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Purkinje cell giant spikes in the trout, Salmo gairdneri (Richardson)
Comp. Biochem. Physiol., 1975, Vol. 5OA,pp. 253 to 257. Pergamon Press. Printed in Great Britain
PURKINJE CELL GIANT SPIKES IN THE TROUT, SALMO GAIRDNERI (RICHARDSON)* MIRIAM D. REID? AND R. A. WESTERMAN Department of Physiology, Monash University, Clayton, Victoria 3168, Australia (Received
8 November 1973)
Abstract-l. Purkinje cell giant spikes were recorded in the trout cerebellum. 2. Lesioning experiments indicated these giant spikes were dendritic rather than soma potentials. 3. Giant spikes could be evoked by deep cerebellar afferent fibre stimulation, but not by stimulation of parallel fibres alone. Antidromic excitation of giant spikes was not observed. 4. Intracellular recordings suggested the trout giant spike was a synaptic potential, not an action potential. 5. It was concluded that the giant spike, as seen in a.c. recordings in the present study, represented the differentiated waveform of the large excitatory post-synaptic potential produced in the Purkinje cell by excitation of its climbing fibre synapses.
INTRODUCTION
MATERIALS AND METHODS
RECENTcomparative studies have shown that there is a basic pattern in cerebellar histology and electrophysiology underlying the similarities in cerebellar function throughout the vertebrate kingdom (see Llinas, 1969). A consistent electrophysiological finding has been that Purkinje cells generate giant spikes (Granit & Phillips, 1956; Matthews et al., 1958; Nacimiento, 1969) which may exhibit two waveforms. These have been termed simple and complex spikes (Thach, 1968, 1970) and are considered to arise from the parallel and climbing fibre inputs respectively (Eccles et al., 1966a, b). Previous experiments in this laboratory have indicated that the giant spikes generated by Purkinje cells in the teleost, Salmo gairdneri (Richardson), are of only one waveform: a positive or positive-negative spike followed by a slow wave (Waks & Westerman, 1970). In the present study, the nature of the giant spike in the trout was further examined. It was concluded that this spike is probably not an action potential but rather the large summed excitatory post-synaptic potential produced in the Purkinje cell dendritic tree by the synchronous excitation of the many climbing fibre synapses located on the dendrites. The “spike-like” appearance of the giant spike resulted from differentiation of the synaptic potential by the a.c. recording procedure.
Trout weighing 200-600 g were anaesthetized in a solution containing 0.2 mg/ml MS 222 (tricaine methanesulphonate, Sandoz) and then injected intramuscularly with Flaxedil (gallamine triethiodide, May and Baker) in order to abolish gill movements. The fish were wrapped in wet paper towels and held in place by clamps. Oxygenated water, maintained at lO-14”C, was passed over the gills through a mouthpiece. The cranium was removed and the exposed cerebellum covered with cold mineral oil. Extracellular recordings in the cerebellum were made using glass-coated, tungsten microelectrodes with platinum-plated tips, connected to a high-input impedance amplifier. The output of the amplifier was passed through a preamplifier, displayed on a dual-beam oscilloscope and could be photographed with a kymographic camera. All extracellular recording was a.c. with the upper and lower frequency limits of the preamplifier set at 40 or 10 kHz and 80 or 8 Hz respectively. Intracellular d.c. records were obtained using glass micropipettes (5-15 M) filled with 3 M KCl, and an electrometer, the output of which was led directly into the oscilloscope. Bipolar stimulating electrodes were placed on the surface of the cerebellum in order to excite parallel fibres preferentially (Eccles et al., 1966a), and deep in the cerebellum to activate Purkinje cells antidromically and to stimulate cerebellar afferent fibres. The surface stimulating electrodes consisted of either a pair of fine (0.004 in. dia) platinum wires insulated with Epoxylite enamel except for the flat surface in contact with the cerebellum, or a pair of fine (O+KKlSin. dia) glass-coated wires (platinum 8% tungsten alloy, English Electric Co.). The deep cerebellar stimulating electrodes were paired electrodes of the same type as those used for extracellular recording.
