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Inhibition of Purkinje cells in the frog cerebellum. I. Evidence for a stellate cell inhibitory pathway
BRAIN RESEARCH
83
INHIBITION OF P U R K I N J E CELLS IN THE F R O G CEREBELLUM. I. EVIDENCE FOR A STELLATE CELL INHIBITORY PATHWAY
D. S. RUSHMER* ANDD. J. WOODWARD Center for Brain Research, and Department of Physiology, University of Rochester, Rochester, N.Y. 14642 (U.S.A.)
(Accepted April 4th, 1971)
INTRODUCTION Stellate and basket cell interneurons in the molecular layer of the cerebellar cortex in cat have been shown 2 to inhibit strongly the Purkinje cells beneath and lateral to a beam of excited parallel fibers. Llin~s et al. is,14, using field potential analysis and single unit recording, have failed to find electrophysiological evidence for strong inhibition on Purkinje cells during similar experiments in the frog (Rana catesbiana). It has therefore been suggested that the cerebellum of the frog, as an early phylogenetic form, may exhibit little of such postsynaptic inhibition. Sotelo 18,19 has recently presented evidence revealing widespread anatomical distribution of endings from molecular layer interneurons (stellate cells) onto the Purkinje cells, in Rana esculenta, whereas previous reports emphasized the scarcity of such connections in Rana catesbiana~O, 11. Moreover Faber and Korn 7 have recently found evidence indicating the presence of long term inhibition of Purkinje cells in Rana pipiens after surface stimulation. In this report, these electrophysiological findings regarding Purkinje cell responses are confirmed and evidence is presented that this inhibition is due to activity of stellate cells. METHODS Frogs (Rana pipiens and Rana clamitans) were paralyzed with 1.5 mg of D-tubocurarine. Xylocaine (4 ~o) was applied topically to wound margins. Access to the cerebellum was obtained by retracting the tectum bilaterally with hooks, following a craniotomy and excision of the dura. For respiration, intermittent positive pressure was applied to the trachea of the frog (15/min for 200-500 msec at 2 psi). Our experience has been that ventilation is essential for maintenance of good circulation in the frog cerebellum. Amphibians are known to exchange CO2 adequately through skin but employ lungs for absorption of one-half to two-thirds of 02 consumed a. With an acceptable circulation, all Purkinje cells in our preparations showed a spontaneous * Presently at the Laboratory of Neurophysiology, Good Samaritan Hospital and Medical Center, Portland, Ore. 97210, U.S.A. Brain Research, 33 (1971) 83-90
84
I). S. R U S H M E R A N D I). .l. W O O D W A R D
activity of about 5-20/sec. Maintenance of adequate circulation was considered to be essential in view of a possible selective effect of hypoxia on small inhibitory interneurons, as has been shown to occur in mammalian spinal cord 4. Stimuli (0.2 msec duration at 0.5/sec) were applied to the surface of the molecular layer through bipolar electrodes made from twisted 40-gauge enameled nichrome wire. Glass micropipettes filled with 3 M NaCl were used for extracellular recording while intracellular recording was done using electrodes filled with 0.5 M K2SO4. A PDP-8 computer was used to construct poststimulus-time (PST) histograms. Methods for recording and applying drugs with multi-barrel electrodes are described in the subsequent paper 20. RESULTS
Purkinje cells were identified by their location at about 250 #m depth and the spontaneous occurrence of bursts or 'inactivation' responses generated by activity in climbing fibers. As has been shown previously5,7,14 stimulation of a beam of parallel fibers caused excitation of Purkinje cells on the beam with a short latency. This excitatory response was followed by a cessation of spike activity lasting up to several hundred msec (Fig. 1A-G). This series of histograms shows further that both the probability of initial excitation and duration of the cessation of activity were smoothly graded with increasing stimulus intensity. In contrast to results reported previously 14, in over 100 cells examined, the initial excitation rarely yielded more than 2-3 spikes at any stimulus strength. The importance of using PST histograms in evaluating this inhibition is especially evident in these slowly firing cells. The histogram (Fig. 1G) reveals a distinct inhibition despite the rather subtle manifestation in the action potential record of Fig. 1A. Lateral spread of the inhibition is apparent and hence is preserved as a functional property in this primitive animal. Recordings from cells far lateral (500 #m or more) to the beam (Fig. 1I) showed no alteration of spontaneous activity, whereas cells 150-200 #m lateral to the beam exhibited cessation of spontaneous activity without initial excitation (Fig. 1H). A postexcitatory rebound mechanism for the inhibition is unlikely in view of this off-beam inhibition found in the absence of driven spikes. Intracellular records of cells stimulated 'on-beam' (Fig. 1B) exhibited an initial EPSP and spike followed by a hyperpolarization. Such hyperpolarizations originated from a flat baseline following an initial spike and could be observed in cells when spiking did not occur. Also, they decreased in size gradually as the cell deteriorated. This response can be readily distinguished from a similar hyperpolarization occasionally observed originating without a delay after a climbing fiber EPSP. The short duration of these penetrations prevented an examination of the permeability characteristics of the response by means of chloride injection, so it could not be positively identified as an IPSP. Stellate cells have been observed in the molecular layer of several species of frog 11,~s. It is reasonable that such cells may serve a function similar to that in mammals and cause inhibition in regions on and lateral to an excited beam of parallel Brain Research, 33 (1971) 83-90
