European Journal of Pharmacology, 187 (1990) 127-130
127
Elsevier EJP 20711 Short communication
Ethanol potentiates the GABAA-activated CI- current in mouse hippocampal and cortical neurons Luis G. Aguayo Section of Electrophysiology, Laboratory of Physiologic and Pharmacologic Studies, National Institute on Alcohol Abuse and Alcoholism, 12501 Washington Avenue, Rockville, MD 20852, U.S.A.
Received 3 August 1990, accepted 7 August 1990
The effects of ethanol (1-80 mM) on GABA-activated C1- c u r r e n t (IGABA) were studied in cultured mammalian hippocampal and cortical neurons. Patch-clamp recordings revealed that ethanol potentiated the C1- current in a concentration-dependent manner (1-40 mM) in the majority of the cells studied. No higher degree of potentiation was found by increasing the concentration of ethanol to 80 mM. This study demonstrates that ethanol can potentiate IGABA in mammalian central neurons. GABA A receptors; C1- currents; Hippocampus; Cortex; Ethanol; (Neuron)
1. Introduction -/-Aminobutyric acid (GABA) is a main inhibitory transmitter in the mammalian brain, and barbiturates and benzodiazepines are thought to exert their primary effects in the brain by potentiating a C1- current linked to G A B A A receptor activation (Bormann, 1988). Although ethanol is the oldest and most frequently abused drug, the cellular mechanisms by which it affects brain function have remained largely unknown. Recent studies have shown that ethanol inhibits N-methyl-D-aspartate action in central and peripheral neurons (Lovinger et al., 1989; White et al., 1990). It has also been suggested that some effects of ethanol may be related to its capacity to alter the responses to activation of central G A B A A receptors (Suzdak et al., 1986; for review see Ticku, 1989; Shefner, 1990).
Correspondence to: L.G. Aguayo, Section of Electrophysiology, L.P.P.S., NIAAA, 12501 Washington Avenue, Rockville, MD 20852, U.S.A.
Ethanol, however, does not affect the GABAA-induced C1- current (IGABA) in peripheral neurons in intoxicating concentrations (White et al., 1990). This study shows that intoxicating concentrations of ethanol, which shares anxiolytic and s e d a t i v e / hypnotic activity with barbiturates and benzodiazepines, can potentiate IGABA in mammalian central neurons. 2. Materials and methods Mouse (E17-E18, C57B16J) hippocampal and cortical neurons (1.5-3.0 x 10 5 cells/dish) were grown in culture in a medium containing minim u m essential media (MEM, Gibco, G r a n d Island, NY, U.S.A.), 5% horse serum (Gibco) and a mixture of neurotrophic supplements on a background cell layer. Recordings were done with conventional whole-cell patch-clamp techniques. The external solution contained (in m M ) 150 NaC1, 5.4 KCI, 2 CaCI2, 1 MgC12, 10 glucose and 10 H E P E S ( p H 7.4). Tetrodotoxin (TTX) was added at a concentration of 200 n M to block sodium dependent spikes. The patch pipette contained (in mM)
0014-2999/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
128
140 CsC1, 4 MgC12, 10 HEPES (pH 7.37); ATPN a 2 (2 mM) and BAPTA (1,2-bis(2-aminophenoxy) hethane-N,N,N',N"-tetra-acetic acid; 10 mM) were added to the intracellular solution to reduce the 'run-down' of IGABA observed in their absence and to tightly control intracellular C a 2+. Under these conditions, about 80% of the neurons had stable IGABA for periods in excess of 20 min. In most experiments, a minimally desensitizing IGABA w a s achieved with a concentration of 2.5 # M GABA. This allowed for better temporal resolution of the potentiation of IGABA produced by ethanol. Serial application of increasing drug concentrations was accomplished using a 200 /xm diameter pipette connected to a 10 ml reservoir and moved relative to the neuron. Currents were displayed on a chart recorder (Gould 2400) and stored in a VCR tape recorder (Vetter 240B) after filtering the signal at 3.15 kHz with a four-pole low-pass Bessel filter (Ithaco 4302).
