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Synaptic transmission is required for initiation of long-term potentiation
Brain Research, 150 (1978) 413--417 © ElsevierfNorth-HollandBiomedicalPress
413
Synaptic transmission is required for initiation of long-term potentiation
THOMAS DUNWIDDIE, DANIEL MADISONand GARY LYNCH Department of Psychobiology, University of California, 1trine, Calif. 92717 ( U.S. A. )
(Accepted January 18th, 1978)
Hippocampal long-term potentiation (LTP) represents a unique form of synaptic plasticity, induced by moderate levels of stimulation (as few as 10-20 pulses), and persisting for indefinite periods of time (months according to some reports) ~,4,5. These characteristics raise the intriguing possibility that the same mechanisms which are responsible for LTP might be the means by which the brain achieves lasting modification of behavior. Although it has been established that the changes underlying LTP have a synaptic location, and are confined to the afferents which have received trains of repetitive stimulation1,6, little is known of the manner in which these trains modify subsequent synaptic transmission. There are two intertwined aspects of this problem; (1) which of the elements of the synaptic complex (terminal, spine or synaptic junction) are altered by the stimulation, and (2) which aspects of the repetitive stimulation are actually responsible for 'triggering' the potentiation effect. Because the synapses involved in LTP are located some distance from the cell bodies of the target neurons, intracellular recording and classical neurophysiological analyses (such as quantal analysis of transmitter release) have been of limited use in answering mechanistic questions of the above type. Therefore, in the experiments detailed in the present paper, we utilized an alternative approach, which was to test for the occurrence of LTP following repetitive stimulation administered during periods in which the transmission process was interrupted at various points. Two very different modulators were used for this purpose: (1) reduction of extracellular calcium, a treatment widely acknowledged to block release of the neurotransmitter and (2) the application of 2-amino-4-phosphonobutyric acid (APB), a drug which competes for glutamic acid receptors in the insect nervous system3. In this report, we demonstrate that both of these manipulations produce a rapid and reversible blockade of synaptic transmission in hippocampal slices, and that synaptic strength remains unchanged by conditioning stimulation applied during such a blockade, even after reinstatement of normal physiology by a return to control medium. These experiments were conducted in the CA 1 field of the in vitro rat hippocampus, prepared according to procedures used in our laboratory for several years7. In order to effect a rapid exchange of medium containing different levels of calcium or APB, slices
414 TABLE I Effects o f A P B and calciumzfree medium on L TP
Data are shown for slices in which two populations of afferents to the CA1 region were activated one pathway ('potentiated') received a high-frequency train under one of two experimental treatments ; the second afferent ('control') received only widely-spaced test stimulation (1 or 2/rain). For the first two rows ('APB' and 0.0 Ca ~ ') pre-measures are the amplitudes of population spikes (average of 2 responses evoked at 5 sec intervals) immediately preceding the beginning of perfusion with either calcium-free or APB (1-3 mM) medium; post-treatment values were taken between 10 and 15 min following the return to control medium. In the third row ('normal'), potentiation was elicited by identical trains of stimulation in control medium, and postpotentiation responses were measured at times corresponding to the intervals used in experimental conditions (i.e., also between 10 and 15 min following the potentiation train). All differences were tested for significance with t-tests for repeated measures on the pre vs. post scores, and then normalized to percentages for presentation. No differences were significant except for the potentiated pathway in control medium, P less than 0.001. Medium
APB 0Ca 2+ Normal
Potentiated p a t h w a y
Control pathway
Pre
t
Post
n
Pre
t
Post
n
100~ 100~ 100K
0.478 0.455 15.23
105~ 116~ 296~
14 9 12
100% 100K 100K
0.297 1.615 0.023
98~ 88% 102%
13 9 11
were perfused constantly in a c h a m b e r o f relatively small volume (approx. 3 ml) at a fairly high flow rate (at least 4 ml/min), and were submerged so that the medium was in contact with both sides of the tissue. Slices o f 500/~m thickness were prepared and perfused until stable responses were obtained; separate control and experimental pathways to the CA1 cells, evoked t h r o u g h stimulation of either stratum radiatum or oriens 5,6, were used. At this point, the flow f r o m the source of control medium was interrupted, and substituted with medium containing either (a) no calcium, or (b) A P B at the appropriate concentration (between 1.5 and 3 m M ) at an equivalent flow rate. This resulted in a complete suppression o f synaptic responses within 5-10 min, at which time one or two tetani of 1 sec duration at 100/sec were given to one pathway, while the control afferents remained unstimulated. During this period, the presynaptic volleys of the stimulated axons were readily recorded, and with appropriate placements of stimulation electrodes in the alveus an additional antidromic response could be elicited from the CA1 target cells. Following the administration of the high frequency trains, the flow of control medium was immediately reinstated, and allowed to continue until well after the responses had returned to baseline amplitudes. In several experiments, paired-pulse interactions were measured as the A P B or low calcium medium was introduced, at times when the field potentials were only partially blocked. At normal levels o f calcium, two pulses delivered 20 msec apart to the same pathway result in diminished population spike amplitudes in the second response; as the level o f calcium is lowered, the first spike becomes correspondingly reduced, while the second response is actually enhanced. This is not simply a consequence of the reduction in the first response (and hence, in the corresponding recurrent I P S P it generates in the pyramidal cell layerg), because it cannot be mimicked by re-