INHIBITION OF PURKINJE CELLS IN FROG CEREBELLUM. I
85
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Fig. 1. A - I are responses of Purkinje cells after parallel fiber stimulation. A, Action potential recording made from tape, spikes about 1 mV in amplitude. B, Intracellular recording, the spike caused by the initial EPSP rises off screen. C - G are poststimulus histograms obtained from the same Purkinje cell stimulated at different intensities. C is subthreshold; D, E, F, and G are 1.2, 1.5, 2.0 and 3.0 times threshold respectively. H and I were obtained from different Purkinje cells 200 and 500 # m lateral to the beam and stimulated with the same intensity in the same experiment as in G. The schematic outline of the half-cerebellum in K illustrates the recording positions at 1, 2, and 3 for G, H and I, respectively, while S is the stimulating position with the beam of excited parallel fibers represented by the horizontal line. J is a typical interspike interval histogram record from a Purkinje cell. Histograms C-I are 400 msec full scale; J is 500 msec full scale. Stimuli were applied at time zero, at 0.5/see and 0.2 msec duration.
Brain Research, 33 (1971) 83-90
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I). S. R U S H M E R A N D l). .I. W O O D W A R D
A
40
msec
B Fig. 2. Activity of a presumed stellate cell. A, Action potential record from tape, spikes about 1 mV. Poststimulus-time histogram 400 msec full scale. Stimuli at time zero. Cell was located 30/~m below the surface and above the Purkinje cell in Fig. 1C-G.
B,
fibers. Evidence of possible involvement of the stellate cells is provided by direct recording of these cells (Figs. 2, 3). Such cells were distinguished from Purkinje cells because they (a) never displayed spontaneous climbing fiber bursts (over a period as long as 20 min), (b) were found high in the molecular layer above the level of Purkinje cell bodies, (c) were lost after slight advances of the microelectrode, and (d) exhibited low rates of spontaneous activity (1-2/sec v e r s u s 5-20/sec for Purkinje cells). Such units were further distinguished by their response to parallel fiber stimulation, which often caused an excitation lasting 60-100 msec, usually represented by a group of 2-6 spikes (Fig. 2) with no sign of a subsequent reduction of activity. Histograms appeared as almost a mirror image of the inhibition exhibited by Purkinje cells. The unit shown in Fig. 2 was found in the molecular layer above the Purkinje cell studied in Fig. 1C-G. Activity was recorded from 21 such units. Further properties are shown in Fig. 3 for another such unit found 30/~m below the surface. The slow firing rate is illustrated in Fig. 3, where the interspike interval histogram reveals a modal interval of about 1 sec. This is in marked contrast with histograms from typical Purkinje cells, as in Fig. 1J where the mean firing rate was 6.5/sec. The spontaneous activity was found to be inhibited by iontophoretic application of GABA (gamma-aminobutyric acid). Fibers are not known to be sensitive to GABA 16 so the electrode was probably near the soma and/or the dendrites. Stimulation of the surface did not result in an inhibition of the spontaneous activity. The Fig. 3B poststimulus histogram consisted of a peak due to 2 or 3 driven spikes followed by a low but even probability of firing at the stimulus intensity employed. The action potential record in Fig. 3B recorded high in the molecular layer shows a driven parallel fiber volley followed by the two spikes. DISCUSSION
The major piece of evidence presented here is that driven activity of presumed stellate cells high in the molecular layer occurs during a period of driven inhibition of spontaneous activity of the Purkinje cells. The presence of a limited lateral spread of Brain Research, 33 (1971) 83-90
INHIBITION OF PURKINJE CELLS IN FROG CEREBELLUM. I
87
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Fig. 3. Activity of presumed stellate cell. A, Action record of slow spontaneous activity is shown to be inhibited by iontophoretic application of GABA. The interspike interval histogram shows a modal interval at about 1 sec. B, Action record shows the shock artifact followed by the parallel fiber negative wave and two spikes. Positive is up. The poststimulus-time histogram shows a high peak due to triggering on parallel fiber volley; a low peak due to evoked spikes and a low and even density of subsequent spontaneous activity. Stimuli applied at 10 msec indicated by arrowhead, applied at 0.5/sec and 0.2 msec duration.
inhibition 7 is confirmed here, this spatial distribution being consistent with the expected distribution of axons from inhibitory interneurons is. Further, a hyperpolarization was observed in Purkinje cells following a stimulus to the afferent parallel fibers. In summary, these results, though circumstantial in some respects, support the simple scheme that parallel fibers excite stellate cells which in turn postsynaptically inhibit P u r k i n j e cells.