3. Results Whole-cell patch-clamp recordings in all central neurons (n > 100) tested with G A B A (1-200 p M )
A
B
GABA ( P M )
5
GABA (2.5 p M)
50
revealed an inward current. Figure 1A shows the current activated by superfusion of 5 and 50/~M G A B A to a single hippocampal neuron. With 50 /LM G A B A the current desensitized by about 60% during the application of the neurotransmitter. Figure 1B illustrates the effect of the G A B A receptor antagonist bicuculline (20 /~M). The current records show that when bicuculline was applied in combination with G A B A (2.5 /~M) it reversibly and completely inhibited the current. Ethanol clearly potentiated IGABA in 70% of the cells tested with ethanol (n = 70). Figure 1C shows the effects of ethanol (1-80 mM) o n IGABA in a hippocampal neuron. As illustrated, the application of 1 m M ethanol potentiated the peak amplitude of IGABA by a factor of 1.3 above control. The potentiation of IGABAreached a factor of 1.65 above control when the cell was perfused with 10 m M ethanol. N o larger extent of potentiation was found with 40 mM, and when the concentration of ethanol was increased to 80 m M the degree of potentiation with 80 m M was close to that obtained with 1 m M ethanol. The potentiating effect of ethanol o n IGABAwas reversible. Interestingly, the study of ethanol at concentrations above 50 m M on C1- flux in rat brain synaptoneurosomes
GABA (2.5 ~. M) + BICUCULLINE (20 I~ M)
GABA (2.5 ~ M)
....
20 s
C
CONTROL
¢~
1 mM ETHANOL
20 s
10 mM ETHANOL
40 mM ETHANOL
80 mM ETHANOL
o
CONTROL
Fig. 1. (A) GABA-activated inward current (IGABA)in cultured hippocampal neurons. Inward current activated by application of 5 and 50 #M GABA. (B) IGABAwas reversibly blocked by bicuculline (20 #M). (C) Ethanol produced a concentration-dependent potentiation of IGABA.The concentration of GABA was 2.5/tM in all records in (C). Holding potential was - 10 mV in (A) and - 50 in (B) and (C). The holding potential of - 1 0 mV was necessary to avoid saturation of the current amplifier with high GABA concentrations. All records are from hippocampal neurons.
129 A CONTROL
ETH A N OL
C ON TR OL
B
20 s CONTROL
FLURAZEPAM
PENTOBARBITAL
ETHANOL
20 s
CONTROL
o
Fig. 2. (A) Ethanol induced a reversible potentiation of IGABAin cerebral cortical neurons. The traces show the response to 2.5 gM GABA (control traces) and GABA plus 20 mM ethanol (middle trace). (B) IGABAin neurons that were insensitive to ethanol was still potentiated by pentobarbital (20 vM) and flurazepam (20 gM). The concentration of GABA was 2.5/tM. The data in (B) are from a hippocampal neuron. Holding potential - 50 mV in all records.
showed a similar biphasic response (Suzdak et al., 1986) and this m a y suggest an antagonistic action of ethanol on the G A B A A receptor-C1- channel complex. Ethanol (40 mM) did not induce any C1- current in the absence of G A B A (not shown). It was previously reported that local and systemic administration of ethanol alters GABAmediated inhibition of cortical neurons (Nestoros, 1980), but these experiments did not provide any indication of the mechanism by which ethanol affected these neurons. A possible mechanism to explain this neuronal inhibition is that ethanol potentiates the GABA-activated C1- current in cortical neurons. Indeed, ethanol (20 mM) was also effective in potentiating IGABA in cortical neurons (fig. 2A). In the presence of ethanol, the peak amplitude of IGABA w a s potentiated by more than 100% above control and the potentiation was fully reversible (fig. 2A, right trace). Previous electrophysiological studies on the effects of ethanol on G A B A responses are conflicting and have suggested that ethanol does not affect all central neurons equally (Mereu and Gessa, 1985; Siggins et al., 1987; Shefner, 1990). In the present experiments, IGABA elicited in a group of hippocampal neurons was not affected by ethanol. Figure 2B shows the lack of effect of 40 m M ethanol o n IGABA activated in one of these neurons. The same neurons, however, had IGABA that was potentiated by pentobarbital and flurazepam (fig. 2B).
4. Discussion This study provides direct evidence of the cellular mechanism by which ethanol affects G A B A A responses in mammalian central neurons. The results also suggest that the modulation of IGABAby benzodiazepine, barbiturates and ethanol m a y occur through distinct mechanisms. The finding that ethanol potentiates IGABA in some neurons but not others suggests that ethanol exerts its cellular actions in a more selective manner than previously believed. The nature for this selectivity is presently unknown. It is possible, however, that similar to benzodiazepines, which are more effective in p o t e n t i a t i n g IGABA in the presence of the 72 subunit (Pritchett et al., 1989), ethanol m a y also require a particular configuration or state of the receptor to exert its action.