* Supported by the Australian Research Grants Committee, Grant No. D66/15991. t Present address : Department of Physiology, School of Medicine, University of California, San Francisco, California 94143, U.S.A. 9
253
MIIUAM D. REIDANDR. A. WESTERMAN
254
To deternine the region of the Purkinje cell from which giant spikes were recorded, current (5 PA for 10 set) was passed through the recording electrode at the depth at which giant spikes reached maximal amplitude. The brains were later fixed, sectioned and stained using conventional histological techniques, and the cerebellar sections examined with a light microscope. RESULTS 1. Types of Purk~nje cell potentials
Several types of spontaneous spike discharge were recorded from the trout Purkinje cell : (i) A dual discharge consisting of a single spike and a short high-frequency (500/set) burst of spikes superimposed on a slow wave. This type of discharge was recorded at depths corresponding to the layer of Purkinje cell somas, and was similar to the dual discharge typical of Purkinje cells in other species. (ii) Large, short d~~ut~o~(1 msec) ~egfftj~e spikes, regularly recorded in the layer of Purkinje cell axons which, in the trout, lies immediately below the Purkinje cell somas (Waks, 1971). It was thought that these spikes were generated by Purkinje cells for the following reasons : (a) Electrical stimulation of the cerebellar surface (parallel fibres) evoked one to three spikes followed by a period of suppression of spike activity (Eccles et al., 19&a). (b) A conditioning surface stimulus inhibited the response to a test surface stimulus at short test intervais (Eccles et al., 1966a). (c) Appropriate placement of stimulating electrodes deep within the cerebellum produced antidromic activation. (iii) Giant spikes: these were recorded more superficially than (i) and (ii) and were the largest and most f~quently recorded form of unit activity. The present paper is concerned only with this form of Purkinje discharge. 2. Giant spikes (i) Extracellular recording. (a) Spontaneous activity. Each giant spike consisted of a large 3-5 msec positive or positive-negative deflection, followed by a smaller slow wave 10-40 msec in duration (Fig. 1). Giant spikes fired at average rates ranging from 0.5 to 2*5/set, the most common rate being about llsec. Multiple firing was never observed, there being at least 100 msec between consecutive giant spikes. (b) Evoked activity. Giant spike activity could be evoked orthodromically by deep cerebellar stimulation but not by stimulation of the parallel fibres. Antidromically activated giant spikes could not be produced by deep cerebellar stimulation. (c) Source. Lesions made at the depths at which giant spikes reached their maximum amplitude were found to be in the molecular layer immediately above
the Purkinje cell somas, in the region of the main dendrites of the Purkinje cells. (d) Relationship to other Purkinje cell potentials. Giant spikes were usually not associated with other single unit activity, but in a few instances, a second, smaller spike was simultaneously recorded (Fig. 2). A 20-100 msec pause in the discharge of the smaller spike always occurred immediately after the initial rapid deflection of the giant spike, and the amplitude of the smaller spike was reduced when the unit fired during the giant spike slow wave, (b)
(a)
+ 4-Y ImV
I
ImV I,
I
1
I 50
msec
40 m3ec
J
Fig. 1. Trout Purkinje cell giant spikes. (a) and (b) show photographs of giant spikes recorded from two different cells. Extracellular a.c. recording, 0302 set coupling time constant.
An additional effect consistently observed was related to a form of injury discharge (Fig. 2) produced when the electrode was advanced from the site of maximal giant spike recording in order to obtain intracellular recordings. This injury discharge was similar to that reported by Fujita (1968) in the rabbit cerebellum, and took the form of slow waves similar in amplitude and duration to the slow wave component of the giant spike. The frequency of the injury discharge increased with time and became rhythmical. At the onset of the injury potentials there was an increase in the firing frequency of the smaller spike, a decrease in its amplitude and finally a complete suppression of its activity. At the termination of the injury potential, there was a gradual recovery in the amplitude of the smaller unit. The frequency of the giant spike discharge appeared to be independent of the smaller spike and the injury potential. However, when a giant spike occurred during an injury potential, it was reduced in amplitude (Fig. 2). These findings suggested that the giant spike, the smaller unit and the injury potential were all generated by the same Purkinje cell, but that the giant spike and the smaller unit
Purkinje cell giant spikes in Salmo gairdneri
I
I
200msec
Fig. 2. Relationship of a giant spike to other Purkinje cell potentials. Extracellular a.c. recording (as Fig. 1). Each trace shows the background activity of a giant spike (large unit) and a simultaneously recorded smaller spike considered, for reasons outlined in the text, to be generated by the same Purkinje cell. The traces are not continuous in time but were recorded at increasing intervals after the units were first isolated. Note that activity of the smaller spike is suppressed during the giant spike slow wave. In the three lower traces, a biphasic slow wave (presumed to be an injury potential) is evident, increasing in frequency and rhythmicity with time. Activity of the small spike is initially increased and then completely suppressed by the injury potential. Note that when the giant spike fires during an injury potential (*), its amplitude is reduced.
