Introduction

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Ethanol is the most abused substance in the United States. There is now compelling evidence that ethanol directly and/or indirectly affects many receptor/ion channels, including N-methyl-D-aspartate (NMDA), non-NMDA (AMPA), GABA_A, glycine, 5-HT₃, and nicotinic acetylcholine receptors (Narahashi et al., 2001). Modulation of these receptors by ethanol may be responsible for its behavioral effects. Because ethanol acts at many sites in the central nervous system, studies of the effects of ethanol on interactions between excitatory and inhibitory synaptic mechanisms are crucial.

The ventral tegmental area (VTA) contains the cells of origin of the mesolimbic system, which is important for the rewarding properties of drugs of abuse like ethanol (Gatto et al., 1994; Wise, 1996). There are two main types of neurons in the VTA: dopamine and nondopamine neurons (Lacey et al., 1989; Johnson and North, 1992). Both receive monosynaptic glutamatergic innervation from prefrontal cortex and have NMDA and non-NMDA receptors (Wang and French, 1993, 1995). According to Floresco et al. (2001), glutamatergic afferents from the hippocampus to the nucleus accumbens strongly excite VTA dopamine neurons.

We have already reported that glycine-activated current (I_Gly) can be recorded in most VTA neurons, and that ethanol (0.1-40 mM) potentiates I_Gly in VTA neurons of 5- to 14-day-old rats and thus alters their excitability (Ye et al., 2001a). Bearing in mind that ethanol alters intracellular Ca²⁺ (for review, see Little, 1991; Simasko et al., 1999; Mennerick and Zorumski, 2000), interactions between ethanol and glycine receptors may involve mechanisms linked to intracellular Ca²⁺. Three recent studies have reported that glutamate-induced Ca²⁺ entry greatly potentiates I_Gly in spinal neurons or oocytes expressing glycine receptors (Xu et al., 1999, 2000; Fucile et al., 2000). However, the effects of glutamate on VTA glycine receptors have not been examined. In view of the important function of glycine receptors in the VTA and the pivotal role of the VTA in drug addiction, we initiated the current study on freshly dissociated VTA neurons to examine: 1) the enhancement of I_Gly by glutamate and 2) the effects of ethanol on this potentiating action of glutamate.

	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 number 00074). Sprague-Dawley rats (5- to 14-day-old) were decapitated as described earlier (Ye et al., 2001a). The brains were quickly excised, placed into ice-cold saline saturated with 95% O₂ and 5% CO₂, glued to the chilled stage of a Vibratome (Campden Instruments Ltd., Loughborough, Leicestershire, UK), and sliced to a thickness of 300 to 400 µm. Slices were transferred to the standard external solution containing 1 mg of pronase/6 ml and saturated with O₂ and incubated at 31°C for 20 min. After 20 min of additional 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.

Chemical Applications. Solutions of agonist, antagonists, and ethanol were prepared on the day of experimentation. Glycine, strychnine, glutamate, BAPTA, and EGTA were obtained from Sigma-Aldrich (St Louis, MO), and 1-[N,O-bis-(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62) was obtained from Calbiochem (San Diego, CA). Ethanol (95%, prepared from grain), obtained from Pharmco Products Inc. (Brookfield, CT), was stored in glass bottles. Solutions were applied via a multibarreled pipette (as described previously: Ye et al., 2001a), the tip of which was usually placed 50 to 100 µm from a dissociated cell. While maintaining recording stability, this system allows complete exchange of solutions in its vicinity within 10 ms. A conditioning pulse of glutamate (1 mM for 1-3 s) was immediately followed by a brief pulse of glycine (30 µM, unless otherwise indicated). In some experiments, the extracellular Ca²⁺ concentration ([Ca²⁺]_o) was raised to 12 mM locally by superfusing the cell body only with a solution containing 12 mM Ca²⁺ (only these barrels and their respective reservoir syringes contained 12 mM Ca²⁺; the other barrels and syringes contained 2 mM Ca²⁺).

