- Email: [email protected]
Electrophysiological analysis of the trigemino-olivo-cerebellar (crura I and II, lobulus simplex) projection in the rat
Brain Res'earch, 482 (19891 402-4116 Elsevier
402 BRE 23431
Electrophysiological analysis of the trigemino-olivo-cerebellar (crura I and II, Iobulus simplex) projection in the rat Tadashi Akaike Department of Physiology, Nagoya University School of Medicine, Nagoya (Japan) (Accepted 13 December 1988)
Key words: Cerebellum; Trigeminal; Inferior olive; Climbing fiber response; Crura I and II; Lobulus simplex
In albino rats the whisker area was electrically stimulated while climbing fiber responses were surveyed in the cerebellar hemisphere on the ipsilateral side. They were identified both deep in the intercrural sulcus, and in the posterior superior fissure. Histological examination has revealed that the response areas extend longitudinally from the dorsal surface of crus II to the ventral surface of crus I in the intercrural sulcus, and from the rostral surface of crus I to the caudal surface of lobulus simplex in the posterior superior fissure. These are supposed to be transmitted through direct trigemino-olivary projections. In individual species of animals there is at least one modality of afferent which is most important and commonly utilized in exploring and guiding behaviors such as the tactile in the rat, the visual in the pigeon, and the auditory in the bat. Besides the cerebrum, wide areas of the cerebellar cortex are involved in processing that kind of information 8' 14,19,32. Specifically in the rat, tactile afferents from the face and the perioral region activate, via mossy fibers, granule cells in the cerebeUar cortex in wide areas of lobule IXa, crura I and II, paramedian lobule and iobulus simplex b 8'22'31"32. On the other hand, vibrissal afferents are transmitted, via climbing fibers, to Purkinje cells in crus II on the contralateral side through the trigemino-tecto-olivocerebellar projections 5"8. In the cat and monkey climbing fiber responses of Purkinje cells, evoked by natural stimulation of the face area on the ipsilateral side, have been identified in mass in lobule HVI, V, and c r u s 17'15"25'26'28"29. In the present study, by stimulation of the whisker area, I investigated climbing fiber responses of Purkinje cells in the cerebellar hemisphere on the ipsilateral side, specifically crura 1 and II, and lobulus simplex. They were identified deep in the cerebellar fissure, both in the lobulus simplex, and crura I and II. Trigeminal
(vibrissal) afferents are supposed to be transmitted through direct trigemino-olivary projections, not via the superior colliculus 12'13"2°'21"33. Twelve albino rats were used (Wister, male adult, 250-350 g). Experimental procedures were described elsewhere 1-3. Briefly the animals were anesthetized with chloral hydrate (400 mg/kg, i.p.). The trachea was cannulated, and the animals were artificially respirated after being immobilized with gallamine triethiodide. Body temperature was maintained by a heating pad. After an animal was mounted on a stereotaxic apparatus, the caudal portion of the skull was removed. The occipital region of the cerebrum, the cerebellum and the upper part of the spinal cord were exposed and covered with a mixture of warmed vaselline and liquid paraffin. A monopolar electrode was placed in the deep layers of the superior colliculus (A 1.0-1.5, L 1.0-2.0) 24. Using current of 50-200/,tA, I adjusted the depth of the tectal electrode to evoke the maximum amplitude of climbing fiber responses in crus II and/or lobule VII on the ipsilateral side. Bipolar metal electrodes, separated 2-5 mm, were inserted subcutaneously in the large whisker area on both sides. Single or double shocks for electrical stimulation (rectangular pulses, duration of 0.2 ms,
Correspondence: T. Akaike, Department of Physiology, Nagoya University; School of Medicine, Nagoya Aichi 466, Japan. 0006-8993/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
403 interval of 0.5-2 ms and 50-800 pA) were derived from isolation units, and delivered at rates of 0.3-1.0 Hz. Effectiveness of stimulation of the whisker area was described in the previous paper 5. Recording micropipettes were filled with 2 M NaCI (usually saturated with Fast green FCF) and had an electrical resistance of 2-5 Mff2. They were connected to a neutralized preamplifier and then to an ATAC 250 (storage type of oscilloscope, Nihonkhoden). Responses were photographed, and recording and stimulating sites were marked by ejecting dye or by making small lesions with electric current. They were identified histologically after experiments. As described in the previous papers 2'4'5, the tectal stimulation evoked climbing fiber responses of Purkinje cells in a longitudinal zone in the medial region of crus II and Iobulus simplex b on the ipsilateral side. Furthermore climbing fiber responses were evoked in the medial portion of the tecto-olivorecipient zone in crus II by stimulation of the whisker area on the contralateral side. Both the tectal and the trigeminal climbing fiber responses showed the same typical laminar profiles4"5; that is, sharp positive potential at the surface of the molecular layer, large negative potentials with a steeply falling phase at the deeper portion of the molecular layer, and again reversed positive potentials with longer duration at the boundary