior olivary complex in rats. An anterograde study using autoradiographic and axonal degeneration techniques, Neuroscience, 8 (1983) 259-275. 49 Voogd, J., The Cerebellum of the Cat. Structure and Fibre Connexions, Thesis, van Gorcum, Assen, The Netherlands, 1964. 50 Wiklund, L., Toggenburger, G. and Cuenod, M., Selective retrograde labelling of the rat olivo-cerebellar climbing fiber system with D-[3H]-aspartate, Neuroscience, 13 (1984) 441-468. 51 Williams, M.N. and Faull, R.L.M., The striatonigral projection and nigrotectal neurons in the rat. A correlated light and electron microscopic study demonstrating a monosynaptic striatal input to identified nigrotectal neurons using a combined degeneration and horseradish peroxidase procedure, Neuroscience, 14 (1985) 991-1010. 52 Wise, S.P. and Jones, E.G., Cells of origin and terminal distribution of descending profections of the rat somatosensory cortex, J. Comp. Neurol., 175 (1977) 129-158.
371
BRE 22417
Electrophysiological analysis of the tecto-olivo-cerebellar (Iobulus simplex) projection in the rat Tadashi Akaike Department of Physiology, Nagoya UniversitySchool of Medicine, Nagoya (Japan) (Accepted 5 May 1987)
Key words: Cerebellum; Superior colliculus; Medial accessory olive; Climbing fiber response; Lobulus simplex; Rat
In albino rats the deep layers of the superior colliculus were stimulated, and climbing fiber responses of Purkinje cells were explored in the medial region of the lobulus simplex. They were identified in a strip (ca. 0.7-1.0 mm wide) in the most medial region of folium b. In the tectorecipient zone of the medial accessory olive antidromically evoked potentials from the lobulus simplex were recorded laterally to those from lobule VII. Evidence is presented that climbing fibers innervating the zone in the lobulus simplex are axon collaterals of the inferior olivary neurons which project to crus II.
In mammals the olivocerebellar projection is characterized by clear longitudinal zonations of their termination in the cerebellar cortex 12A8'37'49. It was demonstrated in the cat that climbing fibers originating from inferior olivary neurons in a certain sector of the nucleus terminate in one longitudinal zone across many folia, lobules or lobes 9'13'37'5°. Inferior olivary neurons in a tectorecipient zone in the caudomedial quadrant of the medial accessory olive are so far supposed to project exclusively to the vermal region of lobule VII of Larsell in the cat and rat 3,3°'34. Recently it was reported in the rat that climbing fiber responses could be evoked in a longitudinal zone in the medial region of crus II by stimulation of the deep layers of the superior colliculus 5,6. Purkinje cells in these two areas are innervated respectively by climbing fibers originating from different inferior olivary neurons in the tectorecipient zone. In the present study climbing fiber responses were surveyed in the lobulus simplex, which were evoked by stimulation of the deep layers of the suPerior colliculus. A new zone is identified in the most medial region of the lobulus simplex (folium b). The area is supposed to be a longitudinal extention of the tecto-olivo-recipient zone in crus II. Evidence is presented that the new zone in
the lobulus simplex is innervated by axon collaterals of inferior olivary neurons projecting to the zone in crus II. Eleven albino rats were used (Wistar, male adult, 200-350 g). Experimental procedures were described elsewhere 2-6. Briefly, the animals were anesthetized with chloral hydrate (400 mg/kg, i.p.). The trachea was cannulated, but usually the animals were not, artificially respirated except when immobilized by gallamine triethiodide. The same results were obtained with or without immobilization. Body temperature was maintained by a heating pad. A burr hole was made at the midline (A 8.0, L 0.0) 4°. The caudal part of the cerebrum, cerebellum and the upper part of the spinal cord were exposed and were covered with a mixture of warmed vaseline and liquid paraffin. A bipolar electrode insulated except the tips (separated 1.0-1.5 mm) was placed through the cerebrum at the optic nerve (A 8.0, L 0.0). A monopolar electrode was placed in the deep layers of the superior colliculus (A 1.0-1.5, L 1.5-2.0). Using a current stimulus of 100-300/~A, I adjusted the depth of the tectal electrode to evoke maximum climbing fiber responses in crus II and lobule VII in the ipsilateral side 3'5,6. In 6 animals bipolar or monopolar elec-