Data Analyses. Whole-cell current decays were fitted by a Chebychev algorithm (pCLAMP). Concentration-response data were analyzed with a nonlinear curve-fitting program (Sigma Plot; Jandel Scientific, San Rafael, CA). Data were statistically compared using Student's t test at a significance level of P < 0.05, unless otherwise indicated. For all experiments, average values are expressed as mean ± S.E.M., with the number of neurons indicated in brackets.

	Results

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Glutamate Potentiates I_Gly. In agreement with our previous observations, most neonatal VTA neurons (82%) were sensitive to glycine. Glycine-induced current (I_Gly) was antagonized by 0.1 µM strychnine (Ye et al., 1998). Glutamate (0.1 and 1 mM) elicited inward currents in all VTA neurons tested. At a holding potential of -50 mV, larger peak currents were induced by 30 µM glycine (520 ± 68 pA, n = 42) than by 1 mM glutamate (338 ± 44 pA, n = 41). To examine the effect of glutamate on I_Gly, a pulse of glycine (10-1000 µM) was preceded by a brief conditioning pulse of glutamate (1-3 s). For this purpose, 1 mM glutamate was routinely used, because of its very predictable action and its previous use in comparable experiments (Fucile et al., 2000). However, substantial potentiation of I_Gly could be obtained with 100 µM glutamate (see below).

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Glutamate prepulse enhances IGly by a non-voltage-dependent mechanism. A, conventional whole-cell voltage-clamp recording with pipette containing 11 mM EGTA from a VTA neuron of a 13-day-old rat. IGly was elicited by 30 µM glycine alone (a and c) and immediately after a conditioning prepulse of glutamate (Glu) (b; 1 mM, 2 s). Dotted lines d, e, and f indicate how current amplitude was measured: IGlu between baseline d and current peak e, peak IGly between baseline d and f. B, similar recording from another cell shows rapid return of glutamate current to baseline after glutamate washout; in B-c, traces a and b are superimposed. This demonstrates the minimal overlap of glutamate tail current with the initial portion of IGly. Calibration: 10 s for traces a and b, 2.5 s for c. C, histogram shows variability of 1 mM glutamate-induced potentiation of IGly (evoked by 30 µM glycine) in a population of 72 cells, all recorded with 11 mM EGTA-containing pipettes. D and E, glycine current-voltage relation was studied in a whole-cell recording with pairs of voltage ramps (from +40 to -80 mV), applied at a rate of 1 mV/10 ms. In each trace, the first, very small current ramp measured background/leakage current; the second measured the total current during 30 µM glycine application. The data plotted in F and G were obtained by subtracting the currents elicited by the first ramp from the currents elicited by the second ramp during glycine application, without (F) and with (G) the 1 mM glutamate prepulse. These current-voltage plots show that glutamate did not change the reversal potential of IGly. For this and the following figures, all the current traces were recorded at a holding potential of -50 mV. The boxes above or below each current trace indicate the duration of drug applications: blank and black boxes represent glycine and glutamate, respectively.

Involvement of Ca²⁺ in the Glutamate-Induced Potentiation of I_Gly. According to recent reports, Ca²⁺ exerts a powerful and rapid modulation of glycine receptor/channels (Xu et al., 1999, 2000; Fucile et al., 2000). The following results suggest that Ca²⁺ also plays a role in I_Gly potentiation in VTA neurons.

Potentiation Was Greater when Extracellular Ca²⁺ Was Increased to 12 mM. As shown in Fig. 2, when [Ca²⁺]_o was raised locally to 12 mM, I_Glu increased by 65 ± 4% (P < 0.01, n = 4) and the magnitude of I_Gly potentiation by glutamate by 55 ± 10% of control tests of glutamate in 2 mM [Ca²⁺]_o (Fig. 2B, P < 0.01, n = 4). This effect of [Ca²⁺]_o was reversible: both I_Glu and the potentiation of I_Gly returned to control values when 2 mM [Ca²⁺]_o was restored.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Glutamate-induced potentiation of IGly is enhanced by raising [Ca2+]o but is unaffected by KN-62, a blocker of CAMKII. A, current traces from a neuron of an 8-day-old rat show IGly elicited by 30 µM glycine before (a and c) and immediately after a brief conditioning pulse of glutamate, in the presence of 2 mM (b) or 12 mM (d) [Ca2+]o. Note, both IGlu and the potentiation are larger in the presence of 12 mM [Ca2+]o. B, bar graphs summarize increases in IGlu and its potentiation produced by 12 mM [Ca2+]o in 4 neurons; 100% represents corresponding data recorded in 2 mM [Ca2+]o. Note that [Ca2+]o was raised to 12 mM only in the vicinity of the cell body. All these recordings were made with 11 mM EGTA-containing pipettes. C, current traces from neuron of a 7-day-old rat show that 8-min applications of 5 µM KN-62 did not alter the glutamate-induced potentiation of IGly (recordings with 4 mM EGTA-containing pipettes). D, bar graphs summarize data obtained in similar tests on another five VTA cells. For this and the following figures, bar graphs and error bars represent means and S.E.M.