between the molecular and the granular layers. The triad is so specific that it can be used as evidence of climbing fiber responses of Purkinje cells. When a recording electrode was lowered in crus I or lobulus simplex, vertically to the horizontal plane, 1.0-2.0 mm lateral to paravermal vein, a typical laminar profile of climbing fiber responses was recorded by stimulation of the whisker area on the ipsilateral side, at a depth of 1.5-2.0 mm from the dorsal surface of cerebellum (Fig. 1D). Specimen records show, in sequence, sharp positive potentials (a: 3, 7-13, c: 2, 5, 6), large negative potentials (b: 1-14), and long positive potentials (a: 1, 2, 4-6, c: 3, 7-14). Occasional intracellular recordings showed climbing fiber-evoked depolarization in presumed Purkinje cells. Latencies were 9-12 ms. Current stimuli with single shocks of 100-300/~A were effective on occasion, but those with double shocks 300-800 pA were usually necessary. They were repeated at rates of below 1.0 Hz. Amplitudes of the responses usually
decreased with the higher frequency of repetition of the stimulation. No climbing fiber responses were evoked by stimulation of the whisker area on the contralateral side, of the optic nerve or of the superior colliculus. The negative climbing fiber responses were always preceded by large negative potentials with short latency (3-5 ms), which were related to granule cell activities evoked by the trigeminal mossy fiber projections8"32. I will call the climbing fiber responses on the ipsilateral side 'direct trigeminal (vibrissal) climbing fiber responses' in contrast to the 'indirect trigeminal climbing fiber responses' which were recorded in the tecto-olivo-recipient zone in crus II on the contralateral side 5. To investigate the extent of the response area of direct trigeminal climbing fiber responses, I moved single recording electrodes rostrocaudally or mediolaterally at every 100 or 200/~m. Fig. 1 shows a result of one animal in which one recording electrode was moved rostrocaudally. At the caudal penetrations (7-14), and at the rostral one (3), surface positive potentials were recorded dorsally to negative potentials, and deep positive potentials most ventrally. In contrast, at the rostral (1-2) and the middle (4-6) penetrations, the responses were in reverse sequence, i.e. deep positive potentials most dorsally and surface positive potentials, ventrally. Histological examination revealed that the rostral response areas (1-3) were deep in the posterior superior fissure; the most rostral area (1-2) was in the caudal surface of lobulus simplex, and the next one (3) in the rostral surface of crus I, and the caudal response areas (4-14) were deep in the intercrural sulcus: the middle area (4-6) was in the ventral surface of crus I, and the most caudal response area (7-14) was in the dorsal surface of crus II. It indicates that the response area extends longitudinally from the dorsal surface of crus II to the ventral surface of crus I in the sulcus. Probably the response area also extends longitudinally and further rostrally, deep in the posterior superior fissure; that is, both in the caudal surface of lobulus simplex and in the rostral surface of crus I. In other experiments I moved single electrodes mediolaterally to investigate width of the zone. The results suggest that the response area is 0.5-1.0 mm wide. In 3 animals I explored in the inferior olive multiunit potentials which were evoked orthodromi-
404
VYYVVY
B
m
2 ..545 7 8 9 1 1 1 4
CRI
D
I 2 5 4 5 6 7 8 9 I 0 II 12 1514 0 "¢~ ,~'., ,,,. w~. ,w~ ,~,.. ,,'~ , ~ ,~..'V. ,~, ~ . ~
b ~p "r '~ ~" '~e "'r '~ '~¢""r '~F "r 'mr "t "e C
1"4~ ~,"
,',~ ,;~, ,¢~ .\~ ,~, ,e.. , V , . . ~
.¢- .~,
Fig. 1. Laminar analysis of climbing fiber responses of Purkinje cells and the response areas in a rat cerebellum, evoked by stimulation of the whisker area on the ipsilateral side. A: a photomontage of a parasagittal section of the cerebellar hemisphere (ca. 4 mm from the midline). Black and white arrowheads indicate traces of a recording electrode (400 # m interval), and recording sites (staining spots), respectively. In the last part of the experiment the tip of the recording micropipette was broken a little to eject dye by current (30-40 #A). It caused traces of the electrode to become visible. Calibration bar = lmm. B: sites of penetration of the recording electrode (dots: 1-14) and level of the parasagittal section in A (bars) were indicated on a dorsal view of the rat cerebellum. Obliquely shaded areas indicate tecto-olivo-recipient zone (lobule VII, crus II, and lobulus simplex b). Filled circles indicate trigemino-tecto-olivo-recipient zone. C and D: recording areas including lobulus simplex (LS), and crura I and II (CR I and CR II) in A are traced using camera lucida. Sites of penetration of the recording electrode were indicated by vertical bars and numbered (1-14). Field potentials, evoked by stimulation of the vibrissal area on the ipsilateral side (600 # A , single shock), were indicated at the recording sites by symbols (open circles = surface positive potentials, filled circles = negative potentials; crosses = deep positive potentials), and their records are shown in D. Records in (a) were obtained most dorsally, (b) in the middle, and (c) ventrally, f.p.s., the posterior superior fissure; s.i.c., the intercrural sulcus. Calibration bar = 2 mV, 50 ms.
