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Journal of Physiology (2002), 539.1, pp. 191-200
© Copyright 2002 The Physiological Society
DOI: 10.1113/jphysiol.2001.013147

-Alanine and taurine as endogenous agonists at glycine receptors in rat hippocampus in vitro

Masahiro Mori, Beat H. Gähwiler and Urs Gerber

	ABSTRACT

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Electrophysiological and pharmacological properties of glycine receptors were characterized in hippocampal organotypic slice cultures. In the presence of ionotropic glutamate and GABA_B receptor antagonists, pressure-application of glycine onto CA3 pyramidal cells induced a current associated with increased chloride conductance, which was inhibited by strychnine. Similar chloride currents could also be induced with -alanine or taurine. Whole-cell glycine responses were significantly greater in CA3 pyramidal cells than in CA1 pyramidal cells and dentate granule cells, while responses to GABA were similar among these three cell types. Although these results demonstrate the presence of functional glycine receptors in the hippocampus, no evidence for their activation during synaptic stimulation was found. Gabazine, a selective GABA_A receptor antagonist, totally blocked evoked IPSCs in CA3 pyramidal cells. Glycine receptor activation is not dependent on transporter-controlled levels of extracellular glycine, as no chloride current was observed in response to sarcosine, an inhibitor of glycine transporters. In contrast, application of guanidinoethanesulfonic acid, an uptake inhibitor of -alanine and taurine, induced strychnine-sensitive chloride current in the presence of gabazine. These data indicate that modulation of transporters for the endogenous amino acids, -alanine and taurine, can regulate tonic activation of glycine receptors, which may function in maintenance of inhibitory tone in the hippocampus.

(Received 15 August 2001; accepted after revision 2 November 2001)
Corresponding author M. Mori: Brain Research Institute, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland. Email: mori{at}hifo.unizh.ch

	INTRODUCTION

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Glycine is a major inhibitory neurotransmitter in the nervous system (Langosch et al. 1990). Inhibition occurs by activation of glycine receptors that gate integral chloride channels consisting of pentamers of or combinations of and subunits (Kuhse et al. 1995). In the brainstem, glycinergic inhibitory signalling is dominant (Kandler & Friauf, 1995; Golding & Oertel, 1996; Stevens et al. 1996; Singer et al. 1998; Donato & Nistri, 2000; Smith et al. 2000), whereas in cortical and subcortical brain areas GABA is the major inhibitory neurotransmitter (Sivilotti & Nistri, 1991). Strychnine-sensitive glycine receptors are, however, widely expressed throughout the brain (Betz, 1991). In the hippocampus, where glycinergic inhibitory synaptic signalling has not been observed, in situ hybridization has revealed the presence of 2, 3, and -glycine receptor subunits (Malosio et al. 1991). Immunohistochemical studies have provided conflicting results as to whether glycine or glycine receptors are, in fact, expressed in the hippocampus (Wenthold et al. 1987; van den Pol & Gorcs, 1988; Araki et al. 1988; Pourcho et al. 1992; Fujiwara et al. 1998). Nevertheless, electrophysiological responses to exogenously applied glycine have consistently been observed in the hippocampus both in young rats (up to postnatal day (P)14) (Ito & Cherubini, 1991; Shirasaki et al. 1991; Trombley & Shepherd, 1994; Schönrock & Bormann, 1995) and in adult rats (from P21) (Ye et al. 1999; Chattipakorn & McMahon, 2000). In addition to glycine, at least two other endogenous amino acids activate glycine receptors, -alanine and taurine. Both agonists can bind to either glycine or GABA_A receptors to increase membrane chloride conductance (Krishtal et al. 1988; Horikoshi et al. 1988; Tokutomi et al. 1989; Langosch et al. 1990; Kuhse et al. 1995; Boehm et al. 1997; Shen et al. 2000). Moreover, the extracellular concentrations of these amino acids in the hippocampus are relatively high, in the micromolar range (Shibanoki et al. 1993), and may thus tonically activate glycine receptors. Further indication for a role of -alanine and taurine in modulating neuronal responses is suggested by the expression of glial and neuronal transporters, which control their concentrations in the brain (Smith et al. 1992b; Liu et al. 1993a). Thus, although functional glycine receptors are clearly present in the hippocampus, the endogenous agonists and the possible role of these receptors remain unknown. A difficult problem in answering these questions has been the lack of specific pharmacological tools, in particular antagonists which unequivocally differentiate between GABA_A and glycine receptors.

