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Phorbol esters broaden the action potential in CA1 hippocampal pyramidal cells
Neuroscienee Letters, 75 (1987) 71 74
71
Elsevier Scientific Publishers Ireland Ltd.
NSL 04464
Phorbol esters broaden the action potential in C A 1 hippocampal pyramidal cells Johan F. Storm D~Tartment of Neurobiology and Behavior, State University of New York at Stony Brook, Stony Brook, NY 11794 (U.S.A.) (Received 17 September 1986; Revised version received and accepted 14 October 1986)
Key words: Phorbol ester; Protein kinase C; Hippocampus; Pyramidal cell; Action potential; Repolarization; Rat Intracellular recordings were made from CAI pyramidal cells in rat hippocampal slices. Single action potentials were elicited by injection of brief current pulses. Bath application of phorbol esters (4fl-phorbol12,13-diacetate, 0.3 5 pM; or 4fi-phorbol-12,13-dibutyrate, 5 10/~M) broadened the action potential in each of the cells tested (n=9). The broadening reflected slowing of the repolarization, whereas the upstroke of the spike was unchanged. This effect may enhance transmitter release from synaptic terminals, and contribute to enhancement of synaptic transmission through activation of protein kinase C, a mechanism which has been associated with long term potentiation.
It has recently been shown that phorbol esters, which directly activate protein kinase C [4], enhance synaptic transmission between hippocampal pyramidal cells, probably via a presynaptic mechanism [12]. This effect mimics long-term potentiation (LTP), a long-lasting increase in synaptic efficacy evoked by synaptic stimulation [3]. In addition, LTP has been found to be associated with translocation of protein kinase C [1]. In Aplysia sensory neurons, the cyclic AMP-dependent protein kinase A mediates the synaptic enhancement which underlies certain forms of learning, by broadening the presynaptic action potential [9]. The present study was undertaken to test whether activation of protein kinase C by phorbol esters in hippocampal pyramidal cells, can cause spike broadening, like the one seen in Aplysia. Hippocampal slices were prepared from male rats and maintained as described elsewhere [15]. The slices were submerged in a perfusion medium containing (in mM) NaCI 122, NaHCO3 22.5, KCI 3.0, MgCI2 1.0, CaCI2 2.0 and glucose 10, and saturated with 95% 02/5% CO2 (pH 7.35), at: 30 or 33°C. Intracellular recordings were made from the cell bodies of CAI pyramidal cells. The microelectrodes were filled with 3 M KCI (tip resistance 30-50 M£2) and connected to a Dagan 8100 amplifier Correspondence: J.F. Storm, Department of Neurobiology and Behavior, State University of New York at Stony Brook, Stony Brook, NY 11794, U.S.A. 0304-3940/87/$ 03.50 (~) 1987 Elsevier Scientific Publishers Ireland Ltd.
