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Local synaptic circuits in rat hippocampus: interactions between pyramidal cells
220
Brain Research, 184 (1980) 220-223 © Elsevier/North-Holland Biomedical Press
Local synaptic circuits in rat hippocampus: interactions between pyramidal cells
BRrAN A. MacVICAR and F. EDWARD DUDEK Department of Zoology and Erindale College, University of Toronto, Mississauga, Ont. LSL 1C6 (Canada)
(Accepted October 25th, 1979)
Key words: hippocampus -- local circuits - - pyramidal cells -- hippocampal slice
Local neuronal circuits reach their maximum complexity and abundance in man TM and their interactions might play an important role in several forms of higher brain function and dysfunction 4,9,12. However, technical difficulties have limited most studies to principal neurons and their projections to other areas of the brain. Rigorous electrophysiological analysis of local circuits requires data from simultaneous intracellular recording of two neighbouring cells so that one can be stimulated in isolation while membrane potential is recorded in the other. In this report simultaneous intracellular recordings in slices of rat hippocampus were used to study directly the local synaptic interactions of CA3 pyramidal cells. The hippocampus has highly organized pathways that show remarkable physiological and anatomical plasticity2, 8,1v. The basic trisynaptic circuit is lamellar and can be preserved in hippocampal slices. These studies were conducted in CA3, which receives input from the granule cells and projects to CA1. We have addressed the question: how do neighbouring pyramidal cells interact with each other? Our strategy has been to analyze the effect that action potentials evoked in one cell have on another cell. Hippocampal slices (500/~m thick) were prepared from female Wistar rats that were 40-70 days old. The slices, cut transverse to the longitudinal axis of the hippocampus, were maintained under conditions described in detail elsewhere 13,~s. Intracellular recording techniques were conventional. Two intracellular micropipettes filled with 2.7 M potassium chloride or 1.3 M potassium citrate and 0.2 M potassium chloride (40-100 M~), were positioned less than 200/~m apart in the cell body layer of CA3 under direct microscopic observation. Mossy fiber stimulation using bipolar extracellular electrodes (4 M sodium chloride-agarose, tips of 30 /~m) evoked the expected sequence of excitatory and inhibitory postsynaptic potentials (EPSPs and 1PSPs); the response appeared typical of those previously reported for pyramidal cellsS,13,14,16 and unlike the interneurons described by Schwartzkroin and Mathers 15. After obtaining two simultaneous intracellular impalements, action potentials
22l m
A
2
..
I q 11!I
1T
I!11! ! !1!
Fig. 1. Inhibitory interactions between pyramidal cells. In a pair of simultaneously impaled cells current-evoked action potentials (A1) caused a period of spike inhibition in a spontaneously active pyramidal cell (A2). Both records in A show 54 superimposed traces. B: the averaged IPSP of one pyramidal cell (onset indicated by arrow) that resulted from activity in another. The trace illustrated is the average of 21 responses (bin width = 250/~sec). The bar indicates the time that current was injected into the presynaptic celles to elicit action potentials. were evoked by intracellularly-injected current (through a bridge circuit) to determine whether synaptic connections existed between the pyramidal cells. The current pulse usually evoked a short burst of spikes; CA3 pyramidal cells often fire in this mode spontaneously. In 10 out of over 88 pairs of cells, spikes in one cell resulted in inhibition of the other cell. Inhibition was usually seen as a period of reduced occurrence of spontaneous action potentials in the follower cell (Fig. 1A). IPSPs were occasionally observed (Fig. IB). In 5 cases current-evoked action potentials in a pyramidal cell resulted in EPSPs and/or spikes in the postsynaptic cell. The latencies of the excitatory responses were as short as 0.5 msec (Fig. 2A) but the maximum observed was 22 msec (Fig. 2B). The responses were probably mediated by chemical synaptic connections since: (i) latencies were too long and variable for electrotonic transmission; (ii) in two out of two cases hyperpolarizing current injection in the postsynaptic cell increased the amplitude of the underlying EPSP (Fig. 2C); and (iii) the interactions were elicited in only one direction for a given pair of cells. Therefore these results demonstrate the dual effect spikes in one pyramidal cell can have on other