were produced by different mechanisms, possibly in different parts of the Purkinje cell. (ii) Zntracellular recording. Difficulty was experienced in obtaining stable intracellular recordings from Purkinje cells. In most instances, membrane potential declined rapidly and it was not possible to make prolonged observations. As the recording electrode was advanced from the extracellular position of maximal giant spike amplitude to a point within the cell membrane (as indicated by the negative d.c. shift in recorded potential), there was no sign of giant spike injury discharge. The giant spike continued to fire at prepenetration rates, but its amplitude rapidly decreased. No action potentials were recorded. However, two types of depolarizing potentials, each followed by a slight hyperpolarization, were seen in intracellular d.c. records (Fig. 3). These differed primarily in their rise-time; one rose rapidly to its peak amplitude (fast rise-time potential, FRTP) while the other had a gradual onset (slow rise-time potential, SRTP). The FRTP corresponded to the greatly diminished giant spike (as seen in the a.c. record in Fig. 3) and the SRTP appeared to correspond to the injury potential described earlier. The FRTP had an additional depolarizing potential superimposed on its repolarizing phase. This delayed
depolarization corresponded to the giant spike slow wave in a.c. records. Although action potentials arising from the FRTP were not observed, it was thought that the initial component of the FRTP was probably an excitatory post-synaptic potential. Because of the difficulty in maintaining stable intracellular recordings, it was not possible to manipulate membrane potential and determine an equilibrium potential for the FRTP. However, it was noted that whenever the FRTP occurred at the peak of one of the SRTPs, the amplitude of the FRTP was reduced, a finding consistent with the interpretation of the FRTP as a synaptic potential rather than an action potential. When comparing simultaneous a.c. and d.c. records (Fig. 3 paired traces), it became apparent that the a.c. recordings of both the giant spike and the slow wave were not true representations of the actual waveform as revealed by the d.c. traces. Since the amplitude of an a.c.-coupled recording is proportional to the rate of change of the d.c. potential, the sharp rise of the d.c.-recorded FRTP was translated into a large positive deflection in the a.c. recording. The subsequent plateau of the FRTP (zero change in slope) caused the a.c. record to return to its zero reference level. Thus the initial portion of the FRTP was differentiated by a.c. coupling into what appears
MIRIAMD. REID AND R. A. WESTERMAN
256
+ 8CmV
i
Fig. 3. Intracellular recording from another Purkinje cell. The first two traces and the upper record in each of the bottom three pairs are d.c. recordings. The lower record in each of the three paired traces
was recorded a.c. simultaneously with the d.c. trace. In the d.c. records, two types of depolarizing potentials, each followed by a slight hyperpolarization, can be seen. One type of&c. potential has a slow rise-time (SRTP) and the other a fast rise-time (FRTP). The FRTP has a delaved deoolarizine wave (peak indicated by a dot) superimposed on its r&polarizing phase. The arrowed-potentials in the a.c. records are greatly deteriorated giant spikes. It can be seen that the a.c. recorded giant spike corresponds to the initial part of the FRTP, the giant spike slow wave to the delayed depolarization of the FRTP and the a.c. recorded slow wave (injury potential) to the SRTP. It was concluded that all the a.c. recorded potentials represented the differentiated waveforms of the d.c. potentials, and that this distortion arose from the short coupling time constant (0402 set) of the preamplifier used in a.c. recording.