72
with a bridge circuit tbr current injection. Only cells with resting potentials negative to - 6 0 mV and action potentials of at least 85 mV were used. The data were stored on tape (Racal 4DS; bandwidth 0 5 kHz) and plotted on an x-y plotter. 4fl-Phorbol12,13-dibutyrate (PDBu) was dissolved in 95% ethanol to a concentration of 5 mM and then added to the perfusing medium to a final concentration of 5-10/~M (0.05% ethanol). The same concentration of ethanol (0.05%) was added to the control medium, to test tbr solvent effects. No such effects were observed. The more watcrsoluble 4#-phorbol-12,13-diacetate (PDAc) was dissolved in distilled water (2 mM), and added to the medium to yield concentrations of 0.3-5/zM. The phorbol esters were shielded from degradation by light by wrapping the vessels in aluminium foil, and the illumination of the slice chamber was turned off. All chemicals were obtained from Sigma. Single action potentials were elicited by injecting brief (1-4 ms) depolarizing current pulses, once every 3-5 s. The membrane potential was held constant at a level close to rest (usually - 6 5 or - 7 0 mV) by steady-current injection. The strength of the stimulus pulse was adjusted so that an action potential was elicited just after turnoff of the pulse (Fig. I A). In all the cells tested (n=9), PDAc ( 0 . 3 - 5 / t M ) or PDBu ( 5 - 1 0 / t M ) broadened the action potential (Fig. 1). The onset of the effect was relatively slow: e.g, the effect of 5/tM PDAc was first detectable after 7-9 min and reached maximum after 20-23 min. Washing with control medium for 40-50 min did not reverse the effect. In contrast, the effects of ionic channel blockers like manganese or tetraethylammonium started within 2-4 rain, were complete after 10-15 rain, and reversed substantially after 20-30 min of washing. The spike broadening was entirely due to slowing of the repolarizing phase of the action potential, with no detectable effect on the spike upstroke. The spike amplitude
A
control
B
PDBu
D
C
~<, PDAc _]
2ms
I I
0.10mV 5hA
I
ms
Fig. 1. Spike broadening caused by 4fl-phorbol-12,13-dibutyrate (PDBu; 10 aM) and 4fl-phorbol-12,13diacetate (PDAc; 1 aM) in CAt pyramidal cells. A: the action potential in control medium, elicited by injection of a 3-ms long current pulse (lower trace). B: the action potential after 13 min in PDBu is broadened, but the fast afterhyperpolarization (fAHP; arrow) is not Mocked. C: superimposed action potentials on a fast time base: 4 of the spikes were obtained in control media (2 without ethanol, and 2 with 0.05% ethanol), and the 3 others after 13 min in PDBu (with 0.05% ethanol). D: effect of 1 pM PDAc (23 rain) in another cell. Temperature 30°C. (In A and B, a weak depolarizing current was given after the stimulus pulse, to enhance the afterpotentials.)
73 was also usually unaffected. In 3 cells, however, there was a slight (1-4%) reduction in the amplitude with 5-10/tM PDBu. Since PDAc and PDBu are known to activate protein kinase C in other systems [4], their spike-broadening effect may be mediated by protein phosphorylation. This conclusion was supported by the observation that 4//-phorbol (10 ltM), which does not activate protein kinase C [4], did not affect the spike. The action potential of CA1 cells is followed by 3 different afterhyperpolarizations (AHPs): (1) a fast AHP (fAHP) lasting 2-5 ms, (2) a medium AHP (mAHP; 50--100 ms) and (3) a slow AHP (sAHP; 0.5-2 s) [13, 15]. The fAHP is primarily due to the fast Ca-dependent K current, Ic [10, 14,. 