EXPERIMENTAL
NEUROLOGY
Ionic
46, 147-155
Mechanisms in Thin
(1975)
of the Depolarization Hippocampal Slices
Shift
~TOBUKUNIOGATA Dcpavtrnext
of Pharmacology,
Faculty of Medicine, Fukzloka 812, Japart
Received
August
Ky2~shu
Vniversit~~,
21,197-f
Mechanisms underlying the generation of “depolarization shift” in pyramidal neurons were studied ilz vitYo in thin slices from the guinea pig hippocampus. In high K+ (10 mM) medium, stimulation of the mossy fibers elicited a depolarization shift followed by a large hyperpolarization, even when the pyramidal neuron responded initially with an IPSP. A direct depolarizing current pulse applied to the soma membrane elicited only a single action potential in high K’ medium, whereas it evoked the same response as the transynaptic stimulation did in Cl--deficient medium. In field potentials, high Ca2+ abolished the high ‘&+-induced slow oscillatory wave, which seemed to be an extracellular manifestation of the depolarization shift, whereas it further enhanced the augmenting effect of Cl--deficiency on the synaptic transmission between the mossy fiber and pyramidal neurons. From these results it was concluded that generation of the depolarization shift was attributed to the extracellular K’ accumulation. INTRODUCTION
Experimental epileptic activities in mammalian brain are characterized by a specific pattern of intracellular electrophysiological events called depolarization shift (9). A number of experiments in V&O indicated that an increase in synaptic excitation plays an important role in generation of the depolarization shift (2, 5, 9). On the other hand, it was also indicated that an accumulation of extracellular potassium ion, (K’),, either initiates seizure or sustains it (6, 13). It seems, therefore, to be important to determine whether the depolarization shift is due to a synaptic mechanism, e.g., blockade of inhibitory mechanisms, or to an extrasynaptic mechanism, e.g., excessive depolarization caused by an endogenous process intrinsic to the neuronal membrane. Yamamoto (12) reported that a depolarization shift was generated in thin hippocampal slices incubated in chloride-deficient medium. However, no study was made of the role of (K’), in the 147 Copyright All rights
0 1975 by Academic Press, Inc. of reproduction in any form reserved.
148
NOBUKUNI
OGATA
mechanism of generation of the depolarization shift. In this series of experiments, therefore, the effects were investigated of (K+)O changes on electrical activities in guinea pig hippocampal slices with particular reference to generation of the depolarization shift. MATERIALS
AND
METHODS
Slices of hippocampal formation (350-450 pm thick) were prepared from adult guinea pigs and incubated in a medium according to the technique of Yamamoto ( 12). The normal incubating medium contained (in mM) : NaCl 124 ; KC1 5 ; KHsPOl 1.24 ; MgS04 1.3 ; CaCla 2.6 ; and glucose 10. In high K+ medium, K+ concentration was maintained at 10 mM. In Cl--deficient medium, NaCl was completely replaced by equivalent amounts of sodium propionate. A supramaximal electrical stimulation with 40-80 psec rectangular pulse was applied to the mossy fibers through a pair of stainless-steel needles insulated except for the tips. Field potentials were recorded from the pyramidal layer in CA3 region with a glass pipette of 20 pm in tip diameter filled with the normal incubating medium. For intracellular potential recordings, glass microelectrodes filled with 2 M potassium citrate were used. The resistance of the electrodes was about 10-20 megohms. A membrane depolarizing current of lo- lo --lOeg amp was applied through a conventional bridge circuit. RESULTS Field Potentials is CA3 Region Evoked by Mossy Fiber Stiwwlation. In normal medium, stimulation of the mossy fibers evoked a field potential of 20-30 msec duration (Fig. lA), which was termed the primary response (12). The primary response usually consisted of two main constituents : one or two sharp negative deflections and a slow positive wave, the earlier portion of which was masked by the negative deflection superposed on it (see also field potentials in Fig. 2). When high K+ medium was used, the primary response was relatively unaffected, and a slow oscillatory wave of 40-100 msec duration appeared following the primary response (Fig. 1B). On the other hand, in Cl--deficient medium, the primary response was augmented particularly in its amplitude, while the duration of the response was slightly increased (Fig. 1C). The slow oscillatory wave which was seen in high K+ medium was not observed. In high K+ plus Cl--deficient medium, the responses were explosively augmented, i.e., the primary response was potentiated and the amplitude of the slow oscillatory wave observed in high K+ medium was increased more than tenfold in amplitude (Fig. 1D). The rhythmicity of
DEPOLARIZATlON
SH 1FT
149
FIG. 1. Field potentials recorded from CA3 region in response to mossy fiber stimulation. A : normal medium ; B : high K’ medium ; C : Cl--deficient medium ; D : high K’ plus Cl--deficient medium ; E: upper trace, high K’ medium; lower trace, high K+ plus Cl--deficient medium. Upward deflections represent positive polarity in this and in the following illustrations. Calibrations: 2 mv and 40 msec.