pyramidal cells in the same sub-field. Studies using extracellular stimulation have shown that antidromic activation of pyramidal cells evokes powerful recurrent inhibition which is believed to be mediated by the basket cells 3. The infrequency and weakness of inhibitory interactions observed here is puzzling. This observation is probably not due to damage of the inhibitory system since significant recurrent inhibition could be evoked with extracellular stimulation. It probably reflects the limitations of a single pyramidal cell in activating inhibitory interneurons. However, our experiments demonstrate that activity in one cell can influence local inhibitory circuits. On the other hand, Lorente de No 7 and R a m 6 n y Caja111 both showed collaterals from pyramidal cells that presumably synapse directly on other
222
C
"1
10 mV
ms
4 mV
s
! i'
7 mV
s
Fig. 2. Excitatory interactions between pyramidal cells. Current-evoked action potentials (A1), which were used to trigger the traces, consistently elicited a single short-latency spike in another pyramidal cell (A2). The latency (peak-to-peak, 0.5 1.0 msec) combined with the unidirectional nature of the connection imply monosynaptic chemical transmission; however, we could not see an underlying EPSP in this cell. In another pair of cells 3 superimposed traces show that depolarizing current pulses in one cell consistently elicited 4 action potentials (Ba, peaks not shown), which caused EPSPs and spikes (B2, peaks not shown) 2 out of 3 times in another pyramidal cell. EPSPs were followed by IPSPs. The long latency and intermittent nature of this synaptic interaction suggest that an excitatory interneuron is being driven by the stimulated cell and produces a response in the postsynaptic cell. In a different pair of ceils hyperpolarizing current injection revealed an underlying EPSP (C). Depolarizing current pulses (Ca) in one cell generated presynaptic spikes which then evoked spikes in another cell that originally looked similar to the responses shown in A2. Three superimposed traces show that an imposed hyperpolarization in the postsynaptic cell uncovered an EPSP (C2), which was increased in amplitude with more intense hyperpolarizing currents (0.3 hA).
nearby pyramidal cells. Interneurons which are not basket cells also exist within the hippocampus 1,15. The only previous physiological evidence for recurrent excitation in the hippocampus 6 was limited by the use o f extracellular stimulation and recording and the potent inhibition. Our evidence from simultaneous intracellular stimulation and recording o f pyramidal cells now provides the strongest possible evidence for recurrent excitation, which was postulated by Dichter and Spencer 4. The strength and importance of recurrent excitation are unknown, but our studies show that spikes in one pyramidal cell can evoke spikes in another pyramidal cell. A l t h o u g h these cells had the electrophysiological characteristics o f pyramidal cells, further studies using intracellular staining are needed to identify the impaled cells unequivocally with morphological criteria and determine the anatomical pathway(s) for these interactions. The range o f latencies (0.5-22 msec) suggests both mono- and polysynaptic connections. The hippocampus is noted for its susceptibility to seizures. Although the depolarization shift recorded intracellularly during epileptiform activity probably arises from intrinsic m e m b r a n e properties 9, recurrent excitatory circuits could trigger and synchronize these events. In conclusion, the strategy o f simultaneous intracellular recording, previously used in 'simple' nervous systems and tissue culture, has been combined with the in vitro slice technique to study local synaptic circuits in the m a m m a l i a n brain. Action potentials in a single pyramidal cell are capable o f exciting and inhibiting other pyramidal cells within the immediate area. This suggests that the CA3 region is not merely a point transferring information through the hippocampus but that substantial processing may be occurring in the sub-field.
223 We t h a n k G. Weir for technical assistance a n d K. C o n n o r a n d C. Fugle for p r e p a r i n g the manuscript. We are grateful to Drs. H. A t w o o d , J. Blankenship, G. H a t t o n , B. P o m e r a n z a n d L. R e n a u d for providing constructive criticisms o f an earlier draft of the m a n u s c r i p t . This work was supported by grants from the A t k i n s o n , B a n t i n g a n d C o n n a u g h t F o u n d a t i o n s a n d the N a t u r a l Sciences a n d Engineering Research Council of C a n a d a .