405 cally by stimulation of the whisker area on the contralateral side. They were recorded with latencies of 7-10 ms, and were relatively labile when compared to those evoked in the caudal half of the nucleus by stimulation of the optic nerve or of the superior colliculus s. The response area extends rostrocaudally 0.5-0.8 mm in the medial quarter of the rostral half of the nucleus. Though the precise extent of the response area was not investigated in the present study, it seems to be the same area as that described by Cook and Wiesendanger ~6, Swenson and Castro 33, and Huerta et ai. 2°. Since it can be estimated to take approximately 2-6 ms to activate trigeminal nuclear neurons 17'18'27, the remaining time (2-5 ms) may be ascribed to conduction time from the trigeminal nucleus to the inferior olive. Direct trigeminal climbing fiber responses and multiunit potentials evoked in the inferior olive were both labile, and they depend on depth of anesthesia, general condition and others. These may be ascribed both to response properties of trigeminal nuclear neurons and of the inferior olivary neurons, and to transmission from the trigeminal nucleus to the inferior olive, because of intranuclear inhibition in the trigeminal nucleus 3° and in the inferior olive 6"9, and of repetitive discharge of trigeminal nuclear neurons ~7"23. It is plausible, based on the above findings, that direct trigeminal climbing fiber responses are evoked through a direct trigemino-olivary projection: that is, trigeminal (vibrissal) afferents change neurons in spinal nuclei of the trigeminal nerve on the ipsilateral side. Trigeminal nuclear neurons cross the midline and project to the inferior olive. Inferior olivary neurons, receiving trigeminal afferents, again cross the midline and project to the cerebellar cortex as climbing fibers. The schema is in good agreement with anatomical and physiological observations ~13.16,2(1.21,33
two units which responded to natural stimulation of a mystacial vibrissa on the ipsilateral side with climbing fiber responses: one in the upper portion of the dorsal surface of crus II, and the other in the ventral surface of crus I deep in the intercrural sulcus. They seem to be in the response area in the present study. Miles and Wiesendanger 25'26, and Robertson and Laxter 28 identified climbing fiber responses by natural stimulation of the face area in Iobulus simplex and lobule V in the cat and monkey. Huerta et al. e~ suggest 4 longitudinal trigeminoolivo-recepient zones spanning the vermis of the anterior lobe, lobulus simplex, crus I and paramedian lobule, by considering the topographical relation between the trigemino-olivary and the olivocerebellar projections in the cat. In the present study, direct trigeminal climbing fiber responses were recorded both deep in the posterior superior fissure and in the intercrural sulcus. However, I could not record them anywhere at the exposed surface of the cerebellar cortex in crura I or II, or lobulus simplex. It is not determined whether or not there are other more lateral longitudinal response areas in the cerebellar hemisphere, whether or not the response area in the present study extends more rostrally in the anterior lobe or more caudally in the paramedian lobule, or whether or not it belongs to the same longitudinal zone as the tecto-olivo-recipient zone of crus II and lobulus simplex. Armstrong and Schild 1°, using the autoradiographic method, revealed that the medial region of crus II including the tecto-olivo-recipient zone projected to the dorsolateral protuberance of the medial nucleus (Fdlp), while the more lateral region probably including the trigemino-olivo-recipient zones projected to the interpositus and/or the lateral nucleus. Functional differences between the direct and the indirect trigemino-olivo-recipient zones are still not known. These are now under investigation.