415
I I B
~------.-
~-----.---
E .J/,~-~---- y - . - - - - . ~
"~~ ' ~
F--.~
I
I o
I "a~
I 15
30
I
I
45
60
L
I
75
I
90
I
105 Min.
Fig. 1. Effects of calcium-free medium (upper figure) and medium containing 2 m M APB (lower) on long-term potentiation of population spike amplitude. In both figures, dark bars along the lower margin indicate periods during which the flow of control medium was replaced with either calcium-free or drug-containing medium. Large arrows at the bottom of both figures indicate the interpolation of conditioning trains to the corresponding pathways (filled arrows denote stimulation delivered to the pathway indicated by filled circles, unfilled arrows to open circles). Figures in the upper right hand panel illustrate records derived from the test (filled circles) pathway in the left column, and control pathway in the right column. The population spike (reflecting synchronous discharge of the CA1 neurons) is the small downward deflection on the peak of the positive field potential in both pathways (trace A), and is essentially unaffected by a conditioning train in 0.0 Ca 2+ medium, but shows marked enhancement following an identical train given 12 min later in control medium, and was unchanged following a subsequent period of perfusion with calcium-free medium. Traces B and E demonstrate the complete suppression of synaptic responses observed in the absence of calcium. (Downward deflections of the trace represent negative polarity, calibration marks indicate 8 mV, 5 msec.) In the lower figure, longterm potentiation elicited during perfusion with control medium (filled arrow) persists throughout two subsequent interruptions of transmission by medium containing APB. A conditioning train to the control pathway (open circles) during the second period of APB perfusion is ineffective in 17roducing potentiation, while a subsequent tl ain under normal conditions (third arrow) provokes an essentially normal increase. (Heterosynaptic depression is seen in the control pathway following the first train 5, and aprears to be diminished by the first APB perfusion.) The time scale for both top and bottom figures is the same, and the vertical axis represents arbitrary scale units for the measure of population spike amplitude; each point represents the average of 2 responses evoked 5 sec apart at the indicated time points.
416 ducing the stimulation voltage. On the other hand, when APB was used to reduce synaptic responses, parallel decrements in both responses were seen, and this reversal of paired-pulse inhibition to facilitation was not observed. As can be seen from Table I, repetitive stimulation administered under either experimental condition failed to produce any evidence of long-lasting changes in synaptic efficacy. Neither experimental nor control pathways showed any consistent changes when compared to their pretreatment values as a result of either the blockade of transmission or conditioning stimulation. Some of these slices were then used to demonstrate that LTP could be initiated by subsequent stimulation of the same pathway, but interposed under conditions of normal transmission, and that such LTP was unaffected when the 'potentiated' responses were abolished and reinstated by the sequence of experimental and control medium changes (Fig. 1). Thus, the effectiveness of either APB or calcium-free medium in blocking potentiation is strictly limited to the period of reduced transmission, and the maintenance of potentiation is immune to such treatments. These results clearly demonstrate that LTP cannot be obtained in the absence of synaptic transmission. The significance of this result for our understanding of the locus and mechanisms of potentiation requires a consideration of which aspects of the transmission process were disrupted by the experimental treatments. The effects of diminished extracellular calcium seen in our experiments accord well with those found in classical neurophysiological investigations of transmission at the neuromuscular junction, and presumably reflect the requirement for calcium influx into the terminal in transmitter release. The mechanism by which APB blocks transmission is less certain. The drug inhibits the binding of glutamate to 