as a spike. In a similar fashion, the slow rise and decay of the SRTP and of the delayed depolarization superimposed on the repolarizing phase of the FRTP were differentiated by a.c. coupling into a biphasic slow wave with a much steeper gradient than the original potential. These distortions of the true waveforms were probably accentuated by the very short coupling time constants (0.02 or 0.002 set) used. DISCUSSION
In common with Purkinje cells in other species, the trout Purkinje cell produces simple and complex spikes. However, in addition to this characteristic dual discharge, trout Purkinje cells also generate giant spikes. The consistent recording of giant spikes in the area immediately above, rather than in, the layer of Purkinje cells bodies suggested the giant spike was a dendritic rather than a soma potential. Stimulation of afferent fibres deep within the cerebellum, but not of parallel fibres alone, was effective in evoking giant spikes. This finding suggested that the generation of giant spikes was related to activity within the climbing fibre rather than the mossy fibre pathway. The suppression of Purkinje cell spike activity by the giant spike was similar to that typically seen following the climbing fibre-induced complex spike in other species (Granit & Phillips, 1956; Thach, 1968; Nacimiento, 1969),
further suggesting a link between trout giant spikes and the climbing fibre system. In a previous communication we considered the possibility that the trout giant spike was an action potential (Waks & Westerman, 1970). The present data, however, suggest that the giant spike is not an action potential. It was not possible to produce an antidromically activated giant spike. Multiple giant spike firing did not occur. In addition, an increase in giant spike firing frequency due to injury of the cell membrane was not observed. Indeed, the rate of giant spike discharge was remarkably constant at all times, suggesting a dependence on extracellular rather than intracellular events. The amplitude of the extracellularly recorded giant spike and the intracellularly recorded FRTP (which appeared to correspond to the giant spike) was reduced by the depolarizing injury potentials. However, giant spike firing frequency was not affected by the injury potentials. Such variation in amplitude with cell without any concomitant membrane potential, change in firing frequency, is consistent with the giant spike being a synaptic potential rather than an action potential. On the basis of this evidence, we suggest that the trout giant spike is a very large exitatory postsynaptic potential produced upon excitation of the Purkinje cell by its climbing fibre, and recorded from the Purkinje cell main dendrite. Its “spike-like” appearance in a.c. records is probably an artifact
Purkinje cell giant spikes in Salmo gairdneri of the short coupling time constant used in the recording apparatus, and may simply represent the differentiated waveform of the EPSP. Intracellular recordings revealed that the giant spike slow wave corresponded to a delayed depolarization occurring on the repolarizing phase of the climbing fibre EPSP. It is possible that this delayed depolarization represents electrotonic spread of dendritic action potentials produced by climbing fibre excitation in the dendritic tree. Such dendritic action potentials have been recorded from Purkinje cells in the rabbit (Fujita, 1968) and alligator (Llinas et al., 1968). The injury potential may arise in a similar fashion from dendritic action potentials resulting from injury to the dendrites by the advancing electrode.
REFERENCES
ECCLESJ. C., LLINAS R. & SASAKIK. (1966a) Parallel fibre stimulation and the responses induced thereby in the Purkinje cells of the cerebellum. Exp. Brain Res. 1, 17-39. ECCLESJ. C., LLINASR. & SASAKIK. (1966b) The excitatory synaptic action of climbing fibres on the Purkinje cells of the cerebellum. .Z.Physiol., Lond. 182,268296. FUJITAY. (1968) Activity of dendrites of single Purkinje cells and its relationship to so-called inactivation response in the rabbit cerebellum. .Z. Neurophysiol. 31,
257
LLINAS R. (Editor) (1969) Neurobiology of Cerebellar Evolution and Development. Am. Med. Assn. Educ.
and Res. Fdn., Chicago. LLINAS R., NICHOLSONC., FREEMANJ. A. & HILLMAN D. E. (1968) Dendritic spikes and their inhibition in alligator Purkinje cells. Science, Wash. 160, 11321135. MATTHEWSP. B. C., PHILLIPSC. G. & RUSHWORTHG. (1958) Afferent systems converging upon cerebellar Purkinje cells in the frog. Q. J. exp. Physiol. 43, 38-52. NACIMIENTOA. C. (1969) Spontaneous and evoked discharges of cerebellar Purkinje cells in the frog. In Neurobiology of Cerebellar Evolution and Development (Edited by LLINASR.), pp. 373-395. Am. Med. Assn. Educ. and Res. Fdn., Chicago. THACHW. T. (1968) Discharge of Purkinje and cerebellar nuclear neurones during rapidly alternating arm movements in the monkey. J. Neurophysiol. 31, 785797. THACH W. T. (1970) Discharge of cerebellar neurons related to two maintained postures and two prompt movements-II. Purkinje cell output and input. J. Neurophysiol. 33, 537-547. WAKS M. D. (1971) Cerebellar neurones in the rainbow trout, Salmo gairdneri (Richardson): structural and electrophysiological observations. Ph.D. thesis. Monash University, Australia. WAKS M. D. & WESTERMANR. A. (1970) Inhibition of Purkinje cells in the cerebellum of the teleost Salmo gairdneri (Richardson). Camp. Biochem. Physiol. 33, 465-469.
