Ethanol (EtOH) has a number of behavioral effects, including intoxication, amnesia, and/or sedation, that are thought to relate to the activation of GABAA receptors. However, GABAA receptors at different cellular locations have different sensitivities to EtOH. The present study used the “synaptic bouton” preparation where we could stimulate nerve endings on mechanically dissociated single rat hippocampal CA1 and CA3 pyramidal neurons and investigate the effects of EtOH on presynaptic and postsynaptic GABAA receptors. Low concentrations of EtOH (10 mM) had no effect on postsynaptic GABAA and glutamate receptors or voltage-dependent Na+ and Ca2+ channels. Higher concentrations (≥100 mM) could significantly inhibit these current responses. EtOH at 10 mM had no direct effect on inhibitory postsynaptic currents (IPSCs) and excitatory postsynaptic currents (EPSCs) evoked by focal stimulation of single boutons [evoked IPSCs (eIPSCs) and evoked EPSCs (eEPSCs)]. However, coapplication of 10 mM EtOH with muscimol decreased the amplitude of eIPSCs and eEPSCs and increased their paired-pulse ratio. The effects on eEPSCs were reversed by bicuculline. Coapplication of muscimol and EtOH significantly increased the frequency of spontaneous IPSCs and EPSCs. The EtOH effects on the postsynaptic responses and eEPSCs were similar in neurons from neonatal and mature rats. These results revealed that low concentrations of EtOH can potentiate the activation of presynaptic GABAA receptors to inhibit evoked GABA and glutamate release. These results indicate a high sensitivity of presynaptic GABAA receptor to EtOH, which needs to be accounted for when considering the cellular mechanisms of EtOH's physiological responses.
Numerous studies have examined the effects of ethanol (EtOH) on the excitatory and inhibitory receptors of the central nervous system by using patch-clamp techniques. In cultured cell or isolated nerve cells, EtOH acts on extrasynaptic GABAA receptors to potentiate GABA-induced Cl− currents (Reynolds and Prasad, 1991; Weight et al., 1992; Aguayo et al., 1994), by increasing their open probability caused by an increase in the open time of single GABAA receptor channels (Tatebayashi et al., 1998). EtOH also acts on excitatory glutamate receptors where it has been shown to inhibit glutamate-induced currents in rat cultured hippocampal and cerebral cortex neurons (Lovinger et al., 1989; Weight at al., 1992; Fischer et al., 2003) and in rat ventral tegmental area neurons (Zhu et al., 2002). These postsynaptic effects are considered to be the major cellular mechanisms mediating the physiological effects of EtOH in reducing anxiety and causing sedation, motor impairment, cognitive impairment, and, at higher doses, amnesia and general anesthesia (Mihalek et al., 2001; Hanchar et al., 2005). However, there have also been a number of reports that physiological concentrations of EtOH have no effect on GABA responses in mouse and rat hippocampal neurons (Weight et al., 1992; Marszalec et al., 1998), in rat nucleus accumbens neurons (Nie et al., 2000), and at synaptic receptors in the basolateral amygdala (Zhu and Lovinger, 2006). Furthermore, other reports have shown that EtOH actually inhibits GABA responses in nucleus accumbens neurons (Nie et al., 2000) and hippocampal CA1 and CA3 neurons (Siggins et al., 1987). Hence there still remain some conflicting data about the effects of EtOH on native neuronal GABAA receptors.
Previous work suggests that GABAA receptors containing the δ subunit and/or mediating tonic extrasynaptic GABA responses (tonic current) may be especially sensitive to EtOH (Wallner et al., 2006). Differences in EtOH sensitivity of various GABAA receptor subunit compositions have been invoked as a potential explanation for the inconsistent findings. However, disagreements have also arisen over these findings (Borghese et al., 2006).
In addition to having direct effects on postsynaptic receptors, EtOH has been shown to modulate GABA and glutamate release from their nerve terminals (Hendricson et al., 2004; Kelm et al., 2007; Jia et al., 2008). EtOH can enhance GABA release in hippocampal slices via a number of mechanisms: increasing spontaneous action potential firing in GABAergic interneurons, decreasing the kainate-driven firing of these neurons, and enhancing presynaptic voltage-dependent Ca2+ channels (VDCCs) by inhibiting the voltage-dependent K+ channels. In addition, EtOH may indirectly activate presynaptic and/or postsynaptic G protein-coupled receptors (Weiner and Valenzuela, 2006). However, again there are conflicting reports that EtOH has no effect on spontaneous or evoked GABA release (Jia et al., 2008) or that it can even inhibit release (Siggins et al., 1987). Generally, pharmacologically relevant concentrations of EtOH have been reported to have minimal effects on glutamate release (Roberto et al., 2004). According to Xiao et al. (2009), however, EtOH can also act on presynaptic dopamine D1 receptors in the ventral tegmental area, resulting in facilitation of glutamate release. In contrast, others studies have reported EtOH decreases glutamate release by blocking either L-type VDCCs in the hippocampal CA1 neurons (Hendricson et al., 2003) or N-type ones in the central amygdala (Zhu et al., 2007). In addition, Mameli et al. (2005) reported that EtOH blocked N-type VDCCs in neonate rats but had no effect on juvenile rat VDCCs, indicating an age dependence of EtOH action. Together, the results suggest that the effects of EtOH on GABA or glutamate release mechanisms are complex.