'receptor-like' proteolipids extracted from insect muscle, and is a potent antagonist of the excitatory actions of glutamate applied to the receptors thought to be present in the membranes of these muscles 3. In view of the evidence that amino acids act as transmitters in hippocampal pathways8, a competitive inhibition of postsynaptic receptors is one possible explanation of the actions of APB in hippocampus. This would also accord well with the rapid onset and reversibility of this drug's actions. However, recent experiments have shown that APB does not block glutamic acid applied iontophoretically to the dendrites of pyramidal cells (Spencer, Dunwiddie and Lynch, in preparation); while this result is open to several interpretations (e.g. 'synaptic receptors' for glutamate are distinct from those activated by iontophoretically applied glutamate) it does indicate the need for caution in ascribing a postsynaptic site to the actions of APB in our experiments. Nevertheless, it is clear that APB blocks transmission by a means other than a reduction in calcium influx, since the effect of reduced calcium on paired-pulse facilitation could not be reproduced with APB. Therefore, the ability of these treatments to block long-term potentiation is presumably due to their common action in interrupting transmission. The failure to initiate long-term potentiation during a blockade of synaptic transmission with APB suggests the involvement of synaptic receptors for amino acids in this effect (see Van Harreveld and Fifkovit1° for an earlier version of this hypothesis). The high-frequency generation of EPSPs in the postsynaptic element, or an unusual aspect of rapid transmitter release (such as its accumulation in abnormal quantities in
417 the cleft o r a ' d e p o l a r i z a t i o n - b l o c k ' t y p e o f effect in the p o s t s y n a p t i c cell) m i g h t be m e c h a n i s m s by which this r e c e p t o r - m e d i a t e d process is induced. A n o b v i o u s i m p l i c a t i o n o f the h y p o t h e s i s t h a t L T P results f r o m the a c t i o n o f a m i n o acids on synaptic receptors is t h a t this is a p o s t s y n a p t i c effect. However, the presence o f p r e s y n a p t i c receptors in certain types o f a x o n terminals suggests that they m a y exist in the h i p p o c a m p u s a n d be involved in LTP.
1 Andersen, P., Sundberg, S. H., Sveen, O. and WigstrOm, H., Specific long-lasting potentiation of synaptic transmission in hippocampal slices, Nature (Lond.), 266 (1977) 736-737. 2 Bliss, T. V. P. and Gardner-Medwin, A. R., Long-lasting potentiation of synaptic transmission in the dentate area of the unanaesthetized rabbit following stimulation of the perforant path, J. Physiol. (Lond.), 232 (1973) 357-374. 3 Cull-Candy, S. G., Donnellan, J. F., James, R. W. and Lunt, G. G., 2-amino-4-phosphonobutyric acid as a glutamate antagonist on locust muscle, Nature (Lond.), 262 (1976) 408-409. 4 Douglas, R. M. and Goddard, G. V., Long-term potentiation of the perforant path granule cell synapse in the rat hippocampus, Brain Research, 86 (1975) 205-215. 5 Dunwiddie, T. V. and Lynch, G. S., Long-term potentiation aad depression of synaptic responses in the hippocampus: localization and frequency dependency, J. Physiol. (Lond.), 276 (1978) 353-367. 6 Lynch, G. S., Dunwiddie, T. V. and Gribkoff, V. K., Heterosynaptic depression: a postsynaptic correlate of long-term potentiation, Nature (Lond.), 266 (1977) 737-739. 7 Lynch, G. S., Smith, R. L., Browning, M. D. and Deadwyler, S. A., Evidence for bidirectional dendritic transport of horseradish peroxidase. In G. W. Kreutzberg (Ed.), Advances in Neurology, Vol. 12, Raven Press, New York, 1975. 8 Nadler, J V., Vaca, K. W., White, W. F., Lynch, G. S. and Cotman, C. W., Aspartate and glutamate as possible transmitters of excitatory hippocampal afferents, Nature (Lond.), 260 (1976) 538-540. 9 Spencer, W. A. and Kandel, E. R., Hippocampal neuron responses to selective activation of recurrent collaterals of hippocampal neurons, Exp. Neurol., 4 (1961) 149-161. 10 Van Harreveld, A. and Filkovfi, E., Swelling of dendritic spines after stimulation of the perforant path as a mechanism of post-tetanic potentiation, Exp. Neurol., 49 (1975) 736-749.