Axelrad and Crepel tl showed recording sites of 1 Akaike, T., Electrophysiological analysis of the tectoolivo-cerebellar (lobule VII) projection in the rat, Brain Research, 340 (1985) 369-372. 2 Akaike, T., Electrophysiological analysis of the tectoolivo-cerebellar (crus II) projection in the rat, Brain Research, 378 (1986) 186-190. 3 Akaike, T., Differential localization of inferior olivary neurons projecting to the tecto-olivo-recipient zones of Iobule VII or crus II in the rat cerebellum, Brain Research,
386 (1986) 400-404. 4 Akaike, T., Electrophysiological analysis of the tectoolivo-cerebellar (lobulus simplex) projection in the rat, Brain Research, 417 (1987) 371-378. 5 Akaike, T., Electrophysiological analysis of the trigeminotecto-olivo-cerebellar (crus II) projection in the rat, Brain Research, 442 (1988) 373-378. 6 Andersson, G., Mutual inhibition between olivary cell groups projecting to different cerebellar microzones in the
406 cat, Exp. Brain Res., 54 (1984) 293-3(t3. 7 Andersson, G. and Erickson, D., Spinal, trigeminal and cortical climbing fiber paths to the lateral vermis of the cerebellar anterior lobe in the cat, Exp. Brain Res., 44 (1981) 71-81. 8 Armstrong, D.M. and Drew, T., Responses in the posterior lobe of the rat cerebellum to electrical stimulation of cutaneous afferents to the snout, J. Phvsiol. (Lond.). 3[19 (1980) 357-374. 9 Armstrong, D.M. and Harvey, R.J., Responses in the inferior olive to stimulation of the cerebellar and cerebral cortices in the cat, J. Physiol. (Lond.), 187 (1966) 553-574. 10 Armstrong, D.M. and Schild, R.E, An investigation of the cerebellar corticonuclear projections in the rat using an autoradiographic tracing method. II. Projections from the hemispheres, Brain Research, 141 (1978) 232-249. 11 Axelrad, H. and Crepel, E, Representation selective des vibrisses mystaciales au niveau des cellules de Purkinje du cervelet par la voie des fibres grimpantes chez le Rat, C.R. Acad. Sci. Paris, 41 (1977) 1321-1324. 12 Berkley, K.J., Projections to the inferior olive of the cat. II. Comparisons of input from the gracile, cuneate and the spinal trigeminal nuclei, J. Comp. Neurol., 180 (1978) 253-264. 13 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 axonal transport of horseradish perioxidase, J. Comp. Neurol., 176 (1977) 1-22. 14 Clarke, P.G.H., The organization of visual processing in the pigeon cerebellum, J. Physiol. (Lond. L 243 (1974) 267-285. 15 Cody, F.W.J. and Richardson, H.C., Mossy and climbing fibre mediated responses evoked in the cerebellar cortex of the cat by trigeminal afferent stimulation, J. Physiol. (Lond.). 287 (1979) 1-14. 16 Cook, J.R. and Wiesendanger, M., Input from trigeminal cutaneous afferents to neurones of the inferior olive in rats, Exp. Brain Res., 26 (1976) 193-202. 17 Darian-Smith, I., Neurone activity in the cat's trigeminal main sensory nucleus elicited by graded afferent stimulation, J. Physiol. (Lond.), 153 (1960)52-73. 18 Erickson, R.P., King, R.L. and Pfaffmann, C., Some characteristics of transmission through the spinal trigeminal nucleus of rat, J. Neurophysiol.. 24 (1961) 621-632. 19 Horikawa, J. and Suga, N., Biosonar signals and cerebellar auditory neurons of the mustached bat, J. Neurophysiol., 55 (1986) 1247-1267. 20 Huerta, M.F., Frankfurter, A. and Harting, J.K., Studies of the principal sensory and spinal trigeminal nuclei of the rat: projections to the superior colliculus, inferior olive, and cerebellum, J. Comp. Neurol., 220 (1983) 147-167.
21 Huerta, M.F., Hashikawa, T., Gayoso, M.J. and Harting, J.K., The trigemino-olivary projection in the cat: contributions of individual subnuclei, J. Comp. Neurol., 241 (1985) 180-190. 22 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. 23 Khayyat, G.F'., Yu, Y.J. and King, R.B., Response patterns to noxious and non-noxious stimuli in rostral trigeminal relay nuclei, Brain Research, 97 (1975) 47-60. 24 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. 25 Miles, T.S. and Wiesendanger, M., Organization of climbing fibre projections to the cerebellar cortex from trigeminal cutaneous afferents and from the SI face area of the cerebral cortex in the cat, J. Physiol. (Lond.). 245 (1975) 409-425. 26 Miles, T.S. and Wiesendanger, M., Climbing fibre inputs to cerebellar Purkinje cells from trigeminal cutaneous afferents and the SI face area of the cerebral cortex in the cat, J. Physiol. (Lond.), 245 (1975) 425-445. 27 Nord, S.G., Responses of neurons in rostral and caudal trigeminal nuclei to tooth pulp stimulation, Brain Res. Bull.. 1 (1976) 489-492. 28 Robertson, L.T. and Laxter, K.D., Localization of cutaneously elicited climbing fiber responses in lobule V of the monkey cerebellum, Brain Behav. Evol., 18 (1981) 157168. 29 Rushmer, D.S., Woollacott, M.H., Robertson, L.T. and Laxter, K.D., Somatotopic organization of climbing fiber projections from low threshold cutaneous afferents to pars intermedia of cerebellar cortex in the cat, Brain Research, 181 (1980) 17-30. 30 Sessle, B.J. and Greenwood, L.F., Inputs to trigeminal brain stem neurones from facial, oral, tooth pulp and paryngolaryngeal tissues. I. Responses to innocuous and noxious stimuli, Brain Research, 117 (1976) 211-226. 31 Shames, 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. 32 Shambes, G.M.. Gibson, J.M. and Welker, W., Fractured somatotopy in granule cell tactile area of rat cerebellar hemispheres revealed by micromapping, Brain Behav. Evol., 15 (1978) 94-140. 33 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.