This is in apparent contradiction with previous reportsla, 14 contending that in frog there is an 'absence of long term inhibition upon Purkinje cells.' However, the possible inhibitory function of stellate cells was not ruled out in those papers 13,14. Such differences in interpretation may result from differences in the physiological state of the preparations. Purkinje cells in our preparation always exhibited spontaneous activity and never yielded more than 2 or 3 spikes in response to local surface stimulation. Lack of inhibition could result if an alteration of state, via anesthetic or circulation, led to increased excitability of Purkinje cells la. Alterations of state may also have selective effects on inhibitory interneurons 4. We have noted considerable variation in Brain Research, 33 (1971) 83-90
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D. S. R U S H M E R A N D D. J. W O O D W A R D
the duration of pause after parallel fiber activation both in different cells and within the same cell at different times, suggesting that the inhibition may be prone to influence by such factors. Variations in function between species of frogs (see Introduction) seem possible. However, as has been noted 18, optical microscopic studies 12 of the cerebellum of 4 different frog species were unable to demonstrate morphological variations according to the species. Rana clamitans in particular was employed here because, in comparison with our experience with Rana catesbiana, it was possible to penetrate through the pial surface with notably less dimpling and possible injury. We feel moreover that methodological variations in physiological studies are as important as species variations in this regard. Several considerations indicate that activity such as shown in Figs. 2 and 3 is in fact from stellate neurons. The location high in the molecular layer, slow firing rate without climbing fiber inactivation responses, and long term activation without inhibition following stimulation of parallel fibers distinguish the units from Purkinje cells. One possibility is that such activity is in fact from aberrant mossy fibers known to penetrate the molecular layer in amphibialL This is ruled out by the experiment shown in Fig. 3 since fibers are not known to be influenced by the application of GABA. This result is also of interest since only parallel and climbing fiber excitatory synapses have been described on frog stellate cells. It may be that the GABA sensitivity indicates the presence of an undescribed class of inhibitory synapses. Alternately, sensitivity to GABA may be characteristic of this type of neuron independent of the presence of a functional mechanism for making use of GABA sensitivity, as in the case of newborn rat cerebellum where Purkinje cells are sensitive to GABA even before onset of synaptogenesislL A limitation of the present results is that it is difficult to evaluate what contribution disfacilitation plays in generating the pause after parallel fiber stimulation 14. The role of the Golgi type cells described by Sotelo 18 has not been evaluated as well as a possible remote disfacilitation of mossy fiber inputs. Faber and Korn 7 strongly supported the existence of inhibition in frog cerebellum with a series of experiments involving an analysis of field potential and single unit data. Section of the cerebellar peduncles provided evidence against disfacilitation of tonic mossy fiber input as a means of producing a localized zone of inhibition. In Purkinje cells off-beam and close to the stimulating electrode, we have observed inhibition at least by 5 msec after the stimulus, a latency which is probably too short for such remote disfacilitation to occur. The minimum latency yet found for inhibition of cerebellar afferents in frog presumably by Purkinje cells by vestibular mossy fiber input of 1.8 msec 15. Lastly, direct evidence for involvement of a local cortical mechanism within the molecular layer rather than disfacilitation is provided in the accompanying paper 20 in which the bulk of the inhibitory pause is shown to be antagonized by direct microiontophoretic application of drugs to the region of the Purkinje cell dendritic tree. Our contention that inhibition exists does not indicate whether it is strong or weak in a normal functional context. A puzzling feature of frog cerebellar cortex is that paired stimuli to the parallel fibers result in either no effect on or a slight facilitaBrain Research, 33 (1971) 83-90
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89
tion of the response of Purkinje cells to the second pulse. In cat, where inhibition has been shown most unequivocally in barbiturate preparations, parallel fiber stimulation in unanesthetized decerebrate animals also has been shown to result in facilitation of Purkinje cells 3. One possibility is that parallel fiber stimulation may result in some facilitation which antagonizes the ongoing inhibition during the suppression of spontaneous firing, as has been suggested in the selachian cerebellum a. Such effects indicate that double pulse experiments, as other purely electrophysiological tests, are unreliable as assays of the lack of or the strength of inhibition. The simplest test, inhibition of spontaneous activity on- and off-beam after surface stimulation, would seem to indicate that inhibition in frog is as strong in relation to the background excitation as in any other vertebrate. If phylogeny is recapitulated by ontogeny in cerebellar development, then the concept of an inhibition-free cerebellum in lower forms may be groundless. Fujita 9 and Altman 1 have shown with autoradiographic pulse labeling techniques in mice and rats that mitosis and differentiation of the basket and stellate cells somewhat precede that of the granule cell proliferation within the external granular layer during the first and second postnatal weeks. The developing mammalian cerebellum thus has an identified inhibitory cell population from the earliest stages. One might hypothesize on these grounds that inhibitory interneurons ought to be present and effective in the earliest evolutionary forms. The results of the present experiments suggest that this may be the case in the frog cerebellum. SUMMARY Depression of spontaneous activity on- and off-beam and a hyperpolarization of Purkinje cells was observed after excitation of parallel fibers by local surface stimulation in curarized frogs. An activation of presumed stellate cell interneurons high in the molecular layer was found during the period of inhibition of Purkinje cells. It is proposed that stellate cells postsynaptically inhibit Purkinje cells in the frog cerebellum. ACKNOWLEDGEMENTS This work was supported by U.S. Public Health Service Grant MH-08034 and National Science Foundation Grant GB-12350. We would like to acknowledge the helpful advice of Dr. Karl Lowy.