Correspondence: T. Akaike, Department of Physiology, Nagoya University School of Medicine, Nagoya Aichi 466, Japan. 0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
372 tive potentials). By lowering the tip of the electrode for 100 or 200 # m , evoked potentials were reversed to negative field potentials having a steeply falling phase and a longer duration (ca. 7 - 1 5 ms). When the tip of the electrode was lowered a little further, amplitudes of the negative field potentials increased, but their duration decreased a little, and they had a more steeply falling phase. It suggests that at the most superficial layer there are sources of the synaptic current, and at the middle layer where the negative field potentials are recorded, there are sinks of synaptic current evoked by climbing fibers, and at the deepest layer the tip of the recording electrode picks up action currents which are generated at the dendrites of Purkinje cells. When it was lowered 50-100 # m deeper into the cerebellar cortex, it reached another layer where the negative potentials were again reversed to positive field potentials with long duration (ca. 7-15 ms: deep positive potentials). The latter were usually preceded by short negative potentials. There, unit discharges were often observed, and occasionally the glass micropipettes impinged intracellularly. It disclosed large excitatory postsynap-
trodes were placed in the tecto-olivo-recipient zones of lobule VII, crus II and/or the lobulus simplex. For electrical stimulation single rectangular pulses (0.2 ms in duration, 50-500 # A of current intensity) were derived through isolation units and delivered at a rate of 0.5-1.0 Hz. For recording glass micropipettes filled with 2 N NaC1 solution (usually saturated with Fast green) and having an electric resistance of 3 - 5 Mr2 were used. They were connected to a neutralized preamplifier and then to an A T A C 250 (a storage type of oscilloscope, Nihonkohden). Responses were photographed and analyzed after experiments. Recording and stimulating sites were marked by ejecting dye or by making a small lesion with electric current. These were identified histologically after experiments. Recording electrodes were lowered perpendicularly in the cerebellum. When a tip of the recording electrode touched the cerebellar surface in the medial region of folium b in the lobulus simplex (Fig. 1), positive field potentials were obtained (latency; 9-11 ms), which had steeply rising and falling phases, and a relatively short duration (ca. 3 - 5 ms: surface posi-
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2 4. J Fig. 1. Laminar analysis of climbing fiber responses of Purkinje cells in the cerebellar cortex of an albino rat. They were evoked by stimulation of the deep layers of the superior colliculus with single pulses (200 #A, 0.2 ms), and were recorded in the medial region of folium b of the lobulus simplex by a perpendicular penetration of a recording electrode (vertical line). Evoked responses (1-24) were reco~ed every 50 #m in depth. Twenty responses were superposed. The recording sites were numbered, and were indicated by short horizontal bars in a camera lucida drawing of a parasagittal section of the cerebellum. Purkinje cell somas were indicated by dots. Inset: a camera lucida drawing of a parasagittal section of the cerebellum and the lower brainstem, in which the recording area was indicated by a square. Calibration: 2 mV, 50 ms (see text for further explanation).