15], the sAHP is due to a slow Ca-activated K current, IAttP [10], whereas the m A H P is Ca-independent [8, 13, 15]. As reported by others [2], the phorbol esters blocked the sAHP, but not the mAHP. Interestingly, the fAHP was not blocked (Fig. IB, arrow). This suggests that the phorbol esters do not eliminate the sAHP by blocking Ca entry, since the fAHP is also Ca-dependent. Furthermore, it suggests that phorbol esters distinguish between the two different Ca-activated K currents Ic and IAttP [10], and probably inhibits IAHp directly and specifically. Spike repolarization in CAx pyramidal cells seems to involve at least two different K currents [13 15]: the fast Ca-activated current, Ic, and the fast transient current, 1A [7, 10]. In addition, an inward Ca current is active during the downstroke of the spike [14, 15], whereas the Na current causes the upstroke. Thus there seem to be at least 4 possible mechanisms for spike broadening by phorbol esters through modulation of these currents: (1) Ic might be reduced. This seems, however, unlikely since the fAHP, which is primarily due to It, [14, 15], was not blocked, and the spike did not show the pronounced 'shoulder' on the falling phase which appears after blockade of It, with tetraethylammonium or the scorpion toxin CTX [14, 15]. (2) IA may be reduced. (3) Ic'a may be enhanced through direct modulation of the Ca channels. Such an effect of phorbol esters has, indeed, been reported to cause spike broadening in Aplysia bag cells [5]; and protein kinase C modulates Ica, IA and a Ca-activated K current in Hermissenda photoreceptors [6]. (4) Finally, changes in the Na current itself may possibly underlie the spike broadening, by slowing of the inactivation. However, since phorbol esters are known to inhibit a hippocampal chloride current, Icl~v), which seems to be located in the dendrites [11], its effect on the somatic spike could be more indirect. Thus, although Icily) appears to have little effect at the soma [11], it may be important for the repolarization of action potentials (e.g. Ca spikes) in the dendrites, and the somatic effect of phorbol esters may reflect a larger dendritic spike broadening, However, the use of KC1 electrodes would be expected to strongly attenuate the repolarizing effect of Icily), due to Cl-loading of the cell. Still, changes in the electrotonic structure of the cell, due to inhibition of l~'llvl, may broaden the somatic spike. Thus, improved soma~lendritic coupling may cause the somatic Na spike to activate more inward (e.g. Ca) current in the dendrites; and any given dendritic inward current may also contribute more to the spike seen in the soma. Finally, the present results do not rule out the possibility that the effects of PDAc and PDBu were mediated by synapses. Some of these possibilities will be tested in a subsequent study.
74 The spike b r o a d e n i n g effect o f p h o r b o l esters m a y c o n t r i b u t e to the e n h a n c e m e n t of synaptic t r a n s m i s s i o n by p h o r b o l esters [12], provided a similar m e c h a n i s m operates in presynaptic cells o f the synapses which show L T P (i,e. the CA3 cells in thc case of the synapse between CA3 a n d CA1 cells [12]). Thus, a b r o a d e n e d spike in the synaptic terminal will allow more influx of Ca ions d u r i n g the action potential, and increase the release of t r a n s m i t t e r substance. This could come a b o u t , either through direct m o d u l a t i o n of Ca c h a n n e l s [5, 6], or t h r o u g h reduction of an o p p o s i n g o u t w a r d current. Either way, this hypothesis assumes that similar m e c h a n i s m s control the action potential in the synaptic terminals as in the cell body, a n a s s u m p t i o n which has been useful in the analysis o f synaptic plasticity in Aplysia n e u r o n s [9]. I t h a n k Dr. Paul R. A d a m s for p r o v i d i n g excellent w o r k i n g c o n d i t i o n s a n d for c o m m e n t s on the manuscript. This work was s u p p o r t e d by a F o g a r t y i n t e r n a t i o n a l fellowship a n d by N I H G r a n t NS 18579. 