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A subset of local interneurons generate slow inhibitory postsynaptic potentials in hippocampal neurons
Brain Research, 511 (1990) 163-164
163
Elsevier BRES 24011
A subset of local interneurons generate slow inhibitory postsynaptic potentials in hippocampal neurons Menahem Segal Center for Neuroscience, The Weizmann Institute of Science, Rehovot (Israel)
(Accepted 5 December 1989) Key words: 4-Aminopyridine;Slow inhibitory postsynapticpotential; Saclofen; Hippocampal slice
Topical application of 4-aminopyridine (4-AP) onto hippocampal slices produced spontaneous repetitive large hyperpolarizingpotentials in CA1 neurons. This effect of 4-AP was blocked by a new GABAB receptor antagonist, 2-hydroxy-saclofen.2-Hydroxy-saclofenalso blocked slow IPSPs evoked by stimulation of stratum radiatum. It is suggested that 4-AP-evoked slow hyperpolarizingpotentials are in fact slow IPSPs evoked by activation of a selective subset of interneurons which do not produce fast IPSPs.
Potent local inhibitory synapses regulate the activity of the hippocampus 4's. Activation of these synapses produce a fast GABAA-mediated increase in C1- conductance and a slow, GABAB-mediated increase in K conductance 7'9. Local electrical stimulation produces both types of inhibitory postsynaptic potentials (IPSPs) recorded intracellularly in hippocampal neurons. Despite extensive research 4"5 it is not yet known whether the two types of IPSPs are generated by activation of a single set of interneurons or that different sets of interneurons are dedicated to generate fast or slow IPSPs in the pyramidal neurons. We now demonstrate that large repetitive slow IPSPs mediated by G A B A B receptor activation can be generated in isolation in hippocampal neurons, indicating that a subset of interneurons is probably dedicated to produce these slow IPSPs. Intracellular activity was recorded from 16 CA1 hippocampal neurons in an interface slice preparation with potassium-acetate or KCl-containing micropipettes as reported elsewhere s. Topical application of a microdrop of buffered 4-aminopyridine (4-AP) produced with a short delay (<5 s), large (5-10 mV) slow (2-5 s) repetitive hyperpolarizing potentials (HPs) (Fig. 1A). These potentials are Ca dependent, q-TX sensitive and reverse near K equilibrium potential whether recorded with KCI- or acetate-containing pipettes indicating that they are likely to be mediated by activation of large K-mediated synaptic potentials 8. 4-AP also triggered spontaneous fast IPSPs and EPSPs but these were seen at a longer delay after application of the drug. Electrical stimulation of stratum radiatum produced a fast EPSP
followed by a fast and subsequently a slow IPSP. 4-AP caused a marked prolongation of the slow IPSP in these cells (data not shown). To examine the possibility that the 4-AP evoked slow HPs are mediated by activation of G A B A s receptors, we used the new G A B A s antagonist, 2-hydroxy-saclofen (saclofenl,3). We found this to be a more potent G A B A B antagonist than a similar compound, phaclofen 2 which produced similar but smaller effects in our hands. Topical application of saclofen blocked the repetitive HPs in all 6 cells studied and prevented generation of these in 4 drug-naive slices. Saclofen also blocked selectively the slow IPSPs without affecting much the fast IPSPs, or the EPSPs generated in these cells. The selectivity of 4-AP action was examined in 6 slices incubated in a mixture of picrotoxin and kynurenic acid (KyA) to block the fast IPSP and the EPSP, respectively. In such a slice electrical stimulation produced a smaller EPSP (depending on the concentration of KyA used) and, primarily, a slow IPSP. In these slices 4-AP was as effective as it was in normal slices in generating the HPs. Furthermore, saclofen blocked the HPs and the slow IPSPs in these slices, as in normal ones (Fig. 1B), without having any other effects on the recorded cells. Finally, the effects of saclofen on neuronal responses to GABA were examined in slices incubated in picrotoxin/KyA. Topical application of G A B A produced in 5 cells recorded from such slices a simple hyperpolarizing response which is different from the complex response to G A B A seen in undrugged cells. Surprisingly, saclofen could only partially antagonize these responses (Fig. 1C)
Correspondence: M. Segal, Center for Neuroscience, The Weizmann Institute, 76 100 Rehovot, Israel.