373 tic potentials, often superposed by a few action potentials, which were characteristic of climbing fiber responses of Purkinje cells 2,3,5,2i. Histological examination revealed that the first reversal points were within the molecular layer, and the second reversal points were between the molecular and the granular layers of the cerebellar cortex. The profile of the evoked potentials across the cortical layers of the cerebellum is characteristic of climbing fiber responses of Purkinje cells in the rat. The laminar analysis of climbing fiber responses has been described in detail in the cat 22, and it is essentially the same as in the rat. Climbing fiber responses evoked by the tectal stimulation, which had the laminar profile described above, were surveyed in the lobulus simplex, mainly in its medial half. They were obtained in the most medial region of folium b, often adjacent to and lateral to the paravermal vein. The responsive area in the cerebellar surface extended longitudinally; usually the caudal region was wider (ca. 0.7-1.0 mm) than the rostral region (ca. 0.5-0.7 mm). Furthermore, these potentials were surveyed and identified deeply in the fissure of folium b. The responsive area seems to extend longitudinally throughout the cerebellar cortex not only in the cerebellar surface but also in the fissure in the medial region of folium b of the lobulus simplex. The amplitudes of the evoked potentials were almost of the same size within the zone, and they decreased steeply at the boundary and disappeared outside the zone. Electrical stimulation of the optic nerve was of no effect. Stimulation of the face region, specifically the vibrissae area in the contralateral side with subcutaneously placed metal electrodes, sometimes evoked climbing fiber responses in the area, but with a much longer latency. Under deep anesthesia they fluctuated or were not evoked. In 6 animals the inferior olivary nucleus was surveyed to record field potentials which were evoked either orthodromically by tectal stimulation, or antidromically by stimulation of the cerebellar cortices within the tecto-olivo-recipient zone of lobule VII, crus II or the lobulus simplex. The tectorecipient zone was, as described in previous papers 4'6 immediately rostral to a caudal, visually responsive area. It extended from the midline to 0.4-0.5 mm laterally, 0.4-0.5 mm long rostrocaudally and 0.1-0.2 m m thick in depth. Histologically it corresponded to subnucleus c in the caudomedial quadrant of the medial
accessory olive at the rostrocaudal level of the middle one-third of nucleus ft. Antidromic field potentials evoked by stimulation of lobule V I I were recorded in the most medial region (from the midline to 0.2-0.3 mm laterally) in the tectorecipient zone, while those evoked by stimulation of crus II were recorded in the lateral region (from 0.2-0.3 m m to 0.4-0.5 m m laterally) in the zone of the inferior olive. Antidromic field potentials, evoked by stimulation of the medial region of folium b of the lobulus simplex were recorded in the lateral region of the tectorecipient zone. The area for the lobulus simplex was included in and almost overlapped with the area for crus II. However, amplitudes of antidromically evoked potentials by stimulation of the lobulus simplex were smaller or larger than those evoked by crus II stimulation at certain sites within the tectorecipient zone. In 3 animals I investigated whether or not these climbing fiber responses of Purkinje cells in the lobulus simplex were evoked by axon collaterals of the inferior olivary neurons projecting to the lobule VII and/or crus II (Fig. 2). When the tip of a recording electrode was in the upper portion of the molecular layer in the lobulus simplex, it recorded surface positive potentials in response to the tectal stimulation (latency; 9-11 ms); the same form of field potentials was obtained with a little shorter latency (ca. 8 - 1 0
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3 4Fig. 2. Climbing fiber responses of Purkinje cells evoked by axonal reflex in the lobulus simplex of a rat cerebellum. Experimental procedures were drawn in the dorsal view of the cerebellum. The cerebellar cortices within the tecto-olivo-recipient zones (blackened areas) of crus II (Cr II), or lobule VII (VII) were stimulated with bipolar electrodes (150 #A, 0.2 ms). The evoked responses (1-3) were recorded at 3 different depths of a penetration of the recording electrode in the cerebellar cortex in the zone of folium b of the lobulus simplex (LS b). When the electrode picked up the surface positive (1), negative (2) or deep positive potentials (3) in response to tectal stimulation (SC: 200 #A, 0.2 ms), the same response types were obtained by crus II stimulation at the same depths. Twenty responses were superposed. Calibration: 1 mV, 25 ms.