1 Akers, R.F., Lovinger, D.M., Colley, P.A., Linden, D.J. and Routtenberg, A., Translocation of protein kinase C activity may mediate hippocampal long-term potentiation, Science,231 (1986) 587- 589. 2 Baraban, J.M., Snyder, S.H. and Alger, B.E., Protein kinase C regulates ionic conductance in hippocampal pyramidal neurons: electrophysiologicaleffects of phorbol esters, Proc. Natl. Acad. Sci. USA, 82 (1985) 2538 -2542. 3 Bliss,T.V.P. and Lomo, T., Long-lasting potentiation of synaptic transmission in the dentate area of the anaesthetized rabbit following stimulation of the perforant path, J. Physiol. (London), 232 (1973) 331 356. 4 Castagna, M., Takai, Y., Kaibuchi, K., Sano, K., Kikkawa, U. and Nishizuka, Y., Direct activation of calcium-activated, phospholipid-dependent protein kinase by tumor-promoting phorbol esters, J. Biol. Chem., 257 (1982) 7847-785!. 5 DeRiemer, Strong, J.A., Albert, K.A., Greengard, P. and Kaczmarek, L.K., Enhancement of calcium current in Aplysia neurones by phorbol esters and protein kinase C, Nature (London), 313 (1985) 313 316. 6 Farley, J. and Auerbach, S., Protein kinase C activation induces conductances changes in Hermissenda photoreceptors like those seen in associative learning, Nature (London), 319 (1986) 220 223. 7 Gustafsson, B., Galvan, M., Grafe, P. and Wigstrom, H.A., Transient outward current in a mammalian central neurone blocked by 4-aminopyridine, Nature (London), 299 (1982) 252-254. 8 Gustafsson, B. and Wigstrom, H., Evidence for two types of afterhyperpolarization in CA~ pyramidal cells in the hippocampus. Brain Res., 206 (1981) 462 468. 9 Klein, M., Shapiro, E. and Kandel, E.R., Synaptic plasticity and the modulation of the Ca2~ current, J. Exp. Biol., 89 (1980) 117 157. l0 Lancaster, B. and Adams, P.R., Calcium-dependent current generating the afterhyperpolarization of hippocampal neurons, J. Neurophysiol., 55 (1986) 1268-1282. I 1 Madison, D.V., Malenka, R.C. and Nicoll, R.A., Phorbol esters block a voltage-sensitivechloride current in hippocampal pyramidal cells, Nature (London), 321 (1986) 695-697. 12 Malenka, R.C., Madison, DV. and Nicoll. R.A., Potentiation of synaptic transmission in the hippocampus by phorbol esters, Nature (London), 321 (1986) 175-177. 13 Storm, J., Calcium-dependent spike repolarization and three kinds of afterhyperpolarization (AHP) in hippocampal pyramidal cells, Soc. Neurosci. Abstr., 11 (1985) 1183. 14 Storm, J.F., Evidence that C-current and A-current contribute to repolarization of the action potential in CA~ pyramidal cells of rat hippocampus, Soc. Neurosci, Abstr., 12 (1986) 764. 15 Storm, J.F., Action potential repolarization and a fast afterhyperpolarization in rat hippocampal pyramidal cells, J. Physiol. (London), in press.
71
Elsevier Scientific Publishers Ireland Ltd.
NSL 04464
Phorbol esters broaden the action potential in C A 1 hippocampal pyramidal cells Johan F. Storm D~Tartment of Neurobiology and Behavior, State University of New York at Stony Brook, Stony Brook, NY 11794 (U.S.A.) (Received 17 September 1986; Revised version received and accepted 14 October 1986)
Key words: Phorbol ester; Protein kinase C; Hippocampus; Pyramidal cell; Action potential; Repolarization; Rat Intracellular recordings were made from CAI pyramidal cells in rat hippocampal slices. Single action potentials were elicited by injection of brief current pulses. Bath application of phorbol esters (4fl-phorbol12,13-diacetate, 0.3 5 pM; or 4fi-phorbol-12,13-dibutyrate, 5 10/~M) broadened the action potential in each of the cells tested (n=9). The broadening reflected slowing of the repolarization, whereas the upstroke of the spike was unchanged. This effect may enhance transmitter release from synaptic terminals, and contribute to enhancement of synaptic transmission through activation of protein kinase C, a mechanism which has been associated with long term potentiation.