In this electrophysiological study we have characterized the properties of strychnine-sensitive glycine receptors in the hippocampus. Experiments were designed to evaluate the possible role of glycine receptors both as mediators of synaptic transmission and as sensors of ambient levels of endogenous agonists that might tonically modulate neuronal activity.

	METHODS

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Preparation of slice cultures

Slice cultures were prepared from 6- to 7-day-old Wistar rat pups killed by decapitation following a protocol approved by the Veterinary Department of the Canton of Zurich, and maintained as previously described (Gähwiler et al. 1998). In brief, 400 µm thick hippocampal slices were attached to glass coverslips using clotted chicken plasma, placed in sealed test tubes with serum-containing medium, and maintained in a roller-drum incubator at 36 °C for 14-28 days. After this time, the cultured hippocampus has differentiated, displaying a cytoarchitecture closely resembling that in situ (Gähwiler et al. 1997).

Electrophysiological recordings

Cultures were then transferred to a recording chamber mounted under an upright microscope (Axioskop FS1; Zeiss, Jena, Germany) and superfused with an external solution (pH 7.4) containing 148.8 mM Na⁺, 2.7 mM K⁺, 149.2 mM Cl^-, 2.8 mM Ca²⁺, 2.0 mM Mg²⁺, 11.6 mM HCO₃^-, 0.4 mM H₂PO₄^-, 5.6 mM D-glucose and 10 mg l^-1 Phenol Red (pH 7.4). In all experiments, neurones were voltage clamped at 0 mV at a temperature of 28 °C. Glycine, GABA, -alanine or taurine (all at 0.3 mM pipette concentration) was pressure-applied locally via a glass pipette positioned about 50 µm from the soma of the recorded cell to obtain a maximal peak response (49.0 kPa for 0.5-1 s; Neuro Phore; Medical Systems, Greenvale, NY, USA). Synaptic currents were evoked with monopolar metal electrodes using single pulses (100 µs, 0.5-30 µA). Recordings were obtained from CA1 and CA3 pyramidal cells, and dentate granule cells (Axopatch 200B amplifier; Axon Instruments, Foster City, CA, USA) with patch pipettes (2-5 M) filled with a solution containing: 130 mM caesium methanesulphonate; 10 mM Hepes; 10 mM ethylene glycol-bis (2-aminoethyl)-N,N,N'N'tetraacetic acid (EGTA); 5 mM QX-314-Cl; 2 mM Mg-ATP, pH 7.25. The actual membrane potentials were corrected for the liquid junction potential of -8.3 mV for the internal solution. Series resistance (typically between 5 and 15 M) was regularly monitored, and if a change of more than 10 % occurred, cells were excluded.

Drugs and chemicals

6-Cyano-7-nitroquinoxaline-2,3(1H,4H)-dione (CNQX, 40 µM), 3-((R)-2-carboxypiperazin-4yl)-propyl-1-phosphonic acid (CPP, 40 µM) and CGP 62349 (5 µM) were always present in the bathing fluid. Tetrodotoxin (TTX, 0.5 µM) was also present in the bath, except for the experiments in which IPSCs were recorded (Fig. 6). CNQX was purchased from Tocris Cookson (Bristol, UK); ATP, EGTA, GABA, glycine, -alanine, taurine, picrotoxin, sarcosine and strychnine from Sigma/Fluka (Buchs, Switzerland); TTX from Latoxan (Rossans, France); gabazine (SR 95531) from Sanofi-Synthelabo (Paris, France); QX-314 from Alomone Labs (Jerusalem, Israel), and guanidinoethanesulfonic acid (GES) from Toronto Research Chemicals (Toronto, Canada). CPP and CGP 62349 were kindly provided by Novartis AG (Basel, Switzerland).