374 ms) by stimulation of the cerebellar cortex within the tecto-olivo-recipient zone of crus II. When the tip of the electrode was moved deeper, and it recorded negative field potentials or deep positive potentials in response to tectal stimulation, the same forms of the field potentials were evoked by crus II stimulation. The reverse relation was also true; that is, climbing fiber responses were evoked in the tecto-olivo-recipient zone of crus II in response to stimulation of the zone in the lobulus simplex. Intracellular recordings also confirmed typical climbing fiber responses of Purkinje cells in crus II by stimulation of the lobulus simplex. Stimulation of lobule VII was of no effect in the present study. These findings suggest that at least some climbing fibers innervating Purkinje cells in the most medial region of folium b of the lobulus simplex are axon collaterals of inferior olivary neurons projecting to the tecto-olivo-recipient zone of crus II. The last question concerns the extent to which climbing fibers innervating the lobulus simplex (folium b) are axon collaterals. Climbing fiber responses were surveyed every 100 ktm across the tecto-olivorecipient zone of the lobulus simplex while the cerebellar cortex within the zone of crus II was stimulated. They were always recorded in any sector in the zone of the cerebellar surface of the lobulus simplex. Although I have not investigated systematically the responses in the cerebellar cortex deep in the fissure of folium b, they seemed to be evoked. However, the reverse relation was not always true: stimulation of the lobulus simplex evoked climbing fiber responses in the zone of crus II, except for its medial portion. This is reasonable since the area of the zone in crus II seems to be wider than that in the lobulus simplex. These findings suggest that a large part of climbing fibers innervating Purkinje cells in the tecto-olivo-recipient zone of the lobulus simplex are axon collaterals. Anatomical investigations by Wiklund et al. 5°, using the anterograde axonal transport techniques, provided evidence in the rat that there were axonal branches of inferior olivary neurons; one projects to the lobulus simplex, the other to crus II. The possibility of current spread from the tectal stimulating electrode to the visual and other afferent pathways and/or various mesencephalo-olivary pathways may be eliminated because the intensity of stimulus current (50-100 ktA) was low enough to evoke climbing fiber responses in the lobulus simplex, and
because all mesencephalo-olivary pathways except the tecto-olivary projection, were described exclusively to terminate in the ipsilateral side 3,5,14,48. Furthermore, the tectorecipient zone in the inferior olive, identified in the present study, was almost the same area as that described by Hess 3°, and by Swenson and Castro 48, using anterograde axonal transport techniques in the rat. Shambes et al. 35'45'46reported that there are extensive tactile projections to the granule layers of the rat cerebellum, specifically in crus I, crus II, paramedian lobule, lobulus simplex (folium b) and the uvula (folia 9a) of the posterior vermis. When the tecto-olivorecipient zone of the lobulus simplex in the present study is referred to their Fig. 1 (ref. 45), it is composed of 3 regions where the granule cells have receptive fields in the contralateral and ipsilateral lower lip, and the ipsilateral vibrissae, respectively. The zone in crus II is also composed of 3 regions where the granule cells have receptive fields in the contralateral, bilateral and ipsilateral perioral areas, respectively46. Purkinje cells in the medial region of crus II are described to innervate neurons in the dorsolateral protuberance of the cerebellar medial nucleus (Fdlp) 1°. It is probable that Purkinje cells in the zone of the lobulus simplex also project to the Fdlp 26. In a previous paper s, I discussed differences in modalities of their afferents such as mossy fibers between the zone of lobule VII and that of crus II, and speculated a sequence of the information processing starting from Purkinje cells in those zones. It may also apply to the zone of the lobulus simplex. Recently Redgrave et al. 44 disclosed that the deep layers of the superior colliculus in rats can be divided into two laminae which contain different neuronal groups, respectively. That is, in the upper layer (stratum album intermediale according to his nomenclature) are located most neurons of origin of the contralateral predorsal bundle, which include neurons of origin of the tecto-olivary projection 33. He suggested that most of the tectal cells of origin of the predorsal bundle also project rostrally to targets in the ipsilateral caudal diencephalon. This is consistent with the electrophysiological findings of Chevalier and Deniau 19. Anatomical and electrophysiological investigations 1'8'17'20'23'24'27'28'32'38'39'41'42indicate that neurons in the deep layers of the superior colliculus, which include neurons of origin of the tecto-olivary