Recently, we have been investigating mechanisms and the modulation of synaptic transmission at single excitatory and inhibitory synapses by using the synaptic bouton preparation. This preparation involves mechanically dissociated neurons with adherent functional presynaptic terminals (Akaike and Moorhouse, 2003) and offers advantages for determining the locus of action of a neuromodulator such as EtOH because surrounding tissues, such as adjacent neurons and glia cells, are absent. We have identified presynaptic GABAA receptors on both GABAergic and glutamatergic nerve terminals projecting to hippocampal CA1 and CA3 neurons and spinal sacral dorsal commissural nucleus neurons that can modulate GABA and glutamate release evoked by focal electrical stimulation (Matsuura et al., 2011; Yamamoto et al., 2011). To clarify more precisely EtOH's actions on excitatory and inhibitory transmission, we report here the effects of EtOH on presynaptic GABAA receptors at single GABAergic and glutamatergic synapses in rat hippocampal CA1 and CA3 pyramidal neurons, using this synaptic bouton preparation.
Materials and Methods
All experiments were performed in accordance with the Guiding Principles for Care and Use of Animals in The Field of Physiological Sciences of The Physiological Society of Japan. Wistar rats [11–18 (immature) and 30–35 days and 2 months old] were decapitated under pentobarbital anesthesia (50 mg · kg−1 i.p.). The brain was quickly removed and immersed in an ice-cold incubation medium (see below), saturated with 95% O2 and 5% CO2. Hippocampal slices at a thickness of 400 μm were prepared with a vibrating microtome (VT 1200S; Leica, Wetzlar, Germany) and then incubated in a medium oxygenated with 95% O2 and 5% CO2 at room temperature (21–24°C) for at least 1 h before mechanical dissociation. For mechanical dissociation, slices were transferred into a 35-mm culture dish (Primaria 3801; BD Biosciences, San Jose, CA) containing a standard external solution (see below), and the region of CA1 or CA3 was identified under a binocular microscope (SMZ345; Nikon, Tokyo, Japan). Details of the mechanical dissociation procedure have been described previously (Akaike and Moorhouse, 2003). In brief, mechanical dissociation was accomplished with a fire-polished glass pipette coupled to a vibration device (Sl-10 Cell Isolator; K.T. Labs, Tokyo, Japan). The tip of the glass pipette was lightly placed on the surface of the CA1 or CA3 region and vibrated horizontally (0.2-to 2.0-mm displacement) at approximately 50 Hz. Thereafter, the slices were removed from the dish, and the mechanically dissociated neurons were left to settle and adhere to the bottom of the dish for at least 15 min before electrophysiological measurements.
Spontaneous and evoked GABAergic inhibitory and glutamatergic excitatory postsynaptic currents (sIPSCs, eIPSCs, sEPSCs, and eEPSCs, respectively) were recorded from the nerve cell body of the isolated hippocampal CA1 (for IPSCs) and CA3 neurons (for EPSCs) by using conventional whole-cell patch recording mode under voltage clamp at a holding potential (VH) of 0 mV (for IPSCs) and −65 mV (for EPSCs). Voltage-dependent Na+ current (INa) and voltage-dependent Ca2+ current (IBa) were also recorded in whole-cell mode at a VH of −70 mV for INa and −60 mV for IBa (Multiclamp 700B; Molecular Devices, Sunnyvale, CA).
Patch pipettes were made from borosilicate capillary glass by a vertical pipette puller (PP-830; Narishige, Tokyo, Japan). The resistance of the recording pipettes filled with the internal (patch pipette) solution (see below) was 3 to 6 MΩ. Isolated neurons were observed under phase contrast on an inverted microscope (Diapot; Nikon) and recorded from CA1 or CA3 pyramidal neurons but not other neurons (e.g., basket cells). Current and voltage were continuously monitored on an oscilloscope (DCS-7040; Kenwood, Melrose, MA). All membrane currents were filtered at 3 kHz (E-3201A Decade Filter; NF Electronic Instruments, Tokyo, Japan) and stored on a computer by using pCLAMP 10.2 (Molecular Devices). Hyperpolarizing step pulses of 5 mV (30-ms duration) were used to monitor the access resistance; if the resistance changed by more than 20%, the recordings were rejected.
