Introduction

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-Aminobutyric acid (GABA) and glycine are main inhibitory neurotransmitters in the mammalian central nervous system. Activation of ionotropic receptors by these ligands opens an integral Cl channel, leading to fast postsynaptic inhibition (Bechade et al., 1994; Rabow et al., 1995; Betz et al., 1999). The GABA_A receptor is modulated by several agents, including sedative/hypnotic drugs such as benzodiazepines and barbiturates, as well as the antagonists bicuculline and Zn²⁺ (Macdonald and Olsen, 1994). In addition, several studies have shown that short (seconds) applications of pharmacologically relevant ethanol concentrations (1-100 mM) can enhance the GABA_A receptor activity (Suzdak et al., 1986; Ticku et al., 1986; Mehta and Ticku, 1988; Reynolds et al., 1992; Aguayo et al., 1994; Yeh and Kolb, 1997). Similarly, glycine receptors in central nervous system neurons are also sensitive to low concentrations of ethanol (Aguayo et al., 1996).

It is known that acute alcohol intoxication causes several behavioral alterations, including severe motor impairment. Furthermore, chronic alcohol leads to neuroadaptive phenomena (Fadda and Rossetti, 1998), as well as pharmacodynamic tolerance associated to adjustments in motor behavior (O'Brien, 1996). Inhibitory (GABA_A and glycine) receptors in areas related to motor control, such as cortex, cerebellum, and spinal cord, are therefore likely sites of action for the development of tolerance (O'Brien, 1996). Indeed, studies with chronic ethanol demonstrated a selective reduction in GABA_A receptor mRNA for ₁- and ₂-subunits and an increase in that for ₆-subunits in cerebral cortex and cerebellum (Morrow et al., 1990; Buck et al., 1991). At the same time, a reduced ethanol action on GABA-induced Cl flux was reported in brain synaptosomes (Allan and Harris, 1987). In spite of these previous studies, no data are available on the repercussion that these changes might have on the functional properties of GABA_A receptors. We suggest that it is very relevant to learn whether ethanol changes the behavior of these receptors because several physiological events such as firing pattern, integration, synaptic plasticity, and development are dependent on their overall properties. Furthermore, it is currently unknown whether glycine receptors are altered after chronic ethanol treatment. Therefore, the present study was undertaken to obtain further insights into the cellular effects of chronic ethanol on GABA_A and glycine receptors.

	Materials and Methods

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Cultured Neurons. Mouse (C57BL/J6) spinal cord neurons obtained from five to six embryos (13-14 days) were plated at 300,000 cells/ml into 35-mm tissue culture dishes coated with poly(L-lysine) (molecular mass >350 kDa; Sigma Chemical Co., St. Louis, MO). The neuronal feeding medium consisted of 90% minimal essential medium (Life Technologies Inc., Grand Island, NY), 10% heat-inactivated horse serum (Life Technologies Inc., Rockville, MD), and a mixture of nutrient supplements (Aguayo and Pancetti, 1994). The feedings were made every 3 days. In the chronic ethanol experiments, 100 mM ethanol was added at day 5. Enzymatic measurement of ethanol concentrations revealed that after 24 h approximately 60% of the added ethanol evaporated in the incubator. Therefore, during the days that the culture medium was not replaced, 50 mM ethanol was added to the medium.

Immunocytochemistry. Cells were fixed in 4% paraformaldehyde for 30 min, washed in PBS, and permeabilized with 0.3% Triton X-100 for 30 min. After washing, normal goat serum (5%) was applied to block nonspecific binding sites and the cells were incubated overnight at 4°C with a rabbit antibody against glycine (Chemicon, Temecula, CA) at a 1:200 dilution. The cells were washed and then incubated with a secondary biotinylated anti-rabbit IgG (Vector Laboratories, Burlingame, CA) diluted 1:100 for 30 min. After washing with PBS, Vectastain Elite ABC solution (Vector Laboratories) was applied for 1 h, washed, and the cells were preincubated with nonactivated 3'3-diaminobenzidine (DAB; Sigma Chemical Co.) for 10 min. Nonactivated DAB was replaced by DAB containing 0.3% H₂O₂ and the color development during 5 to 8 min was visualized under a Zeiss microscope. The reaction was stopped with cold PBS and the cells were dehydrated two times for 2 min in absolute ethanol and xylol before mounting. Parallel negative controls, in the absence of primary antibody, revealed no positive reaction.