Acknowledgements I wish to thank my colleagues Drs. F.F. Weight, G. Kunos and G. White for valuable discussion during these experiments.
References Bormann, J., 1988, Electrophysiology of GABA A and GABA B receptor subtypes, Trends Neurosci. 11, 112. Lovinger, D.M., G. White and F.F. Weight, 1989, Ethanol inhibits NMDA-activated ion current in hippocampal neurons, Science 243, 1721.
130 Mereu, G. and G.L. Gessa, 1985, Low doses of ethanol inhibit the firing of neurons in the substantia nigra, pars reticulata: a gabaergic effect?, Brain Res. 360, 325. Nestoros, N.J., 1980, Ethanol specifically potentiates GABAmediated neurotransmission in feline cerebral cortex. Science 209, 708. Pritchett, D.B., H. Sontheimer, B.D. Shivers, S. Ymer, H. Kettenmann, P.R. Schofield and P.H. Seeburg, 1989, Importance of a novel GABA A receptor subunit for benzodiazepine pharmacology, Nature 338, 582. Shefner, S.A., 1990, Electrophysiological effects of ethanol on brain neurons, in: Focus on Biochemical and Physiology of Substance Abuse, Vol. 2, ed. R.R. Watson (CRC Press, Boca Raton, FL) p. 25. Siggins, G.R., Q.J. Pittman and E.D. French, 1987, Effects of
ethanol on CA1 and CA3 pyramidal cells in the hippocampal slice preparation: an intracellular study, Brain Res. 414, 22. Suzdak, P.D., J.R. Glawa, J.N. Crawley, R.D. Schwartz, P. Skolnick and S.M. Paul, 1986, Ethanol stimulates yaminobutyric acid receptor-mediated chloride transport in rat brain synaptoneurosomes. Proc. Natl. Acad. Sci. 83, 4071. Ticku, M.K., 1989, Ethanol and the benzodiazepine-GABA receptor-ionophore complex, Experientia 45, 413. White, G., D.L. Lovinger and F.F. Weight, 1990, Ethanol inhibits NMDA-activated current but does not alter GABA-activated current in an isolated adult mammalian neuron, Brain Res. 507, 332.
127
Elsevier EJP 20711 Short communication
Ethanol potentiates the GABAA-activated CI- current in mouse hippocampal and cortical neurons Luis G. Aguayo Section of Electrophysiology, Laboratory of Physiologic and Pharmacologic Studies, National Institute on Alcohol Abuse and Alcoholism, 12501 Washington Avenue, Rockville, MD 20852, U.S.A.
Received 3 August 1990, accepted 7 August 1990
The effects of ethanol (1-80 mM) on GABA-activated C1- c u r r e n t (IGABA) were studied in cultured mammalian hippocampal and cortical neurons. Patch-clamp recordings revealed that ethanol potentiated the C1- current in a concentration-dependent manner (1-40 mM) in the majority of the cells studied. No higher degree of potentiation was found by increasing the concentration of ethanol to 80 mM. This study demonstrates that ethanol can potentiate IGABA in mammalian central neurons. GABA A receptors; C1- currents; Hippocampus; Cortex; Ethanol; (Neuron)
1. Introduction -/-Aminobutyric acid (GABA) is a main inhibitory transmitter in the mammalian brain, and barbiturates and benzodiazepines are thought to exert their primary effects in the brain by potentiating a C1- current linked to G A B A A receptor activation (Bormann, 1988). Although ethanol is the oldest and most frequently abused drug, the cellular mechanisms by which it affects brain function have remained largely unknown. Recent studies have shown that ethanol inhibits N-methyl-D-aspartate action in central and peripheral neurons (Lovinger et al., 1989; White et al., 1990). It has also been suggested that some effects of ethanol may be related to its capacity to alter the responses to activation of central G A B A A receptors (Suzdak et al., 1986; for review see Ticku, 1989; Shefner, 1990).
Correspondence to: L.G. Aguayo, Section of Electrophysiology, L.P.P.S., NIAAA, 12501 Washington Avenue, Rockville, MD 20852, U.S.A.