Reducing the Internal Concentration of EGTA Enhanced Both the Magnitude and Duration of Glutamate's Effect. In recordings with pipettes containing 4 mM EGTA (instead of the usual 11 mM), the potentiation of peak I_Gly induced by a 3-s glutamate prepulse reached 405 ± 128% (n = 14; median 164%) of control. Note the unusually large effects of glutamate in traces A of Fig. 3, obtained with an electrode containing 4 mM EGTA; the potentiation persisted for more than 8 s (Fig. 3B). When I_Gly was recorded with such pipettes, even 100 µM glutamate induced a marked potentiation (146 ± 9%, n = 4; Fig. 3, C and D).

View larger version (17K): [in this window] [in a new window]   Fig. 3.   When recording with pipettes containing 4 instead of 11 mM EGTA, potentiation by glutamate was enhanced, could be elicited with 100 µM glutamate, but was reduced when pipettes also contained 10 mM BAPTA. A, current traces obtained with pipette containing 4 mM EGTA from a VTA neuron of a 10-day-old rat. B, time course of change of peak IGly after glutamate prepulse from neuron illustrated in A. Similar data were obtained from another five cells. C, current traces obtained from a VTA neuron of a 15-day-old rat produced by 30 µM glycine alone (a and c) and immediately after a conditioning prepulse of 100 µM glutamate (b). D, histogram shows mean potentiation of IGly (evoked by 30 µM glycine) by 100 µM glutamate (n = 4). E, current traces obtained from a VTA neuron of an 11-day-old rat with 10 mM BAPTA-containing pipette illustrate progressive reduction of glutamate-induced potentiation after 4 min of whole-cell recording (traces a and b) and after 16 min (traces c and d). F, mean data from four cells show significantly reduced potentiation by 16 min. All data in this figure were obtained with 4 mM EGTA pipette solution.

There Was Less Potentiation when Recording with Electrodes Containing 10 to 30 mM BAPTA. In these recordings, the potentiation induced by glutamate decreased with time: 4 min after the start of whole-cell recording, I_Gly was potentiated to 153 ± 8% of control, but by 16 min, to only 126 ± 4% of control (paired t test, P = 0.029, n = 4; cf. traces in Fig. 3E and histograms in Fig. 3F). Presumably, this reflects the time required for BAPTA to equilibrate at the intracellular site of Ca²⁺ action.

Glutamate-Induced Potentiation Is Sensitive to Glycine Concentration. Glutamate could augment I_Gly either by increasing the number or conductance of functional glycine receptor channels or by modifying their sensitivity to glycine. To distinguish between these possibilities, we examined the effect of glutamate on I_Gly induced by 10 to 1000 µM glycine. The traces in Fig. 4 show I_Gly evoked by 30, 100, and 300 µM glycine, in the absence (Fig. 4A) and presence (Fig. 4B) of prepulses of glutamate (1 mM). Glutamate strongly potentiated I_Gly induced by submaximal concentrations of glycine (30 and 100 µM, Fig. 4, a and b); but it had a weaker effect on I_Gly induced by supramaximal concentrations of glycine (300 µM; Fig. 4C). On average, 1 mM glutamate potentiated peak I_Gly elicited by 30, 100, 300, and 1000 µM glycine to 180 ± 21, 152 ± 22, 118 ± 22, and 121 ± 20%, respectively (n = 4, Fig. 4D)

