Introduction

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The nucleus accumbens (NAcc), an interface region between limbic structures (hippocampus, amygdala, and prefrontal cortex) and the extrapyramidal motor system, modulates cognitive and motivational aspects of behavior that are translated into motor activity (Dunah et al., 1996). Projecting GABAergic neurons, also containing mostly either substance P or enkephalin, account for 90% of the neuronal population of NAcc (Meredith, 1999). These neurons receive massive dopaminergic input from the ventral tegmental area, glutamatergic input from cortical and subcortical regions, and GABA innervation from interneurons. The physiology of the receptors for these transmitters is relatively well known, because they have been heavily studied with regard to addiction to psychostimulants, opiates, and alcohol (Koob et al., 1998). NAcc-projecting neurons also receive inputs from acetylcholine- and somatostatin-containing interneurons (Groenewegen et al., 1991; Meredith, 1999), but relatively little is known about the physiology of receptors for these ligands in NAcc. Furthermore, some data suggest that other receptors, including glycine receptors, remain to be identified and characterized.

Glycine receptors, along with GABA receptors, represent the primary fast inhibitory mechanisms in the central nervous system. Activation of glycine receptors opens anionic channels that hyperpolarize neurons. Until recently, it was generally believed that glycine receptors were almost exclusively found in the spinal cord and brainstem of adult rats (Rajendra et al., 1997). However, recent findings suggest that these receptors may also be expressed in upper brainstem regions (Rampon et al., 1996). Recent studies also reported the presence of glycine receptors in forebrain structures (Yoon et al., 1998; Mori et al., 2002).

Glycine receptors are heteromultimeric receptors. Molecular approaches have revealed a certain diversity of glycine receptor subunits. To date, four different  and one  subunit have been identified (Legendre, 2001). Examination of the organization of glycine receptors revealed that glycine receptors are pentameric with an invariant stoichiometry of three  and two  subunits (Langosch et al., 1988). Although the expression of the  subunit matches that of  subunits in spinal cord, pons, and midbrain, there is evidence that  mRNA expression does not fully match that of  subunits in the brain (Fujita et al., 1991; Malosio et al., 1991). Thus, it was established that forebrain regions such as nucleus accumbens and striatum express  subunits, although in moderate levels (Fujita et al., 1991; Kirsch and Betz, 1993; Kirsch et al., 1993), but lack the  subunit (Sato et al., 1991; Malosio et al., 1991) that carries the binding site for glycine (Pfeiffer et al., 1982).

This absence of a systematic overlap between  and  subunit expression has prompted some investigators to suggest that additional  subunits, which could confer different properties for glycine receptors, remain to be discovered (Betz et al., 1999). It was also suggested that  subunits may form part of another receptor complex. Herein, we demonstrate for the first time in a subpopulation of NAcc neurons the existence of a functional receptor with electrophysiological properties reminiscent of glycine receptors but with a distinctive pharmacological profile.

	Materials and Methods

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Animals, Slice Preparation, and Experimental Solutions. We used male Sprague-Dawley rats (100-200 g) to prepare NAcc slices. The rats were anesthetized with 4% halothane, decapitated, and the brains rapidly transferred into a cold (4°C) oxygenated, low-calcium HEPES-buffered salt solution: 234 mM sucrose, 2.5 mM KCl, 2 mM NaH₂PO₄, 11 mM glucose, 4 mM MgSO₄, 2 mM CaCl₂, and 1.5 mM HEPES. We glued a tissue block containing NAcc to a Teflon chuck and cut it transversally with a Vibroslicer (Campden Instruments, Loughborough, UK). Then we incubated the slices (400 µm in thickness) for up to 6 h at room temperature (20-22°C) in a gassed (95% O₂ and 5% CO₂) NaHCO₃-buffered saline solution: 116.4 mM NaCl, 1.8 mM CaCl₂, 0.4 mM MgSO₄, 5.36 mM KCl, 0.89 mM NaH₂PO₄, 5.5 mM glucose, 24 mM NaHCO₃, 100 mM glutathione, 1 mM nitro-arginine, and 1 mM kynurenic acid. pH was adjusted to 7.35 with NaOH (osmolarity 300-305 mOsM/l). After 1 h of incubation, we dissected out the region of the nucleus accumbens with the aid of a dissecting microscope. We incubated the tissue for 25 min in an oxygenated (100% O₂ and constant stirring) HEPES-buffered solution in the inner chamber of a Cell-Stirr flask (Wheaton, Millville, NJ) that contained papain (1 mg/ml) and 136 mM NaCl, 0.44 mM KH₂PO₄, 2.2 mM KCl, 0.35 mM NaH₂PO₄, 5.5 mM glucose, 10 mM HEPES, 100 mM glutathione, 1 mM nitro-arginine, 1 mM kynurenic acid, and 1 mM pyruvic acid (pH 7.35 with NaOH, osmolarity 300-305 mOsM). The temperature of this solution was kept constant (36°C) by a circulating water bath in the outer chamber of the flask (Stefani et al., 1994; Martin et al., 2002).