0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
164
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Fig. 1. A: responses to topical application of 4-aminopyridine (4-AP, A1 and A2, arrowhead) and to afferent stimulation (3 and 4) are blocked by 2-hydroxysaclofen (Sac). Cells were recorded in normal medium with no added drugs. Topical application of a microdrop containing 10 mM pH-adjusted 4-AP produced repetitive, large, spontaneous hyperpolarizing potentials (HPs). In presence of saclofen (applied via a microdrop containing l-5 mM drug solution (A2)) only minor HPs could be detected. Recording was made with potassium-acetate containing micropipettes. A3 and 4, stimulation of stratum radiatum produced a typical 3 component response consisting of a fast EPSP, followed by a fast IPSP followed by a slow, K-mediated IPSP. Only the latter was totally eliminated in the presence of saclofen. The initial EPSP was slightly larger in the presence of the drug. B: responses to 4-AP in slices treated with 100 /~M picrotoxin and 100/~M kynurenic acid, where O--mediated fast IPSPs were eliminated. B l: 4-AP produced a series of HPs associated with marked conductance changes estimated by passage of 0.15 nA constant hyperpolarizing current pulses (current, lower trace). B2: in presence of saclofen the response to 4-AP was totally blocked. B3: saclofen blocked ongoing 4-AP-induced repetitive HPs without affecting other properties of the recorded cell membrane. C: attenuation by saclofen of responses to GABA applied via a microdrop on a picrotoxin/kynurenic acid treated slice. Responses are presented before, left, during, middle, and after saclofen (right). C2 and 3: computer averages of responses to afferent stimulation before and after topical application of saclofen (2) and the net saclofen sensitive potential obtained by subtracting the two averages (3). Scale in A 5 s for A1 and 2; and B 10 s for C1; 0.25 s for A3 and 4; and 0.4 s for C2 and 3. Current and voltage scales same for all traces.
while, in the same cells it completely blocked the slPSPs. Taken together with previous results which illustrate that 4-AP causes putative interneurons to burst s, the present results indicate that a subset of interneurons is likely to be highly sensitive to 4-AP, responding to the drug by bursts of action potentials which produce repetitive HPs in hippocampal pyramidal neurons. These neurons do not activate postsynaptic Cl-mediated IPSPs, since saclofen, which did not affect fast IPSPs, totally blocked the initial responses to 4-AP. The synchronized pattern of the HPs implies that few cells, perhaps one in a small region, are activated to produce HPs in all other cells. This may explain the rarity of encountering these cells in a recording session.
It is interesting to note that saclofen did not completely block the hyperpolarizing responses to GABA even at a dose that blocked the slow IPSPs. This may indicate that the non-synaptic hyperpolarizing K-mediated GABAB receptors that are resistant to picrotoxin, are less sensitive to saclofen than the synaptic ones. The present experiments indicate that few cells possess the powerful ability to regulate activity of populations of neurons in the hippocampus, making their investigation by indirect means, e.g. 4-AP, an intriguing means of studying local control of neural activity. The segregation of GABA-containing interneurons into those producing fast inhibitory potentials and those producing slow potentials has immense significance for our understanding of local neural circuits.
1 Curtis, D.R., Gynther, B.D., Beattie, D.T., Kerr, D.I.B. and Prager, R.H., Baelofen antagonism by 2-hydroxysaclofen in the cat spinal cord, Neurosci. Lett., 92 (1988) 97-101. 2 Dutar, P. and Nicoll, R.A., A physiological role for GABA B receptors in central nervous system, Nature (Lond.), 332 (1988) 156-158. 3 Kerr, D.I.B., Ong, J., Johnston, G.A.R., Abbenante, J. and Prager, R.H., 2-Hydroxy-saclofen: an improved antagonist at central and peripheral GABAa receptors, Neurosci. Lett., 92 (1988) 92-96. 4 Knowles, W.D. and Schwartzkroin, P.A., Local circuit synaptic interactions in hippocampal brain slices, J. Neurosci., 1 (1981) 318-322. 5 Miles, R. and Wong, R.K.S., Unitary inhibitory synaptic
potentials in the Guinea-pig hippocampus in vitro, J. Physiol. (Lond.), 356 (1984) 97-113. Newberry, N.R. and Nicoll, R.A., A bicucuUine-resistant inhibitory post synaptic potential in rat hippocampal pyramidal cells in vitro, J. Physiol. (Lond.), 348 (1984) 239-254. Newberry, N.R. and Nicoll, R.A., Comparison of the action of baclofen with gamma aminobutyric acid on rat hippocampal pyramidal cells in vitro, J. Physiol. (Lond.), 360 (1985) 161-185. Segal, M., Repetitive inhibitory postsynaptic potentials evoked by 4-aminopyridine in hippoeampal neurons in vitro, Brain Research, 414 (1987) 285-293. Thalman, R.H., Reversal properties of an EGTA-resistant late hyperpolarization that follows synaptic stimulation of hippocampal neurons, Neurosci. Lett., 46 (1984) 103-108.