Paired-Pulse Focal Electrical Stimulation on a Single GABAergic or Glutamatergic Bouton Using θ Glass Pipette.
Focal electrical stimulation of a single bouton adherent to mechanically dissociated central nervous system neurons has been described previously (Akaike and Moorhouse, 2003). The stimulating pipette was made from a glass tube and filled with normal external test solution (see below). The stimulating electrode was placed as close as possible to the soma of a single CA1 or CA3 neuron from which a whole-cell recording had been successfully obtained. The stimulating pipette was then carefully moved along the surface membrane of the soma and proximal dendrites, while applying stimulus pulses and monitoring for an evoked response. Responses were evoked by using a paired-pulse stimulus protocol, with each stimulus pulse of 100-μs duration and the same intensity, applied at a frequency of 0.2 Hz by using a stimulus isolator (SS-202 J; Nihon Koden, Tokyo, Japan). For eIPSCs, the stimulus intensity ranged from 0.05 to 0.15 mA, and an interstimulus interval of 30 to 60 ms was used. For eEPSCs, stimulus intensity was 0.05 to 0.08 mA, and the interstimulus interval was 20 to 30 ms. As the stimulation pipette was moved, GABA-gated outward currents (eIPSCs) and glutamate-gated inward currents (eEPSCs) appeared in an all-or-none fashion, indicating that the stimulating pipette was positioned just above a single GABAergic or glutamatergic bouton, respectively.
The ionic composition of the incubation medium was 124 mM NaCl, 5 mM KCl, 1.2 mM KH2PO4, 24 mM NaHCO3, 2.4 mM CaCl2, 1.3 mM MgSO4, and 10 mM glucose saturated with 95% O2 and 5% CO2. The pH was adjusted to 7.4. The standard external solution used for recordings contained 150 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM glucose, and 10 mM HEPES. The modified external solutions for recording INa contained 60 mM NaCl, 100 mM choline-Cl, 10 mM CsCl, 10 mM glucose, 0.01 mM LaCl3, 5 mM tetraethylammonium (TEA)-Cl, and 10 mM HEPES, and for recording IBa they contained 145 mM choline-Cl, 5 mM CsCl2, 5 mM BaCl2, 1 mM MgCl2, 10 mM glucose, and 10 mM HEPES. All external solutions were adjusted to a pH of 7.4 by using Tris base. The composition of the internal pipette solution for glutamatergic EPSCs was 5 mM CsCl, 135 mM CsF, 5 mM TEA-Cl, 2 mM EGTA, 10 mM HEPES, and 5 mM 2-((2,6-dimethylphenyl)amino)-N,N,N-triethyl-2-oxoethanaminium (QX-314) bromide, and for GABAergic IPSCs it was 5 mM CsCl, 70 mM CsF, 65 mM Cs-methanesulfonate, 5 mM TEA-Cl, 2 mM EGTA, 10 mM HEPES, and 4 mM ATP-Mg. The internal pipette solution for INa measurements was 105 mM CsF, 30 mM NaF, 5 mM CsCl, 5 mM TEA-Cl, 2 mM EGTA, 10 mM HEPES, and 2 mM ATP-Mg, and for IBa it was 80 mM Cs-methanesulfonate, 60 mM CsCl, 5 mM TEA-Cl, 2 mM EGTA, 10 mM HEPES, and 2 mM ATP-Mg. All pipette solutions were adjusted to a pH of 7.2 with Tris base. ATP-Mg was dissolved in the internal solution just before use.
GABAergic eIPSCs were isolated from the glutamatergic ones by using 6-cyano-7-nitroquinoxaline-2,3-dione and d-(−)-2-amino-5-phosphonovaleric acid, and recording them at a VH of 0 mV, close to the reversal potential of glutamate response (Matsuura et al., 2011). Likewise, glutamatergic eEPSCs were isolated from GABAergic eIPSCs by recording them at a VH of −65 mV, close to the Cl− equilibrium potential (ECl) (Yamamoto et al., 2011).