the oscillation in high K+ plus Cl--deficient medium was somewhat different from that in high K+ medium (Fig. 1E). Intracellular Recordbgs frona Pyramidal Neurons i+z CA3 Region. In normal medium, pyramidal neurons usually responded to the mossy fiber stimulation with an IPSP of 5-10 mv amplitude and 40-100 msec duration (Fig. 2A) or a small EPSP followed by an IPSP. Occasionally, the generation of a single action potential on the EPSP was observed (Fig. 2BC). Simultaneous intracellular and extracellular recordings in Fig. ZA-C indicate followings : (a) the time course of the extracellular slow positive wave differs from that of IPSP but coincides with that of EPSP; (b) the extracellular sharp negative deflection closely mirrors an action potential on the EPSP. In high K+ medium, a characteristic depolarization shift with 20-30 mv amplitude and 40-100 msec duration succeededby a large hyperpolarization
FIG. 2. Three examples of intracellular recordings from the pyramidal neuron ill CA3 region in response to mossy fiber stimulation. Simultaneous field recordings (lower traces) are also illustrated. In C, three traces are superimposed. Voltage calibration: 10 mv for intracellular recordings, and 1 mv for field potentials. Time calibration : 40 msec.
150
NOBLJKUNI
OGATA
-
,
I
3. Effects of high K’ on the intracellular potentials recorded from the pyramidal neuron in response to mossy fiber stimulation. Broken line in A-C: resting membrane potential level. In D, two successive recordings from the same neuron are illustrated. In E, lower trace, field potential. Calibration: 10 mv (1 mv for lower trace in E) and 40 msec. FIG.
with 10-20 mv amplitude and 200-500 msec duration (hyperpolarization after the depolarization shift) was elicited by the mossy fiber stimulation (Fig. 3A-C). Frequently, a full-sized spike appeared preceeding the depolarization shift, and stereotyped and abortive spikes, which could easily be distinguished from the first full-sized spike, were superposed on the depolarization shift (Fig. 3A). The first interspike interval was markedly variant ranging from a few msec to more than 10 msec. On the contrary, the intervals between each spike on the depolarization shift were relatively constant with an average of 5 msec, though they showed a slight tendency of successive increase. A depolarization shift without any membrane potential oscillation was also frequently observed (Fig. 3B). In some cases the depolarization shift could be triggered by mossy fiber stimulation even when the pyramidal neuron initially responded with an IPSP (Fig. 3C). Biphasic IPSP (Fig. 3D, upper trace) consisted of the ordinary IPSP (Fig. 3D, lower trace) and the second large hyperpolarization was occasionally observed. The time course of the second hyperpolarization was identical with the hyperpolarization after the depolarization shift. In such a case, an extraordinarily small depolarization shift was often picked up spontaneously in successiverecordings (Fig. 3D, lower trace). The amplitude of hyperpolarization after the depolarization shift was approximately in an inverse relation to the maturity of the spikes on the depolarization shift,
being
largest
when
no spike
was superposed
on the depolarization
shift. Simultaneous intracellular and extracellular recordings in Fig. 3E show that the extracellular oscihation coincides with depolarization shift in the time course.
DEPOLARIZATION
SHIFT
151
FIG. 4. Effects of Cl--deficiency on the intracellular potentials in response to mossy fiber stimulation. A-B : most commonly observed types. Upper traces in C-E: field potentials (in C, the polarity was reversed). D-E : continuous recordings from the same neuron; the second stimulus was delivered SO msec after the first one. D: Cl--deficient medium, E: low K+ plus Cl--deficient medium, F : high K+ plus Cl--deficient medium. Dots : mossy fiber stimulation. Voltage calibration: 1 mv for field potentials, 10 mv for intracellular recordings. Time calibration: 40 msec.
In Cl--deficient medium, the IPSP observed in normal medium was replaced by an augmented EPSP with 5-10 mv amplitude which was smaller than the depolarization shift, and one or a few full-sized spikes were triggered (Fig. 4A-B) . The interspike intervals were somewhat longer than those of the stereotyped spikes of depolarization shifts obtained in high K+ medium (Fig. 4B-C) . A depolarization shift was rarely evoked by a single stimulus. To elicit a depolarization shift in Cl-deficient medium, very strong (Fig. 4C) or a paired stimuli (Fig. 4D) were needed. When K+ concentration was lowered to 2 mM from 5 mM, however, a depolarization shift could no longer be elicited (Fig. 4E). In Cl--deficient plus high K+ medium, a depolarization shift with nearly
FIG. 5. Effects of Ca’* on the field potentials evoked by mossy fiber stimulation in high K+ or Cl-deficient medium. A: normal Ca*+ (2.6 mM>, B: high Ca% (5.2 mM), C: Iow Ca- (1 mM). Upper recordings: high K+ medium, lower recordings : Cl--deficient medium. Calibration : 1 mv and 40 msec.
152
NOBUKUNI
0
OGATA
.