REFERENCES 1 ALTMAN,J., Autoradiographic and histological studies of postnatal neurogenesis. III. Dating the time of production and onset of differentiation of cerebellar microneurons in rat, J. comp. Neurol., 136 (1969) 269-294. 2 ANDERSON,P., ECCLES, J., ANDVOORHOEVE,P. E., Postsynaptic inhibition of cerebellar Purkinje cells, J. Neurophysiol., 27 (1964) 1138-1153. 3 BLOEDEL,J. R., AND ROBERTS,W. J., Functional relationship among neurons of the cerebellar cortex in the absence of anesthesia, J. Neurophysiol., 32 (1969) 75-84. Brain Research, 33 (1971) 83-90
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4 DAVIDOFF,R., GRAHAM,L., SHANK, R., WERMAN,R., AND APRISON, M., Changes in amino acid concentration associated with loss of spinal interneurons, J. Neurochem., 14 (1967) 1025-103 I. 5 ECCLES,J. C., LLINAS, R., AND SASAKI,K., Parallel fiber stimulation and the responses induced thereby in the Purkinje cells of the cerebellum, Exp. Brain Res., l (1966) 17-39. 6 ECCLES,J. C., T.~BOP,IKOV.A,,H., AND TSUKAHARA,T., Responses of Purkinje cells of a selachian cerebellum, Brain Research, 17 (1970) 57-86. 7 FABER, D., AND KORN, H., Inhibition in the frog cerebellar cortex following parallel fiber activation, Brain Research, 17 (1970) 506-510. 8 FOXON, G. E. H., Blood and respiration. In J. A. MOORE (Ed.), Physiology of the Amphibia, Academic Press, New York, 1964, pp. 151-209. 9 FUJITA, S., Quantitative analysis of cell proliferation and differentiation in the cortex of the postnatal mouse cerebellum, J. Cell Biol., 32 (1967) 277-288. 10 GLEES, P., PEARSON, C., AND SMITH, A. G., Synapses on the Purkinje cells of the frog. Quart. J. exp. Physiol., 43 (1958) 52-60. 11 HILLMAN, D. E., Morphological organization of frog cerebellar cortex: a light and electron microscopic study, J. Neurophysiol., 32 (1969) 818-846. 12 LARSELL, O., The cerebellum of the frog, J. comp. NeuroL, 36 (1923) 89-112. 13 LLIN~S, R., AND BLOEOEL,J. R., Frog cerebellum: absence of long-term inhibition upon Purkinje cells, Science, 155 (1967) 601-603. 14 LLINAS, R., BLOEDEL, J. R., AND HILLMAN, D. E., Functional characterization of neuronal circuitry of frog cerebellar cortex, J. Neurophysiol., 32 (1969) 847-870. 15 LL1N~S, R., AND PRECH]-, W., The inhibitory vestibular efferent system and its relation to the cerebellum in the frog, Exp. Brain Res., 9 (1969) 16-29. 16 NICOLL, R., AND BARBER,J., Personal communication. 17 SIGGINS, G. R., WOODWARD,D. J., HOFFER, B. J., AND BLOOM,F. E., Responsiveness of cerebellar Purkinje cells to norepinephrine, cyclic AMP and prostaglandin E1 during synaptic morphogenesis in neonatal rat, Pharmacologist, 12 (1970) 198. 18 SOTELO, C., Ultrastructural aspects of the cerebellar cortex of the frog. In R. LLtN/,S (Ed.), Neurobiology of Cerebellar Evolution and Development, AMA Press, Chicago, 1969, pp. 327-370. 19 SOTELO,C., Stellate cells and their synapses on Purkinje cells in the cerebellum of the frog, Brain Research, 17 (1970) 510--515. 20 WOODWARD,D. J., HOFFER,B. J., SIGGINS, G. R., AND OLIVER,A. P., Inhibition of Purkinje cells in frog cerebellum. 11. Evidence for GABA as the inhibitory transmitter, Brain Research, 33 (1971) 91-100.