375 projection, have multimodal sensory properties. That is, they are responsive to somatosensory, visual and/or auditory inputs. They have many axonal branches: one projects rostrally to targets in the ipsilateral side, which are supposed to be the center of attention 19'36'43'44, and the other caudally to the precerebellar nucleus in the tegmental reticular nucleus ~5,43, to the p r e o c u l o m o t o r region in the bulbar reticular formation 29,43, and to the medial accessory olive in the contralateral side 3'6'3°'33'4s. In the bulbar reticular formation they may have many axonal branches 29. Thus, in b o t h afferent and efferent aspects these neurons m a y have integrative functions. The presumptive integrative p r o p e r t i e s of neurons of origin of the tecto-olivary p r o j e c t i o n clearly contrast with the visual pretecto-olivary projection. The latter has definite properties of the receptive field, such as direction and velocity of m o v e m e n t of the receptive fieldt6,47. 1 Abrahams, V.C. and Rose, P.K., Projections of extraocular, neck muscle and retinal afferents to superior colliculus in the cat: their connections to cells of origin of tectospinal tract, J. Neurophysiol., 38 (1975) 10-18. 2 Akaike, T., Lobular distribution of visual climbing fiber responses in the cerebellum, Brain Research, 327 (1985) 359-361. 3 Akaike, T., Electrophysiological analysis of the tecto-olivocerebellar (lobule VII) projection in the rat, Brain Research, 340 (1985) 369-372. 4 Akaike, T., Spatial distribution of evoked potentials in the inferior olivary nucleus by stimulation of the visual afferents in the rat, Brain Research, 368 (1986) 183-187. 5 Akaike, T., Electrophysiological analysis of the tecto-olivo-cerebellar (crus II) projection in the rat, Brain Research, 378 (1986) 186-190. 6 Akaike, T., Differential localization of inferior olivary neurons projecting to the tecto-olivo-recipient zone of lobule VII or crus II in the rat cerebellum, Brain Research, 386 (1986) 400-404. 7 Anderson, G., Demonstration of a cuneate relay in a cortico-olivo-cerebellar pathway in the cat, Neurosci. Lett., 46 (1984) 47-52. 8 Antonetty, C.M. and Webster, K.E., The organization of the spino-tectal projection. An experimental study in rat, J. Comp. Neurol., 163 (1975) 449-466. 9 Armstrong, D.M., Harvey, R.J. and Schild, R.F., The spatial organization of climbing fibre branching in the cat cerebellum, Exp. Brain Res., 18 (1973) 40-58. 10 Armstrong, D.M. and Schild, R.F., An investigation of the cerebellar corticonuclear projections in the rat using an autoradiographic tracing method. II. Projections from the hemispheres, Brain Research, 141 (1978) 235-249. 11 Berkley, K.J. and Worden, I.G., Projection to the inferior olive of the cat. I. Comparisons of the input from the dorsal column nuclei, the lateral cervical nucleus, the spino-olivary pathways, the cerebral cortex and the cerebellum, J.
The superior colliculus is now focused as an output stage of the basal ganglia through the striatonigral, and nigrotectal projections 25'31'5t. The ipsilateral nigrotectal projections in rats were identified in the full extent of the stratum album intermediale 25. Therefore it is most p r o b a b l e that neurons of origin of the predorsal bundle are targets of the nigrotectal fibers in rats. Thus, the p e r i p h e r a l sensory and central motor information converged in and g e n e r a t e d in the cerebral cortex is t r a n s f o r m e d and t r a n s p o r t e d to the cerebellum as climbing fiber inputs, not only via cortico-olivary, cortico-tectal and other cortieo-preolivary nuclear projections 7'11'24'4t'48'52, but also via the cerebral cortex-striatum-substantia nigra-superior colliculus-inferior olive. To u n d e r s t a n d the function of climbing fiber afferents to the cerebellum it is important to clarify physiological properties of neurons identified as origin of the tecto-olivary projections.
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