Whole-Cell Recordings. We used standard whole-cell recording methods (Hamill et al., 1981). Briefly, we pulled patch electrodes from borosilicate capillary glass (Sutter Instruments, Novato, CA) on a Flaming-Brown puller (Sutter Instruments) to a final resistance of 1.8 to 2.2 M. We filled the electrodes with a solution that consisted of 120 mM CsF, 10 mM CsCl, 11 mM EGTA, 10 mM HEPES, and 0.5 mM CaCl₂ (pH 7.35 with CsOH; osmolarity 270-275 mOsM). The capillaries were first filled through the tip and then backfilled with the recording solution. We recorded in voltage-clamp mode with an Axopatch 1D amplifier and a DAC Digidata 1200 interface from Axon Instruments (Union City, CA). The signal was filtered at 5 kHz and digitized at 1 kHz. The series resistance was compensated (70-80%) only when the ramp protocol was used. We subtracted the NMDA current obtained with the ramp from the membrane response recorded in absence of NMDA and glycine. Potentials were not corrected for the liquid junction potential but are estimated to be +4 mV.

Superfusion and Drug Application. Control and drug-containing solutions were applied by gravity at a rate of 1.5 ml/s, using a rapid three-barrel capillary superfusion device (Warner Instrument, Hamden, CT) with the pipette tips placed about 200 µm from the recorded cell. The flow of solutions was controlled by solenoid valves. Each capillary had a tip diameter of 500 µm, and the distance from center to center was 700 µm. The barrel was attached to a motor, allowing fast lateral motions controlled by pClamp 6 (Axon Instruments), our data acquisition program. A drug onset time of 20 ms for the application system was determined by measuring the changes in the tip potential of the recording pipette filled with intracellular solution as the perfusion was switched from a normal to a 1:2 dilution of the extracellular recording solution.

	Results

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Freshly Isolated NAcc Neurons. Mechanical trituration after enzymatic treatment of NAcc slices yielded cells of various sizes. Most neurons had a small soma (Fig. 1, A-C) and only a small population had a larger cell body (Fig. 1, D and E). Neuronal shape also varied markedly; some neurons showed multipolar processes (Fig. 1B), whereas others were bipolar and ovoid (Fig. 1, A and C). Nevertheless, most of the small neurons recorded were likely to be medium spiny cells, because they represent up to 90% of the total number of cells in NAcc (Meredith and Totterdell, 1999). The larger neurons (Fig. 1D) are likely to be interneurons due to their size; few of them are seen after an incubation of 30 min with papain (Bargas et al., 1994; Stefani et al., 1994). Only neurons presenting a smooth and shiny membrane were recorded, because these criteria usually correlate with healthy cells.

View larger version (91K): [in this window] [in a new window]   Fig. 1.   Phase-contrast micrographs of neuron types acutely isolated from the nucleus accumbens of 100- to 140-g rats. A to C, typical neurons representing the majority of isolated cells in NAcc. These had a relatively small soma and were either bipolar (A) or multipolar (B and C). D and E, neurons with a larger cell body believed to be a cholinergic interneurons. These neurons represent only a small fraction of isolated NAcc cells. Scale bars (D and E), 10 µm.

