Abstract
The nucleus accumbens (NAcc) may be a key area in the rewarding effects of abused drugs. We previously showed that low ethanol concentrations decreased both N-methyl-d-aspartate (NMDA)-induced and kainate-induced currents in NAcc core neurons (Nie et al., 1994). To explore the effects of ethanol on γ-aminobutyric acid (GABA) responses in NAcc, we used intracellular voltage-clamp recordings and locally applied GABA in a slice preparation containing the NAcc. Ethanol (11–200 mM) had no effect on resting membrane properties, but 11, 22, 44, 100, and 200 mM ethanol increased GABA currents in 17, 33, 45, 50, and 22% of cells, respectively. Superfusion of low glutamate concentrations that had no direct effect on membrane properties enhanced ethanol potentiation of GABA currents in more than half the NAcc cells. Neither α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid/kainate receptor nor NMDA receptor antagonists affected the percentage of cells showing ethanol enhancement of GABA responses or the degree of ethanol enhancement of GABA currents in NAcc neurons. However, in ethanol-sensitive cells, the metabotropic receptor antagonist α-methyl-4-carboxyphenylglycine (MCPG) blocked the ethanol enhancement of GABA currents. In addition, the metabotropic receptor agonist trans-1-aminocyclopentane-1,3-dicarboxylic acid enhanced GABA responses in 50% of cells tested, an effect blocked by MCPG. These data suggest that NAcc core neurons possess both ethanol-sensitive and -insensitive GABA receptors and that glutamate can mimic and enhance the ethanol potentiation of GABA currents in many of these neurons. Furthermore, the ethanol potentiation of GABA currents may involve metabotropic glutamate receptors, perhaps via a phosphorylation mechanism that regulates ethanol sensitivity of GABA receptors in some NAcc neurons.
The neuronal mechanisms underlying the central actions of acute ethanol exposure are not understood completely, although numerous ethanol effects have been investigated (Bloom and Siggins, 1987; Siggins et al., 1987a; Harris and Allan, 1989; Siggins et al., 1990; Harris et al., 1995b). In the central nervous system (CNS), much attention has focused on the effects of ethanol on synaptic transmission (Berry and Pentreath, 1980; Siggins et al., 1987a; Shefner, 1990; Nie et al., 1993, 1994); many studies suggest that the synapse is the neuronal site most sensitive to ethanol. It is generally agreed that glutamate and γ-aminobutyric acid (GABA) are the principal neurotransmitters mediating, respectively, excitatory and inhibitory synaptic transmission in the brain. Ethanol has been found to reduce the activity of the glutamatergic system (Lovinger et al., 1989,1990; Lovinger, 1993; Nie et al., 1993, Nie et al., 1993, 1994; Martin et al., 1995) and, in some CNS regions, to enhance the activity of GABAergic systems (Celentano et al., 1988; Deitrich et al., 1989;Aguayo and Pancetti, 1994; Mehta and Ticku, 1994); these effects probably contribute to ethanol-elicited neuronal depression in the CNS.
GABA acts on at least two classes of receptors: GABAA and GABAB receptors. Molecular studies have revealed a complex heterogeneity in the structure and pharmacology of GABAA receptors, because at least five different subunit families (α, β, γ, δ, and ρ) have been isolated (Schofield et al., 1987; Pritchett et al., 1989; Shivers et al., 1989). The differences in subunit composition have important functional implications for the pharmacology of GABAA receptors and the action of ethanol because ethanol enhancement of GABAA receptor activation has been controversial (Mancillas et al., 1986; Siggins et al., 1987b;Celentano et al., 1988; Deitrich et al., 1989; White et al., 1990;Proctor et al., 1992; Aguayo and Pancetti, 1994). Often, positive findings depend on satisfaction of certain conditions, such as the activation of protein kinase C (PKC; Weiner et al., 1994; Harris et al., 1995a; Macdonald, 1995) or the study of different brain areas, neuron types or regions (Proctor et al., 1992; Soldo et al., 1994), species, or GABAA subunit compositions (Wafford and Whiting, 1992; Harris et al., 1995c). Recently, our laboratory reported that ethanol enhancement of GABAAergic inhibitory postsynaptic potentials (IPSPs) in hippocampal pyramidal neurons only occurred after blockade of GABABreceptors (Wan et al., 1996), suggesting a complex interaction between ethanol and the two GABA receptor subtypes.