Brain Research, 33 (1971) 83-90
83
INHIBITION OF P U R K I N J E CELLS IN THE F R O G CEREBELLUM. I. EVIDENCE FOR A STELLATE CELL INHIBITORY PATHWAY
D. S. RUSHMER* ANDD. J. WOODWARD Center for Brain Research, and Department of Physiology, University of Rochester, Rochester, N.Y. 14642 (U.S.A.)
(Accepted April 4th, 1971)
INTRODUCTION Stellate and basket cell interneurons in the molecular layer of the cerebellar cortex in cat have been shown 2 to inhibit strongly the Purkinje cells beneath and lateral to a beam of excited parallel fibers. Llin~s et al. is,14, using field potential analysis and single unit recording, have failed to find electrophysiological evidence for strong inhibition on Purkinje cells during similar experiments in the frog (Rana catesbiana). It has therefore been suggested that the cerebellum of the frog, as an early phylogenetic form, may exhibit little of such postsynaptic inhibition. Sotelo 18,19 has recently presented evidence revealing widespread anatomical distribution of endings from molecular layer interneurons (stellate cells) onto the Purkinje cells, in Rana esculenta, whereas previous reports emphasized the scarcity of such connections in Rana catesbiana~O, 11. Moreover Faber and Korn 7 have recently found evidence indicating the presence of long term inhibition of Purkinje cells in Rana pipiens after surface stimulation. In this report, these electrophysiological findings regarding Purkinje cell responses are confirmed and evidence is presented that this inhibition is due to activity of stellate cells. METHODS Frogs (Rana pipiens and Rana clamitans) were paralyzed with 1.5 mg of D-tubocurarine. Xylocaine (4 ~o) was applied topically to wound margins. Access to the cerebellum was obtained by retracting the tectum bilaterally with hooks, following a craniotomy and excision of the dura. For respiration, intermittent positive pressure was applied to the trachea of the frog (15/min for 200-500 msec at 2 psi). Our experience has been that ventilation is essential for maintenance of good circulation in the frog cerebellum. Amphibians are known to exchange CO2 adequately through skin but employ lungs for absorption of one-half to two-thirds of 02 consumed a. With an acceptable circulation, all Purkinje cells in our preparations showed a spontaneous * Presently at the Laboratory of Neurophysiology, Good Samaritan Hospital and Medical Center, Portland, Ore. 97210, U.S.A. Brain Research, 33 (1971) 83-90
84
I). S. R U S H M E R A N D I). .l. W O O D W A R D
activity of about 5-20/sec. Maintenance of adequate circulation was considered to be essential in view of a possible selective effect of hypoxia on small inhibitory interneurons, as has been shown to occur in mammalian spinal cord 4. Stimuli (0.2 msec duration at 0.5/sec) were applied to the surface of the molecular layer through bipolar electrodes made from twisted 40-gauge enameled nichrome wire. Glass micropipettes filled with 3 M NaCl were used for extracellular recording while intracellular recording was done using electrodes filled with 0.5 M K2SO4. A PDP-8 computer was used to construct poststimulus-time (PST) histograms. Methods for recording and applying drugs with multi-barrel electrodes are described in the subsequent paper 20. RESULTS
Purkinje cells were identified by their location at about 250 #m depth and the spontaneous occurrence of bursts or 'inactivation' responses generated by activity in climbing fibers. As has been shown previously5,7,14 stimulation of a beam of parallel fibers caused excitation of Purkinje cells on the beam with a short latency. This excitatory response was followed by a cessation of spike activity lasting up to several hundred msec (Fig. 1A-G). This series of histograms shows further that both the probability of initial excitation and duration of the cessation of activity were smoothly graded with increasing stimulus intensity. In contrast to results reported previously 14, in over 100 cells examined, the initial excitation rarely yielded more than 2-3 spikes at any stimulus strength. The importance of using PST histograms in evaluating this inhibition is especially evident in these slowly firing cells. The histogram (Fig. 1G) reveals a distinct inhibition despite the rather subtle manifestation in the action potential record of Fig. 1A. Lateral spread of the inhibition is apparent and hence is preserved as a functional property in this primitive animal. Recordings from cells far lateral (500 #m or more) to the beam (Fig. 1I) showed no alteration of spontaneous activity, whereas cells 150-200 #m lateral to the beam exhibited cessation of spontaneous activity without initial excitation (Fig. 1H). A postexcitatory rebound mechanism for the inhibition is unlikely in view of this off-beam inhibition found in the absence of driven spikes. Intracellular records of cells stimulated 'on-beam' (Fig. 1B) exhibited an initial EPSP and spike followed by a hyperpolarization. Such hyperpolarizations originated from a flat baseline following an initial spike and could be observed in cells when spiking did not occur. Also, they decreased in size gradually as the cell deteriorated. This response can be readily distinguished from a similar hyperpolarization occasionally observed originating without a delay after a climbing fiber EPSP. The short duration of these penetrations prevented an examination of the permeability characteristics of the response by means of chloride injection, so it could not be positively identified as an IPSP. Stellate cells have been observed in the molecular layer of several species of frog 11,~s. It is reasonable that such cells may serve a function similar to that in mammals and cause inhibition in regions on and lateral to an excited beam of parallel Brain Research, 33 (1971) 83-90
INHIBITION OF PURKINJE CELLS IN FROG CEREBELLUM. I
85
A
(/
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ol
20 m~¢
G
., ,.,n . . . . . . . . . . .