6-Cyano-7-nitroquinoxaline-2,3-dione, d-(−)-2-amino-5-phosphonovaleric acid, and bicuculline were purchased from Tocris Bioscience (Ellisville, MO). Muscimol, TEA-Cl, EGTA, 2-((2,6-dimethylphenyl)amino)-N,N,N-triethyl-2-oxoethanaminium (QX-314) bromide, ATP-Mg, and saclofen were purchased from Sigma-Aldrich (St. Louis, MO). All test solutions containing drugs were applied by a Y-tube system that allowed rapid solution exchange, within approximately 20 ms.
For analysis of the effects of exogenous muscimol- or glutamate-induced responses and the effects of different EtOH concentrations on these responses, the peak current amplitudes were normalized to the currents obtained in response to 100 nM muscimol or 10 μM glutamate, concentrations that elicit little desensitization. Concentration-response curves for muscimol and glutamate were fitted by a sigmoidal dose-response equation with Origin Pro 7.5 software (OriginLab Corp., Northampton, MA). The relative INa and IBa responses with various concentrations of EtOH were similarly normalized to the control peak currents in standard solution without EtOH.
The amplitude, failure rate (Rf), and paired-pulse ratio (PPR) (P2/P1) of eIPSCs and eEPSCs were analyzed with pCLAMP 10.2 (Yamamoto et al., 2011). The effects of drugs on the current amplitude, Rf, and PPR for eIPSCs or eEPSCs were normalized to their respective controls in the absence of EtOH.
sIPSCs and sEPSCs were counted and analyzed in preset epochs before, during, and after each test condition by using the MiniAnalysis Program (Synaptosoft, Decatur, GA). In brief, the events were initially screened automatically by using an amplitude threshold of 10 pA, and then visually accepted or rejected based on their 10 to 90% rise and 90 to 37% decay times. The interevent intervals (frequencies) and amplitudes of both sIPSCs and sEPSCs were examined by constructing and comparing cumulative probability distributions under different conditions using the Kolmogorov-Smirnov test within the MiniAnalysis Program. The frequency and amplitude of synaptic events during the control period (3–5 min) were averaged, and the frequency and amplitude of all events during of drug application were normalized to these values. The effects of drugs were quantified as relative changes in frequency and amplitude as synaptic events and compared with the individual control.
Data are reported as the means ± S.E.M. of these normalized values. Possible differences in the current amplitude, frequency, Rf, and PPR distribution were tested by analysis of variance and post hoc Dunnet's test. Values of p < 0.05 were considered significant.
Effects of EtOH on Extrasynaptic GABAA and Glutamate Receptor-Mediated Responses.
In our experimental conditions using an internal solution containing low internal Cl− and TEA and high Cs+, exogenous application of muscimol, a selective GABAA receptor agonist, induced outward currents in isolated CA1 pyramidal neurons at a holding potential (VH) of 0 mV. The GABAA receptor-mediated outward currents increased in a concentration-dependent manner (EC50 0.92 μM) (Fig. 1A). Application of glutamate to isolated CA3 pyramidal neurons held at a VH of −65 mV (close to the ECl of −65 mV) induced concentration-dependent inward currents with a mean EC50 of 26.1 μM (Fig. 1B).
The effects of EtOH on these extrasynaptic GABAA receptor-mediated responses in mechanically dissociated hippocampal CA1 neurons from rats 11 to 18 days old were examined by coapplying various concentrations of EtOH with 100 nM muscimol. Application of 100 nM muscimol alone induced little desensitization and resulted in readily reversible outward currents. Coapplication of EtOH at concentrations higher than 30 mM slightly, but significantly, inhibited the muscimol-induced outward currents (88.9 ± 2.3% of control at 30 mM, n = 5, p < 0.01; 84.4 ± 1.0% of control at 100 mM, n = 5, p < 0.001) (Fig. 2). At a VH of −65 mV, application of 10 μM glutamate to isolated hippocampal CA3 neurons (in 11- to 18-day-old rats) caused a nondesensitizing and reversible inward current (Fig. 2). Coapplication of various concentrations of EtOH with 10 μM glutamate markedly inhibited this glutamate response in a concentration-dependent manner (Fig. 2; 81.5 ± 4.4% of control at 30 mM, n = 6, p < 0.01; 62.2 ± 1.2% of control at 100 mM, n = 6, p < 0.001; 24.7 ± 1.9% of control at 300 mM, n = 5, p < 0.001). These results indicate that EtOH at concentrations at and above 30 mM inhibits both extrasynaptic GABA and glutamate responses, with the inhibition being more efficacious on the glutamate-induced responses. We also examined the EtOH effects on these extrasynaptic GABAA and glutamate receptor responses in neurons isolated from rats at 30 to 35 days old and 2 months old, using the same experimental protocols. The results obtained from these more mature rats were virtually the same as those obtained from the immature rats (Fig. 2).