Currents Evoked by Coapplication of NMDA and Glycine. For 80% of medium spiny neurons tested (class I; n = 54), coapplication of 200 µM NMDA and 100 µM glycine (the NMDA receptor coagonist) evoked characteristic NMDA currents that were inward at negative holding potentials and outward at positive ones (Fig. 2A), with the reversal potentials of both peak and desensitizing currents near 0 mV (Fig. 1B). Interestingly, in the remaining medium spiny neurons (class II; Fig. 2C), as well as in all large interneurons (Fig. 2E, arrow), an equivalent coapplication of NMDA and glycine evoked a transient inward current that reversed around 0 mV, followed by a desensitizing current that became outward around 30 mV. To determine the nature of this desensitized outward current, we examined its sensitivity to various ions. Because NMDA receptors can be coupled to calcium-dependent chloride channels (Scott et al., 1995), we studied the NMDA effects in a Cl-free solution in class II neurons. In control solution, coapplication of NMDA and glycine onto these neurons evoked an outward current around 20 mV. One minute after switching to a Cl-free solution the outward current was almost entirely blocked, revealing the underlying NMDA current, whose amplitude increased at negative holding potentials, possibly because the shunt exerted by the chloride conductance was blocked (Fig. 3A). In addition, both peak and desensitized currents reversed around 0 mV, and the NMDA current seemed similar in shape to that of class I neurons (Fig. 2A). Because Ca²⁺-dependent Cl channels are blocked by Ba²⁺ (Scott et al., 1995), we substituted Ba²⁺ for Ca²⁺. Surprisingly, Ba²⁺ did not prevent the outward current (Fig. 3A). Averaged over six cells, the Cl-free solution clearly shifted the reversal potential of the outward current to around 0 mV, whereas Ba²⁺ had no effect (Fig. 3B), suggesting the absence of Ca²⁺-dependent Cl channels in class II NAcc neurons.

View larger version (27K): [in this window] [in a new window]   Fig. 2.   Heterogeneity of responses evoked by local coapplication of 200 µM NMDA plus 100 µM glycine onto isolated NAcc neurons in a Mg2+-free solution. A, typical currents evoked by a 5-s NMDA application to a class I medium spiny neuron held between 70 and +20 mV. The current direction reversed at 0 mV. B, current-voltage relationship of the peak (filled circles) and desensitized (continuous line) currents of the cell shown in A. The peak amplitude was measured at the potentials shown in A and the desensitized current amplitude was measured using a ramp protocol (see Materials and Methods). Both currents reversed near 0 mV. C, NMDA + glycine currents evoked from a typical class II medium spiny neuron. Whereas the fast component of the current reversed at 0 mV (arrow), the desensitized one did not, but became outward at much more negative potentials (30 mV). D, current-voltage relationship of the cell shown in C illustrates the discrepancy between the early and late reversal potentials. Note the strong outward rectification at positive potentials. Inset E a similar class II-type NMDA + glycine current in an NAcc interneuron.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Effects of Cl-free, Na+-free, and Ba2+-containing solutions on NMDA/glycine currents of class II medium spiny neurons. A, representative NMDA/glycine currents recorded between 60 and +30 mV holding potentials. In control solution, the desensitized current became outward at around 20 mV. The Cl-free solution shifted the desensitized NMDA/glycine current equilibrium potential to the right, whereas Ba2+-containing and Na+-free solutions had little effect. The solid bar shown at 60 mV represents the coapplication of NMDA and glycine for all traces. Note the dramatic change of the current kinetics at +30 mV with the Cl-free solution. B, current-voltage relationship of the effects of Cl- and Na+-free and Ba2+-containing solutions on the mean (n = 6) current amplitudes induced by NMDA + glycine between 60 and +30 mV. Note the shift of the reversal potential by a Cl-free solution (open circles).