402 BRE 23431
Electrophysiological analysis of the trigemino-olivo-cerebellar (crura I and II, Iobulus simplex) projection in the rat Tadashi Akaike Department of Physiology, Nagoya University School of Medicine, Nagoya (Japan) (Accepted 13 December 1988)
Key words: Cerebellum; Trigeminal; Inferior olive; Climbing fiber response; Crura I and II; Lobulus simplex
In albino rats the whisker area was electrically stimulated while climbing fiber responses were surveyed in the cerebellar hemisphere on the ipsilateral side. They were identified both deep in the intercrural sulcus, and in the posterior superior fissure. Histological examination has revealed that the response areas extend longitudinally from the dorsal surface of crus II to the ventral surface of crus I in the intercrural sulcus, and from the rostral surface of crus I to the caudal surface of lobulus simplex in the posterior superior fissure. These are supposed to be transmitted through direct trigemino-olivary projections. In individual species of animals there is at least one modality of afferent which is most important and commonly utilized in exploring and guiding behaviors such as the tactile in the rat, the visual in the pigeon, and the auditory in the bat. Besides the cerebrum, wide areas of the cerebellar cortex are involved in processing that kind of information 8' 14,19,32. Specifically in the rat, tactile afferents from the face and the perioral region activate, via mossy fibers, granule cells in the cerebeUar cortex in wide areas of lobule IXa, crura I and II, paramedian lobule and iobulus simplex b 8'22'31"32. On the other hand, vibrissal afferents are transmitted, via climbing fibers, to Purkinje cells in crus II on the contralateral side through the trigemino-tecto-olivocerebellar projections 5"8. In the cat and monkey climbing fiber responses of Purkinje cells, evoked by natural stimulation of the face area on the ipsilateral side, have been identified in mass in lobule HVI, V, and c r u s 17'15"25'26'28"29. In the present study, by stimulation of the whisker area, I investigated climbing fiber responses of Purkinje cells in the cerebellar hemisphere on the ipsilateral side, specifically crura 1 and II, and lobulus simplex. They were identified deep in the cerebellar fissure, both in the lobulus simplex, and crura I and II. Trigeminal
(vibrissal) afferents are supposed to be transmitted through direct trigemino-olivary projections, not via the superior colliculus 12'13"2°'21"33. Twelve albino rats were used (Wister, male adult, 250-350 g). Experimental procedures were described elsewhere 1-3. Briefly the animals were anesthetized with chloral hydrate (400 mg/kg, i.p.). The trachea was cannulated, and the animals were artificially respirated after being immobilized with gallamine triethiodide. Body temperature was maintained by a heating pad. After an animal was mounted on a stereotaxic apparatus, the caudal portion of the skull was removed. The occipital region of the cerebrum, the cerebellum and the upper part of the spinal cord were exposed and covered with a mixture of warmed vaselline and liquid paraffin. A monopolar electrode was placed in the deep layers of the superior colliculus (A 1.0-1.5, L 1.0-2.0) 24. Using current of 50-200/,tA, I adjusted the depth of the tectal electrode to evoke the maximum amplitude of climbing fiber responses in crus II and/or lobule VII on the ipsilateral side. Bipolar metal electrodes, separated 2-5 mm, were inserted subcutaneously in the large whisker area on both sides. Single or double shocks for electrical stimulation (rectangular pulses, duration of 0.2 ms,
Correspondence: T. Akaike, Department of Physiology, Nagoya University; School of Medicine, Nagoya Aichi 466, Japan. 0006-8993/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
403 interval of 0.5-2 ms and 50-800 pA) were derived from isolation units, and delivered at rates of 0.3-1.0 Hz. Effectiveness of stimulation of the whisker area was described in the previous paper 5. Recording micropipettes were filled with 2 M NaCI (usually saturated with Fast green FCF) and had an electrical resistance of 2-5 Mff2. They were connected to a neutralized preamplifier and then to an ATAC 250 (storage type of oscilloscope, Nihonkhoden). Responses were photographed, and recording and stimulating sites were marked by ejecting dye or by making small lesions with electric current. They were identified histologically after experiments. As described in the previous papers 2'4'5, the tectal stimulation evoked climbing fiber responses of Purkinje cells in a longitudinal zone in the medial region of crus II and Iobulus simplex b on the ipsilateral side. Furthermore climbing fiber responses were evoked in the medial portion of the tecto-olivorecipient zone in crus II by stimulation of the whisker area on the contralateral side. Both the tectal and the trigeminal climbing fiber responses showed the same typical laminar profiles4"5; that is, sharp positive potential at the surface of the molecular layer, large negative potentials with a steeply falling phase at the deeper portion of the molecular layer, and again reversed positive potentials with longer duration at the boundary between the molecular and the granular layers. The triad is so specific that it can be used as