413
Synaptic transmission is required for initiation of long-term potentiation
THOMAS DUNWIDDIE, DANIEL MADISONand GARY LYNCH Department of Psychobiology, University of California, 1trine, Calif. 92717 ( U.S. A. )
(Accepted January 18th, 1978)
Hippocampal long-term potentiation (LTP) represents a unique form of synaptic plasticity, induced by moderate levels of stimulation (as few as 10-20 pulses), and persisting for indefinite periods of time (months according to some reports) ~,4,5. These characteristics raise the intriguing possibility that the same mechanisms which are responsible for LTP might be the means by which the brain achieves lasting modification of behavior. Although it has been established that the changes underlying LTP have a synaptic location, and are confined to the afferents which have received trains of repetitive stimulation1,6, little is known of the manner in which these trains modify subsequent synaptic transmission. There are two intertwined aspects of this problem; (1) which of the elements of the synaptic complex (terminal, spine or synaptic junction) are altered by the stimulation, and (2) which aspects of the repetitive stimulation are actually responsible for 'triggering' the potentiation effect. Because the synapses involved in LTP are located some distance from the cell bodies of the target neurons, intracellular recording and classical neurophysiological analyses (such as quantal analysis of transmitter release) have been of limited use in answering mechanistic questions of the above type. Therefore, in the experiments detailed in the present paper, we utilized an alternative approach, which was to test for the occurrence of LTP following repetitive stimulation administered during periods in which the transmission process was interrupted at various points. Two very different modulators were used for this purpose: (1) reduction of extracellular calcium, a treatment widely acknowledged to block release of the neurotransmitter and (2) the application of 2-amino-4-phosphonobutyric acid (APB), a drug which competes for glutamic acid receptors in the insect nervous system3. In this report, we demonstrate that both of these manipulations produce a rapid and reversible blockade of synaptic transmission in hippocampal slices, and that synaptic strength remains unchanged by conditioning stimulation applied during such a blockade, even after reinstatement of normal physiology by a return to control medium. These experiments were conducted in the CA 1 field of the in vitro rat hippocampus, prepared according to procedures used in our laboratory for several years7. In order to effect a rapid exchange of medium containing different levels of calcium or APB, slices
414 TABLE I Effects o f A P B and calciumzfree medium on L TP
Data are shown for slices in which two populations of afferents to the CA1 region were activated one pathway ('potentiated') received a high-frequency train under one of two experimental treatments ; the second afferent ('control') received only widely-spaced test stimulation (1 or 2/rain). For the first two rows ('APB' and 0.0 Ca ~ ') pre-measures are the amplitudes of population spikes (average of 2 responses evoked at 5 sec intervals) immediately preceding the beginning of perfusion with either calcium-free or APB (1-3 mM) medium; post-treatment values were taken between 10 and 15 min following the return to control medium. In the third row ('normal'), potentiation was elicited by identical trains of stimulation in control medium, and postpotentiation responses were measured at times corresponding to the intervals used in experimental conditions (i.e., also between 10 and 15 min following the potentiation train). All differences were tested for significance with t-tests for repeated measures on the pre vs. post scores, and then normalized to percentages for presentation. No differences were significant except for the potentiated pathway in control medium, P less than 0.001. Medium
APB 0Ca 2+ Normal
Potentiated p a t h w a y
Control pathway
Pre
t
Post
n
Pre
t
Post
n
100~ 100~ 100K
0.478 0.455 15.23
105~ 116~ 296~
14 9 12
100% 100K 100K
0.297 1.615 0.023
98~ 88% 102%
13 9 11
were perfused constantly in a c h a m b e r o f relatively small volume (approx. 3 ml) at a fairly high flow rate (at least 4 ml/min), and were submerged so that the medium was in contact with both sides of the tissue. Slices o f 500/~m thickness were prepared and perfused until stable responses were obtained; separate control and experimental pathways to the CA1 cells, evoked t h r o u g h stimulation of either stratum radiatum or oriens 5,6, were used. At this point, the flow f r o m the source of control medium was interrupted, and substituted with medium containing either (a) no calcium, or (b) A P B at the appropriate concentration (between 1.5 and 3 m M ) at an equivalent flow rate. This resulted in a complete suppression o f synaptic responses within 5-10 min, at which time one or two tetani of 1 sec duration at 100/sec were given to one pathway, while the control afferents remained unstimulated. During this period, the presynaptic volleys of the stimulated axons were readily recorded, and with appropriate placements of stimulation electrodes in the alveus an additional antidromic response could be elicited from the CA1 target cells. Following the administration of the high frequency trains, the flow of control medium was immediately reinstated, and allowed to continue until well after the responses had returned to baseline amplitudes. In several experiments, paired-pulse interactions were measured as the A P B or low calcium medium was introduced, at times when the field potentials were only partially blocked. At normal levels o f calcium, two pulses delivered 20 msec apart to the same pathway result in diminished population spike amplitudes in the second response; as the level o f calcium is lowered, the first spike becomes correspondingly reduced, while the second response is actually enhanced. This is not simply a consequence of the reduction in the first response (and hence, in the corresponding recurrent I P S P it generates in the pyramidal cell layerg), because it cannot be mimicked by re-