It has recently been shown that phorbol esters, which directly activate protein kinase C [4], enhance synaptic transmission between hippocampal pyramidal cells, probably via a presynaptic mechanism [12]. This effect mimics long-term potentiation (LTP), a long-lasting increase in synaptic efficacy evoked by synaptic stimulation [3]. In addition, LTP has been found to be associated with translocation of protein kinase C [1]. In Aplysia sensory neurons, the cyclic AMP-dependent protein kinase A mediates the synaptic enhancement which underlies certain forms of learning, by broadening the presynaptic action potential [9]. The present study was undertaken to test whether activation of protein kinase C by phorbol esters in hippocampal pyramidal cells, can cause spike broadening, like the one seen in Aplysia. Hippocampal slices were prepared from male rats and maintained as described elsewhere [15]. The slices were submerged in a perfusion medium containing (in mM) NaCI 122, NaHCO3 22.5, KCI 3.0, MgCI2 1.0, CaCI2 2.0 and glucose 10, and saturated with 95% 02/5% CO2 (pH 7.35), at: 30 or 33°C. Intracellular recordings were made from the cell bodies of CAI pyramidal cells. The microelectrodes were filled with 3 M KCI (tip resistance 30-50 M£2) and connected to a Dagan 8100 amplifier Correspondence: J.F. Storm, Department of Neurobiology and Behavior, State University of New York at Stony Brook, Stony Brook, NY 11794, U.S.A. 0304-3940/87/$ 03.50 (~) 1987 Elsevier Scientific Publishers Ireland Ltd.
72
with a bridge circuit tbr current injection. Only cells with resting potentials negative to - 6 0 mV and action potentials of at least 85 mV were used. The data were stored on tape (Racal 4DS; bandwidth 0 5 kHz) and plotted on an x-y plotter. 4fl-Phorbol12,13-dibutyrate (PDBu) was dissolved in 95% ethanol to a concentration of 5 mM and then added to the perfusing medium to a final concentration of 5-10/~M (0.05% ethanol). The same concentration of ethanol (0.05%) was added to the control medium, to test tbr solvent effects. No such effects were observed. The more watcrsoluble 4#-phorbol-12,13-diacetate (PDAc) was dissolved in distilled water (2 mM), and added to the medium to yield concentrations of 0.3-5/zM. The phorbol esters were shielded from degradation by light by wrapping the vessels in aluminium foil, and the illumination of the slice chamber was turned off. All chemicals were obtained from Sigma. Single action potentials were elicited by injecting brief (1-4 ms) depolarizing current pulses, once every 3-5 s. The membrane potential was held constant at a level close to rest (usually - 6 5 or - 7 0 mV) by steady-current injection. The strength of the stimulus pulse was adjusted so that an action potential was elicited just after turnoff of the pulse (Fig. I A). In all the cells tested (n=9), PDAc ( 0 . 3 - 5 / t M ) or PDBu ( 5 - 1 0 / t M ) broadened the action potential (Fig. 1). The onset of the effect was relatively slow: e.g, the effect of 5/tM PDAc was first detectable after 7-9 min and reached maximum after 20-23 min. Washing with control medium for 40-50 min did not reverse the effect. In contrast, the effects of ionic channel blockers like manganese or tetraethylammonium started within 2-4 rain, were complete after 10-15 rain, and reversed substantially after 20-30 min of washing. The spike broadening was entirely due to slowing of the repolarizing phase of the action potential, with no detectable effect on the spike upstroke. The spike amplitude
A
control
B
PDBu
D
C
~<, PDAc _]
2ms
I I
0.10mV 5hA
I
ms
Fig. 1. Spike broadening caused by 4fl-phorbol-12,13-dibutyrate (PDBu; 10 aM) and 4fl-phorbol-12,13diacetate (PDAc; 1 aM) in CAt pyramidal cells. A: the action potential in control medium, elicited by injection of a 3-ms long current pulse (lower trace). B: the action potential after 13 min in PDBu is broadened, but the fast afterhyperpolarization (fAHP; arrow) is not Mocked. C: superimposed action potentials on a fast time base: 4 of the spikes were obtained in control media (2 without ethanol, and 2 with 0.05% ethanol), and the 3 others after 13 min in PDBu (with 0.05% ethanol). D: effect of 1 pM PDAc (23 rain) in another cell. Temperature 30°C. (In A and B, a weak depolarizing current was given after the stimulus pulse, to enhance the afterpotentials.)
