386
Br~dll Restart~i, 103 11976) 3~, 31-;:, Elsevier Sciczltitic Publishiilg Conapany, Amsterdam
Printed ixa The Netheri~mds
Mechanisms of the stereotyped high-frequency burst in hippocampal pyramidal neurons in vitro
NOBUKUNI OGATA
Department o/" Pharmacology, Faculty o/' Medicine, Kyushu University, Fukuoka 812 (Japan) (Accepted October 27th, 1975)
Extracellular recordings from cortical neurons in mammalian chronic epileptogenic foci have shown highly structured interictal burst patterns 2,3. Some of these bursts exhibited a peculiar pattern of firings termed the 'long first-interval' burst which has a tong interval between the first and second spikes, and the mechanism for this peculiar pattern of firings has not been defined yet. Intracellular recordings from the pyramidal neurons in thin hippocampal slices also revealed similar burst patterns in high K ~ incubating mediumL In the present study, the interspike intervals of pyramidal neurons were studied in various media modified in ionic concentration in order to clarify the genesis of the 'long first-interval' burst and to investigate the modes of repetitive firings of hippocampat pyramidal neurons. Slices of hippocampal formation (350-450/zm thick) were prepared from adult guinea pigs and incubated in an artificial medium according to the technique of YamamotolL The normal incubating medium contained (in raM): NaCI, 124; KCI, 5; NaHCO3, 26; KHzPO4, [.24; MgSO4, 1.3; CaCI2, 2.6; and glucose 10. In high K ~ medium, K + concentration was maintained at 10 mM. In Cl--deficient medium, NaCI was completely replaced by equivalent amounts of sodium propionate. A single electrical stimulation with 40-80 #sec rectangular pulse was applied to the mossy fibers through a pair of stainless-steel needles insulated except for the tips. Extracellular unit activities or intracellular potentials were recorded from the CA3 pyramidal layer with glass microelectrodes filled with 3 M NaCI or 2 M potassium citrate, respectively. For the intuitive recognition of discharge patterns, 10 consecutive recordings of extracellular spike discharges in response to the mossy fiber stimulation were aligned on the stimulus point as dot rasters in which each dot represented the peak of a spike. In normal medium, spikes were rarely evoked by the mossy fiber stimulation as previously reported 7. When high K + medium was perfused, the burst similar to that reported by Calvin e t al. 3 was triggered, i.e. a stereotyped high-frequency burst. The burst usually consisted of the first spike and the later spikes time-locked to the second spike (afterburst) as shown in Fig. 1A. The first spike and the afterburst could be easily distinguished by their responses to repetitive stimuli; the former could follow to high frequency (to 10/sec or higher), whereas the latter followed to no more than
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Fig. I. Four representative patterns of the repetitive spike discharges in pyramidal neurons. Each pattern was illustrated in the dot raster display and the interspike interval histogram. At the bottom, a sample of intracellular recordings in corresponding medium is presented. In the dot raster display, 10 consecutive responses were aligned on the stimulus point. Each dot represents the peak of a spike, and vertical lines indicate the mossy fiber stimulations. Histograms were compiled from the respective neurons shown in the dot rasters. Ordinate, relative value of the number of intervals counted with 2 msec bin width. Abscissa, duration of intervals. The column at 12-14 msec includes the intervals greater than 14 msec. The dotted line in each histogram represents a mean interspike interval. A, B: high K + medium. C: el--deficient medium. D: high K + plus CI -deficient medium.
0.5/see. The interval between the first and second spikes was relatively long and markedly variant. In the interspike interval histogram in Fig. IA, the column in the 12-14 msec bin indicates the long first-interval. Afterbursts lacking a long first-interval were occasionally recorded (Fig. I B). In CI -deficient medium, a high-frequency burst was not elicited, and the mean interspike interval was particularly long (7.97 msec) as shown in Fig. I C. In high K + plus el--deficient medium, the dot rasters were regularly adjusted in timing, and the long first-interval was not observed (Fig. 1D). These results and intracellular recordings from the neurons exhibiting the corresponding patterns illustrated at the bottom of Fig. I indicate that the high-frequency burst is generated by the extracellular K + accumulation, and it keeps step with the depolarization shift (a slow depolarization shown in Fig. IA, B, and D) which is a characteristic intracellulac event in epileptic neurons 7,8. It is also indicated that the axo-somatic excitation which is brought about by the deprivation of e l - from the incubating medium1, 6 is independent of the generation of the high-frequency burst.