evidence of climbing fiber responses of Purkinje cells. When a recording electrode was lowered in crus I or lobulus simplex, vertically to the horizontal plane, 1.0-2.0 mm lateral to paravermal vein, a typical laminar profile of climbing fiber responses was recorded by stimulation of the whisker area on the ipsilateral side, at a depth of 1.5-2.0 mm from the dorsal surface of cerebellum (Fig. 1D). Specimen records show, in sequence, sharp positive potentials (a: 3, 7-13, c: 2, 5, 6), large negative potentials (b: 1-14), and long positive potentials (a: 1, 2, 4-6, c: 3, 7-14). Occasional intracellular recordings showed climbing fiber-evoked depolarization in presumed Purkinje cells. Latencies were 9-12 ms. Current stimuli with single shocks of 100-300/~A were effective on occasion, but those with double shocks 300-800 pA were usually necessary. They were repeated at rates of below 1.0 Hz. Amplitudes of the responses usually
decreased with the higher frequency of repetition of the stimulation. No climbing fiber responses were evoked by stimulation of the whisker area on the contralateral side, of the optic nerve or of the superior colliculus. The negative climbing fiber responses were always preceded by large negative potentials with short latency (3-5 ms), which were related to granule cell activities evoked by the trigeminal mossy fiber projections8"32. I will call the climbing fiber responses on the ipsilateral side 'direct trigeminal (vibrissal) climbing fiber responses' in contrast to the 'indirect trigeminal climbing fiber responses' which were recorded in the tecto-olivo-recipient zone in crus II on the contralateral side 5. To investigate the extent of the response area of direct trigeminal climbing fiber responses, I moved single recording electrodes rostrocaudally or mediolaterally at every 100 or 200/~m. Fig. 1 shows a result of one animal in which one recording electrode was moved rostrocaudally. At the caudal penetrations (7-14), and at the rostral one (3), surface positive potentials were recorded dorsally to negative potentials, and deep positive potentials most ventrally. In contrast, at the rostral (1-2) and the middle (4-6) penetrations, the responses were in reverse sequence, i.e. deep positive potentials most dorsally and surface positive potentials, ventrally. Histological examination revealed that the rostral response areas (1-3) were deep in the posterior superior fissure; the most rostral area (1-2) was in the caudal surface of lobulus simplex, and the next one (3) in the rostral surface of crus I, and the caudal response areas (4-14) were deep in the intercrural sulcus: the middle area (4-6) was in the ventral surface of crus I, and the most caudal response area (7-14) was in the dorsal surface of crus II. It indicates that the response area extends longitudinally from the dorsal surface of crus II to the ventral surface of crus I in the sulcus. Probably the response area also extends longitudinally and further rostrally, deep in the posterior superior fissure; that is, both in the caudal surface of lobulus simplex and in the rostral surface of crus I. In other experiments I moved single electrodes mediolaterally to investigate width of the zone. The results suggest that the response area is 0.5-1.0 mm wide. In 3 animals I explored in the inferior olive multiunit potentials which were evoked orthodromi-
404
VYYVVY
B
m
2 ..545 7 8 9 1 1 1 4
CRI
D
I 2 5 4 5 6 7 8 9 I 0 II 12 1514 0 "¢~ ,~'., ,,,. w~. ,w~ ,~,.. ,,'~ , ~ ,~..'V. ,~, ~ . ~
b ~p "r '~ ~" '~e "'r '~ '~¢""r '~F "r 'mr "t "e C
1"4~ ~,"
,',~ ,;~, ,¢~ .\~ ,~, ,e.. , V , . . ~
.¢- .~,
Fig. 1. Laminar analysis of climbing fiber responses of Purkinje cells and the response areas in a rat cerebellum, evoked by stimulation of the whisker area on the ipsilateral side. A: a photomontage of a parasagittal section of the cerebellar hemisphere (ca. 4 mm from the midline). Black and white arrowheads indicate traces of a recording electrode (400 # m interval), and recording sites (staining spots), respectively. In the last part of the experiment the tip of the recording micropipette was broken a little to eject dye by current (30-40 #A). It caused traces of the electrode to become visible. Calibration bar = lmm. B: sites of penetration of the recording electrode (dots: 1-14) and level of the parasagittal section in A (bars) were indicated on a dorsal view of the rat cerebellum. Obliquely shaded areas indicate tecto-olivo-recipient zone (lobule VII, crus II, and lobulus simplex b). Filled circles indicate trigemino-tecto-olivo-recipient zone. C and D: recording areas including lobulus simplex (LS), and crura I and II (CR I and CR II) in A are traced using camera lucida. Sites of penetration of the recording electrode were indicated by vertical bars and numbered (1-14). Field potentials, evoked by stimulation of the vibrissal area on the ipsilateral side (600 # A , single shock), were indicated at the recording sites by symbols (open circles = surface positive potentials, filled circles = negative potentials; crosses = deep positive potentials), and their records are shown in D. Records in (a) were obtained most dorsally, (b) in the middle, and (c) ventrally, f.p.s., the posterior superior fissure; s.i.c., the intercrural sulcus. Calibration bar = 2 mV, 50 ms.