1 Amaral, D. G. and Woodward, D. J., A hippocampal interneuron observed in the inferior region, Brain Research, 124 (1977) 225-236. 2 Andersen, P., Organization of hippocampal neurons and their interconnections. In R. L. Isaacson and K. H. Pribam (Eds.), The Hippocampus, Vol. 1: Structure and Development, Plenum Press, New York, 1975, pp. 155-175. 3 Andersen, P., Eccles, J. C. and Loyning, Y., Pathway of postsynaptic inhibition in the hippocampus, J. Neurophysiol., 27 (1964) 608-619. 4 Dichter, M. and Spencer, W. A., Penicillin-inducedinterictal discharges from the cat hippocampus. II. Mechanisms underlying origin and restriction, J. Neurophysiol., 32 (1969) 663-687. 5 Kandel, E. R., Spencer, W. A. and Brinley, J. F., Electrophysiology of hippocampal neurons. I. Sequential invasion and synaptic organization, J. NeurophysioL, 24 (1961) 224-242. 6 Lebovitz, R. M., Dichter, M. and Spencer, W. A., Recurrent excitation in the CA3 region of cat hippocampus, Int. J. Neurosci., 2 (1971) 99-108. 7 Lorente de No, R., Studies on the structure of the cerebral cortex. II. Continuation of the study of the Ammonic system, J. Psychol. Neurol. (Lpz.), 46 (1934) 113-177. 8 Lynch, G. and Cotman, C. W., The hippocampus as a model for studying anatomical plasticity in the adult brain. In R. L. Isaacson and K. H. Pribram (Eds.), The Hippocampus, Vol. I: Structure and Development, Plenum Press, New York, 1975, pp. 123-254. 9 Prince, D. A., Neurophysiology of epilepsy, Ann. Rev. Neurosci., 1 (1978) 395-415. 10 Raki6, P., (Ed.), Local Circuit Neurons, MIT Press, Cambridge, Mass., 1975. 11 Ram6n y Cajal, S., The Structure of Ammon's Horn, translated from the Spanish by L. M. Kraft, Charles C. Thomas Press, Springfield, Illinois, 1968. 12 Schmitt, F. O., Dev, P. and Smith, B. H., Electrotonic processing of information by brain cells, Science, 193 (1976) 114-120. 13 Schwartzkroin, P. A., Characteristics of CA1 neurons recorded intracellularly in the hippocampal in vitro slice preparation, Brain Research, 85 (1975) 423-436. 14 Schwartzkroin, P. A., Further characteristics of hippocampal CA1 cells in vitro, Brain Research, 128 (1977) 53-68. 15 Schwartzkroin, P. A. and Mathers, L. H., Physiological and morphological identification of a nonpyramidal hippocampal cell type, Brain Research, 157 (1978) 1-10. 16 Spencer, W. A. and Kandel, E. R., Electrophysiology of hippocampal neurons. IV. Fast prepotentials, J. Neurophysiol., 24 (1961) 272-285. 17 Spencer, W. A. and Kandel, E. R., Cellular and integrative properties of the hippocampal pyramidal cell and the comparative electrophysiology of cortical neurons, Int. J. Neurol., 6 (1968) 236-296. 18 Yamamoto, C., Activation of hippocampal neurons by mossy fiber stimulation in thin brain sections in vitro, Exp. Brain Res., 14 (1972) 423~J,35.
Brain Research, 184 (1980) 220-223 © Elsevier/North-Holland Biomedical Press
Local synaptic circuits in rat hippocampus: interactions between pyramidal cells
BRrAN A. MacVICAR and F. EDWARD DUDEK Department of Zoology and Erindale College, University of Toronto, Mississauga, Ont. LSL 1C6 (Canada)
(Accepted October 25th, 1979)
Key words: hippocampus -- local circuits - - pyramidal cells -- hippocampal slice
Local neuronal circuits reach their maximum complexity and abundance in man TM and their interactions might play an important role in several forms of higher brain function and dysfunction 4,9,12. However, technical difficulties have limited most studies to principal neurons and their projections to other areas of the brain. Rigorous electrophysiological analysis of local circuits requires data from simultaneous intracellular recording of two neighbouring cells so that one can be stimulated in isolation while membrane potential is recorded in the other. In this report simultaneous intracellular recordings in slices of rat hippocampus were used to study directly the local synaptic interactions of CA3 pyramidal cells. The hippocampus has highly organized pathways that show remarkable physiological and anatomical plasticity2, 8,1v. The basic trisynaptic circuit is lamellar and can be preserved in hippocampal slices. These studies were conducted in CA3, which receives input from the granule cells and projects to CA1. We have addressed the question: how do neighbouring pyramidal cells interact with each other? Our strategy has been to analyze the effect that action potentials evoked in one cell have on another cell. Hippocampal slices (500/~m thick) were prepared from female Wistar rats that were 40-70 days old. The slices, cut transverse to the longitudinal axis of the hippocampus, were maintained under conditions described in detail elsewhere 13,~s. Intracellular recording techniques were conventional. Two intracellular micropipettes filled with 2.7 M potassium chloride or 1.3 M potassium citrate and 0.2 M potassium chloride (40-100 M~), were positioned less than 200/~m apart in the cell body layer of CA3 under direct microscopic observation. Mossy fiber stimulation using bipolar extracellular electrodes (4 M sodium chloride-agarose, tips of 30 /~m) evoked the expected sequence of excitatory and inhibitory postsynaptic potentials (EPSPs and 1PSPs); the response appeared typical of those previously reported for pyramidal cellsS,13,14,16 and unlike the interneurons described by Schwartzkroin and Mathers 15. After obtaining two simultaneous intracellular impalements, action potentials
22l m
A
2
..