J ....
i
i
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j j _ ~_..
Fig. 1. A - I are responses of Purkinje cells after parallel fiber stimulation. A, Action potential recording made from tape, spikes about 1 mV in amplitude. B, Intracellular recording, the spike caused by the initial EPSP rises off screen. C - G are poststimulus histograms obtained from the same Purkinje cell stimulated at different intensities. C is subthreshold; D, E, F, and G are 1.2, 1.5, 2.0 and 3.0 times threshold respectively. H and I were obtained from different Purkinje cells 200 and 500 # m lateral to the beam and stimulated with the same intensity in the same experiment as in G. The schematic outline of the half-cerebellum in K illustrates the recording positions at 1, 2, and 3 for G, H and I, respectively, while S is the stimulating position with the beam of excited parallel fibers represented by the horizontal line. J is a typical interspike interval histogram record from a Purkinje cell. Histograms C-I are 400 msec full scale; J is 500 msec full scale. Stimuli were applied at time zero, at 0.5/see and 0.2 msec duration.
Brain Research, 33 (1971) 83-90
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I). S. R U S H M E R A N D l). .I. W O O D W A R D
A
40
msec
B Fig. 2. Activity of a presumed stellate cell. A, Action potential record from tape, spikes about 1 mV. Poststimulus-time histogram 400 msec full scale. Stimuli at time zero. Cell was located 30/~m below the surface and above the Purkinje cell in Fig. 1C-G.
B,
fibers. Evidence of possible involvement of the stellate cells is provided by direct recording of these cells (Figs. 2, 3). Such cells were distinguished from Purkinje cells because they (a) never displayed spontaneous climbing fiber bursts (over a period as long as 20 min), (b) were found high in the molecular layer above the level of Purkinje cell bodies, (c) were lost after slight advances of the microelectrode, and (d) exhibited low rates of spontaneous activity (1-2/sec v e r s u s 5-20/sec for Purkinje cells). Such units were further distinguished by their response to parallel fiber stimulation, which often caused an excitation lasting 60-100 msec, usually represented by a group of 2-6 spikes (Fig. 2) with no sign of a subsequent reduction of activity. Histograms appeared as almost a mirror image of the inhibition exhibited by Purkinje cells. The unit shown in Fig. 2 was found in the molecular layer above the Purkinje cell studied in Fig. 1C-G. Activity was recorded from 21 such units. Further properties are shown in Fig. 3 for another such unit found 30/~m below the surface. The slow firing rate is illustrated in Fig. 3, where the interspike interval histogram reveals a modal interval of about 1 sec. This is in marked contrast with histograms from typical Purkinje cells, as in Fig. 1J where the mean firing rate was 6.5/sec. The spontaneous activity was found to be inhibited by iontophoretic application of GABA (gamma-aminobutyric acid). Fibers are not known to be sensitive to GABA 16 so the electrode was probably near the soma and/or the dendrites. Stimulation of the surface did not result in an inhibition of the spontaneous activity. The Fig. 3B poststimulus histogram consisted of a peak due to 2 or 3 driven spikes followed by a low but even probability of firing at the stimulus intensity employed. The action potential record in Fig. 3B recorded high in the molecular layer shows a driven parallel fiber volley followed by the two spikes. DISCUSSION
The major piece of evidence presented here is that driven activity of presumed stellate cells high in the molecular layer occurs during a period of driven inhibition of spontaneous activity of the Purkinje cells. The presence of a limited lateral spread of Brain Research, 33 (1971) 83-90
INHIBITION OF PURKINJE CELLS IN FROG CEREBELLUM. I
87
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j,,,I
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Z 0 ¢J
L1._. 125 msec
Fig. 3. Activity of presumed stellate cell. A, Action record of slow spontaneous activity is shown to be inhibited by iontophoretic application of GABA. The interspike interval histogram shows a modal interval at about 1 sec. B, Action record shows the shock artifact followed by the parallel fiber negative wave and two spikes. Positive is up. The poststimulus-time histogram shows a high peak due to triggering on parallel fiber volley; a low peak due to evoked spikes and a low and even density of subsequent spontaneous activity. Stimuli applied at 10 msec indicated by arrowhead, applied at 0.5/sec and 0.2 msec duration.
inhibition 7 is confirmed here, this spatial distribution being consistent with the expected distribution of axons from inhibitory interneurons is. Further, a hyperpolarization was observed in Purkinje cells following a stimulus to the afferent parallel fibers. In summary, these results, though circumstantial in some respects, support the simple scheme that parallel fibers excite stellate cells which in turn postsynaptically inhibit P u r k i n j e cells.