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Glutamate prepulse reduces EC50 and increases maximal IGly. A and B, current traces (obtained from a neuron of an 11-day-old rat) induced by increasing glycine concentrations without (A) and with (B) preceding pulse of 1 mM glutamate. C, concentration-response curves for glycine alone (open circles) or glycine after glutamate prepulse (filled circles), from the same neuron as in A and B. Continuous lines are fits of the Hill equation: I = Imax/[1 + (EC50/C)n], where I is IGly, Imax is maximal IGly, C is glycine concentration, EC50 is glycine concentration for half-maximal response, and n is the Hill coefficient. D, mean potentiation of IGly (four neurons) by 1 mM glutamate, at various concentrations of glycine. Before pooling data, amplitudes of peak IGly were normalized to response elicited by 30 µM glycine alone.

Glutamate Alters the Kinetics of I_Gly. Changes in either agonist affinity or channel opening efficacy can alter the EC₅₀ values of agonists (Colquhoun, 1998). Indeed, data from human embryonic kidney-AMPA cells transfected with H1 demonstrate different kinetics of I_Gly before and after a glutamate prepulse (Fucile et al., 2000). Therefore, we examined I_Gly channel activation, deactivation, and desensitization, before and after glutamate conditioning pulses. To allow accurate measurement within the limits of the fast perfusion system (time constant of ~10 ms), we applied glycine at a concentration of 30 µM (Fig. 5A). As previously observed (Ye et al., 2001a), both the onset and the decay of I_Gly could be fitted by a single exponential function (Fig. 5, C and D). The activation time constant (_on) was significantly shortened by a glutamate prepulse, from 340 ± 14 ms to 183 ± 22 ms (paired t test, P < 0.01, n = 8). In contrast, glutamate prolonged the deactivation time constant (_off), from 261 ± 19 ms to 350 ± 31 ms (Fig. 5B; paired t test, P < 0.01, n = 8). The slower decay indicates that glutamate increases the affinity of glycine for its receptor (Fucile et al., 2000).

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Glutamate prepulse reduces deactivation rate and accelerates desensitization of IGly. A, whole-cell currents activated by 30 µM glycine before (a) and after (b) glutamate prepulse (from a VTA neuron of an 11-day-old rat). B, histograms show that glutamate prepulse (1 mM, 2 s) decreased the activation time constant (on) and increased the deactivation time constant (off) of IGly; data are from eight neurons. C and D are from the same neuron as in A. Both activation (C) and deactivation (D) could be fitted by a single exponential (continuous curves). E, current traces from another neuron of an 11-day-old rat. IGly was elicited by 30 µM glycine before (a) and immediately after a conditioning prepulse of 1 mM glutamate (b). Superimposed traces in c illustrate decay of IGly elicited by long application of 30 µM glycine, with and without glutamate prepulse. F, histograms indicate average values of time constants of decay (d) for currents activated by 30 µM glycine; values obtained with and without 1 mM glutamate prepulse differed significantly (P < 0.01, n = 6).

Glutamate Accelerates Glycine Receptor Desensitization. The potentiation of I_Gly by glutamate could result from a slower rate of receptor desensitization. To test for this possibility, we compared I_Gly desensitization in the absence and presence of glutamate prepulses. As shown in Fig. 5E, the current activated by a long pulse of glycine (30 µM) decayed more rapidly when applied after a brief pulse of glutamate. The ratio of the decay time constants (_Glu/_control) in Fig. 5E was 0.56. For six neurons (Fig. 5F), 1 mM glutamate significantly shortened the time constant of desensitization from 6.9 ± 1.4 to 4.7 ± 0.8 s (paired t test, P < 0.05). Because glutamate enhanced the peak more than the steady-state I_Gly, the ratio of steady-state to peak current amplitude declined from 0.92 ± 0.02 to 0.71 ± 0.04 (paired t test, P < 0.01, n = 17).