131-141.
GRANIT R. & PHILLIPS C. G. (1956) Excitatory and inhibitory processes acting upon individual Purkinje cells of the cerebellum in cats. J. Physiol., Land. 133, 520-547.
Key Word Index-Trout, cerebellum, Purkinje cell, giant spike; atferent fibre stimulation; dendritic potentials; excitatory post-synaptic potential; differentiation by a.c. coupling.
PURKINJE CELL GIANT SPIKES IN THE TROUT, SALMO GAIRDNERI (RICHARDSON)* MIRIAM D. REID? AND R. A. WESTERMAN Department of Physiology, Monash University, Clayton, Victoria 3168, Australia (Received
8 November 1973)
Abstract-l. Purkinje cell giant spikes were recorded in the trout cerebellum. 2. Lesioning experiments indicated these giant spikes were dendritic rather than soma potentials. 3. Giant spikes could be evoked by deep cerebellar afferent fibre stimulation, but not by stimulation of parallel fibres alone. Antidromic excitation of giant spikes was not observed. 4. Intracellular recordings suggested the trout giant spike was a synaptic potential, not an action potential. 5. It was concluded that the giant spike, as seen in a.c. recordings in the present study, represented the differentiated waveform of the large excitatory post-synaptic potential produced in the Purkinje cell by excitation of its climbing fibre synapses.
INTRODUCTION
MATERIALS AND METHODS
RECENTcomparative studies have shown that there is a basic pattern in cerebellar histology and electrophysiology underlying the similarities in cerebellar function throughout the vertebrate kingdom (see Llinas, 1969). A consistent electrophysiological finding has been that Purkinje cells generate giant spikes (Granit & Phillips, 1956; Matthews et al., 1958; Nacimiento, 1969) which may exhibit two waveforms. These have been termed simple and complex spikes (Thach, 1968, 1970) and are considered to arise from the parallel and climbing fibre inputs respectively (Eccles et al., 1966a, b). Previous experiments in this laboratory have indicated that the giant spikes generated by Purkinje cells in the teleost, Salmo gairdneri (Richardson), are of only one waveform: a positive or positive-negative spike followed by a slow wave (Waks & Westerman, 1970). In the present study, the nature of the giant spike in the trout was further examined. It was concluded that this spike is probably not an action potential but rather the large summed excitatory post-synaptic potential produced in the Purkinje cell dendritic tree by the synchronous excitation of the many climbing fibre synapses located on the dendrites. The “spike-like” appearance of the giant spike resulted from differentiation of the synaptic potential by the a.c. recording procedure.
Trout weighing 200-600 g were anaesthetized in a solution containing 0.2 mg/ml MS 222 (tricaine methanesulphonate, Sandoz) and then injected intramuscularly with Flaxedil (gallamine triethiodide, May and Baker) in order to abolish gill movements. The fish were wrapped in wet paper towels and held in place by clamps. Oxygenated water, maintained at lO-14”C, was passed over the gills through a mouthpiece. The cranium was removed and the exposed cerebellum covered with cold mineral oil. Extracellular recordings in the cerebellum were made using glass-coated, tungsten microelectrodes with platinum-plated tips, connected to a high-input impedance amplifier. The output of the amplifier was passed through a preamplifier, displayed on a dual-beam oscilloscope and could be photographed with a kymographic camera. All extracellular recording was a.c. with the upper and lower frequency limits of the preamplifier set at 40 or 10 kHz and 80 or 8 Hz respectively. Intracellular d.c. records were obtained using glass micropipettes (5-15 M) filled with 3 M KCl, and an electrometer, the output of which was led directly into the oscilloscope. Bipolar stimulating electrodes were placed on the surface of the cerebellum in order to excite parallel fibres preferentially (Eccles et al., 1966a), and deep in the cerebellum to activate Purkinje cells antidromically and to stimulate cerebellar afferent fibres. The surface stimulating electrodes consisted of either a pair of fine (0.004 in. dia) platinum wires insulated with Epoxylite enamel except for the flat surface in contact with the cerebellum, or a pair of fine (O+KKlSin. dia) glass-coated wires (platinum 8% tungsten alloy, English Electric Co.). The deep cerebellar stimulating electrodes were paired electrodes of the same type as those used for extracellular recording.