In previous studies, acute tolerance to EtOH has been demonstrated on NMDA-mediated EPSPs in hippocampal neurons (Grover et al., 1994). Therefore, we next examined whether the effects of EtOH showed any tolerance in our experimental conditions. This, however, was not the case. As shown in Fig. 3, the repeated coapplication of 100 mM EtOH and 100 nM muscimol or 10 μM glutamate induced almost the same response each time.
Effects of EtOH on Voltage-Gated Na+ and Ca2+ Channels.
Because evoked GABAergic IPSCs and glutamatergic EPSCs are very dependent on voltage-dependent Na+ and Ca2+ channels and their respective currents (INa and IBa), we felt it relevant to also investigate how EtOH affects these currents. The currents were elicited by a depolarizing step pulse from a VH of −70 mV to −20 mV (INa) and from −60 mV to +10 mV (IBa). As shown in Fig. 4, only high concentrations of EtOH inhibited INa (82.7 ± 3.4% of control at 300 mM, n = 5, p < 0.001) and IBa (79.0 ± 4.7% of control at 100 mM, n = 5, p < 0.01).
Effects of EtOH on Presynaptic GABAA Receptors on Single GABAergic Nerve Terminals.
Focal paired-pulse electrical stimuli were applied to single GABAergic nerve terminals (boutons) synapsing on dissociated hippocampal CA1 pyramidal neurons at a frequency of 0.2 Hz, producing GABAergic eIPSCs in response to both the first (P1) and second stimuli (P2) with occasional failures. The application of 10 nM muscimol had no effect on either the P1 current amplitude, the P1 Rf of eIPSCs, or the P2/P1 amplitude ratio (the paired-pulse current ratio) (Fig. 5). However, the subsequent coapplication of 10 mM EtOH significantly decreased the P1 amplitude (85.1 ± 2.0% of control, n = 8, p < 0.001) and increased the PPR (130.7 ± 7.5% of control, n = 8, p < 0.01). The P1 Rf was not significantly affected by 10 mM EtOH, although the effects were quite variable, and the mean Rf showed an increase (122.3 ± 17.8% of control, n = 8, not significant). The subsequent application of only 10 mM EtOH after washout of muscimol produced no effect on P1 amplitude, Rf, and PPR (Fig. 5). Thus, these results suggest that EtOH is acting to potentiate the actions of muscimol on presynaptic GABAA receptors (GABAA autoreceptors). Activation of presynaptic GABAA receptors results in the depolarization of presynaptic terminals because of their high Cl− concentration mediated by the dominance of active Cl− influx pumps (Jang et al., 2006; Yamamoto et al., 2011). A small depolarization of presynaptic terminals by the activation of presynaptic GABAA receptors enhances excitability by bringing the membrane potential closer to the firing threshold. However, a large depolarization of presynaptic terminals reduces excitability by a depolarization block of Na+ channels (inactivation of Na+ channels) and/or a shunt of the membrane conductance of nerve terminals (Jang et al., 2006; Matsuura et al., 2011; Yamamoto et al., 2011). Consequently, the coapplication of EtOH and muscimol induces a presynaptic depolarization sufficient to decrease evoked transmitter release with a reduced P1 amplitude and an increase of Rf and PPR.
We also examined the effects of EtOH and muscimol on GABAergic eIPSCs recorded in CA3 pyramidal neurons (Fig. 6). The obtained results were essentially the same as those obtained from CA1 neurons (compare Fig. 5), indicating little difference regarding EtOH actions on presynaptic GABAA autoreceptors on terminals projecting to CA1 and CA3 neurons.
Effects of EtOH on Presynaptic GABAA Receptors on Single Glutamatergic Terminals.
GABAA receptors also exist on glutamatergic nerve terminals projecting to hippocampal CA3 neurons (Jang et al., 2006), where their activation induces depolarization blockade of action potential-dependent glutamate release (Yamamoto et al., 2011). We next examined the effects of EtOH on these presynaptic GABAA receptors. Focal paired-pulse stimuli were similarly applied to single glutamatergic presynaptic bouton while simultaneously voltage-clamping dissociated CA3 neurons at −65 mV. Muscimol and EtOH were applied alone and in combination, using the same protocol as described above for the GABAergic terminals. Muscimol (10 nM) or EtOH (10 mM) by themselves had no significant effects on the eEPSCs, but simultaneous application of both muscimol and EtOH decreased the amplitude of P1 eEPSCs (70.0 ± 7.0% of control, n = 4, p < 0.05) and increased PPR (167.4 ± 16.6% of control, n = 4, p < 0.05) (Fig. 7). The mean P1 Rf increased but there was marked variability in this effect, and the Rf with and without muscimol and EtOH was not significantly different with muscimol, and EtOH was 144.0 ± 39.4% of the control (n = 8). Hence, the results were similar to those obtained for GABAergic terminals and therefore suggest that EtOH potentiates muscimol activation of presynaptic GABAA receptors, resulting in enhanced presynaptic inhibition of glutamate release.