Ethanol Potentiates I_Gly But Inhibits Glutamate Current. In agreement with our previous findings (Ye et al., 2001a), 0.1 to 100 mM ethanol enhanced I_Gly in 35% of VTA neurons from 5- to 14-day-old rats. This effect is illustrated in Fig. 6A, where I_Gly evoked by 30 µM glycine was potentiated by 0.1, 1, and 10 mM ethanol (Fig. 6, A-b-A-d). After washout of ethanol, I_Gly recovered to control amplitude (Fig. 6, A-e). For a series of neurons, 0.1, 1, 10, and 100 mM ethanol enhanced peak I_Gly to 116 ± 5% (n = 3), 135 ± 4% (n = 34), 127 ± 3% (n = 34), and 117 ± 6% (n = 4) of control, respectively. When a brief pulse of 10 mM ethanol was coapplied during a longer pulse of glycine, there was an immediate and rapidly reversible increase in I_Gly (Fig. 6B). In contrast to this potentiation of I_Gly, 10 mM ethanol depressed glutamate-evoked current to 74 ± 3% (n = 10) of control (Fig. 6C), in agreement with previous reports (Narahashi et al., 2001).

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Ethanol enhances IGly, depresses IGlu, and suppresses glutamate-induced potentiation of IGly. A, traces from a VTA neuron of a 7-day-old rat show IGly elicited by 30 µM glycine alone (a and e, open horizontal bars) or together with 0.1 (b), 1 (c), and 10 mM ethanol (d, hatched bars). B, brief pulse of 10 mM ethanol potentiated IGly induced by 30 µM glycine (from a VTA neuron of an 8-day-old rat). C, brief pulse of 10 mM ethanol inhibited IGlu induced by 1 mM glutamate (from the same neuron as in B). D, in a VTA neuron of a 12-day-old rat, IGly was elicited by 30 µM glycine alone (a), immediately after 1 mM glutamate prepulse (b), together with 10 mM ethanol (c), and immediately after coapplication of 1 mM glutamate and 10 mM ethanol (d). E, mean values of peak IGly activated by 30 µM glycine obtained in the three recording conditions as in D-b, -c, and -d. Before plotting, all values were normalized to the value of peak IGly obtained in the control condition (D-a).

Ethanol Suppresses the Glutamate-Induced Potentiation of I_Gly. Records a and b of Fig. 6D illustrate the usual potentiation of I_Gly by a conditioning prepulse of glutamate. When 10 mM ethanol was coapplied with glutamate and glycine (Fig. 6D-d), the potentiation of I_Gly by glutamate was significantly reduced: from 188 ± 72% of control in the absence of ethanol to 146 ± 26% in its presence (Fig. 6E; P < 0.05, n = 4).

View larger version (18K): [in this window] [in a new window]   Fig. 7.   Ethanol suppressed glutamate-induced potentiation of IGly even when ethanol was applied after glutamate prepulse or when ethanol alone had no effect on IGly. A, in a VTA neuron of a 7-day-old rat, IGly was elicited by 30 µM glycine alone (a), immediately after 1 mM glutamate prepulse (b), coapplication with 10 mM ethanol (c), and coapplication with 10 mM ethanol immediately after 1 mM glutamate prepulse (d). B, mean values of peak IGly activated by 30 µM glycine. There was no significant difference among the three recording conditions shown in A-b, -c, and -d (P > 0.05, n = 13). C, current traces from a VTA neuron of a 5-day-old rat. IGly was elicited by 30 µM glycine alone (a), immediately after 1 mM glutamate pulses (b), coapplication with 10 mM ethanol (c), and coapplication with 10 mM ethanol immediately after 1 mM glutamate alone (d). Note that when ethanol was coapplied with glycine, glutamate did not enhance IGly. D, mean values of peak IGly activated by 30 µM glycine. There was no significant difference among the three recording conditions shown in C-a, C-c, and C-d (P > 0.05, n = 5), although glutamate alone significantly potentiated IGly (P < 0.01, n = 5).