Effects of Glycine Alone on Class II NAcc Neurons. We asked whether glycine alone could activate a similar outward current. At 70 mV, application of 100 µM glycine evoked an inward current that decreased at more depolarized holding potentials and reversed around 50 mV (Fig. 4A). Using a ramp protocol in 10 cells, the mean reversal potential of the glycine current was 50 ± 2 mV (54 mV corrected for the liquid junction potential), a value close to the theoretical value for Cl given by the Nernst equation (55.4 mV) with [Cl]_o of 145 mM and estimated [Cl]_i of 10 mM. This current rectified outwardly at both depolarized and hyperpolarized holding potentials (Fig. 4B). It is possible that part of the rectification may be due to poor space clamping or to the participation of unblocked voltage-gated channels. It is also possible that fluoride, used in the recording pipette, passing through glycine receptors may contribute to some extent to the rectification. However, because the permeability of glycine receptors for this anion is the lowest of the six tested by Fathima-Shad and Barry (1989), its contribution is probably minimal.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Glycine alone evokes a current in NAcc class II neurons. A, typical currents evoked by long (5-s) applications of 100 µM glycine onto a class II neuron. Note the strong and rapid desensitization of the current. The solid bar shown at 70 mV represents the time of glycine application for all records. B, current-voltage relationship of glycine currents evoked during a ramp protocol. The potential was held initially at 100 mV, whereas the cell was superfused with glycine and depolarized at a rate of 10 mV/s. The average reversal potential was 50 ± 2 mV (n = 10). Note the outward rectification. The dashed line represents a theoretical linear current-voltage relationship.

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View larger version (17K): [in this window] [in a new window]   Fig. 5.   Dose-response relationship for glycine-evoked currents. A, representative responses evoked at 0 mV holding membrane potential by 5-s glycine applications (solid column). The cell was exposed to glycine concentrations from 3 µM to 1 mM. Traces obtained with glycine concentrations of 10, 50, and 300 µM are omitted for space reason. Note the change of the kinetics of the desensitization associated with a change of glycine concentrations. B, mean amplitudes averaged from five cells of peak currents in response to different glycine applications. All cells were tested first at 1 mM and responses to subsequent glycine concentrations normalized to this response amplitude. Mean normalized responses were fit to the Hill equation described under Materials and Methods. Apparent EC50 = 110 µM.

View larger version (29K): [in this window] [in a new window]   Fig. 6.   Desensitization and inactivation of glycine-mediated currents. A, 5-s application of 1 mM glycine evoked a strong desensitization of the current recorded at 0 mV. The duration of the superfusion is indicated by the solid bar below the current trace. The desensitization was best fitted by a double exponential. The fast and slow components are represented by a near vertical and a near horizontal line, respectively. B, mean rapid (tau 1) and slow (tau 2) desensitization rates of glycine currents measured at several holding potentials between 80 and + 10 mV (n = 8). Averaged tau values were fit by linear regression (see Materials and Methods). C, current responses to a first conditioning application of glycine followed at various intervals by test glycine applications. Recordings of the conditioning responses seem darker because six traces are superimposed. D, mean amplitude (n = 5) of test responses relative to the peak amplitude of conditioning glycine responses.

Pharmacology of NAcc Glycine-Like Receptors. We first examined the effects of strychnine on the glycine current because this compound is a highly selective and potent (IC₅₀ = 5-10 nM) glycine receptor antagonist (Young and Snyder, 1973, 1974). Surprisingly, glycine currents were strongly antagonized only with high strychnine concentrations (10-100 µM) (Fig. 7A), whereas 1 µM strychnine, coapplied with glycine decreased the mean glycine current amplitudes by only 18 ± 1.8% (n = 8; Fig. 7B), an equivalent strychnine-insensitivity was found in three neurons where strychnine was applied before glycine. The apparent strychnine IC₅₀ was 12 µM. We also examined the effects of picrotoxin, an antagonist of both GABA_A and glycine receptors in some preparations (Rajendra et al., 1997) on glycine currents at 0 mV. A 1-min application of 50 µM picrotoxin moderately decreased glycine current amplitudes (Fig. 8A). At a greater picrotoxin concentration (200 µM) glycine current amplitudes were slightly increased (data not shown). On average, 50 µM picrotoxin decreased mean glycine current amplitudes by 15 ± 4%, whereas 200 µM increased it by 22 ± 6% (Fig. 8C). We also examined the effects of Zn²⁺, known to potentiate and to block glycine currents at a low (1 µM) and high (10 µM) concentrations, respectively (Bloomenthal et al., 1994; Laube et al., 1995); we found that 1-min preincubation with 1 and 10 µM zinc only slightly decreased glycine current amplitudes, by 8 ± 2 and 6 ± 2%, respectively (Fig. 8C).