.c Q&J-. FIG. 6. Effects of depolarizing currents directly applied to the soma membrane of the pyramidal neuron. A-B : high K+ medium, C-D : W-deficient medium, E-F : high K’ plus Cl--deficient medium. Lines: direct depolarizing current pulses (about 80 msec duration). Dots : mossy fiber stimulation. Calibration : 10 my and 40 msec.
full-sized spikes on it was triggered, and the hyperpolarization after the depolarization shift was not observed (Fig. 4F). Efects of Cal&m Ion on the Field Potentials. In high K+ medium, high Caz+blocked the slow oscillatory wave induced by the mossy fiber stimulation, whereas low Ca2+attenuated the primary response and had no effect on the rhythmicity of the oscillation. In Cl-deficient medium, high Ca2+ further potentiated the primary response, while the number of sharp negative deflections was unchanged in contrast to the increase observed when normal medium was replaced by Cl--deficient medium. Low Ca2+ caused an afterdischarge as was seen in high K+ plus Cl-deficient medium. These results are summarized in Fig. 5. Effects of Depolarizing Currents Directly Applied to the Pyramidal Sonul Mevwbrane. In high K+ medium, a depolarizing current pulse applied to the soma membrane could not evoke a depolarization shift and usually, only a single spike was elicited (Fig. 6A-B), whereas the mossy fiber stimulation elicited a depolarization shift as shown in the later half of the traces in Fig. 6A-B. Application of an extremely strong current resulted in the generation of repetitive firings without any depolarization shift. On the contrary, in case of Cl--deficient medium, the current pulse could induce the samespike discharges as those induced by the mossy fiber stimulation (Fig. 6C-D), and a depolarization shift could be triggered neither by the mossy fiber stimulation nor by the depolarizing current. In high K+ plus Cl--deficient medium, a depolarizing current pulse usually elicited several full-sized spikes, interspike intervals of which resembled to those observed in Cl--deficient medium (Fig. 6D), whereas the mossy fiber stimulation triggered prolonged spike discharges with depolarization shift (Fig. 6.E) _ Generation of prolonged spike discharges
DEPOLARIZATION
by a current pulse was also frequently
SHIFT
observed particularly
153
when the
intense current pulse was applied (Fig. 6F). DISCUSSION The present results of intracellular recordings clearly demonstrate that high concentration of K+ in the incubating medium gives rise to depolarization shift generation. Also in the field potentials, high K+ introduced the slow oscillatory wave, which is believed to be an extracellular manifestation of the depolarization shift, because its time course coincides with that of the depolarization shift (Fig. 3E and 4C). The concentration of K+ employed in this study (10 mM) is in close agreement with the (K’),, values estimated in experimental epileptic foci by Futamachi et al. (7) using K+-sensitive electrodes (g-10 mM) , and by Ransom (10) who measured glial membrane potentials (10-17 mM) _ These findings suggest that the depolarization shift may be generated by (K+jo accumulation. On the other hand, high K’ produced little or no excitatory effect on the primary response, which is presumed to be a summed extracellular manifestation of monosynaptic EPSP, action potential and IPSP. In Cl--deficient medium, on the contrary, the slow oscillatory wave could not be elicited though the primary response was markedly potentiated. It seems, therefore, that there is no relationship between depolarization shift generation and the degree of monosynaptic excitation of the pyramidal neuron. The findings that the slow oscillatory wave in high K+ medium was antagonized by high Ca?+, whereas the primary response was uniformly potentiated by high Ca”+ in various media further support above notion. The observation that, in high K’ medium, only the mossy fiber stimulation could elicit a depolarization shift and a direct depolarizing current through the soma membrane failed also implies that DS generation is due to (K+)O accumulation, because the fiber stimulation, which should fire innumerable neurons simultaneously, is expected to cause a drastic increase in (K’),,. If the depolarization shift were triggered by a recurrent mechanism, a single pyramidal firing by the current pulse should have been sufficient to activate the circuit that generates the depolarization shift, because in such a case, the soma of the pyramidal neuron must be a main constituent of the circuit. On the other hand, in Cl--deficient medium. the direct depolarizing current evoked the same spike discharges as those evoked by a transynaptic activation. This fact demonstrates that the mechanism of spike generation in Cl--deficient medium may involve a certain recurrent mechanism. As a result, the somal single firing tends to generate a trait1 of full-sized spikes, As reported by Yamamoto (12)) to elicit a depolarization shift in Cl--deficient medium, very strong or a paired stimuli were needed also in
154
NOBUKUNI
OGATA
this experiment. Such conditions may facilitate the increase in (K’),, particularly when the pyramidal neuron is disinhibited by O-deficiency and tends to fire repetitively. The fact that the second shock of a paired stimuli failed to elicit a depolarization shift when (K+),, was reduced to 2 mM indicates that the generation of the depolarization shift in Cl--deficient medium may be due to (K+)O accumulation. The effect of Cl--deficiency should be essentially restricted to the enhancement of synaptic transmission between the mossy fiber and the pyramidal neuron as a result of blockade of the recurrent inhibition acting on the soma and its proximal portions (1). Prince (9) suggested that the depolarization shift originates at a site remote from the soma on the basis of findings that hyperpolarizing current pulses showed less effect upon the depolarization shift than upon an EPSP. The inability of depolarizing current applied to the soma membrane in depolarization shift generation (Fig. 6A-B) also support this idea. A depolarization shift could be evoked in the course of an IPSP in high K+ medium (Fig. 3C), and was always followed by a large hyperpolarization, which was incomparably larger in amplitude than that observed by Yamamoto in normal or Cl--deficient medium (12), and rather resembled to the hyperpolarization after the depolarization shift in experiments in tivo (4, 8). It is probably, therefore, that a depolarization shift can be evoked independently of the mechanism of postsynaptic inhibition, and that the propagation of the depolarization shift, an abnormal potential, to the soma may result in enhancement of the inhibition. This idea is supported by the observation of biphasic IPSP (Fig, 3D, upper trace), the second large hyperpolarization of which may represent the potentiated IPSP in response to a seemingly small depolarization shift probably elicited in a near-by neuron (Fig. 3D lower trace). In high K+ medium, the spikes on the depolarization shift were always abortive and stereotyped, whereas the spikes in Cl--deficient medium were nearly full-sized. Moreover, these two types of spikes apparently differed each other in the interspike intervals (compare Fig. 3A with Fig. 4B) indicating that these two types of spike discharges were evoked in different origins. The inverse relation between the maturity of spikes on the depolarization shift and the amplitude of hyperpolarization after the depolarization shift suggests that the depolarization shift may exert a facillitatory action on the soma membrane in a similar manner to the generator potential, being counteracted by the hyperpolarization a part of which can be observed as hyperpolarization after the depolarization shift. As a result, only abortive spikes can be evoked in high K+ medium, and spikes are largest when hyperpolarization is nearly abolished by the removal of Cl- from the medium. This seems to be a reason why the field response is so drastically augmented in high K+ plus Cl--deficient medium. The abortiveness of the
DEPOLARIZATION
SHIFT
155
q&es in high K’ medium might not be due to an excessive depolarization, because the first spike was always full-sized and the reduction of Cl-, which is expected to further bring about a membrane depolarization by facilitating neuronal firings, resulted in an increase of the spike amplitude. The spike discharge patterns in high K+ medium resembled in many respects to those observed in alumina-gel induced epileptic foci in monkeys (3). In this respect, Wyler eE al. also suggested, on the basis of analysis of inter-spike intervals in extracellular recordings, the existence of a prolonged depolarization, which is originated at some remote region of the cell and triggers repetitive spike discharges (11). REFERENCES 1. ANDERSEN, P., J. C. ECCLES, and Y. L$YNING. 1964. Location of postsynaptic inhibitory synapses on hippocampal pyramids. J. Ne~roplzysiol. 27 : 592-607. 2. AYALA, G. F., and C. VASCONETTO. 1972. Role of recurrent excitatory pathways in epileptogenesis. Electroencephalogr. Cl&. Neurophysiol. 33 : 96-98. 3. CALVIN, W. H., G. W. SYPERT, and A. A. WARD, JR. 1968. Structured timing patterns within bursts from epileptic neurons in undrugged monkey cortex. Exp. Neural. 21: 535-549. 4. DICHTER, M., and W. A. SPENCER. 1969. Penicillin-induced interictal discharges from the cat hippocampus. I. Characteristics and topographical features. I. Neurofihgsiol. 5. DICHTER, M.,
32 : 649-662.
and W. A. SPENCER. 1969. Penicillin-induced interictal discharges from the cat hippocampus. II. Mechanisms underlying origin and restriction. I. Ncwophysiol.
32 : 663-687.
6. FERTZIGER, A. P., and J. B. RANCK, JR. 1970. Potassium accumulation in interstitial space during epileptiform seizures. Erp. ,Ner#ol. 26: 571-585. 7. FUTAMACHI, J. K., R. MUTANI, and D. A. PRINCE. 1974. Potassium activity in rabbit cortex. Brain Rcs. 75 : 5-25. 8. PRINCE, D. A. 1968. Inhibition in “epileptic” neurons. Ex). NPUYOZ. 21 : 307-3-71. 9. PRINCE, D. A. 1968. The depolarization shift in “epileptic” neurons. Exp. Newel. 21: 467-485. 10. RANSOM, B. R. 1974. The behavior of presumed glial cells during seizure discharge in cat cerebral cortex. Brain Rcs. 69 : 83-100. 11. WYLER, A. R., E. E. FETZ, and A. A. WARD, JR. 1973. Spontaneous firing patterns of epileptic neurons in the monkey motor cortex. Exp. Neurol. 40: 567-585. 12. YAMAMOTO, C. 1972. Intracellular study of seizure-like afterdischarges elicited in thin hippocampal sections in vitro. Erp. Neural. 35: 154-164. 13. ZUCKERMANN, E. C., and G. H. GLASER. 1968. Hippocampal epileptic activity induced by localized ventricular perfusion with high-potassium cerebrospinal fluid. Exp. Neurol. 20 : 87-110.
NEUROLOGY
Ionic
46, 147-155
Mechanisms in Thin
(1975)
of the Depolarization Hippocampal Slices
Shift
~TOBUKUNIOGATA Dcpavtrnext
of Pharmacology,
Faculty of Medicine, Fukzloka 812, Japart
Received
August
Ky2~shu
Vniversit~~,
21,197-f
Mechanisms underlying the generation of “depolarization shift” in pyramidal neurons were studied ilz vitYo in thin slices from the guinea pig hippocampus. In high K+ (10 mM) medium, stimulation of the mossy fibers elicited a depolarization shift followed by a large hyperpolarization, even when the pyramidal neuron responded initially with an IPSP. A direct depolarizing current pulse applied to the soma membrane elicited only a single action potential in high K’ medium, whereas it evoked the same response as the transynaptic stimulation did in Cl--deficient medium. In field potentials, high Ca2+ abolished the high ‘&+-induced slow oscillatory wave, which seemed to be an extracellular manifestation of the depolarization shift, whereas it further enhanced the augmenting effect of Cl--deficiency on the synaptic transmission between the mossy fiber and pyramidal neurons. From these results it was concluded that generation of the depolarization shift was attributed to the extracellular K’ accumulation. INTRODUCTION
Experimental epileptic activities in mammalian brain are characterized by a specific pattern of intracellular electrophysiological events called depolarization shift (9). A number of experiments in V&O indicated that an increase in synaptic excitation plays an important role in generation of the depolarization shift (2, 5, 9). On the other hand, it was also indicated that an accumulation of extracellular potassium ion, (K’),, either initiates seizure or sustains it (6, 13). It seems, therefore, to be important to determine whether the depolarization shift is due to a synaptic mechanism, e.g., blockade of inhibitory mechanisms, or to an extrasynaptic mechanism, e.g., excessive depolarization caused by an endogenous process intrinsic to the neuronal membrane. Yamamoto (12) reported that a depolarization shift was generated in thin hippocampal slices incubated in chloride-deficient medium. However, no study was made of the role of (K’), in the 147 Copyright All rights
0 1975 by Academic Press, Inc. of reproduction in any form reserved.
148
NOBUKUNI
OGATA
mechanism of generation of the depolarization shift. In this series of experiments, therefore, the effects were investigated of (K+)O changes on electrical activities in guinea pig hippocampal slices with particular reference to generation of the depolarization shift. MATERIALS
AND
METHODS
Slices of hippocampal formation (350-450 pm thick) were prepared from adult guinea pigs and incubated in a medium according to the technique of Yamamoto ( 12). The normal incubating medium contained (in mM) : NaCl 124 ; KC1 5 ; KHsPOl 1.24 ; MgS04 1.3 ; CaCla 2.6 ; and glucose 10. In high K+ medium, K+ concentration was maintained at 10 mM. In Cl--deficient medium, NaCl was completely replaced by equivalent amounts of sodium propionate. A supramaximal electrical stimulation with 40-80 psec rectangular pulse was applied to the mossy fibers through a pair of stainless-steel needles insulated except for the tips. Field potentials were recorded from the pyramidal layer in CA3 region with a glass pipette of 20 pm in tip diameter filled with the normal incubating medium. For intracellular potential recordings, glass microelectrodes filled with 2 M potassium citrate were used. The resistance of the electrodes was about 10-20 megohms. A membrane depolarizing current of lo- lo --lOeg amp was applied through a conventional bridge circuit. RESULTS Field Potentials is CA3 Region Evoked by Mossy Fiber Stiwwlation. In normal medium, stimulation of the mossy fibers evoked a field potential of 20-30 msec duration (Fig. lA), which was termed the primary response (12). The primary response usually consisted of two main constituents : one or two sharp negative deflections and a slow positive wave, the earlier portion of which was masked by the negative deflection superposed on it (see also field potentials in Fig. 2). When high K+ medium was used, the primary response was relatively unaffected, and a slow oscillatory wave of 40-100 msec duration appeared following the primary response (Fig. 1B). On the other hand, in Cl--deficient medium, the primary response was augmented particularly in its amplitude, while the duration of the response was slightly increased (Fig. 1C). The slow oscillatory wave which was seen in high K+ medium was not observed. In high K+ plus Cl--deficient medium, the responses were explosively augmented, i.e., the primary response was potentiated and the amplitude of the slow oscillatory wave observed in high K+ medium was increased more than tenfold in amplitude (Fig. 1D). The rhythmicity of
DEPOLARIZATlON
SH 1FT
149
FIG. 1. Field potentials recorded from CA3 region in response to mossy fiber stimulation. A : normal medium ; B : high K’ medium ; C : Cl--deficient medium ; D : high K’ plus Cl--deficient medium ; E: upper trace, high K’ medium; lower trace, high K+ plus Cl--deficient medium. Upward deflections represent positive polarity in this and in the following illustrations. Calibrations: 2 mv and 40 msec.
the oscillation in high K+ plus Cl--deficient medium was somewhat different from that in high K+ medium (Fig. 1E). Intracellular Recordbgs frona Pyramidal Neurons i+z CA3 Region. In normal medium, pyramidal neurons usually responded to the mossy fiber stimulation with an IPSP of 5-10 mv amplitude and 40-100 msec duration (Fig. 2A) or a small EPSP followed by an IPSP. Occasionally, the generation of a single action potential on the EPSP was observed (Fig. 2BC). Simultaneous intracellular and extracellular recordings in Fig. ZA-C indicate followings : (a) the time course of the extracellular slow positive wave differs from that of IPSP but coincides with that of EPSP; (b) the extracellular sharp negative deflection closely mirrors an action potential on the EPSP. In high K+ medium, a characteristic depolarization shift with 20-30 mv amplitude and 40-100 msec duration succeededby a large hyperpolarization
FIG. 2. Three examples of intracellular recordings from the pyramidal neuron ill CA3 region in response to mossy fiber stimulation. Simultaneous field recordings (lower traces) are also illustrated. In C, three traces are superimposed. Voltage calibration: 10 mv for intracellular recordings, and 1 mv for field potentials. Time calibration : 40 msec.
150
NOBLJKUNI
OGATA
-
,
I
3. Effects of high K’ on the intracellular potentials recorded from the pyramidal neuron in response to mossy fiber stimulation. Broken line in A-C: resting membrane potential level. In D, two successive recordings from the same neuron are illustrated. In E, lower trace, field potential. Calibration: 10 mv (1 mv for lower trace in E) and 40 msec. FIG.
with 10-20 mv amplitude and 200-500 msec duration (hyperpolarization after the depolarization shift) was elicited by the mossy fiber stimulation (Fig. 3A-C). Frequently, a full-sized spike appeared preceeding the depolarization shift, and stereotyped and abortive spikes, which could easily be distinguished from the first full-sized spike, were superposed on the depolarization shift (Fig. 3A). The first interspike interval was markedly variant ranging from a few msec to more than 10 msec. On the contrary, the intervals between each spike on the depolarization shift were relatively constant with an average of 5 msec, though they showed a slight tendency of successive increase. A depolarization shift without any membrane potential oscillation was also frequently observed (Fig. 3B). In some cases the depolarization shift could be triggered by mossy fiber stimulation even when the pyramidal neuron initially responded with an IPSP (Fig. 3C). Biphasic IPSP (Fig. 3D, upper trace) consisted of the ordinary IPSP (Fig. 3D, lower trace) and the second large hyperpolarization was occasionally observed. The time course of the second hyperpolarization was identical with the hyperpolarization after the depolarization shift. In such a case, an extraordinarily small depolarization shift was often picked up spontaneously in successiverecordings (Fig. 3D, lower trace). The amplitude of hyperpolarization after the depolarization shift was approximately in an inverse relation to the maturity of the spikes on the depolarization shift,
being
largest
when
no spike
was superposed
on the depolarization
shift. Simultaneous intracellular and extracellular recordings in Fig. 3E show that the extracellular oscihation coincides with depolarization shift in the time course.
DEPOLARIZATION
SHIFT
151
FIG. 4. Effects of Cl--deficiency on the intracellular potentials in response to mossy fiber stimulation. A-B : most commonly observed types. Upper traces in C-E: field potentials (in C, the polarity was reversed). D-E : continuous recordings from the same neuron; the second stimulus was delivered SO msec after the first one. D: Cl--deficient medium, E: low K+ plus Cl--deficient medium, F : high K+ plus Cl--deficient medium. Dots : mossy fiber stimulation. Voltage calibration: 1 mv for field potentials, 10 mv for intracellular recordings. Time calibration: 40 msec.
In Cl--deficient medium, the IPSP observed in normal medium was replaced by an augmented EPSP with 5-10 mv amplitude which was smaller than the depolarization shift, and one or a few full-sized spikes were triggered (Fig. 4A-B) . The interspike intervals were somewhat longer than those of the stereotyped spikes of depolarization shifts obtained in high K+ medium (Fig. 4B-C) . A depolarization shift was rarely evoked by a single stimulus. To elicit a depolarization shift in Cl-deficient medium, very strong (Fig. 4C) or a paired stimuli (Fig. 4D) were needed. When K+ concentration was lowered to 2 mM from 5 mM, however, a depolarization shift could no longer be elicited (Fig. 4E). In Cl--deficient plus high K+ medium, a depolarization shift with nearly
FIG. 5. Effects of Ca’* on the field potentials evoked by mossy fiber stimulation in high K+ or Cl-deficient medium. A: normal Ca*+ (2.6 mM>, B: high Ca% (5.2 mM), C: Iow Ca- (1 mM). Upper recordings: high K+ medium, lower recordings : Cl--deficient medium. Calibration : 1 mv and 40 msec.
152
NOBUKUNI
0
OGATA
.