	Discussion

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Our previous research revealed glycine receptors in the majority of freshly dissociated VTA neurons (Ye et al., 1998), all of which also respond to glutamate (Wang and French, 1993; Wu and Johnson, 1996; Ye et al., 2001b). The present results show that in such VTA neurons, glutamate consistently enhances I_Gly, as previously observed in spinal neurons by Xu et al. (1999, 2000), and spinal and transfected cells by Fucile et al. (2000). Our principal new finding is that ethanol suppresses the glutamate-induced potentiation of I_Gly in VTA neurons. In keeping with the previous authors, our results point to the involvement of Ca²⁺, perhaps Ca²⁺ influx, in this phenomenon, although probably not calcium/calmodulin-dependent protein kinase II.

Comparison with Previous Reports of Interactions between Glutamate and Glycine. The previous studies of glutamate-induced fast potentiation of I_Gly (Xu et al., 1999, 2000; Fucile et al., 2000) attributed this effect to a rise in intracellular free Ca²⁺. Although outwardly similar, these reports differed in some important respects. The results of the perforated-patch recordings from freshly dissociated spinal neurons (Xu et al., 1999, 2000) led to the conclusion that the increase in I_Gly is not caused by a change in the affinity of glycine for its receptor and that the potentiation is mediated by activation of CAMKII. In contrast, conventional whole-cell recordings from transfected cells (Fucile et al., 2000) suggested potentiation was due to a higher affinity of glycine receptors and not mediated by known protein kinases. In our experiments, an increase in glycine receptor affinity was indicated by the slower deactivation of I_Gly and the consistent reduction in EC₅₀. However, the glycine concentration-response plots also showed a clear increase in the maximal I_Gly. In VTA neurons, the potentiation thus appeared to be mediated by both mechanisms.

Is the Potentiating Action of Glutamate in VTA Neurons Mediated by Intracellular Ca²⁺? Our findings that glutamate was more effective when [Ca²⁺]_o was raised to 12 mM and less effective when a stronger Ca²⁺ buffer, BAPTA or 11 mM EGTA (as compared with 4 mM EGTA) was present in the electrodes are consistent with some involvement of Ca²⁺. However, the relatively modest effects produced by these manipulations, especially when compared with those seen in the experiments of Xu et al. (1999, 2000) and Fucile et al. (2000), seem more in keeping with a Ca²⁺-sensitive than a Ca²⁺-dependent process. Admittedly, by buffering slow changes in [Ca²⁺]_i, the routine presence of EGTA in the electrodes would tend to reduce Ca²⁺-mediated mechanisms. This may, at least in part, account for the relatively small and transient effects of glutamate observed in the current study as compared with the potentiation observed in the previous studies, where weaker or no buffers were used. Since even 30 mM BAPTA failed to abolish the action of glutamate, the potentiation of glycine receptors may occur at a site that is not entirely intracellular, or at least not easily accessible to intracellular chelators. Judging by the lack of effect of KN-62, CAMKII is probably not the agent of Ca²⁺-mediated modulation. Whether phosphorylation or dephosphorylation plays a significant role should be clarified by further tests of selective blockers.

Mechanisms of Ethanol's Actions. Because ethanol alone inhibits glutamate-induced currents, it could exert its effect by a direct depression of glutamate receptor/channels and consequently a smaller rise in [Ca²⁺]_i (Gruol et al., 1997). This is unlikely because ethanol suppressed glutamate-induced potentiation when ethanol was applied after the end of the glutamate prepulse. Therefore, ethanol probably exerts its suppressant action at a site closer to the glycine receptors; for example, where Ca²⁺-sensitive phosphorylation occurs.

Consequences of Ethanol's Inhibition of Glutamate-Induced Potentiation of Glycine Responses and Other Cellular Activities. Because excitatory and inhibitory receptors coexist in many neurons, it is essential for us to understand how they interact. Modulation of glycine receptors by agents such as glutamate and ethanol is important because changes in the efficacy of glycinergic transmission have pathophysiological implications in nociception and motor behavior (Breitinger and Becker, 1998; Simpson and Huang, 1998). Moreover, Ca²⁺-dependent clustering of glycine receptors during synaptogenesis has been demonstrated (Kirsch and Betz, 1998). Being present in most VTA neurons, glycine receptors are likely to play an important role in modulating the excitability of VTA dopaminergic and nondopaminergic neurons and, consequently, the release of dopamine and other agents in the brain.