415
I I B
~------.-
~-----.---
E .J/,~-~---- y - . - - - - . ~
"~~ ' ~
F--.~
I
I o
I "a~
I 15
30
I
I
45
60
L
I
75
I
90
I
105 Min.
Fig. 1. Effects of calcium-free medium (upper figure) and medium containing 2 m M APB (lower) on long-term potentiation of population spike amplitude. In both figures, dark bars along the lower margin indicate periods during which the flow of control medium was replaced with either calcium-free or drug-containing medium. Large arrows at the bottom of both figures indicate the interpolation of conditioning trains to the corresponding pathways (filled arrows denote stimulation delivered to the pathway indicated by filled circles, unfilled arrows to open circles). Figures in the upper right hand panel illustrate records derived from the test (filled circles) pathway in the left column, and control pathway in the right column. The population spike (reflecting synchronous discharge of the CA1 neurons) is the small downward deflection on the peak of the positive field potential in both pathways (trace A), and is essentially unaffected by a conditioning train in 0.0 Ca 2+ medium, but shows marked enhancement following an identical train given 12 min later in control medium, and was unchanged following a subsequent period of perfusion with calcium-free medium. Traces B and E demonstrate the complete suppression of synaptic responses observed in the absence of calcium. (Downward deflections of the trace represent negative polarity, calibration marks indicate 8 mV, 5 msec.) In the lower figure, longterm potentiation elicited during perfusion with control medium (filled arrow) persists throughout two subsequent interruptions of transmission by medium containing APB. A conditioning train to the control pathway (open circles) during the second period of APB perfusion is ineffective in 17roducing potentiation, while a subsequent tl ain under normal conditions (third arrow) provokes an essentially normal increase. (Heterosynaptic depression is seen in the control pathway following the first train 5, and aprears to be diminished by the first APB perfusion.) The time scale for both top and bottom figures is the same, and the vertical axis represents arbitrary scale units for the measure of population spike amplitude; each point represents the average of 2 responses evoked 5 sec apart at the indicated time points.
416 ducing the stimulation voltage. On the other hand, when APB was used to reduce synaptic responses, parallel decrements in both responses were seen, and this reversal of paired-pulse inhibition to facilitation was not observed. As can be seen from Table I, repetitive stimulation administered under either experimental condition failed to produce any evidence of long-lasting changes in synaptic efficacy. Neither experimental nor control pathways showed any consistent changes when compared to their pretreatment values as a result of either the blockade of transmission or conditioning stimulation. Some of these slices were then used to demonstrate that LTP could be initiated by subsequent stimulation of the same pathway, but interposed under conditions of normal transmission, and that such LTP was unaffected when the 'potentiated' responses were abolished and reinstated by the sequence of experimental and control medium changes (Fig. 1). Thus, the effectiveness of either APB or calcium-free medium in blocking potentiation is strictly limited to the period of reduced transmission, and the maintenance of potentiation is immune to such treatments. These results clearly demonstrate that LTP cannot be obtained in the absence of synaptic transmission. The significance of this result for our understanding of the locus and mechanisms of potentiation requires a consideration of which aspects of the transmission process were disrupted by the experimental treatments. The effects of diminished extracellular calcium seen in our experiments accord well with those found in classical neurophysiological investigations of transmission at the neuromuscular junction, and presumably reflect the requirement for calcium influx into the terminal in transmitter release. The mechanism by which APB blocks transmission is less certain. The drug inhibits the binding of glutamate to 