The neuronal effects of glutamate are brought about by two receptor classes: ionotropic receptors, which are ligand-gated channels passing cationic currents, and metabotropic receptors, which are coupled to transduction systems via G proteins (Watkins et al., 1991; Pin and Duvoisin, 1995). Interestingly, low glutamate concentrations can enhance GABA responses in hippocampus (Stelzer and Wong, 1989), and GABAA receptors can mediate excitatory as well as inhibitory synaptic events in central neurons, depending on the presence of endogenous glutamate at appropriate levels (Michelson and Wong, 1991). These data suggest that important interactions exist between glutamate and GABA receptors in the CNS.
Behavioral studies indicate that the nucleus accumbens (NAcc) is a brain region involved in the rewarding effects of ethanol (Koob and Bloom, 1988). Both glutamate and GABA are major neurotransmitters in this area. Glutamatergic afferents from cortex, subiculum, and amygdala terminate on the most abundant cell type in the NAcc: medium spiny neurons. These neurons are most likely GABAergic (Bolam et al., 1983) and probably project to each other as well as to extrinsic sites. Recently, we reported that low ethanol concentrations significantly reduced glutamatergic synaptic transmission in rat NAcc neurons (Nie et al., 1993, 1994). To further clarify the effects of ethanol on amino acid transmitter systems in the NAcc, we now have examined GABA responses and their possible interactions with ethanol and glutamate in the core NAcc using intracellular recording in a slice preparation. We found that ethanol enhanced responses to exogenous GABA in about half the NAcc neurons studied, suggesting heterogeneity with respect to their GABA receptors. In the ethanol-sensitive neurons, glutamate and a metabotropic receptor agonist also enhanced the ethanol-induced potentiation of GABA responses. These and other results suggest that ethanol and glutamate interactions with GABA receptors may involve metabotropic receptors, perhaps operating through a second messenger linkage.
Materials and Methods
Slice Preparation.
We prepared NAcc slices from male Sprague-Dawley rats (100–170 g) as previously described (Yuan et al., 1992). Briefly, we cut coronal slices 350- to 400-μm thick on a vibrating cutter (Vibroslice; Campden Instruments, Silbey, UK) and placed them in ice-cold (3–5°C) artificial cerebrospinal fluid (ACSF) gassed with carbogen (95% O2, 5% CO2) and of the following composition: NaCl, 130 mM; KCl, 3.5 mM; NaH2PO4, 1.25 mM; MgSO4·7H2O, 1.5 mM; CaCl2·2H2O, 2 mM; NaHCO3, 24 mM; and glucose, 10 mM. We immediately transferred the slices to a recording chamber where they were incubated in an interface configuration for 30 min with their upper surfaces exposed to warmed, humidified carbogen. The slices were then completely submerged and continuously superfused with ACSF at a constant rate (2–4 ml/min) for the remainder of the experiment. The inner chamber had a total volume of ∼0.5 ml; at the superfusion rates used, 90% replacement of the chamber solution could be obtained within 1 to 2 min (Siggins et al., 1987b). During testing, we maintained the bath temperature constant at 31–35°C.
Electrophysiology.
We filled intracellular glass micropipettes with 3 M KCl (tip resistance, 60–110 MΩ), performed single-electrode voltage-clamp studies with an Axoclamp 2A preamplifier (Axon Instruments Inc., Foster City, CA), and, in discontinuous voltage-clamp recording, continuously monitored the electrode settling time and capacitance neutralization on a separate oscilloscope. We took neuronal recordings from the NAcc core region at levels 2.2 to 0.7 mm from bregma and surrounding, but ventromedial to, the anterior commissure, using a “blind” approach. We accepted all successfully impaled [as judged by long-term stability of resting membrane potentials (RMPs) > −60 mV] neurons into the study sample. We applied GABA (1 or 5 mM in the pipette) locally near the recorded neuron by pressure (pipette tip diameter, 2.5–4 μm; pressure, 3–10 psi; duration, 2–4 s). GABA currents were always elicited in the presence of tetrodotoxin (TTX; 1 μM) to minimize presynaptic effects. In some cells, we also superfused 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) ordl-2-amino-5-phosphonovalereate (d-APV) to block non-N-methyl-d-aspartate (NMDA) or NMDA receptors. In most neurons, the GABA currents were highly reproducible for up to 90 min using GABA pressure application at intervals of 2 to 3 min (Fig. 1A). The cells were held near RMPs (approximately −83 mV); thus, with Cl−-containing recording pipettes, GABA currents were inward (depolarizing) in direction. After stable responses were achieved, we took various current and voltage measurements at several time points before, during, and after ethanol superfusion. Continuous d.c. recordings were stored on polygraph paper, and selected records were digitized, stored, and analyzed on an 80486 computer using the pClamp programs (Axon Instruments).