Indirect Fluorescence of the Benzodiazepine Receptor. The stock solution of Bodipy RO-1986 (Molecular Probes) was prepared in distilled, deionized water, and was stored at 20°C. The working concentration (10 nM) was made daily by diluting the stock in normal external solution. We selected 10 nM Bodipy RO-1986 to reduce nonspecific binding. Cells were incubated for 30 min at room temperature (20-22°C) in the dark before starting the measurement. The analysis, including the subtraction of nonspecific fluorescence, was done as described above.

Recordings. The current recordings were made in 12- to 17-day in vitro (DIV) neurons using the patch-clamp technique (Hamill et al., 1981). The culture medium in the dish was changed to an external solution containing 150 mM NaCl, 5.4 mM KCl, 2.0 mM CaCl₂, 1.0 mM MgCl₂, 10 mM HEPES, pH 7.4, and 10 mM glucose. The neurons were perfused continuously with the external solution. In the chronic ethanol-treated cells, 100 mM ethanol was added to the external solution to avoid the development of ethanol withdrawal during the recordings. The standard internal solution contained 120 mM CsCl, 10 mM BAPTA, 10 mM HEPES, 4 mM MgCl₂, and 2 mM ATP-disodium, pH 7.35. The cells were stabilized at room temperature (20-22°C) for at least 30 min before starting the recordings. The whole-cell recordings were done using an Axopatch-1D amplifier (Axon Instruments). The holding potential was 60 mV. Electrodes were pulled from borosilicate capillary glass (World Precision Instruments, Sarasota, FL) in two stages on a vertical puller (Sutter Instruments, Novato, CA). The resistance of the fire-polished patch pipettes was below 4 M when filled with the internal solution. After the whole-cell configuration was established, the capacitance of the cell and the series resistance were compensated by using the patch amplifier (>80%). The current signals were filtered at 2 kHz and stored for off-line analysis on a 386-based PC interfaced with a TL-1 board (Axon Instruments).

Solution Applications and Data Analyses. Stock solutions of glycine, picrotoxin, strychnine, GABA, pentobarbital, Cl-218,872, and ZnCl₂ were prepared each week in distilled, deionized water, and kept refrigerated at 4°C. Stock solutions of diazepam were prepared in dimethyl sulfoxide. The working concentrations were made daily by diluting the stock in the normal external solution. Concentrations of reagent-grade ethanol (Aldrich, Milwaukee, WI) were obtained by directly diluting the stock in external solution. All drugs and reagents were purchased from Sigma Chemical Co. To apply increasing concentrations of the drugs rapidly (time constant <100 ms), we used an array of external tubes (internal diameter, 200 µm) placed within 50 µm of the neuron. The solutions containing the ligands flowed continuously by gravity from the tubes, which were connected to a 20-ml reservoir. Drug application was started and terminated by horizontally moving, with the aid of a micromanipulator, the tubes relative to the neuron under study (Aguayo and Pancetti, 1994). The time between each ligand application was at least 60 s to minimize receptor desensitization. The amplitude of the current was measured at the peak with PClamp (Axon Instruments), and was plotted and analyzed using a commercially available nonlinear regression analysis program (Origin; Microcal, Inc. Northampton, MA). Nonlinear regression analysis of concentration-response relationships from the average peak current amplitude was done with the following equation:
where I_MAX is the maximal response of the current, EC₅₀ is the concentration of the drug that produces 50% of the response, and n_H is the Hill coefficient. The values of EC₅₀ and n_H obtained from the analysis are expressed in the text as mean ± S.E. To analyze the actions of positive (pentobarbital, diazepam, Cl-218,872) and negative (Zn²⁺) modulators, concentrations of GABA to achieve GABA₁₀ (5 µM GABA) and GABA₅₀ (20 µM GABA) were used. A glycine₆₀ (25 µM glycine) was used to analyze the biphasic actions of Zn²⁺.