388 Judging from the study o f intracellular recordings from the pyramidal neurons v whose representative patterns are illustrated in Fig. 1, bottom, the first spike in the stereotyped burst in high K ~ medium may be an axosomatic spike ew~ked by monosynaptic excitation caused by mossy fiber stimulation, whereas the later spikes (afterburst) may be partial spikes evoked in some remote regions from the soma with longer and variable latencies. It seems, therefore, that the long first-interval represents the time devoted for the activation o f the burst-generating mechanisms after somatic excitation, e.g., the time needed for extracellular K - accumulation to reach a critical level or the time needed for antidromic invasion o f a dendrite by the first spike. In this respect, a reduction of the long first-interval by increasing K -~ in the incubating medium (unpublished observation) may support the former speculation. The mean interspike interval within the afterburst preceded by a longfirstinterval in high K ~ medium was 6.08 msec, whereas that without a tong first-interval was 6.43 msec. In high K - plus CI -deficient medium, the mean interspike interval was as short as 4.65 msec. Thus, the interspike interval within the afterburst showed progressive diminution in p r o p o r t i o n to the excitation of the somatic region, i.e., the longest interval was observed when the orthodromic spike (the first spike) was blocked presumably by a recurrent inhibition, and the shortest, when CI was removed from the incubating medium. The level o f somatic excitation, therefore, seems to have a rate-determining effect on the interspike intervals within the afterburst. This ratedetermining effect m a y be the main factor o f the discrepancy between the interspike intervals o f the present study and those reported by other investigators, who obtained comparatively short intervals o f 2-3 msec from chronic epileptic foci in vivo 4,5,9 i:,. The a u t h o r is indebted to Prof. K. Tanaka, Prof. N. Katsuda, and Dr. N. Hori for their unfailing guidance and support in this work. l ANDERSEN,P., ECCLES,J. C., AND LOYNING, Y., Location of postsynaptic inhibitory synapses on
hippocampal pyramids, ,1. Neurophysiol., 27 (1964) 592-607. 2 CALVIN, W. H., Generation of spike trains in CNS neurons, Brain Research, 84 (1975) 1..~. 3 CALVIN, W. H., SYPERT, G. W., AND WARD, A. A., JR., Structured timing patterns within bursts
from epileptic neurons in undrugged monkey cortex, Exp. Neurol., 21 (1968) 535-549. 4 FETZ, E. E., AND WYLER,A. R., Operantly conditioned firing patterns of epileptic neurons in the monkey motor cortex, Exp. Neurol., 40 (1973) 586-607. 5 GLOTZNER,F. L., FETZ, E. E., AND WARD, A. A., JR., Neuronal activity in the chronic and acute epileptogenic focus, Exp. Neurol., 42 (1974) 502-518. 6 KANDEL, E. R., SPENCER, W.A., AND BRINLEY, F.J., JR., Electrophysiology of hippocampal neurons. I. Sequential invasion and synaptic organization, J. Neurophysiol., 24 (1961) 225-242. 70~ATA, N., Ionic mechanisms of the depolarization shift in thin hippocampal slices, Exp. NeuroL, 46 (1975) 147-155. 8 PRINCE, D. A., The depolarization shift in 'epileptic' neurons, Exp. Neurol., 21 (1968) 467-485. 9 WYLER,A. R., Epileptic neurons during sleep and wakefulness, Exp. Neurol., 42 (1974) 593-608~ 10 WYLER, A.R., FETZ, E.E., AND WARD, A.A., JR., Spontaneous firing patterns of epileptic neurons in the monkey motor cortex, Exp. Neurol., 40 (1973) 567-585. 11 WYLER, A. R., FETZ, E. E., AND WARD, A. A., JR., Injury-induced long-first-interval bursts in cortical neurons, Exp. Neurol., 41 (1973) 773-776. 12 WVLER,A. R., FETZ, E. E., AND WARD, A. A., JR. Antidromic and orthodromic activation of epileptic neurons in neocortex of awake monkey, Exp. Neurol., 43 (1974) 59--74. 13 YAMAMOTO,C., Intracellular study of seizure-like afterdischarges elicited in thin hippocampal sections in vitro, Exp. Neurol., 35 (1972) 154464.