We further investigated whether there was a developmental change in this presynaptic effect of EtOH, with the above results being obtained in CA3 neurons from immature (postnatal day 11–18) rats. Using the same experimental protocols, we observed essentially the same EtOH effects on presynaptic GABAA receptors on glutamatergic nerve endings projecting to CA3 neurons isolated from more mature (postnatal day 30–31) rats (Fig. 8).
Bicuculline Removes Presynaptic Inhibition at Glutamatergic Nerve Terminals Induced by EtOH and Muscimol.
If EtOH is potentiating muscimol activation of GABAA receptors on glutamatergic nerve terminals to induce presynaptic inhibition, we should be able to block its effects with 10 μM bicuculline, a selective GABAA receptor antagonist. Bicuculline alone had no effect on the eEPSC P1 amplitude (98.2 ± 1.4% of control, n = 4), Rf (108.3 ± 8.3% of control, n = 4), and PPR (106.7 ± 2.8% of control, n = 4) (Fig. 9). However, as shown in Fig. 9, bicuculline completely reversed the effects of combined muscimol (10 nM) and EtOH (10 mM) on these eEPSC parameters. These results indicate clearly that EtOH is acting on presynaptic GABAA receptors.
Presynaptic Depolarization of Glutamatergic Nerve Terminals Induced by EtOH and Muscimol.
Depolarization of presynaptic nerve terminals in isolated CA1 and CA3 pyramidal neurons enhances spontaneous transmitter release (Jang et al., 2006; Matsuura et al., 2011; Yamamoto et al., 2011). Hence as a test of whether muscimol and EtOH was depolarizing presynaptic nerve terminals via activation of GABAA receptors we investigated the effects of 10 nM muscimol and 10 mM EtOH on the spontaneous release of GABA and glutamate, by quantifying sIPSCs and sEPSCs, respectively. Coapplication of muscimol (10 nM) and EtOH (10 mM) increased the frequency of sIPSCs and sEPSCs, without affecting their current amplitudes (Fig. 10). Application of muscimol (10 nM) alone had no effect on the frequency and amplitude of sIPSCs and sEPSCs (Fig. 10). Application of EtOH (10 mM) also had no effect on the frequency and amplitude of sIPSCs (amplitude, 108.2 ± 3.8% of control, n = 4; frequency, 105.2 ± 3.8% of control, n = 4) and sEPSCs (amplitude, 105.1 ± 8.4% of control, n = 4; frequency, 109.3 ± 10.2% of control, n = 4). Bicuculline (10 μM) reversed the facilitation of sEPSC frequency induced by simultaneous application of muscimol and EtOH (Fig. 10Bb). Bicuculline alone had no effect on frequency (98.2 ± 2.5% of control, n = 4) and amplitude of sEPSCs (102.8 ± 2.8% of control, n = 4).
Effects of EtOH on Presynaptic GABAB Receptors in Single GABAergic and Glutamatergic Terminals.
Presynaptic GABAB receptors have been shown to modulate acute EtOH actions on GABAA receptor-mediated transmission in hippocampal neurons (Ariwodola and Weiner; 2004). Therefore, we examined the EtOH effects on eIPSCs and eEPSCs in the presence of saclofen, a GABAB receptor antagonist. Application of 100 μM saclofen alone had no effect on the P1 amplitude, P1 Rf, and PPR of GABAergic eIPSCs (n = 4) or glutamatergic eEPSCs (n = 4) (Fig. 11). A simultaneous application of 100 μM saclofen and 10 mM EtOH also had no effect on the parameters of the synaptic currents.