Data acquisition and analysis

Signals were filtered at 2 kHz, digitally recorded on a computer using Clampex 7 software (Axon Instruments) and stored on tape for later analysis. Interpolated E_rev of the currents was calculated by fitting data points of the current-voltage relationship with third-order polynominals. Numerical data in the text are expressed as means ± S.E.M. Student's t test or ANOVA with Fisher's least-significant difference test was used to compare values when appropriate. P < 0.05 was considered significant.

	RESULTS

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Glycine receptor-mediated chloride currents in the hippocampus

Pressure-application of glycine induced a transient outward current in CA3 pyramidal cells voltage clamped at 0 mV in the presence of CNQX (40 µM), CPP (40 µM), CGP 62349 (5 µM) and TTX (0.5 µM) in the bath solution to block, respectively, AMPA/kainate, NMDA and GABA_B receptors and voltage-dependent Na⁺ channels (Fig. 1). The amplitude of the current increased with duration of glycine application (Fig. 1A). Plots of the I-V relationship of the glycine-induced current revealed a reversal potential of -88.8 ± 2.4 mV (Fig. 1B) (n = 5), close to the theoretical equilibrium potential calculated for chloride of -88.3 mV (assuming a permeability ratio of HCO₃^- relative to Cl^- of 0.11; Bormann et al. 1987), indicating an underlying increase in chloride conductance (5.35 ± 1.10 nS, n = 5). To determine the specificity of the glycine receptor antagonist strychnine and the GABA_A receptor antagonist gabazine, we examined their effects on currents induced by glycine and GABA applied alternately at 1 min intervals to the same CA3 pyramidal cell. Significantly larger currents were induced by GABA as compared to glycine (GABA, 4158.5 ± 868.7 pA, n = 6; glycine, 698.3 ± 98.1 pA, n = 6; Fig. 2 and Fig. 5). Bath-application of strychnine preferentially inhibited glycine-induced currents with an IC₅₀ of 0.30 µM (n = 4, Fig. 2A). The Hill coefficient of less than 1 (0.81) might be due to the heterogeneity of glycine receptors with different affinities for strychnine. At a concentration greater than 1.0 µM, strychnine also inhibited GABA-induced currents (at 10 µM, by 8.5 ± 2.1 %, P < 0.01, n = 4) as reported previously (Shirasaki et al. 1991; Trombley & Shepherd, 1994; Jonas et al. 1998). Gabazine preferentially inhibited GABA-induced currents with an IC₅₀ of 0.34 µM (n = 8, Fig. 2B). Gabazine did not significantly inhibit glycine-induced currents up to a concentration of 10 µM (at 10 µM, by 5.4 ± 10.6 %, P = 0.64, n = 4; Fig. 2B). These results demonstrate that exogenous application of glycine selectively activates postsynaptic glycine receptors.

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Figure 1. Glycine-induced currents in CA3 pyramidal cells A, currents recorded from a typical cell held at 0 mV in response to three different durations of glycine pressure-application (glycine pipette concentration, 0.3 mM, applied at 1 min intervals at time indicated by arrow). CNQX (40 µM), CPP (40 µM), CGP 62349 (5 µM) and TTX (0.5 µM) were present in the bathing solution. B, an example of the glycine-induced currents at different voltages (range, from -100 to +20 mV in 20 mV increments) (top) and a plot of the current-voltage relationship for this cell (bottom). Each trace and data point represents an average of three sweeps.

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Figure 2. The effects of strychnine and gabazine on glycine- and GABA-induced currents in CA3 pyramidal cells Glycine and GABA (pipette concentration, 0.3 mM) were pressure-applied alternately at 1 min intervals from different pipettes approximately equidistant to the same cell. A, typical responses of glycine- and GABA-induced currents in a cell at a holding potential of 0 mV before, 5 min after strychnine (1 µM) perfusion, and after 30 min of wash (top). Graph shows reduction of glycine- (n = 4) and GABA-induced current (n = 4) as a function of strychnine concentration. The concentration-dependent effect of strychnine on glycine responses was fitted with a curve calculated by the Hill equation (glycine: IC50 = 0.30 µM, Hill coefficient, nH = 0.81). B, typical responses to glycine and GABA recorded before, 5 min after gabazine (1 µM) perfusion, and after 30 min of wash (top). Graph shows reduction of glycine- (n = 4) and GABA-induced currents (n = 8) as a function of gabazine concentration. The concentration-dependent effect of gabazine on GABA responses was fitted with a curve calculated by the Hill equation (IC50 = 0.34 µM, nH = 0.80).