73 was also usually unaffected. In 3 cells, however, there was a slight (1-4%) reduction in the amplitude with 5-10/tM PDBu. Since PDAc and PDBu are known to activate protein kinase C in other systems [4], their spike-broadening effect may be mediated by protein phosphorylation. This conclusion was supported by the observation that 4//-phorbol (10 ltM), which does not activate protein kinase C [4], did not affect the spike. The action potential of CA1 cells is followed by 3 different afterhyperpolarizations (AHPs): (1) a fast AHP (fAHP) lasting 2-5 ms, (2) a medium AHP (mAHP; 50--100 ms) and (3) a slow AHP (sAHP; 0.5-2 s) [13, 15]. The fAHP is primarily due to the fast Ca-dependent K current, Ic [10, 14,. 15], the sAHP is due to a slow Ca-activated K current, IAttP [10], whereas the m A H P is Ca-independent [8, 13, 15]. As reported by others [2], the phorbol esters blocked the sAHP, but not the mAHP. Interestingly, the fAHP was not blocked (Fig. IB, arrow). This suggests that the phorbol esters do not eliminate the sAHP by blocking Ca entry, since the fAHP is also Ca-dependent. Furthermore, it suggests that phorbol esters distinguish between the two different Ca-activated K currents Ic and IAttP [10], and probably inhibits IAHp directly and specifically. Spike repolarization in CAx pyramidal cells seems to involve at least two different K currents [13 15]: the fast Ca-activated current, Ic, and the fast transient current, 1A [7, 10]. In addition, an inward Ca current is active during the downstroke of the spike [14, 15], whereas the Na current causes the upstroke. Thus there seem to be at least 4 possible mechanisms for spike broadening by phorbol esters through modulation of these currents: (1) Ic might be reduced. This seems, however, unlikely since the fAHP, which is primarily due to It, [14, 15], was not blocked, and the spike did not show the pronounced 'shoulder' on the falling phase which appears after blockade of It, with tetraethylammonium or the scorpion toxin CTX [14, 15]. (2) IA may be reduced. (3) Ic'a may be enhanced through direct modulation of the Ca channels. Such an effect of phorbol esters has, indeed, been reported to cause spike broadening in Aplysia bag cells [5]; and protein kinase C modulates Ica, IA and a Ca-activated K current in Hermissenda photoreceptors [6]. (4) Finally, changes in the Na current itself may possibly underlie the spike broadening, by slowing of the inactivation. However, since phorbol esters are known to inhibit a hippocampal chloride current, Icl~v), which seems to be located in the dendrites [11], its effect on the somatic spike could be more indirect. Thus, although Icily) appears to have little effect at the soma [11], it may be important for the repolarization of action potentials (e.g. Ca spikes) in the dendrites, and the somatic effect of phorbol esters may reflect a larger dendritic spike broadening, However, the use of KC1 electrodes would be expected to strongly attenuate the repolarizing effect of Icily), due to Cl-loading of the cell. Still, changes in the electrotonic structure of the cell, due to inhibition of l~'llvl, may broaden the somatic spike. Thus, improved soma~lendritic coupling may cause the somatic Na spike to activate more inward (e.g. Ca) current in the dendrites; and any given dendritic inward current may also contribute more to the spike seen in the soma. Finally, the present results do not rule out the possibility that the effects of PDAc and PDBu were mediated by synapses. Some of these possibilities will be tested in a subsequent study.