View larger version (19K): [in this window] [in a new window]   Fig. 7.   Dose-response relationship for glycine-evoked currents in the presence of increasing concentrations of strychnine. A, representative responses evoked at 0 mV holding potential by application of glycine (1 mM; solid column) and strychnine (0.01-100 µM; gray column). B, mean amplitudes averaged from eight cells of peak glycine currents in the presence of different strychnine concentrations. Strychnine IC50 = 12 µM.

View larger version (24K): [in this window] [in a new window]   Fig. 8.   Neither picrotoxin nor zinc blocked the glycine currents. A, 50 µM picrotoxin only slightly decreased the amplitude of the current evoked by 100 µM glycine. B, in another NAcc cell, 10 µM zinc failed to block glycine currents. C, mean effects of picrotoxin (50 and 200 µM; n = 5), and zinc (1 and 10 µM; n = 5) on glycine-evoked peak current amplitudes. All these antagonists failed to markedly alter glycine current amplitudes. All currents were recorded at 0 mV holding potential. Neurons were preexposed to zinc.

What Is the Endogenous Ligand for NAcc Glycine-Like Receptors? Because glycine-immunoreactive cells and fibers have not been identified to date in forebrain structures such as NAcc (Rampon et al., 1996), we further examined the possibility that GABA could be the endogenous transmitter for the glycine-like receptor. GABA applications (500 µM) onto class II NAcc neurons evoked currents with strong similarities to the glycine-evoked currents in that they displayed a strong desensitization (Fig. 9A) and were also strongly inactivated by a conditioning GABA application (data not shown). Bicuculline (40 µM) moderately decreased the GABA currents and altered the time to peak of the current, with a recovery on washout (Fig. 9A). Averaged over four neurons, bicuculline decreased mean GABA current amplitude by only 17 ± 6% (Fig. 9B). Because of the surprising ineffectiveness of bicuculline we also tested picrotoxin (200 µM), another potent GABA_A receptor antagonist. As with bicuculline, picrotoxin markedly altered the kinetics of the currents, with recovery on washout (Fig. 9C). However, picrotoxin also failed to fully antagonize the response (mean GABA_A current amplitudes decreased by 66 ± 12%; Fig. 9D). The persistence of a sizable GABA current in the presence of a high concentration of picrotoxin suggests that GABA may activate a conductance different from that of GABA_A receptors, although we cannot totally rule out that the concentration of GABA used in our experiments may also partially account for this effect due to competitive agonist/antagonist interactions. This current did not involve GABA_B receptors because it was not altered by the presence in the recording solution of a GABA_B receptor antagonist (CGP 55845) or Cs⁺, known to block potassium currents. Despite the fact that the glycine current was strongly inactivated by a conditioning glycine pulse, currents evoked by 500 µM GABA did not alter glycine current amplitudes at any interapplication interval (Fig. 9E), suggesting little interaction between GABA_A and glycine-like receptors in NAcc.

View larger version (25K): [in this window] [in a new window]   Fig. 9.   High concentrations of GABAA receptor antagonists only partially reduced NAcc GABA currents. A, representative GABA currents evoked by a 5-s GABA (0.5 mM) application at VH = 0 mV in the continued presence of the GABAB antagonist CGP 55845 (1 µM) before (control), during, and after (wash) application of 40 µM bicuculine. B, mean (n = 4) effects of bicuculine on GABA current amplitudes measured at VH = 0 mV. C, effects of 200 µM picrotoxin on GABA currents at 0 mV. D, mean GABA current amplitudes before, during, and after picrotoxin application (n = 5). E, currents obtained by a conditioning application of GABA followed at various intervals by test glycine applications (bars). Recordings of the conditioning responses appear darker because six GABA traces overlapped. Note that the GABA conditioning currents did not inactivate glycine currents.