'receptor-like' proteolipids extracted from insect muscle, and is a potent antagonist of the excitatory actions of glutamate applied to the receptors thought to be present in the membranes of these muscles 3. In view of the evidence that amino acids act as transmitters in hippocampal pathways8, a competitive inhibition of postsynaptic receptors is one possible explanation of the actions of APB in hippocampus. This would also accord well with the rapid onset and reversibility of this drug's actions. However, recent experiments have shown that APB does not block glutamic acid applied iontophoretically to the dendrites of pyramidal cells (Spencer, Dunwiddie and Lynch, in preparation); while this result is open to several interpretations (e.g. 'synaptic receptors' for glutamate are distinct from those activated by iontophoretically applied glutamate) it does indicate the need for caution in ascribing a postsynaptic site to the actions of APB in our experiments. Nevertheless, it is clear that APB blocks transmission by a means other than a reduction in calcium influx, since the effect of reduced calcium on paired-pulse facilitation could not be reproduced with APB. Therefore, the ability of these treatments to block long-term potentiation is presumably due to their common action in interrupting transmission. The failure to initiate long-term potentiation during a blockade of synaptic transmission with APB suggests the involvement of synaptic receptors for amino acids in this effect (see Van Harreveld and Fifkovit1° for an earlier version of this hypothesis). The high-frequency generation of EPSPs in the postsynaptic element, or an unusual aspect of rapid transmitter release (such as its accumulation in abnormal quantities in
417 the cleft o r a ' d e p o l a r i z a t i o n - b l o c k ' t y p e o f effect in the p o s t s y n a p t i c cell) m i g h t be m e c h a n i s m s by which this r e c e p t o r - m e d i a t e d process is induced. A n o b v i o u s i m p l i c a t i o n o f the h y p o t h e s i s t h a t L T P results f r o m the a c t i o n o f a m i n o acids on synaptic receptors is t h a t this is a p o s t s y n a p t i c effect. However, the presence o f p r e s y n a p t i c receptors in certain types o f a x o n terminals suggests that they m a y exist in the h i p p o c a m p u s a n d be involved in LTP.
1 Andersen, P., Sundberg, S. H., Sveen, O. and WigstrOm, H., Specific long-lasting potentiation of synaptic transmission in hippocampal slices, Nature (Lond.), 266 (1977) 736-737. 2 Bliss, T. V. P. and Gardner-Medwin, A. R., Long-lasting potentiation of synaptic transmission in the dentate area of the unanaesthetized rabbit following stimulation of the perforant path, J. Physiol. (Lond.), 232 (1973) 357-374. 3 Cull-Candy, S. G., Donnellan, J. F., James, R. W. and Lunt, G. G., 2-amino-4-phosphonobutyric acid as a glutamate antagonist on locust muscle, Nature (Lond.), 262 (1976) 408-409. 4 Douglas, R. M. and Goddard, G. V., Long-term potentiation of the perforant path granule cell synapse in the rat hippocampus, Brain Research, 86 (1975) 205-215. 5 Dunwiddie, T. V. and Lynch, G. S., Long-term potentiation aad depression of synaptic responses in the hippocampus: localization and frequency dependency, J. Physiol. (Lond.), 276 (1978) 353-367. 6 Lynch, G. S., Dunwiddie, T. V. and Gribkoff, V. K., Heterosynaptic depression: a postsynaptic correlate of long-term potentiation, Nature (Lond.), 266 (1977) 737-739. 7 Lynch, G. S., Smith, R. L., Browning, M. D. and Deadwyler, S. A., Evidence for bidirectional dendritic transport of horseradish peroxidase. In G. W. Kreutzberg (Ed.), Advances in Neurology, Vol. 12, Raven Press, New York, 1975. 8 Nadler, J V., Vaca, K. W., White, W. F., Lynch, G. S. and Cotman, C. W., Aspartate and glutamate as possible transmitters of excitatory hippocampal afferents, Nature (Lond.), 260 (1976) 538-540. 9 Spencer, W. A. and Kandel, E. R., Hippocampal neuron responses to selective activation of recurrent collaterals of hippocampal neurons, Exp. Neurol., 4 (1961) 149-161. 10 Van Harreveld, A. and Filkovfi, E., Swelling of dendritic spines after stimulation of the perforant path as a mechanism of post-tetanic potentiation, Exp. Neurol., 49 (1975) 736-749.