74 The spike b r o a d e n i n g effect o f p h o r b o l esters m a y c o n t r i b u t e to the e n h a n c e m e n t of synaptic t r a n s m i s s i o n by p h o r b o l esters [12], provided a similar m e c h a n i s m operates in presynaptic cells o f the synapses which show L T P (i,e. the CA3 cells in thc case of the synapse between CA3 a n d CA1 cells [12]). Thus, a b r o a d e n e d spike in the synaptic terminal will allow more influx of Ca ions d u r i n g the action potential, and increase the release of t r a n s m i t t e r substance. This could come a b o u t , either through direct m o d u l a t i o n of Ca c h a n n e l s [5, 6], or t h r o u g h reduction of an o p p o s i n g o u t w a r d current. Either way, this hypothesis assumes that similar m e c h a n i s m s control the action potential in the synaptic terminals as in the cell body, a n a s s u m p t i o n which has been useful in the analysis o f synaptic plasticity in Aplysia n e u r o n s [9]. I t h a n k Dr. Paul R. A d a m s for p r o v i d i n g excellent w o r k i n g c o n d i t i o n s a n d for c o m m e n t s on the manuscript. This work was s u p p o r t e d by a F o g a r t y i n t e r n a t i o n a l fellowship a n d by N I H G r a n t NS 18579. 1 Akers, R.F., Lovinger, D.M., Colley, P.A., Linden, D.J. and Routtenberg, A., Translocation of protein kinase C activity may mediate hippocampal long-term potentiation, Science,231 (1986) 587- 589. 2 Baraban, J.M., Snyder, S.H. and Alger, B.E., Protein kinase C regulates ionic conductance in hippocampal pyramidal neurons: electrophysiologicaleffects of phorbol esters, Proc. Natl. Acad. Sci. USA, 82 (1985) 2538 -2542. 3 Bliss,T.V.P. and Lomo, T., Long-lasting potentiation of synaptic transmission in the dentate area of the anaesthetized rabbit following stimulation of the perforant path, J. Physiol. (London), 232 (1973) 331 356. 4 Castagna, M., Takai, Y., Kaibuchi, K., Sano, K., Kikkawa, U. and Nishizuka, Y., Direct activation of calcium-activated, phospholipid-dependent protein kinase by tumor-promoting phorbol esters, J. Biol. Chem., 257 (1982) 7847-785!. 5 DeRiemer, Strong, J.A., Albert, K.A., Greengard, P. and Kaczmarek, L.K., Enhancement of calcium current in Aplysia neurones by phorbol esters and protein kinase C, Nature (London), 313 (1985) 313 316. 6 Farley, J. and Auerbach, S., Protein kinase C activation induces conductances changes in Hermissenda photoreceptors like those seen in associative learning, Nature (London), 319 (1986) 220 223. 7 Gustafsson, B., Galvan, M., Grafe, P. and Wigstrom, H.A., Transient outward current in a mammalian central neurone blocked by 4-aminopyridine, Nature (London), 299 (1982) 252-254. 8 Gustafsson, B. and Wigstrom, H., Evidence for two types of afterhyperpolarization in CA~ pyramidal cells in the hippocampus. Brain Res., 206 (1981) 462 468. 9 Klein, M., Shapiro, E. and Kandel, E.R., Synaptic plasticity and the modulation of the Ca2~ current, J. Exp. Biol., 89 (1980) 117 157. l0 Lancaster, B. and Adams, P.R., Calcium-dependent current generating the afterhyperpolarization of hippocampal neurons, J. Neurophysiol., 55 (1986) 1268-1282. I 1 Madison, D.V., Malenka, R.C. and Nicoll, R.A., Phorbol esters block a voltage-sensitivechloride current in hippocampal pyramidal cells, Nature (London), 321 (1986) 695-697. 12 Malenka, R.C., Madison, DV. and Nicoll. R.A., Potentiation of synaptic transmission in the hippocampus by phorbol esters, Nature (London), 321 (1986) 175-177. 13 Storm, J., Calcium-dependent spike repolarization and three kinds of afterhyperpolarization (AHP) in hippocampal pyramidal cells, Soc. Neurosci. Abstr., 11 (1985) 1183. 14 Storm, J.F., Evidence that C-current and A-current contribute to repolarization of the action potential in CA~ pyramidal cells of rat hippocampus, Soc. Neurosci, Abstr., 12 (1986) 764. 15 Storm, J.F., Action potential repolarization and a fast afterhyperpolarization in rat hippocampal pyramidal cells, J. Physiol. (London), in press.