	Results

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Identification of Cultured Neurons. Based on cell morphology and the availability of specific antibodies, cultured spinal cord neurons can be divided into two main types. Most abundant are small neurons (soma <15 µm), containing two to three neurites, which are reactive to an antibody raised against glycine (Fig. 1, A and B). In addition, we have detected the presence of synaptic transmission that is strongly blocked by strychnine, therefore these cells have been identified as glycinergic interneurons. The cultures also contain a smaller number of large neurons (soma >25 µm) with abundant (4-8) neurites. These neurons are Islet-1 positive and therefore resemble motor neurons (Ericson et al., 1992; J. P. Roa, L. G. Aguayo, and R. Navarrete, unpublished data). For the present study, only small neurons (soma <15 µm) with a few neurites were selected (see arrows in Fig. 1).

View larger version (69K): [in this window] [in a new window]   Fig. 1.   Immunocytochemical identification of mouse spinal neurons in culture. A, Immunocytochemical staining shows that only small neurons (somata <15 µm) containing few (2-3) neurites were strongly labeled with an antibody to glycine (arrow). The larger neurons (soma >20 µm) with four to eight neurites were not intensely marked by the glycine antibody (arrowheads). B, another field under higher magnification. Scale bar, 15 µm.

Sensitivity of Glycine and GABA_A Receptors to Their Agonist in Control and after Chronic Ethanol Treatment. We analyzed whether chronic ethanol affected the sensitivity of the receptor to glycine in neurons derived from parallel cultures. The receptor presented similar affinity to glycine in control and treated neurons; for instance, the value for glycine₅₀ was 19 ± 0.6 and 21 ± 0.3 µM in control (n = 5) and chronic ethanol-treated (n = 5) neurons, respectively. The Hill coefficient also was unchanged and the analysis of the data gave values of 3.1 ± 0.3 for control cells and 2.5 ± 0.1 for ethanol-treated cells (Fig. 2B). Furthermore, the maximal current amplitude activated with 500 µM glycine was 310 ± 80 pA/pF in control and 440 ± 19 pA/pF in treated cells (P > .05). In agreement with the electrophysiological results, analysis of immunoreactivity using an antibody raised against glycine receptors showed no difference in these two groups of cells (Fig. 3).

View larger version (26K): [in this window] [in a new window]   Fig. 2.   Effect of chronic ethanol treatment on the sensitivity of the receptors to their neurotransmitters. A, traces were obtained at the indicated concentrations of glycine in a control neuron. The holding potential was 60 mV. B, data obtained from 12 DIV neurons illustrate the effect of increasing glycine concentrations on the current peak amplitude in control () and treated neurons (). The response at each concentration was normalized to the current obtained with 500 µM glycine. The solid line is the best fit with an EC50 of 19 ± 0.6 µM and a Hill coefficient of 3.1 ± 0.3. In the presence of ethanol these values were 21 ± 0.3 µM and 2.5 ± 0.1, respectively. C, graph shows the normalized amplitude of the current obtained with increasing concentrations of GABA in control () and chronic ethanol treated () neurons. The response at each concentration was normalized to the current obtained with 200 µM GABA. The solid line is the best fit with an EC50 of 16 ± 0.8 µM and a Hill coefficient of 1.6 ± 0.1. In the presence of ethanol these values were 16 ± 2.0 µM and 1.4 ± 0.2, respectively. Each symbol represents the mean ± S.E. obtained from the indicated number of neurons.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Effect of chronic ethanol treatment on glycine receptor immunoreactivity. A, immunofluorescent image was obtained from a control neuron using the monoclonal antibody mAb4a to detect glycine receptors. Note the patchy appearance of the receptors. B, graph shows values for background-subtracted averaged intensity levels obtained by drawing regions of interest (under Materials and Methods), which included the cell's soma under both experimental conditions. The bars are mean ± S.E. obtained from 17 DIV neurons.

Effects of Strychnine on Glycine-Activated Current in Control and Treated Neurons. The traces in Fig. 4A show the inhibitory effect of several concentrations of strychnine on the current elicited by 50 µM glycine. Analysis of the concentration-response relationships showed that the potency of strychnine was reduced by 29% after the treatment (Fig. 4A). For instance, the IC₅₀ obtained in parallel cultures was 14 ± 2 nM in control (n = 5) and 18 ± 6 nM in treated neurons (n = 6, P > .05). The solid lines are the best fit to the data points and show that the effect of strychnine on the glycine receptor was well described by a single-site isoterm.

View larger version (29K): [in this window] [in a new window]   Fig. 4.   Effect of chronic ethanol treatment on the sensitivity of the glycine receptor to strychnine. A, traces show responses activated with 50 µM glycine alone and in the presence of increasing concentrations of strychnine. B, graph illustrates the effect of increasing concentrations of strychnine in control () and treated () 15 DIV neurons. The IC50 values were 14 ± 2 and 18 ± 6 nM in control and treated neurons, respectively. Each symbol represents the mean ± S.E.

Effects of Zn²⁺ on Glycine and GABA_A Currents. Similar to previous studies (Laube et al., 1995; Tapia and Aguayo, 1998), we found that low micromolar concentrations of Zn²⁺ enhanced the amplitude of the glycine current in a concentration-dependent manner (Fig. 5B). For example, at a concentration of 10 µM, Zn²⁺ potentiated the response induced by glycine (25 µM) to 143 ± 33% of control. In parallel cultures with ethanol, Zn²⁺ potentiated the current only to 118 ± 13% of control (P > .05). This apparent reduction on the positive allosteric effect contrasts with the similar inhibitory activity on the receptor obtained at a higher concentration. For example, the glycine current was inhibited to about 60% of control in both groups of cells with 1 mM Zn²⁺.

View larger version (30K): [in this window] [in a new window]   Fig. 5.   Effect of chronic ethanol treatment on the sensitivity of the receptors to Zn2+. A, current traces illustrate the potentiating effect of 10 µM Zn2+ and the inhibiting action of 1000 µM Zn2+ on the glycine-activated current. B, data illustrate the effects of several concentrations of Zn2+ in control () and treated () neurons. Note that although chronic ethanol attenuated the positive effect of low Zn2+ concentrations it did not affect its inhibitory action at higher concentrations. C, columns illustrate the peak amplitude of the GABA current activated by 20 µM GABA in the presence of 50 µM Zn2+ in control and chronic ethanol-treated neurons. Each data point or column represents the mean ± S.E.

Effects of Picrotoxin on Glycine Current in Control and Treated Neurons. Immature neurons display picrotoxin-sensitive glycine receptors (Tapia and Aguayo, 1998). It is thought that this noncompetitive antagonist reports the presence of homomeric glycine receptors (Pribilla et al., 1992). Therefore, to determine whether chronic ethanol can alter the sensitivity of the receptor to this antagonist, we examined the effect of 10 µM picrotoxin on the current activated by 50 µM glycine in 17 DIV neurons (Fig. 6). The amplitude of the glycine current in control neurons was reduced to 79 ± 3% (n = 5) of control, indicating the presence of an immature receptor. Interestingly, cells treated with ethanol in parallel cultures from the same litters displayed a complete insensitivity to picrotoxin (98 ± 4% of control, n = 5, P < .05).

View larger version (22K): [in this window] [in a new window]   Fig. 6.   Effect of picrotoxin on control and treated neurons. A, traces illustrate the inhibitory effect of 10 µM picrotoxin on the glycine current amplitude. B, data were obtained in 17 DIV neurons and illustrate the effect of 10 µM picrotoxin on the current activated with 50 µM glycine. Each symbol represents the mean ± S.E. *P < .05.

Effects of Positive Allosteric Ligands on GABA_A Current in Control and Treated Neurons. Pentobarbital was able to enhance the amplitude of the GABA response in a concentration-dependent manner, with maximal responses at 200 µM (Fig. 7A). The value for the EC₅₀ of pentobarbital was 35 ± 8 and 33 ± 6 µM in control (n = 6) and treated (n = 5) neurons, respectively. The Hill coefficient obtained from individual neurons was 1.5 ± 0.6 for control cells and 1.5 ± 0.5 for ethanol-treated cells. Similar to pentobarbital, diazepam also enhanced the amplitude of the GABA response in a concentration-dependent manner in control and chronic ethanol-treated neurons. We found that 10 µM diazepam produced a maximal response on the receptor and for this reason the effects of chronic ethanol treatment were studied with this concentration. Diazepam potentiated the GABA current by 49 ± 8% (n = 12) above control. After treatment with chronic ethanol, diazepam potentiated the GABA current by 38 ± 5% (n = 8) above control, which was smaller but not statistically different from control neurons. In agreement with these small changes on the activity of diazepam, we found that the binding of Bodipy RO-1986, a fluorescent derivative of the benzodiazepine agonist dediethyl fluorazepam, was unchanged after chronic ethanol treatment (107 ± 5% from control, n = 6).

View larger version (29K): [in this window] [in a new window]   Fig. 7.   Effect of chronic ethanol treatment on the positive modulators of GABAA receptors. A, graph shows the normalized amplitude of the Cl current obtained with increasing concentrations of pentobarbital in control () and chronic ethanol-treated () neurons. The solid line is the best fit with an EC50 of 35 ± 8 µM and a Hill coefficient of 1.5 ± 0.6. In the presence of ethanol these values were 33 ± 6 µM and 1.5 ± 0.5, respectively. Typical current traces obtained with 5 µM GABA in the absence and presence of 50 µM pentobarbital are shown. B, graph shows a plot of the normalized current amplitude obtained with increasing concentrations of Cl-218,872 in control () and treated () neurons. The solid line in the control data is the best fit with an EC50 of 741 ± 15 nM and a Hill coefficient of 1.1 ± 0.03. In treated neurons these values were 1140 ± 112 nM and 1.4 ± 0.2, respectively. Each symbol represents the mean ± S.E. The inset shows typical current traces obtained with 5 µM GABA in the absence and presence of 10 µM Cl-218,872.

	Discussion

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Effects of Chronic Ethanol on Glycine Receptors. To our knowledge this is the first study dealing with the effect of chronic ethanol on glycine receptors. Spinal glycine receptors suffer several changes on their functional properties during development. For instance, it has been reported that while the sensitivity of the receptor to glycine and strychnine increases, the sensitivity to picrotoxin decreases (Tapia and Aguayo, 1998). These changes were previously interpreted in terms of a developmentally regulated switch between ₂-homomeric to ₁-heteromeric receptors (Pribilla et al., 1992; Schmieden et al., 1992). Interestingly, our study suggests that chronic ethanol treatment can alter this developmentally controlled change. First, an interesting finding was the decrease in the sensitivity of the glycine receptor to picrotoxin after treatment. Because it is known that the -subunit is capable of conferring picrotoxin resistance to the glycine receptor (Pribilla et al., 1992), we suggest that this is related to an accelerated expression of the -subunit and current studies in our laboratory are dealing with this possibility. Second, we found that treated neurons showed a small but consistent decrease in the sensitivity of the glycine receptor to strychnine together with a 20% reduction in the sensitivity to low concentrations of Zn²⁺. Therefore, the present results can be viewed in terms of a reduction on the expression of ₁-subunits because this is responsible for the high receptor sensitivity to both agents (Schmieden et al., 1992; Laube et al., 1995). Although experimental confirmation for this possibility is necessary, similar interpretations have been previously used to correlate physiological with structural properties of receptors (Kapur and Macdonald, 1996; Tapia and Aguayo, 1998).

Effects of Chronic Ethanol on GABA_A Receptors. It was surprising to find that the sensitivity of the GABA_A receptor to diazepam, Cl-218,872, and pentobarbital was not markedly altered after chronic ethanol treatment. This is pharmacologically relevant because it is known that ethanol, benzodiazepines, and barbiturates develop significant cross-tolerance after chronic administration of any of these compounds (Harris, 1990). The low sensitivity of spinal GABA_A receptors to acute applications of ethanol (Aguayo et al., 1996), together with the unchanged sensitivity to diazepam and pentobarbital after chronic ethanol exposure, is thus in line with the lack of cross-tolerance at this cellular level.

Physiological Relevance of Chronic Ethanol Effects on Inhibitory Receptors in Spinal Interneurons. Our results dealing with the GABA_A receptor did not provide any cellular mechanism that could explain tolerance after chronic ethanol treatment. This is particularly interesting because GABA_A receptors have been postulated to be important in this phenomenon. For instance, previous studies showed a reduction in ₁- and ₂-subunits and an increase in the ₆-subunit mRNA of the GABA_A receptor in cerebral cortex and cerebellum after chronic ethanol (Morrow et al., 1990; Buck et al., 1991). The lack of physiological changes in our study when compared with these previous reports can be interpreted in two ways. First, changes in the level of the message are unable to alter the physiological properties of the GABA_A receptors. Second, there are regional differences to ethanol sensitivity, with spinal neurons being much more resistant. In any case, our detailed analysis of chronically treated neurons, using an ethanol concentration able to induce tolerance and physical dependence (Pohorecky and Roberts, 1992), failed to show significant changes on the physiological properties of GABA_A receptors. No other studies are presently available to determine whether our finding is ubiquitous.