405 cally by stimulation of the whisker area on the contralateral side. They were recorded with latencies of 7-10 ms, and were relatively labile when compared to those evoked in the caudal half of the nucleus by stimulation of the optic nerve or of the superior colliculus s. The response area extends rostrocaudally 0.5-0.8 mm in the medial quarter of the rostral half of the nucleus. Though the precise extent of the response area was not investigated in the present study, it seems to be the same area as that described by Cook and Wiesendanger ~6, Swenson and Castro 33, and Huerta et ai. 2°. Since it can be estimated to take approximately 2-6 ms to activate trigeminal nuclear neurons 17'18'27, the remaining time (2-5 ms) may be ascribed to conduction time from the trigeminal nucleus to the inferior olive. Direct trigeminal climbing fiber responses and multiunit potentials evoked in the inferior olive were both labile, and they depend on depth of anesthesia, general condition and others. These may be ascribed both to response properties of trigeminal nuclear neurons and of the inferior olivary neurons, and to transmission from the trigeminal nucleus to the inferior olive, because of intranuclear inhibition in the trigeminal nucleus 3° and in the inferior olive 6"9, and of repetitive discharge of trigeminal nuclear neurons ~7"23. It is plausible, based on the above findings, that direct trigeminal climbing fiber responses are evoked through a direct trigemino-olivary projection: that is, trigeminal (vibrissal) afferents change neurons in spinal nuclei of the trigeminal nerve on the ipsilateral side. Trigeminal nuclear neurons cross the midline and project to the inferior olive. Inferior olivary neurons, receiving trigeminal afferents, again cross the midline and project to the cerebellar cortex as climbing fibers. The schema is in good agreement with anatomical and physiological observations ~13.16,2(1.21,33
two units which responded to natural stimulation of a mystacial vibrissa on the ipsilateral side with climbing fiber responses: one in the upper portion of the dorsal surface of crus II, and the other in the ventral surface of crus I deep in the intercrural sulcus. They seem to be in the response area in the present study. Miles and Wiesendanger 25'26, and Robertson and Laxter 28 identified climbing fiber responses by natural stimulation of the face area in Iobulus simplex and lobule V in the cat and monkey. Huerta et al. e~ suggest 4 longitudinal trigeminoolivo-recepient zones spanning the vermis of the anterior lobe, lobulus simplex, crus I and paramedian lobule, by considering the topographical relation between the trigemino-olivary and the olivocerebellar projections in the cat. In the present study, direct trigeminal climbing fiber responses were recorded both deep in the posterior superior fissure and in the intercrural sulcus. However, I could not record them anywhere at the exposed surface of the cerebellar cortex in crura I or II, or lobulus simplex. It is not determined whether or not there are other more lateral longitudinal response areas in the cerebellar hemisphere, whether or not the response area in the present study extends more rostrally in the anterior lobe or more caudally in the paramedian lobule, or whether or not it belongs to the same longitudinal zone as the tecto-olivo-recipient zone of crus II and lobulus simplex. Armstrong and Schild 1°, using the autoradiographic method, revealed that the medial region of crus II including the tecto-olivo-recipient zone projected to the dorsolateral protuberance of the medial nucleus (Fdlp), while the more lateral region probably including the trigemino-olivo-recipient zones projected to the interpositus and/or the lateral nucleus. Functional differences between the direct and the indirect trigemino-olivo-recipient zones are still not known. These are now under investigation.