There are a number of conflicting reports that EtOH, at intoxicating concentrations (1–100 mM), induces a potentiation (Reynolds and Prasad, 1991) or inhibition (Siggins et al., 1987; Nie et al., 2000) of extrasynaptic GABA responses, whereas in other reports EtOH has no effect on GABA responses, even at the higher range of concentrations (Mihic et al., 1992). In the accumbens dorsomedial cells, GABAA receptors containing δ subunits have a high sensitivity to EtOH, with their responses being enhanced at concentrations of 3 to 30 mM (Nie et al., 2011). GABAA receptors containing the δ subunit and/or mediating tonic extrasynaptic GABA responses (tonic current) may be especially sensitive to EtOH (Wallner et al., 2006). Studies of δ-subunit knockout mice determined that α4/6- and δ subunit-containing receptors were involved in some aspects of acute intoxication and drinking behavior; however, many features of intoxication are still intact in these mice (Mihalek et al., 2001). In contrast, EtOH has no effect on the extrasynaptic GABAA response in rat cultured hippocampal neurons at concentrations of 10 to 300 mM (Marszalec et al., 1998), and only small (but significant) inhibitory effects on muscimol-induced extrasynaptic GABAA response were seen at 30 mM or more in the current study (rat, acutely dissociated CA1 neurons). The range of results seen with EtOH might relate to the different sensitivity to EtOH of different GABAA receptor subunit compositions in different animals (mouse or rat), the different preparations used (slice or culture), and/or tissues (hippocampus, cortical, spinal, ganglion cell) and subcellular domains (dendrite or soma) (Wafford et al., 1991; Harris et al., 1995; Criswell et al., 2008). Indeed, GABA response in mouse cultured hippocampal neurons are much more sensitive to EtOH potentiation than in rat (Aguayo et al., 1994). However, even for GABAA receptors containing the δ subunit there are no uniform agreements on EtOH sensitivity (Borghese et al., 2006), suggesting that GABAA receptor sensitivity for EtOH cannot be explained only by differences in subunit composition.
We also show in the current study that EtOH concentration-dependently inhibited the response to bath application of glutamate in hippocampal CA3 pyramidal neurons. The results agree with a large number of previous reports, including in cultured rat hippocampal neurons (Lovinger et al., 1989; Weight et al., 1992), mechanically isolated rat ventral tegmental area neurons (Zhu et al., 2002), and cultured rat cerebral cortex nonpyramidal neurons (Fischer et al., 2003). The inhibitory effects of 30 and 100 mM EtOH were much greater for the extrasynaptic glutamate receptor responses, compared with the extrasynaptic GABAA receptor-mediated muscimol responses.
The effects of EtOH on both the amplitude of extrasynaptic GABAA and glutamate receptor-mediated whole-cell currents (Fig. 2) and the evoked glutamatergic EPSCs (Figs. 7 and 8) showed little difference in neurons isolated from immature and mature rats. Thus, we did not detect any age-dependent effects for EtOH. A previous report in hippocampal slices reported that low doses of EtOH showed a developmental change in glutamate receptor specificity, inhibiting juvenile AMPA receptors and mature NMDA receptors (Mameli et al., 2005). In the same study, a higher EtOH concentration (50 mM) selectively and presynaptically inhibited glutamate mediated synaptic responses only in slices from juvenile rats. Our data suggest that EtOH effects may differ between the slice and isolated synaptic bouton preparations. However, the age dependence of presynaptic effects can differ even in the slice, because the extent of presynaptic facilitation of evoked GABAergic IPSCs by 30 and 60 mM was greater in slices from older rats (Li et al., 2003).
In a previous study, EtOH actions on NMDA-mediated EPSPs showed acute tolerance (Grover et al., 1994), but in this study we could not observe such tolerance (Fig. 3). In our synaptic bouton preparation, there is no surrounding tissue such as adjacent neurons and glia cells. EtOH can be directly applied to the isolated neuron under study. In slice preparations, EtOH has to diffuse to the target neuron and will also affect many surrounding glia cells and neurons. Actions on GABA transporters to alter ambient GABA in response to EtOH, for example, may make interpretations more difficult. These or other factors might contribute to the differences observed in the apparent tolerance to the effects of EtOH.
We also demonstrated that EtOH at high concentrations (100 or 300 mM) inhibited the voltage-dependent Na+ and Ca2+ currents (INa and IBa). Again, these findings concur with results obtained from rat hippocampal slices (Hendricson et al., 2004), cultured superior cervical ganglion neurons (Xiao et al., 2008), and recombinant channels expressed in Xenopus laevis oocytes (Horishita and Harris, 2008). However, such high concentrations of EtOH are beyond those typically encountered by social drinkers and, in fact, bring a significant risk of death. Hence these actions could be considered as adverse and nonselective effects, which are less relevant for physiological responses.