Drug Treatment.
We introduced ethanol and other drugs in known concentrations into the slice chamber by a multiple valve system, without disrupting the flow of the superfusate. To avoid loss of ethanol by evaporation, the solutions were diluted in gassed ACSF from sealed stock solutions of reagent grade 95% ethyl alcohol in water immediately before administration. The usual testing protocol was: recording of membrane currents with periodic local application of GABA for 10 to 15 min during superfusion of ACSF alone (“control”); followed by switching to ACSF with drug [e.g., ethanol, glutamate, CNQX, d-APV, α-methyl-4-carboxyphenylglycine (MCPG)] while repeating GABA application for 5 to 20 min; then switching again to ACSF alone for 15 to 35 min with subsequent GABA current measures (“washout”). We defined ethanol potentiation of GABA responses as at least a 10% increase in peak GABA current. We obtained TTX from Calbiochem (La Jolla, CA), CNQX and MCPG from Tocris Cookson (Bristol, UK), d-APV from Research Biochemicals (Wayland, MA), ethanol from the Remet Corp. (La Mirada, CA), and all other drugs from Sigma Chemical Co. (St. Louis, MO).
Data Analysis.
Data are expressed as mean ± S.E. For statistical analysis, we used ANOVA for repeated measures, followed by the Newman-Keuls post hoc test. We accepted P < .05 as statistically significant.
Results
Neuronal Sample.
We studied a total of 84 neurons within the NAcc core area (see Fig. 1 of Yuan et al., 1992) at depths within the slice of 40 to 380 μm. As reported previously, these neurons had large RMPs averaging −83 ± 1.1 mV (mean ± S.E.; range: −66 to −93 mV; n = 67), and current-evoked spikes averaging 115 mV (range: 100 to 125 mV; n = 67). In most cells, stable recordings could be maintained for up to 2 to 3 h.
Ethanol Effects on Membrane Properties.
In general, superfusion of intoxicating concentrations of ethanol (11, 22, 44, 100, or 200 mM) had little reversible or reproducible effect on membrane potential or input slope resistance of NAcc neurons (Nie et al., 1993). Of 67 neurons, only two showed a small (2–3 mV) hyperpolarizing response, and three showed small (2–3 mV) depolarizing responses. In the remaining 62 cells, ethanol had no measurable effect on membrane potential. Therefore, analysis of ethanol effects on GABA responses could proceed without the confounding effects of direct potential changes.
Ethanol Effects on GABA Currents.
GABA currents were evoked in the presence of 1 μM TTX to minimize presynaptic effects. In our conditions, exogenous GABA evoked reproducible inward currents (Fig.1A) in NAcc core neurons that were nearly totally blocked by 30 μM bicuculline (Fig. 1B), indicating mediation by GABAA receptors. The small residual currents that are sometimes seen are likely due to incomplete blockade of GABAA receptors at these low bicuculline concentrations, rather than GABAB receptor effects (see Fig. 1B legend). With superfusion of ethanol (11–200 mM), GABA currents were increased as follows: in 17% of tested cells with 11 mM ethanol, in 33% of cells with 22 mM ethanol, in 45% with 44 mM ethanol, in 50% of cells with 100 mM ethanol, and in 22% of cells with 200 mM ethanol (Table 1). In the remaining neurons, ethanol had no effect (Fig.2) or slightly decreased GABA currents (<10%). In most cells, ethanol potentiation of GABA currents occurred within 3 to 14 min (usually 3–7 min, counting the 1–2 min “dead-time” of the superfusion tubing) and recovered to control levels on washout for 5 to 15 min, or even disappeared despite continuing superfusion of ethanol. Thus, in four cells, the potentiation occurred within 3 to 5 min, then disappeared, suggesting the development of rapid tolerance to ethanol in these NAcc neurons (Wan et al., 1996).