* Supported by the Australian Research Grants Committee, Grant No. D66/15991. t Present address : Department of Physiology, School of Medicine, University of California, San Francisco, California 94143, U.S.A. 9
253
MIIUAM D. REIDANDR. A. WESTERMAN
254
To deternine the region of the Purkinje cell from which giant spikes were recorded, current (5 PA for 10 set) was passed through the recording electrode at the depth at which giant spikes reached maximal amplitude. The brains were later fixed, sectioned and stained using conventional histological techniques, and the cerebellar sections examined with a light microscope. RESULTS 1. Types of Purk~nje cell potentials
Several types of spontaneous spike discharge were recorded from the trout Purkinje cell : (i) A dual discharge consisting of a single spike and a short high-frequency (500/set) burst of spikes superimposed on a slow wave. This type of discharge was recorded at depths corresponding to the layer of Purkinje cell somas, and was similar to the dual discharge typical of Purkinje cells in other species. (ii) Large, short d~~ut~o~(1 msec) ~egfftj~e spikes, regularly recorded in the layer of Purkinje cell axons which, in the trout, lies immediately below the Purkinje cell somas (Waks, 1971). It was thought that these spikes were generated by Purkinje cells for the following reasons : (a) Electrical stimulation of the cerebellar surface (parallel fibres) evoked one to three spikes followed by a period of suppression of spike activity (Eccles et al., 19&a). (b) A conditioning surface stimulus inhibited the response to a test surface stimulus at short test intervais (Eccles et al., 1966a). (c) Appropriate placement of stimulating electrodes deep within the cerebellum produced antidromic activation. (iii) Giant spikes: these were recorded more superficially than (i) and (ii) and were the largest and most f~quently recorded form of unit activity. The present paper is concerned only with this form of Purkinje discharge. 2. Giant spikes (i) Extracellular recording. (a) Spontaneous activity. Each giant spike consisted of a large 3-5 msec positive or positive-negative deflection, followed by a smaller slow wave 10-40 msec in duration (Fig. 1). Giant spikes fired at average rates ranging from 0.5 to 2*5/set, the most common rate being about llsec. Multiple firing was never observed, there being at least 100 msec between consecutive giant spikes. (b) Evoked activity. Giant spike activity could be evoked orthodromically by deep cerebellar stimulation but not by stimulation of the parallel fibres. Antidromically activated giant spikes could not be produced by deep cerebellar stimulation. (c) Source. Lesions made at the depths at which giant spikes reached their maximum amplitude were found to be in the molecular layer immediately above
the Purkinje cell somas, in the region of the main dendrites of the Purkinje cells. (d) Relationship to other Purkinje cell potentials. Giant spikes were usually not associated with other single unit activity, but in a few instances, a second, smaller spike was simultaneously recorded (Fig. 2). A 20-100 msec pause in the discharge of the smaller spike always occurred immediately after the initial rapid deflection of the giant spike, and the amplitude of the smaller spike was reduced when the unit fired during the giant spike slow wave, (b)
(a)
+ 4-Y ImV
I
ImV I,
I
1
I 50
msec
40 m3ec
J
Fig. 1. Trout Purkinje cell giant spikes. (a) and (b) show photographs of giant spikes recorded from two different cells. Extracellular a.c. recording, 0302 set coupling time constant.