Br~dll Restart~i, 103 11976) 3~, 31-;:, Elsevier Sciczltitic Publishiilg Conapany, Amsterdam
Printed ixa The Netheri~mds
Mechanisms of the stereotyped high-frequency burst in hippocampal pyramidal neurons in vitro
NOBUKUNI OGATA
Department o/" Pharmacology, Faculty o/' Medicine, Kyushu University, Fukuoka 812 (Japan) (Accepted October 27th, 1975)
Extracellular recordings from cortical neurons in mammalian chronic epileptogenic foci have shown highly structured interictal burst patterns 2,3. Some of these bursts exhibited a peculiar pattern of firings termed the 'long first-interval' burst which has a tong interval between the first and second spikes, and the mechanism for this peculiar pattern of firings has not been defined yet. Intracellular recordings from the pyramidal neurons in thin hippocampal slices also revealed similar burst patterns in high K ~ incubating mediumL In the present study, the interspike intervals of pyramidal neurons were studied in various media modified in ionic concentration in order to clarify the genesis of the 'long first-interval' burst and to investigate the modes of repetitive firings of hippocampat pyramidal neurons. Slices of hippocampal formation (350-450/zm thick) were prepared from adult guinea pigs and incubated in an artificial medium according to the technique of YamamotolL The normal incubating medium contained (in raM): NaCI, 124; KCI, 5; NaHCO3, 26; KHzPO4, [.24; MgSO4, 1.3; CaCI2, 2.6; and glucose 10. In high K ~ medium, K + concentration was maintained at 10 mM. In Cl--deficient medium, NaCI was completely replaced by equivalent amounts of sodium propionate. A single electrical stimulation with 40-80 #sec rectangular pulse was applied to the mossy fibers through a pair of stainless-steel needles insulated except for the tips. Extracellular unit activities or intracellular potentials were recorded from the CA3 pyramidal layer with glass microelectrodes filled with 3 M NaCI or 2 M potassium citrate, respectively. For the intuitive recognition of discharge patterns, 10 consecutive recordings of extracellular spike discharges in response to the mossy fiber stimulation were aligned on the stimulus point as dot rasters in which each dot represented the peak of a spike. In normal medium, spikes were rarely evoked by the mossy fiber stimulation as previously reported 7. When high K + medium was perfused, the burst similar to that reported by Calvin e t al. 3 was triggered, i.e. a stereotyped high-frequency burst. The burst usually consisted of the first spike and the later spikes time-locked to the second spike (afterburst) as shown in Fig. 1A. The first spike and the afterburst could be easily distinguished by their responses to repetitive stimuli; the former could follow to high frequency (to 10/sec or higher), whereas the latter followed to no more than
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Fig. I. Four representative patterns of the repetitive spike discharges in pyramidal neurons. Each pattern was illustrated in the dot raster display and the interspike interval histogram. At the bottom, a sample of intracellular recordings in corresponding medium is presented. In the dot raster display, 10 consecutive responses were aligned on the stimulus point. Each dot represents the peak of a spike, and vertical lines indicate the mossy fiber stimulations. Histograms were compiled from the respective neurons shown in the dot rasters. Ordinate, relative value of the number of intervals counted with 2 msec bin width. Abscissa, duration of intervals. The column at 12-14 msec includes the intervals greater than 14 msec. The dotted line in each histogram represents a mean interspike interval. A, B: high K + medium. C: el--deficient medium. D: high K + plus CI -deficient medium.
0.5/see. The interval between the first and second spikes was relatively long and markedly variant. In the interspike interval histogram in Fig. IA, the column in the 12-14 msec bin indicates the long first-interval. Afterbursts lacking a long first-interval were occasionally recorded (Fig. I B). In CI -deficient medium, a high-frequency burst was not elicited, and the mean interspike interval was particularly long (7.97 msec) as shown in Fig. I C. In high K + plus el--deficient medium, the dot rasters were regularly adjusted in timing, and the long first-interval was not observed (Fig. 1D). These results and intracellular recordings from the neurons exhibiting the corresponding patterns illustrated at the bottom of Fig. I indicate that the high-frequency burst is generated by the extracellular K + accumulation, and it keeps step with the depolarization shift (a slow depolarization shown in Fig. IA, B, and D) which is a characteristic intracellulac event in epileptic neurons 7,8. It is also indicated that the axo-somatic excitation which is brought about by the deprivation of e l - from the incubating medium1, 6 is independent of the generation of the high-frequency burst.