We found two apparent types of NAcc core neurons, in terms of their RMPs and response to locally applied GABA. The neuron type represented in Fig. 2A shows several characteristics, including high RMPs (−87 ± 0.5 mV), fast onset of GABA currents, short GABA current duration, and larger current amplitude (132 ± 5.9 pA). This type of neuron (type 1) constitutes ∼79% of the NAcc core area; 67% of these neurons were sensitive to ethanol. The neuron type represented in Fig. 2B had lower RMPs (−73 ± 1.1 mV), slower GABA current onset, longer current duration, and smaller amplitude (48 ± 7.1 pA) than seen in neurons from the group depicted in Fig. 2A. This neuron type (type 2) comprised ∼21% of the NAcc core area; 83% of these neurons were insensitive to ethanol. Because we accepted all successfully impaled neurons into the sample, it is possible (although not easily testable without visual identification) that these type 2 neurons are interneurons, rather than the medium spiny (principal) neurons usually encountered with the “blind” recording approach used in this study.
To test whether the GABA currents evoked in these two neuron types were mediated by GABAA receptors, we superfused the GABAA antagonist bicuculline. In five of five cells (four of the first type of NAcc core neurons, one of the second type), 30 μM bicuculline almost totally blocked the GABA currents (as in Fig. 1B), suggesting that these currents are mediated primarily by GABAA receptors.
As the concentration-response curves of Fig.3 show, 11 to 200 mM ethanol enhanced the GABA currents in a bell-shaped fashion; the effects were significant only at 44 and 100 mM ethanol. We analyzed the whole neuronal population separately from the cells showing ethanol potentiation of GABA currents. Across the whole neuronal population, neither ethanol 11 mM [F(2,10) = 2.434, P = .14], 22 mM [F(2, 10) = 2.585, P = .13], nor 200 mM [F(2,16) = 2.320, P= .13] significantly changed GABA currents, but 44 and 100 mM ethanol significantly enhanced GABA currents to 120 ± 3.91% [F(2,20) = 7.365, P = .004] and 121 ± 7.29% [F(2, 22) = 8.849,P = .0015] of control, respectively. Analyzed across only those cells showing ethanol enhancement of GABA currents, 44 and 100 mM ethanol significantly enhanced GABA currents to 143 ± 3.7% [F(2,8) = 13.496, P = .0027] and 142 ± 6.3% [F(2,10) = 18.985,P = .0004] of control, respectively. In this subpopulation, ethanol 22 mM [F(2,2) = 7.603,P = .12] and 200 mM [F(2,2) = 1.660,P = .38] did not significantly change GABA currents. The highest ethanol concentration, 200 mM, actually enhanced GABA currents to a lesser extent than did 44 and 100 mM ethanol, with ethanol 44 mM near the maximal dose for enhancing GABA currents in these neurons.
Glutamate Enhances GABA Currents and Their Potentiation by Ethanol.
To determine possible mechanisms underlying the variability of interactions between GABA and ethanol, we asked whether GABA responses could be altered by glutamate in NAcc neurons, as reported previously for hippocampal neurons (Stelzer and Wong, 1989). In the presence of 1 μM TTX, 20 μM glutamate enhanced (>10%) GABA responses in 6 of 11 cells studied to a mean of 119 ± 3.3% of control (Fig. 4A). In the remaining cells, glutamate had no measurable effect on GABA responses. At this low concentration, glutamate had no effect on RMPs in any tested neuron, suggesting that enhancement of GABA responses by glutamate occurs at concentrations below those required for its known excitatory (ionotropic) action.