An additional effect consistently observed was related to a form of injury discharge (Fig. 2) produced when the electrode was advanced from the site of maximal giant spike recording in order to obtain intracellular recordings. This injury discharge was similar to that reported by Fujita (1968) in the rabbit cerebellum, and took the form of slow waves similar in amplitude and duration to the slow wave component of the giant spike. The frequency of the injury discharge increased with time and became rhythmical. At the onset of the injury potentials there was an increase in the firing frequency of the smaller spike, a decrease in its amplitude and finally a complete suppression of its activity. At the termination of the injury potential, there was a gradual recovery in the amplitude of the smaller unit. The frequency of the giant spike discharge appeared to be independent of the smaller spike and the injury potential. However, when a giant spike occurred during an injury potential, it was reduced in amplitude (Fig. 2). These findings suggested that the giant spike, the smaller unit and the injury potential were all generated by the same Purkinje cell, but that the giant spike and the smaller unit
Purkinje cell giant spikes in Salmo gairdneri
I
I
200msec
Fig. 2. Relationship of a giant spike to other Purkinje cell potentials. Extracellular a.c. recording (as Fig. 1). Each trace shows the background activity of a giant spike (large unit) and a simultaneously recorded smaller spike considered, for reasons outlined in the text, to be generated by the same Purkinje cell. The traces are not continuous in time but were recorded at increasing intervals after the units were first isolated. Note that activity of the smaller spike is suppressed during the giant spike slow wave. In the three lower traces, a biphasic slow wave (presumed to be an injury potential) is evident, increasing in frequency and rhythmicity with time. Activity of the small spike is initially increased and then completely suppressed by the injury potential. Note that when the giant spike fires during an injury potential (*), its amplitude is reduced.
were produced by different mechanisms, possibly in different parts of the Purkinje cell. (ii) Zntracellular recording. Difficulty was experienced in obtaining stable intracellular recordings from Purkinje cells. In most instances, membrane potential declined rapidly and it was not possible to make prolonged observations. As the recording electrode was advanced from the extracellular position of maximal giant spike amplitude to a point within the cell membrane (as indicated by the negative d.c. shift in recorded potential), there was no sign of giant spike injury discharge. The giant spike continued to fire at prepenetration rates, but its amplitude rapidly decreased. No action potentials were recorded. However, two types of depolarizing potentials, each followed by a slight hyperpolarization, were seen in intracellular d.c. records (Fig. 3). These differed primarily in their rise-time; one rose rapidly to its peak amplitude (fast rise-time potential, FRTP) while the other had a gradual onset (slow rise-time potential, SRTP). The FRTP corresponded to the greatly diminished giant spike (as seen in the a.c. record in Fig. 3) and the SRTP appeared to correspond to the injury potential described earlier. The FRTP had an additional depolarizing potential superimposed on its repolarizing phase. This delayed
depolarization corresponded to the giant spike slow wave in a.c. records. Although action potentials arising from the FRTP were not observed, it was thought that the initial component of the FRTP was probably an excitatory post-synaptic potential. Because of the difficulty in maintaining stable intracellular recordings, it was not possible to manipulate membrane potential and determine an equilibrium potential for the FRTP. However, it was noted that whenever the FRTP occurred at the peak of one of the SRTPs, the amplitude of the FRTP was reduced, a finding consistent with the interpretation of the FRTP as a synaptic potential rather than an action potential. When comparing simultaneous a.c. and d.c. records (Fig. 3 paired traces), it became apparent that the a.c. recordings of both the giant spike and the slow wave were not true representations of the actual waveform as revealed by the d.c. traces. Since the amplitude of an a.c.-coupled recording is proportional to the rate of change of the d.c. potential, the sharp rise of the d.c.-recorded FRTP was translated into a large positive deflection in the a.c. recording. The subsequent plateau of the FRTP (zero change in slope) caused the a.c. record to return to its zero reference level. Thus the initial portion of the FRTP was differentiated by a.c. coupling into what appears
MIRIAMD. REID AND R. A. WESTERMAN
256
+ 8CmV
i
Fig. 3. Intracellular recording from another Purkinje cell. The first two traces and the upper record in each of the bottom three pairs are d.c. recordings. The lower record in each of the three paired traces
was recorded a.c. simultaneously with the d.c. trace. In the d.c. records, two types of depolarizing potentials, each followed by a slight hyperpolarization, can be seen. One type of&c. potential has a slow rise-time (SRTP) and the other a fast rise-time (FRTP). The FRTP has a delaved deoolarizine wave (peak indicated by a dot) superimposed on its r&polarizing phase. The arrowed-potentials in the a.c. records are greatly deteriorated giant spikes. It can be seen that the a.c. recorded giant spike corresponds to the initial part of the FRTP, the giant spike slow wave to the delayed depolarization of the FRTP and the a.c. recorded slow wave (injury potential) to the SRTP. It was concluded that all the a.c. recorded potentials represented the differentiated waveforms of the d.c. potentials, and that this distortion arose from the short coupling time constant (0402 set) of the preamplifier used in a.c. recording.