An indication of the subunit composition of the glycine receptors was obtained by testing the effects of picrotoxin, which is much more effective in blocking homomeric subunit-containing glycine receptors than heteromeric glycine receptors (Pribilla et al. 1992; Yoon et al. 1998). Picrotoxin (100 µM) incompletely inhibited the glycine-induced current (by 81.4 ± 1.9 %, P < 0.01, n = 4) consistent with the expression of not only homomeric subunit-containing glycine receptors but also heteromeric glycine receptors in CA3 pyramidal cells (Fig. 3).

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Figure 3. Effect of picrotoxin on glycine-induced currents Glycine (0.3 mM) was pressure-applied at 1 min intervals. A, traces recorded from a cell at a holding potential of 0 mV before and 5 min after perfusion of picrotoxin (100 µM) are superimposed. B, summary bar graph showing mean for four cells. Asterisk indicates statistical significance (Student's t test).

In addition to glycine, the amino acids -alanine and taurine are reported to be endogenous partial agonists at strychnine-sensitive glycine receptors (Langosch et al. 1990; Boehm et al. 1997). We therefore determined whether this is also the case in hippocampus. Because -alanine and taurine also activate GABA_A receptors (Horikoshi et al. 1988; Puopolo et al. 1998; Shen et al. 2000), the responses of these agonists were examined in the presence of gabazine (10 µM). In CA3 pyramidal cells held at 0 mV, pressure-application of -alanine or taurine induced transient outward chloride currents, which were, however, smaller than the glycine-induced currents (-alanine, 215.7 ± 135.8 pA, n = 4; taurine, 39.4 ± 7.5 pA, n = 5; glycine, 593.5 ± 75.2 pA, n = 4) (Fig. 4). Both -alanine and taurine responses were inhibited by strychnine (1 µM) (data not shown).

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Figure 4. Responses to glycine receptor agonists in CA3 pyramidal cells A, currents induced by the glycine receptor agonists, glycine, -alanine and taurine (concentrations in application pipette: 0.3 mM), at a holding potential of 0 mV. Gabazine (10 µM) was present in the bathing solution. B, bar graph showing mean effects in four to five cells. S.E.M. is indicated and the asterisks denote statistically significant differences for the current amplitude induced by glycine (ANOVA with Fisher's least-significant difference test).

Functional evidence for glycine receptor expression was also obtained for CA1 pyramidal cells and dentate granule cells. A comparison of whole-cell glycine-induced responses among the three principal excitatory hippocampal cells showed significantly larger currents in CA3 pyramidal cells (698.3 ± 98.1 pA; current density, 3.0 ± 0.4 A F^-1; n = 6) versus CA1 pyramidal (274.1 ± 75.1 pA; current density, 1.2 ± 0.2 A F^-1; n = 6) or dentate granule cells (375.4 ± 69.4 pA; current density 3.5 ± 0.6 A F^-1; n = 6) (Fig. 5). As a reference, the much larger GABA-induced currents, which were not significantly different among the three cell types (P = 0.34), are shown superimposed on the glycine responses.

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Figure 5. Glycine-induced currents in different hippocampal cell types Application parameters were set to obtain maximal amplitude glycine and GABA responses. A, typical responses of glycine-induced and GABA-induced currents recorded at a holding potential of 0 mV in a CA3 pyramidal cell (CA3), a dentate granule cell (DG) and a CA1 pyramidal cell (CA1). B, summary bar graphs. Each column shows the mean response in six cells. Cell capacitance was 228.4 ± 18.0 pF in CA3 pyramidal cells, 109.9 ± 9.2 pF in dentate granule cells, and 217.9 ± 34.3 pF in CA1 pyramidal cells. S.E.M. is indicated and asterisks denote statistically significant differences for the current amplitude in CA3 cells (ANOVA with Fisher's least-significant difference test).