In another subset of NAcc neurons, we also asked whether glutamate receptors could be involved in the ethanol enhancement of GABA responses. Glutamate superfusion (20 μM) enhanced ethanol potentiation of GABA currents (Fig. 4) in five of nine cells. In the remaining four cells, ethanol (44 mM) alone did not change the GABA currents. In the five cells showing a glutamate effect, ethanol alone first enhanced GABA currents (to 132% of control in the neuron of Fig.4A), whereupon superfusion of glutamate (20 μM) combined with ethanol further enhanced the GABA currents (to 159% of control in Fig. 4A). When averaged over all nine cells of this subset, ethanol alone increased GABA currents by a mean of 17 ± 5.2%, whereas glutamate combined with ethanol significantly increased GABA currents by a mean of 36 ± 9.2% [F(1,8) = 11.278,P = .01].
To investigate what subtype of glutamate receptor might mediate the enhancement of GABA responses or their interaction with ethanol, we used subtype-specific antagonists to block NMDA and non-NMDA (AMPA/kainate) glutamate receptors. In four of seven cells previously shown to express ethanol-GABA interactions, 44 mM ethanol still enhanced GABA responses by more than 10% after superfusion of the NMDA receptor antagonist d-APV (30 μM) for more than 30 min (Fig. 5A). In the remaining three ethanol-insensitive neurons, d-APV had no effect on GABA receptor insensitivity to ethanol. Similarly, after 30 min of superfusion of the non-NMDA glutamate receptor antagonist CNQX (10 μM), ethanol still enhanced the GABA responses (Fig. 5B) in three of six cells (by more than 10%) but had no effect in the remaining three ethanol-insensitive cells. These data suggest that neither NMDA nor AMPA/kainate receptors mediate the ethanol potentiation of GABA responses in NAcc neurons.
The Role of Metabotropic Receptors.
Because of the lack of effect of d-APV and CNQX, we examined the possible involvement of metabotropic glutamate receptors in the effects of ethanol and glutamate on GABA currents. In eight NAcc cells, superfusion of the metabotropic glutamate receptor agonisttrans-1-aminocyclopentane-1,3-dicarboxylic acid (trans-ACPD; 5 μM) had no effect on RMP, but enhanced GABA currents in four cells (Fig. 6). In testing two of these trans-ACPD-sensitive cells, subsequent superfusion of the metabotropic receptor antagonist MCPG (1 mM) blocked the trans-ACPD enhancement of GABA responses in both cells (Fig. 6). Furthermore, in all five ethanol-sensitive NAcc cells studied, superfusion of MCPG (1 mM) significantly blocked the ethanol enhancement of GABA currents (Fig. 7), suggesting that ethanol enhancement of GABA effects in NAcc could be regulated or mediated by postsynaptic metabotropic glutamate receptors.
Discussion
In this study, we found that ethanol enhanced GABA responses in 17 to 50% of NAcc core neurons, but had no effect or slightly decreased (<10%) GABA responses in the remaining neurons (depending on concentration). These and other data suggest the existence of a subset of NAcc core neurons with ethanol-sensitive GABAAreceptors. In these ethanol-sensitive neurons, ethanol potentiation of GABA responses also could be enhanced further by glutamate. In the ethanol-insensitive neurons, glutamate plus ethanol had no effect on GABA responses. The NMDA and non-NMDA ionotropic glutamate receptor antagonists had no effect on either the percentage of cells showing ethanol enhancement of GABA responses or the degree of ethanol enhancement of GABA currents in these neurons. The metabotropic glutamate receptor agonist trans-ACPD mimicked glutamate and ethanol by enhancing GABA responses in half the NAcc neurons tested. Interestingly, the metabotropic glutamate receptor antagonist MCPG blocked the ethanol enhancement of GABA responses.
As noted in the Introduction, there have been many electrophysiological studies of ethanol effects on GABAergic systems. Although ethanol has been shown to enhance GABAA receptor-activated events in neurons or isolated preparations of several CNS regions (see the Introduction), such an ethanol-GABAergic interaction (e.g., of GABAA-mediated IPSPs or responses to exogenous GABA) in native neurons has not been universally observed (Siggins et al., 1987b; Osmanovic and Shefner, 1990; White et al., 1990; Frye et al., 1994). For example, in the hippocampus, there is still some disagreement about whether ethanol can affect the GABAA receptor/chloride channel complex (see, e.g., Aguayo and Pancetti, 1994, using cultured mouse hippocampal neurons versus the in vivo and in vitro studies of rat hippocampus byHarrison et al., 1987; Siggins et al., 1987b; Morrisett et al., 1991;Proctor et al., 1992; Peoples and Weight, 1994). Our laboratory initially saw no specific effect of systemic ethanol on responses to iontophoretic GABA in rat hippocampal neurons in vivo (Mancillas et al., 1986) and little effect or an inhibitory action on evoked IPSPs or GABA-induced hyperpolarizations in rat hippocampal slices (Siggins et al., 1987b). However, our more recent hippocampal studies using pharmacological isolation of GABAA-IPSPs suggest that ethanol can reproducibly enhance those IPSPs, but only when GABAB receptors are blocked (Wan et al., 1996) and probably via a presynaptic mechanism (Siggins et al., 1999).