as a spike. In a similar fashion, the slow rise and decay of the SRTP and of the delayed depolarization superimposed on the repolarizing phase of the FRTP were differentiated by a.c. coupling into a biphasic slow wave with a much steeper gradient than the original potential. These distortions of the true waveforms were probably accentuated by the very short coupling time constants (0.02 or 0.002 set) used. DISCUSSION
In common with Purkinje cells in other species, the trout Purkinje cell produces simple and complex spikes. However, in addition to this characteristic dual discharge, trout Purkinje cells also generate giant spikes. The consistent recording of giant spikes in the area immediately above, rather than in, the layer of Purkinje cells bodies suggested the giant spike was a dendritic rather than a soma potential. Stimulation of afferent fibres deep within the cerebellum, but not of parallel fibres alone, was effective in evoking giant spikes. This finding suggested that the generation of giant spikes was related to activity within the climbing fibre rather than the mossy fibre pathway. The suppression of Purkinje cell spike activity by the giant spike was similar to that typically seen following the climbing fibre-induced complex spike in other species (Granit & Phillips, 1956; Thach, 1968; Nacimiento, 1969),
further suggesting a link between trout giant spikes and the climbing fibre system. In a previous communication we considered the possibility that the trout giant spike was an action potential (Waks & Westerman, 1970). The present data, however, suggest that the giant spike is not an action potential. It was not possible to produce an antidromically activated giant spike. Multiple giant spike firing did not occur. In addition, an increase in giant spike firing frequency due to injury of the cell membrane was not observed. Indeed, the rate of giant spike discharge was remarkably constant at all times, suggesting a dependence on extracellular rather than intracellular events. The amplitude of the extracellularly recorded giant spike and the intracellularly recorded FRTP (which appeared to correspond to the giant spike) was reduced by the depolarizing injury potentials. However, giant spike firing frequency was not affected by the injury potentials. Such variation in amplitude with cell without any concomitant membrane potential, change in firing frequency, is consistent with the giant spike being a synaptic potential rather than an action potential. On the basis of this evidence, we suggest that the trout giant spike is a very large exitatory postsynaptic potential produced upon excitation of the Purkinje cell by its climbing fibre, and recorded from the Purkinje cell main dendrite. Its “spike-like” appearance in a.c. records is probably an artifact
Purkinje cell giant spikes in Salmo gairdneri of the short coupling time constant used in the recording apparatus, and may simply represent the differentiated waveform of the EPSP. Intracellular recordings revealed that the giant spike slow wave corresponded to a delayed depolarization occurring on the repolarizing phase of the climbing fibre EPSP. It is possible that this delayed depolarization represents electrotonic spread of dendritic action potentials produced by climbing fibre excitation in the dendritic tree. Such dendritic action potentials have been recorded from Purkinje cells in the rabbit (Fujita, 1968) and alligator (Llinas et al., 1968). The injury potential may arise in a similar fashion from dendritic action potentials resulting from injury to the dendrites by the advancing electrode.
REFERENCES
ECCLESJ. C., LLINAS R. & SASAKIK. (1966a) Parallel fibre stimulation and the responses induced thereby in the Purkinje cells of the cerebellum. Exp. Brain Res. 1, 17-39. ECCLESJ. C., LLINASR. & SASAKIK. (1966b) The excitatory synaptic action of climbing fibres on the Purkinje cells of the cerebellum. .Z.Physiol., Lond. 182,268296. FUJITAY. (1968) Activity of dendrites of single Purkinje cells and its relationship to so-called inactivation response in the rabbit cerebellum. .Z. Neurophysiol. 31,
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Key Word Index-Trout, cerebellum, Purkinje cell, giant spike; atferent fibre stimulation; dendritic potentials; excitatory post-synaptic potential; differentiation by a.c. coupling.