Glycine receptors do not mediate synaptic currents in CA3 pyramidal cells

To determine whether glycine receptors, which are expressed most strongly on CA3 pyramidal cells, are involved in synaptic transmission, we characterized the pharmacological profile of the receptors mediating inhibitory postsynaptic currents (IPSCs) evoked by brief extracellular stimulation (100 µs, single current pulse up to 30 µA) in the stratum radiatum of the CA3 region in the presence of CNQX (40 µM), CPP (40 µM) and CGP 62349 (5 µM). First, the sensitivity of the IPSC to GABA_A receptor blockade by gabazine was tested. Evoked IPSCs and responses to GABA puffs were alternately induced. The degree of GABA_A receptor blockade was monitored by examining the effect of gabazine on responses induced by GABA puffs, whose peak amplitudes were adjusted to match those of the alternately evoked IPSCs. As shown in Fig. 6A, bath application of gabazine (10 µM) completely blocked the IPSC and inhibited the GABA-induced currents by 98.7 ± 0.3 % (n = 7). Application of gabazine also abolished spontaneous IPSCs. The incomplete blockade of responses to exogenous GABA by the competitive antagonist gabazine probably reflects the prolonged presence of GABA resulting from the puff applications, which may saturate GABA transporters, since even with addition of gabazine to the GABA application pipette, GABA-induced currents could not be blocked completely. The hippocampus contains a large variety of interneurones, all of which are considered to mediate inhibition by release of GABA (Freund & Buzsáki, 1996). Consistent with these findings, evoked IPSCs always appeared to be mediated entirely by GABA, even when the stimulating electrode was moved from stratum radiatum to stratum oriens (n = 3), stratum pyramidale (n = 2) or dentate hilus (n = 4).

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Figure 6. Glycine receptors do not mediate fast IPSCs in CA3 pyramidal cells A, gabazine (10 µM) completely blocks electrically evoked IPSCs. Pressure applications of GABA and synaptic stimulation were alternated every 60 s in a CA3 pyramidal cell held at 0 mV. The horizontal bar indicates when gabazine was applied. Responses (a-d) are expanded below (a, c: GABA responses; b, d: evoked IPSCs). Transient suppression of spontaneous IPSCs after extracellular electrical stimulation is probably due to depolarization-induced suppression of inhibition (Alger & Pitler, 1995). B, effect of strychnine on the IPSC evoked in a CA3 pyramidal cell at a holding potential of 0 mV. The trace labelled 'strychnine' was recorded after a 5 min perfusion with strychnine (3 µM). Decay time constants for each trace are indicated. Bar graphs for decay time constants (middle) and peak amplitudes (right) show means from four cells. The asterisk indicates statistical significance (Student's t test).

Even though these experiments suggest that the IPSC is mediated entirely by GABA_A receptors, we checked whether a glycine-dependent component could be detected by application of strychnine. Bath-perfusion of strychnine (3 µM, a relatively high concentration) partially inhibited the IPSC (control, 907.3 ± 168.9 pA; after perfusion of strychnine, 631.5 ± 129.2 pA, n = 4) (Fig. 6B). However, the decay time constants of the IPSCs before and after application of strychnine were the same (36.3 ± 6.5 and 37.0 ± 5.9 ms, respectively, P = 0.35; n = 4). Significant differences have been reported for the time constants of decay for glycine receptor-mediated IPSCs versus GABA_A receptor-mediated IPSCs. In spinal cord or brainstem, glycine receptor-mediated IPSCs exhibit a significantly shorter decay time constant than GABA_A receptor-mediated IPSCs (Jonas et al. 1998; Donato & Nistri, 2000), while the opposite is the case in retinal ganglion cells (Protti et al. 1997). The reduction in IPSC amplitude is likely to be due to an action of strychnine at GABA_A receptors, similar to the effect on GABA-induced responses (Fig. 2A). Taken together, these results indicate that activation of glycine receptors does not contribute to IPSCs in CA3 pyramidal cells.

Are glycine receptors activated by endogenous agonists?