The most sensitive EtOH response we observed in the current study involved the presynaptic receptors. Low concentrations of 10 mM EtOH and 10 nM muscimol that by themselves had no effect on extrasynaptic GABAA and glutamate response, INa, IBa, or spontaneous or evoked IPSCs and EPSCs together had synergistic reproducible effects on synaptic responses. Coapplication of 10 mM EtOH and 10 nM muscimol reduced the amplitude of the evoked responses, increased the response failure rate, and enhanced the PPR of P1 amplitude/P2 amplitude of both GABAergic eIPSCs and glutamatergic eEPSCs. In rat hippocampal slices, 75 mM EtOH similarly decreased the PPR of eEPSCs (Hendricson et al., 2004), but that was at a much higher concentration than observed here. Such high concentrations may have additional effects, such as nonsynaptic actions or inhibition of presynaptic IBa as shown in the present study (Fig. 4), and both eIPSCs and eEPSCs are very sensitive to Ca2+ influx passing through voltage-dependent Ca2+ channels. The effects of low doses of EtOH and muscimol seen here indicate a synergistic presynaptic inhibition of GABA and glutamate release. At the same time, the frequency of both spontaneous GABA and glutamate release increased significantly, without any change in their current amplitudes. These effects all are consistent with activation of presynaptic GABAA receptors, resulting in an outwardly directed Cl− flux from the terminals that are known to maintain a high Cl− concentration (Jang et al., 2006; Yamamoto et al., 2011) and a resultant depolarization of presynaptic GABAergic and glutamatergic nerve terminals. A small depolarization of presynaptic terminals results in facilitation of both spontaneous and evoked release, presumably by enhancing action potential firing probability and Ca2+ influx and vesicle exocytosis. In contrast, a larger depolarization of presynaptic terminals results in a reduction of action potential firing by inactivation of Na+ channels and/or a shunt of the membrane conductance of nerve terminals (Jang et al., 2006; Matsuura et al., 2011; Yamamoto et al., 2011). However, a large depolarization induces more Ca2+ influx through Ca2+ channels, resulting in the increase of sIPSCs and sEPSCs frequency, but an inhibition of eEPSCs and eEPSCs with an increase in their Rf. A similar differential effect on spontaneous and evoked release has been observed with increased activation of GABAA receptors by muscimol or other agonists (Yamamoto et al., 2011). Consistently, bicuculline, a selective GABAA receptor antagonist, completely reversed the effects of coapplication of EtOH and muscimol. The results indicate clearly that very low EtOH concentrations potentiate the effects of muscimol on presynaptic GABAA receptors on both GABAergic and glutamatergic nerve terminals, resulting in presynaptic depolarization and inhibition of evoked GABA and glutamate release.
These presynaptic GABAA receptors showed the greatest sensitivity to EtOH, with effects at 10 mM, whereas the extrasynaptic GABAA receptor responses were evident only at 30 mM EtOH. It has been reported that GABAA receptors composed of α4β2δ (Sundstrom-Poromaa et al., 2002) or α4/6β3δ (Wallner et al., 2006) subunits have a high sensitivity to EtOH, with affects seen even at 3 mM EtOH. Such highly sensitive GABAA receptor subunits may also comprise the presynaptic GABAA receptors seen in the present study. δ Subunit-containing GABAA receptors have other unique pharmacological properties, including high affinity for agonist (e.g., neurosteroids) and partial desensitization at low agonist concentrations (Mortensen et al., 2010). We have also observed bicuculline-sensitive presynaptic effects of low doses of the allopregnalone on eEPSCs (unpublished work), suggesting that these presynaptic GABAA receptors also have high affinity for neurosteroids, consistent with them containing the δ subunit. Our previous study (Matsuura et al., 2011) also reported a higher sensitivity of presynaptic GABAA receptors, compared with extrasynaptic receptors, toward α-chloralose. Therefore, GABAA receptors composed of subunits endowing high sensitive to modulators may exist on presynaptic nerve terminals in the hippocampus where they may make important contributions to presynaptic modulation of neuronal excitability (Dittman and Regehr, 1996).
Participated in research design: Wakita and Akaike.
Conducted experiments: Wakita, Shin, Iwata, and Nonaka.
Performed data analysis: Wakita, Shin, Iwata, and Nonaka.
Wrote or contributed to the writing of the manuscript: Wakita, Shin, and Akaike.
We thank Dr. A. Moorhouse (University of New South Wales, Sydney, Australia) for valuable comments and critical reading of the manuscript.
This work was supported by Grants-in Aid from Kumamoto Health Science University (to M.W., M.C.S., K.N., and N.A.).
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
- excitatory postsynaptic current
- spontaneous EPSC
- evoked EPSC
- inhibitory postsynaptic current
- evoked IPSC
- spontaneous IPSC
- paired-pulse ratio
- failure rate
- voltage-dependent Ca2+ channel
- no significant difference
- Received November 5, 2011.
- Accepted March 19, 2012.
- Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics