Introduction

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Protein phosphorylation regulates both voltage- and ligand-gated channels (Swope et al., 1999). For example, the glycine receptor-chloride channel complex is phosphorylated by several protein kinases, including cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) (Song and Huang, 1990; Ruiz-Gomez et al., 1994; Aguayo et al., 1996). The glycine receptor consists of two main subunits,  (48 kDa) and  (58 kDa), forming a pentameric channel structure (Betz, 1991). PKC and PKA appear to phosphorylate specifically the ₁ subunit (Vaello et al., 1994). Functional studies of the phosphorylation of glycine receptors show that PKA and PKC have different effects on glycine responses depending on the type of preparations involved (Huang, 1990; Uchiyama et al., 1994; Vaello et al., 1994; Schonrock and Bormann, 1995; Aguayo et al., 1996; Song and Tapia et al., 1997; Ren et al., 1998; Albarran et al., 2001).

Immunological and molecular cloning studies reveal that glycine receptors are widely distributed not only in the spinal cord and brain stem, but throughout the mammalian central nervous system, including the ventral tegmental area (VTA) (Betz, 1991; Ye et al., 2001a, 2001b). The VTA contains the cells of origin of the mesolimbic system, which plays a pivotal role in the mediation of the rewarding effects of drugs of abuse, including ethanol (Betz, 1991; Gatto et al., 1994; Ye et al., 2001a, 2001b). Recent experiments in this laboratory have revealed that glycine-mediated responses can be recorded in the majority of VTA neurons, and that glycine-mediated responses of VTA neurons are sensitive to pharmacologically relevant concentrations of ethanol (Ye et al., 2001a, 2001b). Because glycine has inhibitory effects on neuronal activity, modulation of glycine-receptor function would contribute to the effects of ethanol on the neuronal excitability. However, despite the importance of the VTA in the reinforcement of drug abuse, ethanol effects on the glycine receptors of the VTA have not been well studied.

Several laboratories have shown that ethanol enhances the function of glycine receptors (Celentano et al., 1988; Engblom and Akerman, 1991; Aguayo et al., 1996; Mascia et al., 1998; Ye et al., 2001a). In addition to the potentiating effect, our recent study showed that ethanol (0.1-10 mM) suppresses glycine-activated current in 45% of the VTA neurons freshly dissociated from neonatal rats (Ye et al., 2001b). Previous studies revealed that PKC plays an important role in ethanol modulation of the function of several receptors, including NMDA (Snell et al., 1994), kainate (Dildy-Mayfield and Harris, 1995), 5-HT_2C (Snell et al., 1994), GABA_A (Wafford and Whiting, 1992), and homomeric ₁ subunits of glycine receptors (Mascia et al., 1998). However, the role of these protein kinases in the ethanol inhibition of glycine-receptor function is not clear. In the present article, we show that PKC is involved in ethanol-induced inhibition of glycine-receptor function of neonatal VTA neurons.

	Materials and Methods

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Isolation of Neurons and Electrophysiological Recording. The care and use of animals and the experimental protocol of this study were approved by the Institutional Animal Care and Use Committee of the University of Medicine and Dentistry of New Jersey (protocol 00074). Sprague-Dawley rats (5-14 days old) were decapitated as described earlier (Ye et al., 2001b). The brain was quickly excised, placed into ice-cold saline saturated with 95% O₂ and 5% CO₂, glued to the chilled stage of a vibratome (Campden Instruments, UK), and sliced to a thickness of 300 to 400 µm. Slices were transferred to the standard external solutioncontaining 1 mg pronase/6 ml and saturated with O₂and incubated at 31°C for 20 min. After an additional 20-min incubation in 1 mg of thermolysin/6 ml, the VTA was identified medial to the accessory optic tract and lateral to the fasciculus retroflexus under a dissecting microscope. Micro-punches of the VTA were isolated and transferred to a 35-mm culture dish. Mild trituration through heat-polished pipettes of progressively smaller tip diameters dissociated single neurons. Within 20 min of trituration, isolated neurons attached to the bottom of the culture dish and were ready for electrophysiological experiments. Based on morphology under the light microscope, the cells acutely isolated from VTA were of two types: bipolar and multipolar. The majority was bipolar with 1 to 3 dendritic processes emerging from each end of the fusiform soma (20-40 µm in length and 15-25 µm in diameter). The multipolar neurons were larger with a diameter of 35 to 60 µm and four to five major dendrites. In agreement with a recent report, most of the cells were tyrosine hydroxylase-positive (Brodie et al., 1999). There were no appreciable differences in the response of these two groups of neurons to ethanol.

Chemical Application. Solution of N-(2[methylamino]-ethyl)-5-isoquinoline sulfonamide dihydrochloride (H-89), PKCI (19-31), and chelerythrine (Calbiochem, San Diego, CA), ethanol (95%, prepared from grain, stored in glass bottles; Pharmco, Brookfield, CT), glycine, gramicidin, ATP, phorbol-12-myristate-13-acetate (PMA), and all other chemicals (Sigma, St. Louis, MO) were prepared on the day of the experiment. PMA was prepared in dimethyl sulfoxide and diluted to its final concentration in standard external solution. The final concentration of dimethyl sulfoxide was always less than 0.1%; it did not induce any ionic current and had no effect on the glycine response at the concentrations used. Solutions were applied to a dissociated neuron with a superfusion system having a multibarreled pipette as described in Ye et al. (2001b). The tip of the superfusion pipette was normally placed 50 to 100 µm away from the cell, a position that allowed rapid and uniform drug application without disturbing the mechanical stability of the neuron. This system allows complete exchange of solutions in the vicinity of the neuron within 20 to 35 ms. Throughout all experimental procedures the bath was continuously perfused with the standard external solution. In our perfusion system, we used glass containers and Teflon tubes, instead of plastic ones, to avoid the production of bis(2,3,6,6,-tetramethyl-4-piperridinyl) sebacate (Tinuvin 770), a sterically hindered amine light and radiation stabilizer used in a wide range of plastics. Tinuvin 770 is known to inhibit some receptors such as nicotinic acetylcholine receptors.

Statistical Analysis. Data were statically compared using ANOVA at a significant level of P < 0.05. For all experiments, average values are expressed as mean ± S.E.M. with the number of neurons (n).

	Results

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Ethanol Pretreatment Enhances Ethanol Inhibition of Glycine-Activated Current. Our previous findings on neonatal VTA neurons can be summarized as follows: Most VTA neurons (82%) are sensitive to glycine. All glycine-induced responses were antagonized by 100 nM strychnine. Application of 0.1 to 40 mM ethanol acutely enhances or depresses glycine responses in 35 and 45%, respectively, of VTA neurons (Ye et al., 2001a, 2001b).

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Preincubation of ethanol enhances ethanol inhibition of glycine-activated current. A, current traces a to d were consecutive records obtained from a voltage-clamped neuron of a 10-day-old rat. Glycine current was elicited by 30 µM glycine alone (a) or by glycine coapplied with 1 mM ethanol without (b) and with (c) preincubation of ethanol for 30 s. For all figures, all current traces were obtained with whole-cell recording with 2 mM Mg2+-ATP in pipette at a holding potential of 50 mV. The bars above the current traces indicate the period of perfusion of the indicated chemicals. B, mean (± S.E.M.) inhibition of peak current activated by 30 µM glycine with (++, n = 43) and without (+, n = 22) preincubation of ethanol. C, dependence of enhanced ethanol inhibition on the preincubation time. Mean (± S.E.M., n = 8) inhibition of peak amplitude of current-activated by 30 µM glycine was plotted as a function of preincubation time.

Intracellular ATP Is Required for the Enhancement of Ethanol Inhibition. Increasing evidence has revealed that phosphorylation regulates the sensitivity to ethanol of several proteins (Wafford and Whiting, 1992; Snell et al., 1994; Dildy-Mayfield and Harris, 1995; Aguayo et al., 1996), and that protein kinases regulate ethanol's effects on several receptors (Stubbs and Slater, 1999). Therefore, phosphorylation may be involved in ethanol inhibition of glycine-activated current. If this is the case, ethanol inhibition would be affected by intracellular ATP necessary for the dephosphorylated protein to be converted to the phosphorylated state. To test this hypothesis, we first examined effects of intracellular ATP on glycine-activated current. In agreement with our earlier report (Ren et al., 1998) that in the presence of intracellular ATP, in the majority (69 of 99) of cells examined, glycine-activated current spontaneously increased with time (run-up) and reached a plateau within 20 min. The effects of ethanol were examined after the current was stabilized.

An Increase in PKC Activity Attenuates Glycine-Activated Current. The requirement of Mg²⁺-ATP in the internal pipette solution suggests that protein phosphorylation is responsible for the enhanced ethanol inhibition of glycine-activated current induced by ethanol preincubation. To assess whether alteration of PKC activity is responsible for ethanol inhibition of glycine-activated current in VTA neurons, the effect of PKC activation on this current was examined first. PMA was widely used as a selective activator of PKC. Using human platelets as a model system, Castagna et al. (1982) reported that PMA directly activated PKC in a concentration-dependent manner. The maximum effect of PMA was seen at 10 ng/ml (16.2 nM). To fully activate PKC, 100 nM PMA was extracellularly applied to cells before its coapplication with the agonist. Glycine current was recorded with pipette solution containing 2 mM Mg²⁺-ATP. PMA suppressed glycine-activated current. This suppression increased with the preincubation time and reached the maximum at 1 min. On average, 0.5- and 1-min preincubation of 100 nM PMA suppressed current activated by 30 µM glycine by 19 ± 4% (n = 4) and 28 ± 4% (n = 9), respectively (P < 0.05). A 5-min preincubation of 100 or 300 nM PMA did not significantly increase the suppression (30 ± 6% and 32 ± 5%, respectively, P > 0.05, n = 4). Thus, in the present experimental conditions, 1 min preincubation of 100 nM PMA induced maximal inhibition of glycine-activated current. Therefore, a 1-min preincubation of PMA was chosen in following experiments.

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Activation of PKC depresses glycine-activated current. A, current traces were recorded from single neuron. From left to right, current induced by 30 µM glycine alone (a and e) or together with 7 µM chelerythrine (b, CLT), 100 nM PMA (c), or 100 nM PMA and 7 µM chelerythrine (d), respectively. PMA was applied after 5 min of pretreatment with chelerythrine. B, mean (± S.E.M., n = 9) inhibition of peak current by chelerythrine, PMA alone, or PMA+ chelerythrine.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   PKCI attenuates PMA inhibition of glycine-activated current. A, current traces recorded from a single cell without PKCI in the pipette solution. From left to right, current induced by 30 µM glycine alone (a, c, and e), or together with 100 nM PMA (b and d, ++ paradigm) recorded at the indicated time after rupturing the membrane. B, current traces records from a single cell with 100 nM PKCI in the pipette solution. From left to right, current induced by 30 µM glycine alone (a, c, and e), or together with 100 nM PMA (b and d, ++ paradigm) recorded at the indicated time after rupturing the membrane. C, mean (± S.E.M., n = 5) PMA inhibition of peak current-activated by 30 µM glycine in the absence (control) and presence of pipette PKCI on indicated time after rupturing the membrane. Twelfth minute after rupturing the membrane, PKCI significantly attenuated the inhibition of peak current induced by PMA.

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Effect of okadaic acid on glycine-activated current. A, current traces recorded from the same cell showing current responses to 30 µM glycine alone (a, c, and e), or together with 100 nM PMA (b), or 1 µM okadaic acid (OA, d). PMA and okadaic acid were applied 2 min before it was coapplied with glycine. B, mean (± S.E.M., n = 4) inhibition of peak current induced by PMA or okadaic acid.

PKC Inhibitors Attenuate Ethanol Inhibition of Glycine-Activated Current. The above experiments demonstrate that an increase in PKC activity attenuated glycine-activated current_. Thus, ethanol may suppress glycine-activated current by increasing PKC activity. If it were the case, an inhibition of intracellular PKC activity would be expected to attenuate ethanol inhibition of glycine-receptor function. To test this hypothesis, effects of ethanol were examined on glycine-activated current in cells before and after incubation with chelerythrine. As shown in Fig. 5, incubation with chelerythrine significantly reduced ethanol inhibition of glycine-activated current. On average, 1 mM ethanol inhibited glycine current by 28 ± 2.6%, and 14 ± 2.8% before and after chelerythrine treatment, respectively (P < 0.01, n = 6). To further assess the effect of PKC inhibition on ethanol suppression of glycine-activated current, ethanol effect on glycine-activated current was studied in the absence and presence of PKCI. PKCI (100 nM) was included in the internal pipette solution for conventional whole-cell recording. Currents activated by 30 µM glycine were recorded in the absence and presence of 1 mM ethanol at 1-min intervals beginning immediately after membrane rupture. As mentioned above, the effects of PKCI were small at the first 4 min after rupturing the membrane. The effects of ethanol during this initial time period were considered as control. As expected, PKCI significantly reduced ethanol suppression of glycine-activated current. On average, 1 mM ethanol inhibited current activated by 30 µM glycine by 34 ± 5% within the first 4 min after rupturing the membrane. Ethanol inhibition significantly reduced to 18 ± 4% 12 min after rupturing the membrane (P < 0.01, n = 5, Fig. 6).

View larger version (23K): [in this window] [in a new window]   Fig. 5.   PKC inhibitor chelerythrine attenuates ethanol inhibition of glycine-activated current. A, current traces in response to 30 µM glycine were recorded in the absence (a) and presence of 1 mM ethanol (++ paradigm), before (b) and after (d) treatment of 7 µM chelerythrine (CLT). Trace c was recorded after 5-min incubation of chelerythrine. Glycine current recovered to control 5 min after ethanol- and chelerythrine-containing solutions were washed out (e). B, mean (± S.E.M.) of inhibitions of peak current induced by ethanol or chelerythrine alone and by the mixture from four to six neurons. After treating with chelerythrine, ethanol inhibition was significantly reduced (**, P < 0.01).

View larger version (22K): [in this window] [in a new window]   Fig. 6.   PKCI attenuates ethanol inhibition of glycine-activated current. A, traces a to e were consecutive records from the same cell recorded without PKCI in the pipette solution. From left to right were currents induced by 30 µM glycine alone (a, c, and e), and together with 1 mM ethanol (b and d, ++ paradigm) recorded at the indicated time after rupturing the membrane. B, current traces recorded from single neuron with 100 nM PKCI in the pipette solution. From left to right were currents induced by 30 µM glycine alone (a, c, and e), and together with 1 mM ethanol (b and d, ++ paradigm), recorded at the indicated time after rupturing the membrane. C, mean (± S.E.M.) of ethanol inhibition of peak current-activated by 30 µM glycine in the absence (control) and presence of pipette PKCI from five to six neurons on indicated time after rupturing the membrane. Twelfth minute after rupturing the membrane, PKCI significantly attenuated the inhibition of peak current induced by ethanol.

Effects of PKC Activation on Ethanol Inhibition of Glycine-Activated Current. We have shown that a decrease in PKC activity attenuated ethanol inhibition of glycine-activated current. An increase in PKC activity, therefore, would be expected to alter ethanol inhibition. To assess this possibility, we applied ethanol with different concentrations of PMA (1 and 100 nM) to neurons after obtaining stable glycine-activated current and ethanol inhibition of glycine-activated current. As shown in Fig. 7, A and B, 1 mM ethanol (++ paradigm, Fig. 7A, b) or 1 nM PMA (++ paradigm, Fig. 7A, d) depressed current activated by 30 µM glycine by 26 ± 7% and 18 ± 5% (++ paradigm, n = 5), respectively. A consequent coapplication of 1 mM ethanol (++ paradigm) after preincubation with 1 nM PMA depressed the glycine current to a significantly greater extent, by 44 ± 5% (Fig. 7A, e, and B; P < 0.01, n = 5).

View larger version (22K): [in this window] [in a new window]   Fig. 7.   Effects of PKC activation on ethanol inhibition of glycine-activated current. A, traces a to f were consecutive records from the same cell showing current response to 30 µM glycine alone (a, c, and f) and ethanol inhibition before (b) and after 1 nM PMA treatment (e). Ethanol or 1 nM PMA alone suppressed glycine-activated current. Ethanol + 1 nM PMA suppressed the current to a significantly greater extent (P < 0.01, n = 5). B, mean (± S.E.M.) inhibition of glycine-current induced by 1 mM ethanol alone (1 mM ethanol), 1 nM PMA, and ethanol + PMA. PMA treatment greatly enhanced ethanol inhibition (**, P < 0.01, n = 5). C, traces a to h were consecutive records from the same cell showing current responses to 30 µM glycine alone (a, d, and h) and ethanol inhibition (+ paradigm) before (b) and after (f) 100 nM PMA treatment, or ethanol inhibition with preincubation of ethanol (++ paradigm) before (c) and after (g) 100 nM PMA treatment. D, Mean (± S.E.M., n = 6) inhibition of glycine-current induced by 1 mM ethanol alone (ethanol + and ethanol ++), 100 nM PMA, ethanol + PMA (+), and ethanol + PMA (++). After treatment of 100 nM PMA, whereas ethanol inhibition induced by coapplication of ethanol with the agonist (+ paradigm) remained, the enhanced ethanol inhibition induced by preincubation of ethanol (++ paradigm) was not seen.

Suppression of PKA Has No Effect on Ethanol Inhibition of Glycine-Activated Current. Previous studies revealed that an activation of PKA increased glycine-activated current in various tissues including VTA neurons (Song and Huang, 1990; Vaello et al., 1994; Ren et al., 1998). Thus, a decrease in PKA activity may also be responsible for ethanol inhibition of glycine-activated current. To assess this possibility, we compared ethanol inhibition before and after incubation of H-89, a specific PKA antagonist. As shown in Fig. 8, H-89 at the concentration of 1 µM, which was reported to induce 90% inhibition of PKA activity (Chijiwa et al., 1990) did not significantly decrease glycine-activated current. This suggests that a decrease in PKA activity did not alter glycine-activated current in the present experiment condition. Ethanol (1 mM) suppressed glycine-activated current to a similar extent, by 30 ± 3% (Fig. 8, A, c, and B, b) and 29 ± 2% (Fig. 8, A, b and d, and B, a and c), before and after 5-min incubation of H-89, respectively (P > 0.05, n = 5).

View larger version (23K): [in this window] [in a new window]   Fig. 8.   PKA inhibitor has no effect on glycine-activated current and ethanol inhibition. A, current traces in response to 30 µM glycine were recorded in the absence (a and e) and presence of 1 mM ethanol before (b) and after (d) application of 1 µM H-89. Trace c was recorded after 5 min of treatment with 1 µM H-89. Glycine current has no appreciable change. B, mean (± S.E.M.) of effects of H-89 on ethanol inhibition of the peak current. H-89 did not significantly change ethanol inhibition of glycine-activated current (P > 0.05, n = 5).

	Discussion

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Our principle finding is that PKC is involved in the ethanol-induced inhibition of glycine-activated currents in VTA neurons of neonatal rats. This is an extension of previous work studying ethanol-glycine interactions.

Several studies on many preparations showed that ethanol and other anesthetics enhance neuronal responses to glycine (Celentano et al., 1988; Engblom and Akerman, 1991; Aguayo et al., 1996; Mascia et al., 1998; Valenzuela et al., 1998). In our own study, ethanol potentiated, depressed, or had no effect on glycine-activated responses of 35, 45, and 20%, respectively, of neonatal VTA neurons (Ye et al., 2001a, 2001b). Likewise, similar studies have revealed that ethanol potentiated, inhibited, or had no effect on GABA_A receptor-mediated synaptic responses of neurons from different brain regions, as well as within a single neuronal population (Weiner et al., 1997; Harris, 1999). Several factors, such as subunit composition of the receptors, its phosphorylation state, and the methods for GABA and ethanol application, may contribute to the variability. These same factors may also be involved in ethanol-glycine receptors interactions.

Molecular cloning studies have revealed a developmental heterogeneity of glycine-receptor subunits. For example, the ₂ subunit is present in fetus for only 2 to 3 weeks after birth. Afterward, the ₁ subunit becomes dominant (Betz, 1991). Recent studies on glycine receptors in expression system revealed difference in sensitivity to ethanol between homomeric ₁ and ₂ receptors (Mascia et al., 1998). In the current study, the VTA cells are clearly immature. The immature glycine receptors may be dominant in these neurons. This immaturity of the glycine receptors may contribute to the various responses to ethanol.

As an alternatively, the various responses to ethanol could indicate regulation of the channel protein by a process such as phosphorylation (Mascia et al., 1998; Stubbs and Slater, 1999; Swope et al., 1999). In VTA neurons, ethanol pretreatment potentiated ethanol's effect on glycine-receptor function. A similar phenomenon was reported for ethanol modulation of the function of NMDA receptors in granule cells (Popp et al., 1999) and of nicotinic acetylcholine receptors in cortical neurons (Aistrup et al., 1999). In VTA neurons, this enhancement depends on both ethanol pretreatment time and intracellular ATP, suggesting the involvement of intracellular factors, such as protein kinases in ethanol-glycine receptor interaction.

The fact that PMA, a PKC activator depressed glycine-activated current and the PKC inhibitors, chelerythrine, and PKCI (19-31) attenuated ethanol inhibition of glycine-activated current, whereas a PKA inhibitor (H-89) had no effect on ethanol inhibition of glycine-activated current, suggested that the activation of PKC, (and not of PKA) mediated ethanol inhibition of glycine-activated current in VTA neurons. These data are consistent with previous studies in Xenopus oocyte (Uchiyama et al., 1994; Vaello et al., 1994) and in spinal neurons (Aguayo et al., 1996; Tapia et al., 1997), showing that activation of PKC inhibited glycine current. However, our data are inconsistent with data from hippocampal (Schonrock and Bormann, 1995) and trigeminal neurons (Gu and Huang, 1998), where PKC potentiated glycine-activated currents. Our result regarding that PKA inhibitors did not alter glycine-receptor sensitivity to ethanol, on the other hand, is consistent with previous studies in mouse spinal cord neurons (Aguayo et al., 1996) and in homomeric ₁ glycine-receptors expressed in Xenopus oocytes (Mascia et al., 1998).

The data of ethanol-PKC-glycine receptor interaction of VTA neurons are consistent with previous finding in homomeric ₁-glycine receptors that PKC inhibitors blocked partially the effect of ethanol on glycine currents (Mascia et al., 1998), and that PKC inhibitors attenuated ethanol action but not the action of glycine on the receptors. One possible explanation, as proposed by Mascia and colleagues (1998) is that ethanol binds to a site on the glycine-receptor subunit, which is formed in part by the 267 residue located between transmembranes 2 and 3 (Mihic et al., 1997) and that receptor phosphorylation alters the affinity or efficacy of this interaction. However, in contrast to chelerythrine, which did not affect the glycine response, PMA significantly suppressed the glycine-activated current. Our results raise the question of why PKC activator can, but PKC inhibitor cannot, modulate the glycine response. One possible explanation is that in VTA neurons, the basic level of PKC activity is relatively low.

The data showed that 1 nM PMA enhanced ethanol inhibition of glycine-activated current and imply that a partial increase of PKC activity had a synergetic effect of ethanol. However, the enhanced ethanol inhibition induced by ethanol pretreatment was not seen after the treatment with 100 nM PMA, suggesting that an occlusion had occurred. Taken together, these results suggest that ethanol and PMA share the same pathway in the inhibition of the glycine receptors.

Although PMA depressed glycine-activated currents in both the VTA and the spinal neurons, the responses to okadaic acid were different. Okadaic acid depressed glycine-activated current of freshly isolated VTA neurons but potentiated glycine current of cultured spinal neurons (Tapia et al., 1997). The reason for the difference is unknown, however, it may be attributable to the difference between the preparations used. The current experiments also noted that the inhibition of glycine currents induced by okadaic acid was weaker than that induced by PMA. Again, the mechanisms underlying the difference are unclear. However, a possible explanation is that because the protein phosphatases inhibited by okadaic acid are not specific to PKC, they may also affect the activity of PKA and other kinases, and that different kinases may have different effects on glycine receptors. The effect of okadaic acid on glycine-activated current may reflect a summation of the activation of all kinases sensitive to the protein phosphatases inhibited by okadaic acid.

Protein phosphorylation is a major mechanism for regulation of receptor function and synaptic transmission in the central nervous system. PKC plays a pivotal role in the biochemical pathways that transfer information into cells by phosphorylating and regulating the function of its target proteins. Activation of PKC has been shown to play a role in ethanol's actions on a number of receptor/channels including Ca²⁺-activated K⁺ channels, BK channels, Ca²⁺ channels (Harris, 1999), NMDA receptors (Snell et al., 1994), -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid/kainate receptors (Dildy-Mayfield and Harris, 1995), and glycine receptors (Mascia et al., 1998). Taken together, the results of this study support the notion that activation of PKC appears to be a common mechanism for ethanol action on several types of ionotropic and metabotropic receptors in the central nervous system. In vivo study (Harris et al., 1995) has provided an important link between the pharmacological action of ethanol and PKC function. PKC may be involved in the processes of ethanol intoxication.

In VTA neurons, however, manipulation of PKC activity affected only a part of the ethanol inhibition. Maximal activation of PKC by 100 nM PMA abolished only the enhanced ethanol inhibition induced by ethanol pretreatment, without affecting the inhibition induced by coapplication of ethanol with the agonist. This is consistent with a previous report that PKC inhibitors and mutagenesis of the phosphorylated residue were not able to completely block the action of ethanol on the glycine responses of homomeric ₁ glycine receptors (Mascia et al., 1998). In VTA neurons, the remaining part of the ethanol-induced inhibition depended on neither intracellular ATP nor ethanol pretreatment, suggesting a direct action of ethanol on the receptors. These data support further the notion that there are direct and indirect components of ethanol-induced inhibition.

Several studies have shown that ethanol may interact directly with ion channels (Harris, 1999). For example, the lack of effect of long-chain alcohols, or so-called "cut-off" phenomenon has been served as a strong evidence for the alcohol to act directly with the receptor proteins (Li et al., 1994). Recent studies on mutated receptors have suggested an alcohol-binding site on the  subunits of glycine receptors (Mihic et al., 1997; Ye et al., 1998). This ethanol-glycine receptor direct interaction mechanism is further supported by our recent experiments showing that n-alcohol inhibition of glycine-receptor function increases with the increase of the carbon number, a cutoff occurred at about nonanol (unpublished observation).

In summary, the present report showed that there are direct and indirect mechanisms underlying ethanol inhibition of glycine responses, and PKC may be involved in the indirect one. Our study confirms and extends upon previous findings of ethanol-glycine receptor interactions from recombinant expression systems or native preparations via electrophysiological recording and neurochemical methods. The mechanisms of ethanol modulation of ligand-gated receptor/channels merit further exploration. Further investigation of ethanol modulation of other protein kinase pathways will unveil more diverse mechanisms underlying its neurotoxicity.