This is in apparent contradiction with previous reportsla, 14 contending that in frog there is an 'absence of long term inhibition upon Purkinje cells.' However, the possible inhibitory function of stellate cells was not ruled out in those papers 13,14. Such differences in interpretation may result from differences in the physiological state of the preparations. Purkinje cells in our preparation always exhibited spontaneous activity and never yielded more than 2 or 3 spikes in response to local surface stimulation. Lack of inhibition could result if an alteration of state, via anesthetic or circulation, led to increased excitability of Purkinje cells la. Alterations of state may also have selective effects on inhibitory interneurons 4. We have noted considerable variation in Brain Research, 33 (1971) 83-90
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the duration of pause after parallel fiber activation both in different cells and within the same cell at different times, suggesting that the inhibition may be prone to influence by such factors. Variations in function between species of frogs (see Introduction) seem possible. However, as has been noted 18, optical microscopic studies 12 of the cerebellum of 4 different frog species were unable to demonstrate morphological variations according to the species. Rana clamitans in particular was employed here because, in comparison with our experience with Rana catesbiana, it was possible to penetrate through the pial surface with notably less dimpling and possible injury. We feel moreover that methodological variations in physiological studies are as important as species variations in this regard. Several considerations indicate that activity such as shown in Figs. 2 and 3 is in fact from stellate neurons. The location high in the molecular layer, slow firing rate without climbing fiber inactivation responses, and long term activation without inhibition following stimulation of parallel fibers distinguish the units from Purkinje cells. One possibility is that such activity is in fact from aberrant mossy fibers known to penetrate the molecular layer in amphibialL This is ruled out by the experiment shown in Fig. 3 since fibers are not known to be influenced by the application of GABA. This result is also of interest since only parallel and climbing fiber excitatory synapses have been described on frog stellate cells. It may be that the GABA sensitivity indicates the presence of an undescribed class of inhibitory synapses. Alternately, sensitivity to GABA may be characteristic of this type of neuron independent of the presence of a functional mechanism for making use of GABA sensitivity, as in the case of newborn rat cerebellum where Purkinje cells are sensitive to GABA even before onset of synaptogenesislL A limitation of the present results is that it is difficult to evaluate what contribution disfacilitation plays in generating the pause after parallel fiber stimulation 14. The role of the Golgi type cells described by Sotelo 18 has not been evaluated as well as a possible remote disfacilitation of mossy fiber inputs. Faber and Korn 7 strongly supported the existence of inhibition in frog cerebellum with a series of experiments involving an analysis of field potential and single unit data. Section of the cerebellar peduncles provided evidence against disfacilitation of tonic mossy fiber input as a means of producing a localized zone of inhibition. In Purkinje cells off-beam and close to the stimulating electrode, we have observed inhibition at least by 5 msec after the stimulus, a latency which is probably too short for such remote disfacilitation to occur. The minimum latency yet found for inhibition of cerebellar afferents in frog presumably by Purkinje cells by vestibular mossy fiber input of 1.8 msec 15. Lastly, direct evidence for involvement of a local cortical mechanism within the molecular layer rather than disfacilitation is provided in the accompanying paper 20 in which the bulk of the inhibitory pause is shown to be antagonized by direct microiontophoretic application of drugs to the region of the Purkinje cell dendritic tree. Our contention that inhibition exists does not indicate whether it is strong or weak in a normal functional context. A puzzling feature of frog cerebellar cortex is that paired stimuli to the parallel fibers result in either no effect on or a slight facilitaBrain Research, 33 (1971) 83-90
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tion of the response of Purkinje cells to the second pulse. In cat, where inhibition has been shown most unequivocally in barbiturate preparations, parallel fiber stimulation in unanesthetized decerebrate animals also has been shown to result in facilitation of Purkinje cells 3. One possibility is that parallel fiber stimulation may result in some facilitation which antagonizes the ongoing inhibition during the suppression of spontaneous firing, as has been suggested in the selachian cerebellum a. Such effects indicate that double pulse experiments, as other purely electrophysiological tests, are unreliable as assays of the lack of or the strength of inhibition. The simplest test, inhibition of spontaneous activity on- and off-beam after surface stimulation, would seem to indicate that inhibition in frog is as strong in relation to the background excitation as in any other vertebrate. If phylogeny is recapitulated by ontogeny in cerebellar development, then the concept of an inhibition-free cerebellum in lower forms may be groundless. Fujita 9 and Altman 1 have shown with autoradiographic pulse labeling techniques in mice and rats that mitosis and differentiation of the basket and stellate cells somewhat precede that of the granule cell proliferation within the external granular layer during the first and second postnatal weeks. The developing mammalian cerebellum thus has an identified inhibitory cell population from the earliest stages. One might hypothesize on these grounds that inhibitory interneurons ought to be present and effective in the earliest evolutionary forms. The results of the present experiments suggest that this may be the case in the frog cerebellum. SUMMARY Depression of spontaneous activity on- and off-beam and a hyperpolarization of Purkinje cells was observed after excitation of parallel fibers by local surface stimulation in curarized frogs. An activation of presumed stellate cell interneurons high in the molecular layer was found during the period of inhibition of Purkinje cells. It is proposed that stellate cells postsynaptically inhibit Purkinje cells in the frog cerebellum. ACKNOWLEDGEMENTS This work was supported by U.S. Public Health Service Grant MH-08034 and National Science Foundation Grant GB-12350. We would like to acknowledge the helpful advice of Dr. Karl Lowy.
REFERENCES 1 ALTMAN,J., Autoradiographic and histological studies of postnatal neurogenesis. III. Dating the time of production and onset of differentiation of cerebellar microneurons in rat, J. comp. Neurol., 136 (1969) 269-294. 2 ANDERSON,P., ECCLES, J., ANDVOORHOEVE,P. E., Postsynaptic inhibition of cerebellar Purkinje cells, J. Neurophysiol., 27 (1964) 1138-1153. 3 BLOEDEL,J. R., AND ROBERTS,W. J., Functional relationship among neurons of the cerebellar cortex in the absence of anesthesia, J. Neurophysiol., 32 (1969) 75-84. Brain Research, 33 (1971) 83-90
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4 DAVIDOFF,R., GRAHAM,L., SHANK, R., WERMAN,R., AND APRISON, M., Changes in amino acid concentration associated with loss of spinal interneurons, J. Neurochem., 14 (1967) 1025-103 I. 5 ECCLES,J. C., LLINAS, R., AND SASAKI,K., Parallel fiber stimulation and the responses induced thereby in the Purkinje cells of the cerebellum, Exp. Brain Res., l (1966) 17-39. 6 ECCLES,J. C., T.~BOP,IKOV.A,,H., AND TSUKAHARA,T., Responses of Purkinje cells of a selachian cerebellum, Brain Research, 17 (1970) 57-86. 7 FABER, D., AND KORN, H., Inhibition in the frog cerebellar cortex following parallel fiber activation, Brain Research, 17 (1970) 506-510. 8 FOXON, G. E. H., Blood and respiration. In J. A. MOORE (Ed.), Physiology of the Amphibia, Academic Press, New York, 1964, pp. 151-209. 9 FUJITA, S., Quantitative analysis of cell proliferation and differentiation in the cortex of the postnatal mouse cerebellum, J. Cell Biol., 32 (1967) 277-288. 10 GLEES, P., PEARSON, C., AND SMITH, A. G., Synapses on the Purkinje cells of the frog. Quart. J. exp. Physiol., 43 (1958) 52-60. 11 HILLMAN, D. E., Morphological organization of frog cerebellar cortex: a light and electron microscopic study, J. Neurophysiol., 32 (1969) 818-846. 12 LARSELL, O., The cerebellum of the frog, J. comp. NeuroL, 36 (1923) 89-112. 13 LLIN~S, R., AND BLOEOEL,J. R., Frog cerebellum: absence of long-term inhibition upon Purkinje cells, Science, 155 (1967) 601-603. 14 LLINAS, R., BLOEDEL, J. R., AND HILLMAN, D. E., Functional characterization of neuronal circuitry of frog cerebellar cortex, J. Neurophysiol., 32 (1969) 847-870. 15 LL1N~S, R., AND PRECH]-, W., The inhibitory vestibular efferent system and its relation to the cerebellum in the frog, Exp. Brain Res., 9 (1969) 16-29. 16 NICOLL, R., AND BARBER,J., Personal communication. 17 SIGGINS, G. R., WOODWARD,D. J., HOFFER, B. J., AND BLOOM,F. E., Responsiveness of cerebellar Purkinje cells to norepinephrine, cyclic AMP and prostaglandin E1 during synaptic morphogenesis in neonatal rat, Pharmacologist, 12 (1970) 198. 18 SOTELO, C., Ultrastructural aspects of the cerebellar cortex of the frog. In R. LLtN/,S (Ed.), Neurobiology of Cerebellar Evolution and Development, AMA Press, Chicago, 1969, pp. 327-370. 19 SOTELO,C., Stellate cells and their synapses on Purkinje cells in the cerebellum of the frog, Brain Research, 17 (1970) 510--515. 20 WOODWARD,D. J., HOFFER,B. J., SIGGINS, G. R., AND OLIVER,A. P., Inhibition of Purkinje cells in frog cerebellum. 11. Evidence for GABA as the inhibitory transmitter, Brain Research, 33 (1971) 91-100.
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