Ethanol, however, does not affect the GABAA-induced C1- current (IGABA) in peripheral neurons in intoxicating concentrations (White et al., 1990). This study shows that intoxicating concentrations of ethanol, which shares anxiolytic and s e d a t i v e / hypnotic activity with barbiturates and benzodiazepines, can potentiate IGABA in mammalian central neurons. 2. Materials and methods Mouse (E17-E18, C57B16J) hippocampal and cortical neurons (1.5-3.0 x 10 5 cells/dish) were grown in culture in a medium containing minim u m essential media (MEM, Gibco, G r a n d Island, NY, U.S.A.), 5% horse serum (Gibco) and a mixture of neurotrophic supplements on a background cell layer. Recordings were done with conventional whole-cell patch-clamp techniques. The external solution contained (in m M ) 150 NaC1, 5.4 KCI, 2 CaCI2, 1 MgC12, 10 glucose and 10 H E P E S ( p H 7.4). Tetrodotoxin (TTX) was added at a concentration of 200 n M to block sodium dependent spikes. The patch pipette contained (in mM)
0014-2999/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
128
140 CsC1, 4 MgC12, 10 HEPES (pH 7.37); ATPN a 2 (2 mM) and BAPTA (1,2-bis(2-aminophenoxy) hethane-N,N,N',N"-tetra-acetic acid; 10 mM) were added to the intracellular solution to reduce the 'run-down' of IGABA observed in their absence and to tightly control intracellular C a 2+. Under these conditions, about 80% of the neurons had stable IGABA for periods in excess of 20 min. In most experiments, a minimally desensitizing IGABA w a s achieved with a concentration of 2.5 # M GABA. This allowed for better temporal resolution of the potentiation of IGABA produced by ethanol. Serial application of increasing drug concentrations was accomplished using a 200 /xm diameter pipette connected to a 10 ml reservoir and moved relative to the neuron. Currents were displayed on a chart recorder (Gould 2400) and stored in a VCR tape recorder (Vetter 240B) after filtering the signal at 3.15 kHz with a four-pole low-pass Bessel filter (Ithaco 4302).
3. Results Whole-cell patch-clamp recordings in all central neurons (n > 100) tested with G A B A (1-200 p M )
A
B
GABA ( P M )
5
GABA (2.5 p M)
50
revealed an inward current. Figure 1A shows the current activated by superfusion of 5 and 50/~M G A B A to a single hippocampal neuron. With 50 /LM G A B A the current desensitized by about 60% during the application of the neurotransmitter. Figure 1B illustrates the effect of the G A B A receptor antagonist bicuculline (20 /~M). The current records show that when bicuculline was applied in combination with G A B A (2.5 /~M) it reversibly and completely inhibited the current. Ethanol clearly potentiated IGABA in 70% of the cells tested with ethanol (n = 70). Figure 1C shows the effects of ethanol (1-80 mM) o n IGABA in a hippocampal neuron. As illustrated, the application of 1 m M ethanol potentiated the peak amplitude of IGABA by a factor of 1.3 above control. The potentiation of IGABAreached a factor of 1.65 above control when the cell was perfused with 10 m M ethanol. N o larger extent of potentiation was found with 40 mM, and when the concentration of ethanol was increased to 80 m M the degree of potentiation with 80 m M was close to that obtained with 1 m M ethanol. The potentiating effect of ethanol o n IGABAwas reversible. Interestingly, the study of ethanol at concentrations above 50 m M on C1- flux in rat brain synaptoneurosomes
GABA (2.5 ~. M) + BICUCULLINE (20 I~ M)
GABA (2.5 ~ M)
....
20 s
C
CONTROL
¢~
1 mM ETHANOL
20 s
10 mM ETHANOL
40 mM ETHANOL
80 mM ETHANOL
o
CONTROL
Fig. 1. (A) GABA-activated inward current (IGABA)in cultured hippocampal neurons. Inward current activated by application of 5 and 50 #M GABA. (B) IGABAwas reversibly blocked by bicuculline (20 #M). (C) Ethanol produced a concentration-dependent potentiation of IGABA.The concentration of GABA was 2.5/tM in all records in (C). Holding potential was - 10 mV in (A) and - 50 in (B) and (C). The holding potential of - 1 0 mV was necessary to avoid saturation of the current amplifier with high GABA concentrations. All records are from hippocampal neurons.