.c Q&J-. FIG. 6. Effects of depolarizing currents directly applied to the soma membrane of the pyramidal neuron. A-B : high K+ medium, C-D : W-deficient medium, E-F : high K’ plus Cl--deficient medium. Lines: direct depolarizing current pulses (about 80 msec duration). Dots : mossy fiber stimulation. Calibration : 10 my and 40 msec.
full-sized spikes on it was triggered, and the hyperpolarization after the depolarization shift was not observed (Fig. 4F). Efects of Cal&m Ion on the Field Potentials. In high K+ medium, high Caz+blocked the slow oscillatory wave induced by the mossy fiber stimulation, whereas low Ca2+attenuated the primary response and had no effect on the rhythmicity of the oscillation. In Cl-deficient medium, high Ca2+ further potentiated the primary response, while the number of sharp negative deflections was unchanged in contrast to the increase observed when normal medium was replaced by Cl--deficient medium. Low Ca2+ caused an afterdischarge as was seen in high K+ plus Cl-deficient medium. These results are summarized in Fig. 5. Effects of Depolarizing Currents Directly Applied to the Pyramidal Sonul Mevwbrane. In high K+ medium, a depolarizing current pulse applied to the soma membrane could not evoke a depolarization shift and usually, only a single spike was elicited (Fig. 6A-B), whereas the mossy fiber stimulation elicited a depolarization shift as shown in the later half of the traces in Fig. 6A-B. Application of an extremely strong current resulted in the generation of repetitive firings without any depolarization shift. On the contrary, in case of Cl--deficient medium, the current pulse could induce the samespike discharges as those induced by the mossy fiber stimulation (Fig. 6C-D), and a depolarization shift could be triggered neither by the mossy fiber stimulation nor by the depolarizing current. In high K+ plus Cl--deficient medium, a depolarizing current pulse usually elicited several full-sized spikes, interspike intervals of which resembled to those observed in Cl--deficient medium (Fig. 6D), whereas the mossy fiber stimulation triggered prolonged spike discharges with depolarization shift (Fig. 6.E) _ Generation of prolonged spike discharges
DEPOLARIZATION
by a current pulse was also frequently
SHIFT
observed particularly
153
when the
intense current pulse was applied (Fig. 6F). DISCUSSION The present results of intracellular recordings clearly demonstrate that high concentration of K+ in the incubating medium gives rise to depolarization shift generation. Also in the field potentials, high K+ introduced the slow oscillatory wave, which is believed to be an extracellular manifestation of the depolarization shift, because its time course coincides with that of the depolarization shift (Fig. 3E and 4C). The concentration of K+ employed in this study (10 mM) is in close agreement with the (K’),, values estimated in experimental epileptic foci by Futamachi et al. (7) using K+-sensitive electrodes (g-10 mM) , and by Ransom (10) who measured glial membrane potentials (10-17 mM) _ These findings suggest that the depolarization shift may be generated by (K+jo accumulation. On the other hand, high K’ produced little or no excitatory effect on the primary response, which is presumed to be a summed extracellular manifestation of monosynaptic EPSP, action potential and IPSP. In Cl--deficient medium, on the contrary, the slow oscillatory wave could not be elicited though the primary response was markedly potentiated. It seems, therefore, that there is no relationship between depolarization shift generation and the degree of monosynaptic excitation of the pyramidal neuron. The findings that the slow oscillatory wave in high K+ medium was antagonized by high Ca?+, whereas the primary response was uniformly potentiated by high Ca”+ in various media further support above notion. The observation that, in high K’ medium, only the mossy fiber stimulation could elicit a depolarization shift and a direct depolarizing current through the soma membrane failed also implies that DS generation is due to (K+)O accumulation, because the fiber stimulation, which should fire innumerable neurons simultaneously, is expected to cause a drastic increase in (K’),,. If the depolarization shift were triggered by a recurrent mechanism, a single pyramidal firing by the current pulse should have been sufficient to activate the circuit that generates the depolarization shift, because in such a case, the soma of the pyramidal neuron must be a main constituent of the circuit. On the other hand, in Cl--deficient medium. the direct depolarizing current evoked the same spike discharges as those evoked by a transynaptic activation. This fact demonstrates that the mechanism of spike generation in Cl--deficient medium may involve a certain recurrent mechanism. As a result, the somal single firing tends to generate a trait1 of full-sized spikes, As reported by Yamamoto (12)) to elicit a depolarization shift in Cl--deficient medium, very strong or a paired stimuli were needed also in
154
NOBUKUNI
OGATA