If glycine receptors are not involved in inhibitory synaptic transmission, the question arises whether they are modulated by ambient levels of extracellular glycine. Bath-perfusion of strychnine (1 µM), however, had no effect on the holding current of CA3 pyramidal cells clamped at 0 mV (n = 4; data not shown). Thus, glycine receptors appear not to be tonically activated under these experimental conditions, in which the basal concentrations of endogenous agonists for glycine receptors are not supplemented to in vivo levels (micromolar range; Shibanoki et al. 1993). In further experiments, we checked for a possible role of glycine transporters in the modulation of glycinergic responses. Bath-application of sarcosine (300 µM), a specific inhibitor of glycine transporter function, did not, however, change the holding current or the kinetics of the current induced by a pressure puff of glycine (n = 4; Fig. 7A). Raising the concentration of sarcosine to 1 mM still had no effect (n = 2; data not shown), indicating that glycine transporters do not modulate extracellular glycine concentrations in a range relevant for gating of chloride currents in vitro.

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Figure 7. Tonic activation of glycine receptors by inhibiting the -alanine/taurine transporter A, currents induced by pressure-application of glycine were recorded in a CA3 pyramidal cell held at 0 mV. Horizontal bar indicates when sarcosine (300 µM) was applied. Traces labelled with letters are shown at an expanded time scale below and the three traces superimposed are shown on the right. B, application of GES (300 µM) induces an outward current in a CA3 pyramidal cell held at 0 mV. Horizontal bars indicate the time of perfusion of GES (300 µM), gabazine (10 µM) and strychnine (1 µM). Strychnine was applied after perfusion of gabazine when currents had maintained a plateau level for 2 min. A portion of the trace after gabazine perfusion is shown enlarged below. C, outward current induced by perfusion of GES (300 µM) in the presence of gabazine (10 µM) (left). The holding potential was alternated between 0 and +10 mV every 5 s. Horizontal bars indicate the time of perfusion of gabazine (10 µM), GES (300 µM) and strychnine (1 µM). D, summary bar graph. Each column represents mean responses in nine cells 1 min before application of strychnine (1 µM) and 2 min after currents attained a steady level, in the presence of gabazine (10 µM) and GES (300 µM). The asterisk indicates statistical significance (Student's t test).

On the other hand, bath-application of GES (300 µM), an amidino analogue of taurine that inhibits a transporter for -alanine, taurine and GABA (Li & Lombardini, 1990; Smith et al. 1992b) without exhibiting agonist activity at glycine receptors (Flint et al. 1998), induced a persistent outward current (725.3 ± 94.1 pA, n = 4). Most of this current was rapidly blocked by gabazine (10 µM), indicating that a major part of the current is mediated by GABA_A receptors activated by extracellular accumulation of GABA (Fig. 7B). In these experiments, however, a residual current persisted (14.5 ± 4.0 pA, n = 4), which was inhibited by strychnine (1 µM). GES applied in the presence of gabazine (10 µM) also induced a strychnine-sensitive outward current (29.0 ± 12.9 pA, n = 5; Fig. 7C). This sensitivity of the GES-induced gabazine-insensitive current to strychnine (before strychnine, 22.6 ± 7.4 pA; after strychnine 5.1 ± 3.5 pA; P < 0.01; n = 9, Fig. 7D) is consistent with glycine receptor activation resulting from extracellular accumulation of -alanine and/or taurine.

	DISCUSSION

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The principal findings of this study are that functional strychnine-sensitive glycine receptors expressed in the hippocampus, predominantly on CA3 pyramidal cells, do not appear to be involved in inhibitory synaptic transmission and that glycine does not attain sufficient ambient concentrations to induce chloride currents. Rather, our experiments suggest that -alanine and taurine are the primary agonists at hippocampal strychnine-sensitive glycine receptors, and that membrane transporters are critical in regulating their extracellular concentration.