388 Judging from the study o f intracellular recordings from the pyramidal neurons v whose representative patterns are illustrated in Fig. 1, bottom, the first spike in the stereotyped burst in high K ~ medium may be an axosomatic spike ew~ked by monosynaptic excitation caused by mossy fiber stimulation, whereas the later spikes (afterburst) may be partial spikes evoked in some remote regions from the soma with longer and variable latencies. It seems, therefore, that the long first-interval represents the time devoted for the activation o f the burst-generating mechanisms after somatic excitation, e.g., the time needed for extracellular K - accumulation to reach a critical level or the time needed for antidromic invasion o f a dendrite by the first spike. In this respect, a reduction of the long first-interval by increasing K -~ in the incubating medium (unpublished observation) may support the former speculation. The mean interspike interval within the afterburst preceded by a longfirstinterval in high K ~ medium was 6.08 msec, whereas that without a tong first-interval was 6.43 msec. In high K - plus CI -deficient medium, the mean interspike interval was as short as 4.65 msec. Thus, the interspike interval within the afterburst showed progressive diminution in p r o p o r t i o n to the excitation of the somatic region, i.e., the longest interval was observed when the orthodromic spike (the first spike) was blocked presumably by a recurrent inhibition, and the shortest, when CI was removed from the incubating medium. The level o f somatic excitation, therefore, seems to have a rate-determining effect on the interspike intervals within the afterburst. This ratedetermining effect m a y be the main factor o f the discrepancy between the interspike intervals o f the present study and those reported by other investigators, who obtained comparatively short intervals o f 2-3 msec from chronic epileptic foci in vivo 4,5,9 i:,. The a u t h o r is indebted to Prof. K. Tanaka, Prof. N. Katsuda, and Dr. N. Hori for their unfailing guidance and support in this work. l ANDERSEN,P., ECCLES,J. C., AND LOYNING, Y., Location of postsynaptic inhibitory synapses on
hippocampal pyramids, ,1. Neurophysiol., 27 (1964) 592-607. 2 CALVIN, W. H., Generation of spike trains in CNS neurons, Brain Research, 84 (1975) 1..~. 3 CALVIN, W. H., SYPERT, G. W., AND WARD, A. A., JR., Structured timing patterns within bursts
from epileptic neurons in undrugged monkey cortex, Exp. Neurol., 21 (1968) 535-549. 4 FETZ, E. E., AND WYLER,A. R., Operantly conditioned firing patterns of epileptic neurons in the monkey motor cortex, Exp. Neurol., 40 (1973) 586-607. 5 GLOTZNER,F. L., FETZ, E. E., AND WARD, A. A., JR., Neuronal activity in the chronic and acute epileptogenic focus, Exp. Neurol., 42 (1974) 502-518. 6 KANDEL, E. R., SPENCER, W.A., AND BRINLEY, F.J., JR., Electrophysiology of hippocampal neurons. I. Sequential invasion and synaptic organization, J. Neurophysiol., 24 (1961) 225-242. 70~ATA, N., Ionic mechanisms of the depolarization shift in thin hippocampal slices, Exp. NeuroL, 46 (1975) 147-155. 8 PRINCE, D. A., The depolarization shift in 'epileptic' neurons, Exp. Neurol., 21 (1968) 467-485. 9 WYLER,A. R., Epileptic neurons during sleep and wakefulness, Exp. Neurol., 42 (1974) 593-608~ 10 WYLER, A.R., FETZ, E.E., AND WARD, A.A., JR., Spontaneous firing patterns of epileptic neurons in the monkey motor cortex, Exp. Neurol., 40 (1973) 567-585. 11 WYLER, A. R., FETZ, E. E., AND WARD, A. A., JR., Injury-induced long-first-interval bursts in cortical neurons, Exp. Neurol., 41 (1973) 773-776. 12 WVLER,A. R., FETZ, E. E., AND WARD, A. A., JR. Antidromic and orthodromic activation of epileptic neurons in neocortex of awake monkey, Exp. Neurol., 43 (1974) 59--74. 13 YAMAMOTO,C., Intracellular study of seizure-like afterdischarges elicited in thin hippocampal sections in vitro, Exp. Neurol., 35 (1972) 154464.