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8 9
163
Elsevier BRES 24011
A subset of local interneurons generate slow inhibitory postsynaptic potentials in hippocampal neurons Menahem Segal Center for Neuroscience, The Weizmann Institute of Science, Rehovot (Israel)
(Accepted 5 December 1989) Key words: 4-Aminopyridine;Slow inhibitory postsynapticpotential; Saclofen; Hippocampal slice
Topical application of 4-aminopyridine (4-AP) onto hippocampal slices produced spontaneous repetitive large hyperpolarizingpotentials in CA1 neurons. This effect of 4-AP was blocked by a new GABAB receptor antagonist, 2-hydroxy-saclofen.2-Hydroxy-saclofenalso blocked slow IPSPs evoked by stimulation of stratum radiatum. It is suggested that 4-AP-evoked slow hyperpolarizingpotentials are in fact slow IPSPs evoked by activation of a selective subset of interneurons which do not produce fast IPSPs.
Potent local inhibitory synapses regulate the activity of the hippocampus 4's. Activation of these synapses produce a fast GABAA-mediated increase in C1- conductance and a slow, GABAB-mediated increase in K conductance 7'9. Local electrical stimulation produces both types of inhibitory postsynaptic potentials (IPSPs) recorded intracellularly in hippocampal neurons. Despite extensive research 4"5 it is not yet known whether the two types of IPSPs are generated by activation of a single set of interneurons or that different sets of interneurons are dedicated to generate fast or slow IPSPs in the pyramidal neurons. We now demonstrate that large repetitive slow IPSPs mediated by G A B A B receptor activation can be generated in isolation in hippocampal neurons, indicating that a subset of interneurons is probably dedicated to produce these slow IPSPs. Intracellular activity was recorded from 16 CA1 hippocampal neurons in an interface slice preparation with potassium-acetate or KCl-containing micropipettes as reported elsewhere s. Topical application of a microdrop of buffered 4-aminopyridine (4-AP) produced with a short delay (<5 s), large (5-10 mV) slow (2-5 s) repetitive hyperpolarizing potentials (HPs) (Fig. 1A). These potentials are Ca dependent, q-TX sensitive and reverse near K equilibrium potential whether recorded with KCI- or acetate-containing pipettes indicating that they are likely to be mediated by activation of large K-mediated synaptic potentials 8. 4-AP also triggered spontaneous fast IPSPs and EPSPs but these were seen at a longer delay after application of the drug. Electrical stimulation of stratum radiatum produced a fast EPSP
followed by a fast and subsequently a slow IPSP. 4-AP caused a marked prolongation of the slow IPSP in these cells (data not shown). To examine the possibility that the 4-AP evoked slow HPs are mediated by activation of G A B A s receptors, we used the new G A B A s antagonist, 2-hydroxy-saclofen (saclofenl,3). We found this to be a more potent G A B A B antagonist than a similar compound, phaclofen 2 which produced similar but smaller effects in our hands. Topical application of saclofen blocked the repetitive HPs in all 6 cells studied and prevented generation of these in 4 drug-naive slices. Saclofen also blocked selectively the slow IPSPs without affecting much the fast IPSPs, or the EPSPs generated in these cells. The selectivity of 4-AP action was examined in 6 slices incubated in a mixture of picrotoxin and kynurenic acid (KyA) to block the fast IPSP and the EPSP, respectively. In such a slice electrical stimulation produced a smaller EPSP (depending on the concentration of KyA used) and, primarily, a slow IPSP. In these slices 4-AP was as effective as it was in normal slices in generating the HPs. Furthermore, saclofen blocked the HPs and the slow IPSPs in these slices, as in normal ones (Fig. 1B), without having any other effects on the recorded cells. Finally, the effects of saclofen on neuronal responses to GABA were examined in slices incubated in picrotoxin/KyA. Topical application of G A B A produced in 5 cells recorded from such slices a simple hyperpolarizing response which is different from the complex response to G A B A seen in undrugged cells. Surprisingly, saclofen could only partially antagonize these responses (Fig. 1C)
Correspondence: M. Segal, Center for Neuroscience, The Weizmann Institute, 76 100 Rehovot, Israel.