I q 11!I
1T
I!11! ! !1!
Fig. 1. Inhibitory interactions between pyramidal cells. In a pair of simultaneously impaled cells current-evoked action potentials (A1) caused a period of spike inhibition in a spontaneously active pyramidal cell (A2). Both records in A show 54 superimposed traces. B: the averaged IPSP of one pyramidal cell (onset indicated by arrow) that resulted from activity in another. The trace illustrated is the average of 21 responses (bin width = 250/~sec). The bar indicates the time that current was injected into the presynaptic celles to elicit action potentials. were evoked by intracellularly-injected current (through a bridge circuit) to determine whether synaptic connections existed between the pyramidal cells. The current pulse usually evoked a short burst of spikes; CA3 pyramidal cells often fire in this mode spontaneously. In 10 out of over 88 pairs of cells, spikes in one cell resulted in inhibition of the other cell. Inhibition was usually seen as a period of reduced occurrence of spontaneous action potentials in the follower cell (Fig. 1A). IPSPs were occasionally observed (Fig. IB). In 5 cases current-evoked action potentials in a pyramidal cell resulted in EPSPs and/or spikes in the postsynaptic cell. The latencies of the excitatory responses were as short as 0.5 msec (Fig. 2A) but the maximum observed was 22 msec (Fig. 2B). The responses were probably mediated by chemical synaptic connections since: (i) latencies were too long and variable for electrotonic transmission; (ii) in two out of two cases hyperpolarizing current injection in the postsynaptic cell increased the amplitude of the underlying EPSP (Fig. 2C); and (iii) the interactions were elicited in only one direction for a given pair of cells. Therefore these results demonstrate the dual effect spikes in one pyramidal cell can have on other pyramidal cells in the same sub-field. Studies using extracellular stimulation have shown that antidromic activation of pyramidal cells evokes powerful recurrent inhibition which is believed to be mediated by the basket cells 3. The infrequency and weakness of inhibitory interactions observed here is puzzling. This observation is probably not due to damage of the inhibitory system since significant recurrent inhibition could be evoked with extracellular stimulation. It probably reflects the limitations of a single pyramidal cell in activating inhibitory interneurons. However, our experiments demonstrate that activity in one cell can influence local inhibitory circuits. On the other hand, Lorente de No 7 and R a m 6 n y Caja111 both showed collaterals from pyramidal cells that presumably synapse directly on other
222
C
"1
10 mV
ms
4 mV
s
! i'
7 mV
s
Fig. 2. Excitatory interactions between pyramidal cells. Current-evoked action potentials (A1), which were used to trigger the traces, consistently elicited a single short-latency spike in another pyramidal cell (A2). The latency (peak-to-peak, 0.5 1.0 msec) combined with the unidirectional nature of the connection imply monosynaptic chemical transmission; however, we could not see an underlying EPSP in this cell. In another pair of cells 3 superimposed traces show that depolarizing current pulses in one cell consistently elicited 4 action potentials (Ba, peaks not shown), which caused EPSPs and spikes (B2, peaks not shown) 2 out of 3 times in another pyramidal cell. EPSPs were followed by IPSPs. The long latency and intermittent nature of this synaptic interaction suggest that an excitatory interneuron is being driven by the stimulated cell and produces a response in the postsynaptic cell. In a different pair of ceils hyperpolarizing current injection revealed an underlying EPSP (C). Depolarizing current pulses (Ca) in one cell generated presynaptic spikes which then evoked spikes in another cell that originally looked similar to the responses shown in A2. Three superimposed traces show that an imposed hyperpolarization in the postsynaptic cell uncovered an EPSP (C2), which was increased in amplitude with more intense hyperpolarizing currents (0.3 hA).
nearby pyramidal cells. Interneurons which are not basket cells also exist within the hippocampus 1,15. The only previous physiological evidence for recurrent excitation in the hippocampus 6 was limited by the use o f extracellular stimulation and recording and the potent inhibition. Our evidence from simultaneous intracellular stimulation and recording o f pyramidal cells now provides the strongest possible evidence for recurrent excitation, which was postulated by Dichter and Spencer 4. The strength and importance of recurrent excitation are unknown, but our studies show that spikes in one pyramidal cell can evoke spikes in another pyramidal cell. A l t h o u g h these cells had the electrophysiological characteristics o f pyramidal cells, further studies using intracellular staining are needed to identify the impaled cells unequivocally with morphological criteria and determine the anatomical pathway(s) for these interactions. The range o f latencies (0.5-22 msec) suggests both mono- and polysynaptic connections. The hippocampus is noted for its susceptibility to seizures. Although the depolarization shift recorded intracellularly during epileptiform activity probably arises from intrinsic m e m b r a n e properties 9, recurrent excitatory circuits could trigger and synchronize these events. In conclusion, the strategy o f simultaneous intracellular recording, previously used in 'simple' nervous systems and tissue culture, has been combined with the in vitro slice technique to study local synaptic circuits in the m a m m a l i a n brain. Action potentials in a single pyramidal cell are capable o f exciting and inhibiting other pyramidal cells within the immediate area. This suggests that the CA3 region is not merely a point transferring information through the hippocampus but that substantial processing may be occurring in the sub-field.