Axelrad and Crepel tl showed recording sites of 1 Akaike, T., Electrophysiological analysis of the tectoolivo-cerebellar (lobule VII) projection in the rat, Brain Research, 340 (1985) 369-372. 2 Akaike, T., Electrophysiological analysis of the tectoolivo-cerebellar (crus II) projection in the rat, Brain Research, 378 (1986) 186-190. 3 Akaike, T., Differential localization of inferior olivary neurons projecting to the tecto-olivo-recipient zones of Iobule VII or crus II in the rat cerebellum, Brain Research,
386 (1986) 400-404. 4 Akaike, T., Electrophysiological analysis of the tectoolivo-cerebellar (lobulus simplex) projection in the rat, Brain Research, 417 (1987) 371-378. 5 Akaike, T., Electrophysiological analysis of the trigeminotecto-olivo-cerebellar (crus II) projection in the rat, Brain Research, 442 (1988) 373-378. 6 Andersson, G., Mutual inhibition between olivary cell groups projecting to different cerebellar microzones in the
406 cat, Exp. Brain Res., 54 (1984) 293-3(t3. 7 Andersson, G. and Erickson, D., Spinal, trigeminal and cortical climbing fiber paths to the lateral vermis of the cerebellar anterior lobe in the cat, Exp. Brain Res., 44 (1981) 71-81. 8 Armstrong, D.M. and Drew, T., Responses in the posterior lobe of the rat cerebellum to electrical stimulation of cutaneous afferents to the snout, J. Phvsiol. (Lond.). 3[19 (1980) 357-374. 9 Armstrong, D.M. and Harvey, R.J., Responses in the inferior olive to stimulation of the cerebellar and cerebral cortices in the cat, J. Physiol. (Lond.), 187 (1966) 553-574. 10 Armstrong, D.M. and Schild, R.E, An investigation of the cerebellar corticonuclear projections in the rat using an autoradiographic tracing method. II. Projections from the hemispheres, Brain Research, 141 (1978) 232-249. 11 Axelrad, H. and Crepel, E, Representation selective des vibrisses mystaciales au niveau des cellules de Purkinje du cervelet par la voie des fibres grimpantes chez le Rat, C.R. Acad. Sci. Paris, 41 (1977) 1321-1324. 12 Berkley, K.J., Projections to the inferior olive of the cat. II. Comparisons of input from the gracile, cuneate and the spinal trigeminal nuclei, J. Comp. Neurol., 180 (1978) 253-264. 13 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 axonal transport of horseradish perioxidase, J. Comp. Neurol., 176 (1977) 1-22. 14 Clarke, P.G.H., The organization of visual processing in the pigeon cerebellum, J. Physiol. (Lond. L 243 (1974) 267-285. 15 Cody, F.W.J. and Richardson, H.C., Mossy and climbing fibre mediated responses evoked in the cerebellar cortex of the cat by trigeminal afferent stimulation, J. Physiol. (Lond.). 287 (1979) 1-14. 16 Cook, J.R. and Wiesendanger, M., Input from trigeminal cutaneous afferents to neurones of the inferior olive in rats, Exp. Brain Res., 26 (1976) 193-202. 17 Darian-Smith, I., Neurone activity in the cat's trigeminal main sensory nucleus elicited by graded afferent stimulation, J. Physiol. (Lond.), 153 (1960)52-73. 18 Erickson, R.P., King, R.L. and Pfaffmann, C., Some characteristics of transmission through the spinal trigeminal nucleus of rat, J. Neurophysiol.. 24 (1961) 621-632. 19 Horikawa, J. and Suga, N., Biosonar signals and cerebellar auditory neurons of the mustached bat, J. Neurophysiol., 55 (1986) 1247-1267. 20 Huerta, M.F., Frankfurter, A. and Harting, J.K., Studies of the principal sensory and spinal trigeminal nuclei of the rat: projections to the superior colliculus, inferior olive, and cerebellum, J. Comp. Neurol., 220 (1983) 147-167.
21 Huerta, M.F., Hashikawa, T., Gayoso, M.J. and Harting, J.K., The trigemino-olivary projection in the cat: contributions of individual subnuclei, J. Comp. Neurol., 241 (1985) 180-190. 22 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. 23 Khayyat, G.F'., Yu, Y.J. and King, R.B., Response patterns to noxious and non-noxious stimuli in rostral trigeminal relay nuclei, Brain Research, 97 (1975) 47-60. 24 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. 25 Miles, T.S. and Wiesendanger, M., Organization of climbing fibre projections to the cerebellar cortex from trigeminal cutaneous afferents and from the SI face area of the cerebral cortex in the cat, J. Physiol. (Lond.). 245 (1975) 409-425. 26 Miles, T.S. and Wiesendanger, M., Climbing fibre inputs to cerebellar Purkinje cells from trigeminal cutaneous afferents and the SI face area of the cerebral cortex in the cat, J. Physiol. (Lond.), 245 (1975) 425-445. 27 Nord, S.G., Responses of neurons in rostral and caudal trigeminal nuclei to tooth pulp stimulation, Brain Res. Bull.. 1 (1976) 489-492. 28 Robertson, L.T. and Laxter, K.D., Localization of cutaneously elicited climbing fiber responses in lobule V of the monkey cerebellum, Brain Behav. Evol., 18 (1981) 157168. 29 Rushmer, D.S., Woollacott, M.H., Robertson, L.T. and Laxter, K.D., Somatotopic organization of climbing fiber projections from low threshold cutaneous afferents to pars intermedia of cerebellar cortex in the cat, Brain Research, 181 (1980) 17-30. 30 Sessle, B.J. and Greenwood, L.F., Inputs to trigeminal brain stem neurones from facial, oral, tooth pulp and paryngolaryngeal tissues. I. Responses to innocuous and noxious stimuli, Brain Research, 117 (1976) 211-226. 31 Shames, 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. 32 Shambes, G.M.. Gibson, J.M. and Welker, W., Fractured somatotopy in granule cell tactile area of rat cerebellar hemispheres revealed by micromapping, Brain Behav. Evol., 15 (1978) 94-140. 33 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.