Our data showing ethanol enhancing GABA responses in some NAcc neurons are generally consistent with those of others studying different native neuronal preparations (Deitrich et al., 1989; Harris and Allan, 1989;Aguayo and Pancetti, 1994) and specific GABAAreceptor subunit compositions expressed in Xenopus oocytes (Wafford et al., 1991; Whitten et al., 1996). It is possible that the existence of ethanol-sensitive and -insensitive NAcc neurons reflect different GABAA receptor subunit compositions, as with studies showing that the potentiating action of ethanol required the γ2L subunit (Wafford et al., 1991; Wafford and Whiting, 1992). One possible explanation for the lack of ethanol-GABA interaction in some NAcc neurons, that rapid tolerance in the ethanol-insensitive cells masks a fleeting increase in GABA currents (see below), could also derive from differences in GABAA receptor subunit compositions.
Another possible mechanism for the inconsistent actions of ethanol on GABAA receptors is that ethanol or some regulator (e.g., a metabotropic link) alters the activity of a calcium- or phospholipid-dependent protein kinase (e.g., PKC), which in turn alters the sensitivity of the GABA receptor via phosphorylation. Several reports suggest that ethanol enhancement of GABA responses requires phosphorylation of a GABAA receptor subunit by PKC (Harris et al., 1995a; Macdonald, 1995). It may be that only half the NAcc core neurons we tested (the ethanol-sensitive neurons) contained the PKC phosphorylation sites or the related metabotropic machinery necessary to render GABAA receptors responsive to ethanol. Our current studies are using protein kinase inhibitors to test this hypothesis.
Based on RMP, magnitude of responses to locally applied GABA, and GABA current kinetics, we found evidence for two different types of neurons in the NAcc core. The GABAA receptor antagonist bicuculline blocked GABA currents in both types, suggesting mediation by GABAA receptors in both. Although we cannot eliminate other factors, based on the amplitude and duration of GABA currents, the amount of GABA and its concentration gradient reaching the neuron may be important factors in these differences. The differences might also indicate that the GABA currents in “ethanol-insensitive” neurons have an early, ethanol-sensitive, but rapidly desensitizing component that is unresolved under our conditions of GABA application by pipette in a slice preparation.
We also found that ethanol enhancement of GABA currents in some cells lasted only 4 to 6 min, despite continued ethanol superfusion, suggesting rapid tolerance for this effect in some NAcc neurons. This finding agrees with several reports (Durand et al., 1981; Gilliam, 1989; Khanna et al., 1990; Ghosh et al., 1991; Palmer et al., 1992) and also with data from our laboratory showing rapid tolerance to the ethanol enhancement of IPSPs in some hippocampal neurons (Wan et al., 1996). This tolerance could explain why ethanol effects were found in some but not all cells, and it contrasts with the ethanol reduction of NMDA-induced currents that shows no apparent acute tolerance in NAcc neurons (Nie et al., 1994).
The inverted U-shaped dose-response curve for ethanol potentiation of GABA responses in NAcc may seem unusual for ethanol effects. This ethanol-GABA receptor interaction is different from the more standard dose-response relationship seen for ethanol inhibition of NMDA currents in the same NAcc slice preparation, which shows no inverted-U shape but rather a standard asymptotic depression at all higher doses (Nie et al., 1994). This may be related to the relative lack of short-term tolerance seen in the ethanol-NMDA interactions (Nie et al., 1994), versus a more obvious short-term tolerance to ethanol-GABA receptor interactions in some NAcc neurons. Thus, the tolerance mechanism in NAcc neurons for GABA receptor-ethanol interactions may be different from that of NMDAR-ethanol interactions, possibly because of the involvement of metabotropic system(s) in the former. This might also suggest that the ethanol inhibition of some NAcc neurons via GABAergic mechanisms might be more fleeting than for the ethanol reduction of NMDA receptor-mediated glutamatergic transmission. The need for a metabotropic step may also explain the sometimes slow development of the ethanol effects seen in our in vitro slice paradigm and in related slice studies (Proctor et al., 1992; Soldo et al., 1994).