129 A CONTROL
ETH A N OL
C ON TR OL
B
20 s CONTROL
FLURAZEPAM
PENTOBARBITAL
ETHANOL
20 s
CONTROL
o
Fig. 2. (A) Ethanol induced a reversible potentiation of IGABAin cerebral cortical neurons. The traces show the response to 2.5 gM GABA (control traces) and GABA plus 20 mM ethanol (middle trace). (B) IGABAin neurons that were insensitive to ethanol was still potentiated by pentobarbital (20 vM) and flurazepam (20 gM). The concentration of GABA was 2.5/tM. The data in (B) are from a hippocampal neuron. Holding potential - 50 mV in all records.
showed a similar biphasic response (Suzdak et al., 1986) and this m a y suggest an antagonistic action of ethanol on the G A B A A receptor-C1- channel complex. Ethanol (40 mM) did not induce any C1- current in the absence of G A B A (not shown). It was previously reported that local and systemic administration of ethanol alters GABAmediated inhibition of cortical neurons (Nestoros, 1980), but these experiments did not provide any indication of the mechanism by which ethanol affected these neurons. A possible mechanism to explain this neuronal inhibition is that ethanol potentiates the GABA-activated C1- current in cortical neurons. Indeed, ethanol (20 mM) was also effective in potentiating IGABA in cortical neurons (fig. 2A). In the presence of ethanol, the peak amplitude of IGABA w a s potentiated by more than 100% above control and the potentiation was fully reversible (fig. 2A, right trace). Previous electrophysiological studies on the effects of ethanol on G A B A responses are conflicting and have suggested that ethanol does not affect all central neurons equally (Mereu and Gessa, 1985; Siggins et al., 1987; Shefner, 1990). In the present experiments, IGABA elicited in a group of hippocampal neurons was not affected by ethanol. Figure 2B shows the lack of effect of 40 m M ethanol o n IGABA activated in one of these neurons. The same neurons, however, had IGABA that was potentiated by pentobarbital and flurazepam (fig. 2B).
4. Discussion This study provides direct evidence of the cellular mechanism by which ethanol affects G A B A A responses in mammalian central neurons. The results also suggest that the modulation of IGABAby benzodiazepine, barbiturates and ethanol m a y occur through distinct mechanisms. The finding that ethanol potentiates IGABA in some neurons but not others suggests that ethanol exerts its cellular actions in a more selective manner than previously believed. The nature for this selectivity is presently unknown. It is possible, however, that similar to benzodiazepines, which are more effective in p o t e n t i a t i n g IGABA in the presence of the 72 subunit (Pritchett et al., 1989), ethanol m a y also require a particular configuration or state of the receptor to exert its action.
Acknowledgements I wish to thank my colleagues Drs. F.F. Weight, G. Kunos and G. White for valuable discussion during these experiments.
References Bormann, J., 1988, Electrophysiology of GABA A and GABA B receptor subtypes, Trends Neurosci. 11, 112. Lovinger, D.M., G. White and F.F. Weight, 1989, Ethanol inhibits NMDA-activated ion current in hippocampal neurons, Science 243, 1721.
130 Mereu, G. and G.L. Gessa, 1985, Low doses of ethanol inhibit the firing of neurons in the substantia nigra, pars reticulata: a gabaergic effect?, Brain Res. 360, 325. Nestoros, N.J., 1980, Ethanol specifically potentiates GABAmediated neurotransmission in feline cerebral cortex. Science 209, 708. Pritchett, D.B., H. Sontheimer, B.D. Shivers, S. Ymer, H. Kettenmann, P.R. Schofield and P.H. Seeburg, 1989, Importance of a novel GABA A receptor subunit for benzodiazepine pharmacology, Nature 338, 582. Shefner, S.A., 1990, Electrophysiological effects of ethanol on brain neurons, in: Focus on Biochemical and Physiology of Substance Abuse, Vol. 2, ed. R.R. Watson (CRC Press, Boca Raton, FL) p. 25. Siggins, G.R., Q.J. Pittman and E.D. French, 1987, Effects of
ethanol on CA1 and CA3 pyramidal cells in the hippocampal slice preparation: an intracellular study, Brain Res. 414, 22. Suzdak, P.D., J.R. Glawa, J.N. Crawley, R.D. Schwartz, P. Skolnick and S.M. Paul, 1986, Ethanol stimulates yaminobutyric acid receptor-mediated chloride transport in rat brain synaptoneurosomes. Proc. Natl. Acad. Sci. 83, 4071. Ticku, M.K., 1989, Ethanol and the benzodiazepine-GABA receptor-ionophore complex, Experientia 45, 413. White, G., D.L. Lovinger and F.F. Weight, 1990, Ethanol inhibits NMDA-activated current but does not alter GABA-activated current in an isolated adult mammalian neuron, Brain Res. 507, 332.