0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
164
2~Sac { 4-AP
T]-i~I
I I I I I I III
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B•-AP
I I I I I I I IFI-
secJ my 0.5nA
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0.2sec
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Fig. 1. A: responses to topical application of 4-aminopyridine (4-AP, A1 and A2, arrowhead) and to afferent stimulation (3 and 4) are blocked by 2-hydroxysaclofen (Sac). Cells were recorded in normal medium with no added drugs. Topical application of a microdrop containing 10 mM pH-adjusted 4-AP produced repetitive, large, spontaneous hyperpolarizing potentials (HPs). In presence of saclofen (applied via a microdrop containing l-5 mM drug solution (A2)) only minor HPs could be detected. Recording was made with potassium-acetate containing micropipettes. A3 and 4, stimulation of stratum radiatum produced a typical 3 component response consisting of a fast EPSP, followed by a fast IPSP followed by a slow, K-mediated IPSP. Only the latter was totally eliminated in the presence of saclofen. The initial EPSP was slightly larger in the presence of the drug. B: responses to 4-AP in slices treated with 100 /~M picrotoxin and 100/~M kynurenic acid, where O--mediated fast IPSPs were eliminated. B l: 4-AP produced a series of HPs associated with marked conductance changes estimated by passage of 0.15 nA constant hyperpolarizing current pulses (current, lower trace). B2: in presence of saclofen the response to 4-AP was totally blocked. B3: saclofen blocked ongoing 4-AP-induced repetitive HPs without affecting other properties of the recorded cell membrane. C: attenuation by saclofen of responses to GABA applied via a microdrop on a picrotoxin/kynurenic acid treated slice. Responses are presented before, left, during, middle, and after saclofen (right). C2 and 3: computer averages of responses to afferent stimulation before and after topical application of saclofen (2) and the net saclofen sensitive potential obtained by subtracting the two averages (3). Scale in A 5 s for A1 and 2; and B 10 s for C1; 0.25 s for A3 and 4; and 0.4 s for C2 and 3. Current and voltage scales same for all traces.
while, in the same cells it completely blocked the slPSPs. Taken together with previous results which illustrate that 4-AP causes putative interneurons to burst s, the present results indicate that a subset of interneurons is likely to be highly sensitive to 4-AP, responding to the drug by bursts of action potentials which produce repetitive HPs in hippocampal pyramidal neurons. These neurons do not activate postsynaptic Cl-mediated IPSPs, since saclofen, which did not affect fast IPSPs, totally blocked the initial responses to 4-AP. The synchronized pattern of the HPs implies that few cells, perhaps one in a small region, are activated to produce HPs in all other cells. This may explain the rarity of encountering these cells in a recording session.
It is interesting to note that saclofen did not completely block the hyperpolarizing responses to GABA even at a dose that blocked the slow IPSPs. This may indicate that the non-synaptic hyperpolarizing K-mediated GABAB receptors that are resistant to picrotoxin, are less sensitive to saclofen than the synaptic ones. The present experiments indicate that few cells possess the powerful ability to regulate activity of populations of neurons in the hippocampus, making their investigation by indirect means, e.g. 4-AP, an intriguing means of studying local control of neural activity. The segregation of GABA-containing interneurons into those producing fast inhibitory potentials and those producing slow potentials has immense significance for our understanding of local neural circuits.
1 Curtis, D.R., Gynther, B.D., Beattie, D.T., Kerr, D.I.B. and Prager, R.H., Baelofen antagonism by 2-hydroxysaclofen in the cat spinal cord, Neurosci. Lett., 92 (1988) 97-101. 2 Dutar, P. and Nicoll, R.A., A physiological role for GABA B receptors in central nervous system, Nature (Lond.), 332 (1988) 156-158. 3 Kerr, D.I.B., Ong, J., Johnston, G.A.R., Abbenante, J. and Prager, R.H., 2-Hydroxy-saclofen: an improved antagonist at central and peripheral GABAa receptors, Neurosci. Lett., 92 (1988) 92-96. 4 Knowles, W.D. and Schwartzkroin, P.A., Local circuit synaptic interactions in hippocampal brain slices, J. Neurosci., 1 (1981) 318-322. 5 Miles, R. and Wong, R.K.S., Unitary inhibitory synaptic
potentials in the Guinea-pig hippocampus in vitro, J. Physiol. (Lond.), 356 (1984) 97-113. Newberry, N.R. and Nicoll, R.A., A bicucuUine-resistant inhibitory post synaptic potential in rat hippocampal pyramidal cells in vitro, J. Physiol. (Lond.), 348 (1984) 239-254. Newberry, N.R. and Nicoll, R.A., Comparison of the action of baclofen with gamma aminobutyric acid on rat hippocampal pyramidal cells in vitro, J. Physiol. (Lond.), 360 (1985) 161-185. Segal, M., Repetitive inhibitory postsynaptic potentials evoked by 4-aminopyridine in hippoeampal neurons in vitro, Brain Research, 414 (1987) 285-293. Thalman, R.H., Reversal properties of an EGTA-resistant late hyperpolarization that follows synaptic stimulation of hippocampal neurons, Neurosci. Lett., 46 (1984) 103-108.
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