Electrophysiological data from Stelzer and Wong (1989) showed that glutamate can enhance GABA responses in central neurons. Recently, this laboratory also reported a suppression of GABAAreceptor responses by NMDA application (Chen and Wong, 1995), suggesting that NMDA receptors do not mediate glutamate's enhancement of GABA responses NAcc. Our results agree with these findings: neitherd-APV nor CNQX blocked glutamate enhancement of GABA responses in NAcc core neurons. In the ethanol-sensitive NAcc neurons, glutamate plus ethanol further potentiated GABA responses, and this type of NAcc neuron was more sensitive not only to ethanol but also to glutamate. The reason for this correlation is not known, but it is possible that the factor(s) responsible for ethanol sensitivity, such as a specific GABAA receptor subunit composition, also confer glutamate sensitivity.
The metabotropic glutamate receptor agonist trans-ACPD enhanced GABA responses in half the NAcc neurons tested, suggesting mediation of the glutamate effect by metabotropic glutamate receptors. We also found that the mGluR antagonist MCPG blocked the ethanol enhancement of GABA responses, further supporting the regulation of ethanol-GABAA receptor interactions by metabotropic receptors, perhaps through endogenously released glutamate. It is possible that these interactions involve protein kinase A or C, an idea consistent with cerebellar data suggesting that adenylyl cyclase or PKA activation is necessary for ethanol potentiation of GABA responses (Freund and Palmer, 1996) and that ethanol potentiation of IPSPs in hippocampal slices may require GABAA receptor subunit phosphorylation by PKC (Weiner et al., 1994). Further studies are needed to determine whether these metabotropic and kinase mechanisms also generalize to GABAA receptors of other central neurons (Siggins et al., 1999).
As to the functional and behavioral significance of our findings, the reported role of the NAcc in alcohol and drug reinforcement and dependence may suggest that ethanol potentiation of inhibitory GABAergic function in some NAcc neurons could underlie some aspect of these phenomena. In addition, ethanol (Nie et al., 1993, 1994) and other reinforcing drugs (Yuan et al., 1992) inhibit excitatory glutamatergic synaptic transmission in accumbens neurons. These combined data suggest that animals may work to inhibit their accumbens neurons, and ethanol may facilitate such inhibition. Furthermore, data in this study suggest that metabotropic mechanisms may be involved in these ethanol actions. Because most NAcc neurons are themselves GABAergic and thus inhibitory, disinhibition of “downstream” neurons (for example, ventral pallidum, thalamus) could play a role in ethanol reinforcement or dependence.
Acknowledgments
We thank Drs. S. Steffensen and G. Martin for helpful comments and F. Bellinger for technical support.
Footnotes
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Send reprint requests to: Dr. G. R. Siggins, CVN-12, Dept. of Neuropharmacology, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037. E-mail:geobob{at}scripps.edu
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↵1 This study was supported by National Institutes of Health Grants AA06420 and DA03665.
- Abbreviations:
- CNS
- central nervous system
- GABA
- γ-aminobutyric acid
- IPSPs
- inhibitory postsynaptic potentials
- NAcc
- nucleus accumbens
- CNQX
- 6-cyano-7-nitroquinoxaline-2,3-dione
- d-APV
- dl-2-amino-5-phosphonovalerate
- AMPA
- α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid
- MCPG
- α-methyl-4-carboxyphenylglycine
- trans-ACPD
- trans-1-aminocyclopentane-1,3-dicarboxylic acid
- RMP
- resting membrane potential
- PKC
- protein kinase C
- NMDA
- N-methyl-d-aspartate
- ACSF
- artificial cerebrospinal fluid
- TTX
- tetrodotoxin
- Received May 25, 1999.
- Accepted December 27, 1999.
- The American Society for Pharmacology and Experimental Therapeutics