View larger version (14K): [in this window] [in a new window]   Fig. 10.   Taurine inactivates glycine currents. A, representative outward currents evoked by 5-s taurine (10 µM-1 mM) and by glycine (100 µM) application in the same NAcc neuron held at 0 mV. B, current responses to a first conditioning application of 1 mM taurine followed at various intervals by test glycine applications (1 mM). Recordings of the conditioning responses appear darker because four traces are superimposed. Note the strong inactivation of glycine currents at short interapplication intervals (1-4 s). C, mean amplitude (n = 6) of test responses relative to the peak amplitude of conditioning taurine responses. D, coapplication of saturating concentrations of glycine and taurine occludes the taurine current. The interval between each application is 20 s.

	Discussion

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In these experiments on acutely isolated NAcc neurons we found that all NAcc interneurons and 20% of medium spiny cells expressed glycine-like receptors. Although their biophysical properties are similar to those of other glycine receptors, their pharmacological properties set them apart. In addition, our data support the possibility that taurine is an endogenous transmitter for NAcc glycine-like receptors.

Identity of NAcc Glycine-Like Receptors. These newly described NAcc receptors exhibited a set of properties reminiscent of strychnine-sensitive glycine receptors. Thus, NAcc glycine-evoked currents rectified outwardly in both the hyperpolarizing and depolarizing range, a phenomenon observed at voltages negative to 50 mV in cultured (Bormann et al., 1987; Fatima-Shad and Barry, 1992, 1993) and acutely isolated hypothalamic (Akaike and Kaneda, 1989) neurons, as well as in recombinant glycine receptors in expression systems (Gundersen et al., 1984; Akagi et al., 1991; Morales et al., 1994). The affinity of glycine for NAcc glycine-like receptors was 110 µM, a value similar to those obtained from glycine receptors in hyothalamic, cerebellar, and ventral cochlear neurons (Akaike and Kaneda, 1989; Virginio and Cherubini, 1997; Harty and Manis, 1998). NAcc glycine currents desensitized rapidly with a biphasic course, in agreement with glycine receptors in other studies (Akaike and Kaneda, 1989; Lewis et al., 1991). However, in contrast to neurons in hypothalamus (Akaike and Kaneda, 1989), only the slow desensitization was clearly voltage-dependent in NAcc neurons. NAcc glycine currents also showed a strong inactivation, as reported for other glycine receptors (Akaike and Kaneda, 1989; Virginio and Cherubini, 1997; Harty and Manis, 1998).

Endogenous Agonist of NAcc Glycine-Like Receptors. Regardless of the exact identity of the subunits forming the NAcc glycine-like receptors, the nature of their endogenous agonist remains to be determined. Although glycine evoked the ionic conductance, this amino acid may not be the endogenous ligand for the NAcc receptors because no glycine-containing terminals have been detected in this region (Rampon et al., 1996). Because the primary structure of GABA_A and glycine receptors shares significant sequence homologies, we examined the possibility that GABA could activate the glycine-like receptors in NAcc. We found that picrotoxin, a potent GABA receptor antagonist, did not totally block the GABA-mediated currents, suggesting the involvement of either a GABA receptor resistant to picrotoxin or a glycine receptor insensitive to picrotoxin. The latter choice has some credibility, because the ability of picrotoxin to block glycine receptors varies dramatically with the subunit composition of the receptor. Thus,  homomeric glycine receptors are much more sensitive to picrotoxin block than heteromeric  plus  glycine receptors (Pribilla et al., 1992). In addition, Fucile et al. (1999) reported that GABA activates homomeric 1 glycine receptors from zebrafish. Similarly, in rat olfactory bulb neurons both glycine and GABA can bind to either glycine or GABA_A receptors (Trombley et al., 1999). However, the fact that GABA failed to inactivate or occlude glycine currents supports the idea that GABA does not bind to the glycine-like receptors.