223 We t h a n k G. Weir for technical assistance a n d K. C o n n o r a n d C. Fugle for p r e p a r i n g the manuscript. We are grateful to Drs. H. A t w o o d , J. Blankenship, G. H a t t o n , B. P o m e r a n z a n d L. R e n a u d for providing constructive criticisms o f an earlier draft of the m a n u s c r i p t . This work was supported by grants from the A t k i n s o n , B a n t i n g a n d C o n n a u g h t F o u n d a t i o n s a n d the N a t u r a l Sciences a n d Engineering Research Council of C a n a d a .
1 Amaral, D. G. and Woodward, D. J., A hippocampal interneuron observed in the inferior region, Brain Research, 124 (1977) 225-236. 2 Andersen, P., Organization of hippocampal neurons and their interconnections. In R. L. Isaacson and K. H. Pribam (Eds.), The Hippocampus, Vol. 1: Structure and Development, Plenum Press, New York, 1975, pp. 155-175. 3 Andersen, P., Eccles, J. C. and Loyning, Y., Pathway of postsynaptic inhibition in the hippocampus, J. Neurophysiol., 27 (1964) 608-619. 4 Dichter, M. and Spencer, W. A., Penicillin-inducedinterictal discharges from the cat hippocampus. II. Mechanisms underlying origin and restriction, J. Neurophysiol., 32 (1969) 663-687. 5 Kandel, E. R., Spencer, W. A. and Brinley, J. F., Electrophysiology of hippocampal neurons. I. Sequential invasion and synaptic organization, J. NeurophysioL, 24 (1961) 224-242. 6 Lebovitz, R. M., Dichter, M. and Spencer, W. A., Recurrent excitation in the CA3 region of cat hippocampus, Int. J. Neurosci., 2 (1971) 99-108. 7 Lorente de No, R., Studies on the structure of the cerebral cortex. II. Continuation of the study of the Ammonic system, J. Psychol. Neurol. (Lpz.), 46 (1934) 113-177. 8 Lynch, G. and Cotman, C. W., The hippocampus as a model for studying anatomical plasticity in the adult brain. In R. L. Isaacson and K. H. Pribram (Eds.), The Hippocampus, Vol. I: Structure and Development, Plenum Press, New York, 1975, pp. 123-254. 9 Prince, D. A., Neurophysiology of epilepsy, Ann. Rev. Neurosci., 1 (1978) 395-415. 10 Raki6, P., (Ed.), Local Circuit Neurons, MIT Press, Cambridge, Mass., 1975. 11 Ram6n y Cajal, S., The Structure of Ammon's Horn, translated from the Spanish by L. M. Kraft, Charles C. Thomas Press, Springfield, Illinois, 1968. 12 Schmitt, F. O., Dev, P. and Smith, B. H., Electrotonic processing of information by brain cells, Science, 193 (1976) 114-120. 13 Schwartzkroin, P. A., Characteristics of CA1 neurons recorded intracellularly in the hippocampal in vitro slice preparation, Brain Research, 85 (1975) 423-436. 14 Schwartzkroin, P. A., Further characteristics of hippocampal CA1 cells in vitro, Brain Research, 128 (1977) 53-68. 15 Schwartzkroin, P. A. and Mathers, L. H., Physiological and morphological identification of a nonpyramidal hippocampal cell type, Brain Research, 157 (1978) 1-10. 16 Spencer, W. A. and Kandel, E. R., Electrophysiology of hippocampal neurons. IV. Fast prepotentials, J. Neurophysiol., 24 (1961) 272-285. 17 Spencer, W. A. and Kandel, E. R., Cellular and integrative properties of the hippocampal pyramidal cell and the comparative electrophysiology of cortical neurons, Int. J. Neurol., 6 (1968) 236-296. 18 Yamamoto, C., Activation of hippocampal neurons by mossy fiber stimulation in thin brain sections in vitro, Exp. Brain Res., 14 (1972) 423~J,35.