this experiment. Such conditions may facilitate the increase in (K’),, particularly when the pyramidal neuron is disinhibited by O-deficiency and tends to fire repetitively. The fact that the second shock of a paired stimuli failed to elicit a depolarization shift when (K+),, was reduced to 2 mM indicates that the generation of the depolarization shift in Cl--deficient medium may be due to (K+)O accumulation. The effect of Cl--deficiency should be essentially restricted to the enhancement of synaptic transmission between the mossy fiber and the pyramidal neuron as a result of blockade of the recurrent inhibition acting on the soma and its proximal portions (1). Prince (9) suggested that the depolarization shift originates at a site remote from the soma on the basis of findings that hyperpolarizing current pulses showed less effect upon the depolarization shift than upon an EPSP. The inability of depolarizing current applied to the soma membrane in depolarization shift generation (Fig. 6A-B) also support this idea. A depolarization shift could be evoked in the course of an IPSP in high K+ medium (Fig. 3C), and was always followed by a large hyperpolarization, which was incomparably larger in amplitude than that observed by Yamamoto in normal or Cl--deficient medium (12), and rather resembled to the hyperpolarization after the depolarization shift in experiments in tivo (4, 8). It is probably, therefore, that a depolarization shift can be evoked independently of the mechanism of postsynaptic inhibition, and that the propagation of the depolarization shift, an abnormal potential, to the soma may result in enhancement of the inhibition. This idea is supported by the observation of biphasic IPSP (Fig, 3D, upper trace), the second large hyperpolarization of which may represent the potentiated IPSP in response to a seemingly small depolarization shift probably elicited in a near-by neuron (Fig. 3D lower trace). In high K+ medium, the spikes on the depolarization shift were always abortive and stereotyped, whereas the spikes in Cl--deficient medium were nearly full-sized. Moreover, these two types of spikes apparently differed each other in the interspike intervals (compare Fig. 3A with Fig. 4B) indicating that these two types of spike discharges were evoked in different origins. The inverse relation between the maturity of spikes on the depolarization shift and the amplitude of hyperpolarization after the depolarization shift suggests that the depolarization shift may exert a facillitatory action on the soma membrane in a similar manner to the generator potential, being counteracted by the hyperpolarization a part of which can be observed as hyperpolarization after the depolarization shift. As a result, only abortive spikes can be evoked in high K+ medium, and spikes are largest when hyperpolarization is nearly abolished by the removal of Cl- from the medium. This seems to be a reason why the field response is so drastically augmented in high K+ plus Cl--deficient medium. The abortiveness of the
DEPOLARIZATION
SHIFT
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q&es in high K’ medium might not be due to an excessive depolarization, because the first spike was always full-sized and the reduction of Cl-, which is expected to further bring about a membrane depolarization by facilitating neuronal firings, resulted in an increase of the spike amplitude. The spike discharge patterns in high K+ medium resembled in many respects to those observed in alumina-gel induced epileptic foci in monkeys (3). In this respect, Wyler eE al. also suggested, on the basis of analysis of inter-spike intervals in extracellular recordings, the existence of a prolonged depolarization, which is originated at some remote region of the cell and triggers repetitive spike discharges (11). REFERENCES 1. ANDERSEN, P., J. C. ECCLES, and Y. L$YNING. 1964. Location of postsynaptic inhibitory synapses on hippocampal pyramids. J. Ne~roplzysiol. 27 : 592-607. 2. AYALA, G. F., and C. VASCONETTO. 1972. Role of recurrent excitatory pathways in epileptogenesis. Electroencephalogr. Cl&. Neurophysiol. 33 : 96-98. 3. CALVIN, W. H., G. W. SYPERT, and A. A. WARD, JR. 1968. Structured timing patterns within bursts from epileptic neurons in undrugged monkey cortex. Exp. Neural. 21: 535-549. 4. DICHTER, M., and W. A. SPENCER. 1969. Penicillin-induced interictal discharges from the cat hippocampus. I. Characteristics and topographical features. I. Neurofihgsiol. 5. DICHTER, M.,
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and W. A. SPENCER. 1969. Penicillin-induced interictal discharges from the cat hippocampus. II. Mechanisms underlying origin and restriction. I. Ncwophysiol.
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6. FERTZIGER, A. P., and J. B. RANCK, JR. 1970. Potassium accumulation in interstitial space during epileptiform seizures. Erp. ,Ner#ol. 26: 571-585. 7. FUTAMACHI, J. K., R. MUTANI, and D. A. PRINCE. 1974. Potassium activity in rabbit cortex. Brain Rcs. 75 : 5-25. 8. PRINCE, D. A. 1968. Inhibition in “epileptic” neurons. Ex). NPUYOZ. 21 : 307-3-71. 9. PRINCE, D. A. 1968. The depolarization shift in “epileptic” neurons. Exp. Newel. 21: 467-485. 10. RANSOM, B. R. 1974. The behavior of presumed glial cells during seizure discharge in cat cerebral cortex. Brain Rcs. 69 : 83-100. 11. WYLER, A. R., E. E. FETZ, and A. A. WARD, JR. 1973. Spontaneous firing patterns of epileptic neurons in the monkey motor cortex. Exp. Neurol. 40: 567-585. 12. YAMAMOTO, C. 1972. Intracellular study of seizure-like afterdischarges elicited in thin hippocampal sections in vitro. Erp. Neural. 35: 154-164. 13. ZUCKERMANN, E. C., and G. H. GLASER. 1968. Hippocampal epileptic activity induced by localized ventricular perfusion with high-potassium cerebrospinal fluid. Exp. Neurol. 20 : 87-110.