The properties of glycine receptors in the hippocampus

A major difficulty in the functional investigation of GABAergic and glycinergic inhibitory responses is the lack of absolutely specific antagonists. It was therefore necessary in our initial experiments to determine the specificity of the best available antagonists, strychnine and gabazine, in the hippocampus. Although brief applications of GABA and glycine were used in our experiments, outside-out patches in combination with a fast-application system would be necessary to avoid all desensitization. Glycine-induced current was preferentially inhibited by strychnine with an IC₅₀ of 0.30 µM (Fig. 2A), which is relatively high compared to the IC₅₀ typically reported for strychnine-sensitive glycine receptors (less than 0.1 µM) (Krishtal et al. 1988; Tokutomi et al. 1989; Shirasaki et al. 1991; Boehm et al. 1997; Jonas et al. 1998). In light of the finding that neonatal forms of the glycine receptor are strychnine resistant (Becker et al. 1988; Langosch et al. 1990), our data suggest that the glycine receptors expressed in 3-week-old hippocampal slice cultures may include the neonatal phenotype. Moreover, the incomplete blockade of responses by picrotoxin (Fig. 3) indicates that these glycine receptors are composed both of homomeric subunits and heteromeric subunits (Pribilla et al. 1992; Yoon et al. 1998).

Glycine receptors do not contribute to synaptic inhibitory responses

The complete blockade of evoked IPSCs by the specific GABA_A receptor antagonist gabazine (Fig. 6A) strongly suggests that this response is mediated entirely by GABA receptors. The glycine receptor antagonist strychnine at a relatively high concentration of 3 µM also reduced the amplitude of evoked IPSCs (Fig. 6B). Strychnine, however, is not an absolutely specific antagonist. As shown in Fig. 2A and as previously reported (Shirasaki et al. 1991; Trombley & Shepherd, 1994; Jonas et al. 1998), strychnine also inhibits GABA_A receptors. In addition, strychnine did not change the kinetics of the IPSC component (Fig. 6B). If the glycine response were inhibited by strychnine, then the residual strychnine-insensitive component should exhibit altered decay kinetics (Protti et al. 1997; Jonas et al. 1998; Donato & Nistri, 2000). These results indicate that glycine receptors are not involved in synaptic transmission in the hippocampus. A similar finding has been reported in rat cerebellar granule cells, where GABA- and glycine-induced responses are observed, but spontaneous IPSCs are mediated exclusively by GABA (Kaneda et al. 1995).

Amino acid transporters and the activation of hippocampal glycine receptors

Extracellular concentrations of endogenous neuroactive agents must be tightly regulated by membrane transporters or catabolic enzymes that terminate signalling (Nelson, 1998). A number of membrane transporters have been identified that control the levels of the amino acids acting as agonists at hippocampal glycine receptors. GLYT1 and GLYT2 are two transporters specific for glycine, with only GLYT1 expressed in the hippocampus by the glial cells (Smith et al. 1992a; Zafra et al. 1995). In our study, however, sarcosine, an inhibitor of GLYT1, did not induce a current reflecting the activation of strychnine-sensitive glycine receptors nor was there a change in the kinetics of the current induced by pressure-application of glycine (Fig. 7A), indicating that GLYT1 does not play a significant role in modulating glycine-dependent chloride currents in the hippocampus.

-Alanine and taurine, the other endogenous agonists activating strychnine-sensitive glycine receptors, are not substrates for GLYT1 and GLYT2 (Smith et al. 1992a; Liu et al. 1993b). These amino acids are transported by a specific membrane transporter (Smith et al. 1992b; Liu et al. 1992) and by the GABA transporters, GAT3 and GAT4 (Liu et al. 1993a), which do not, however, transport glycine. Our observation of a tonic current after inhibition of -alanine and taurine transport (Fig. 7B, C and D) suggests that ambient levels of these amino acids could act at glycine receptors to modulate neuronal processing. Tissue concentrations of taurine are in the millimolar range within most of the brain and both the synthesizing enzyme and transporter for taurine are present in glia (Almarghini et al. 1991; Liu et al. 1992). Thus, glial transport may control extracellular concentrations of taurine as reported in rat neonatal neocortex (Flint et al. 1998), although a hypotonic stimulus (Deleuze et al. 1998) or ischaemia (Saransaari & Oja, 1999) can also lead to taurine release.

Functional role of glycine receptors in the hippocampus

Compared to GABA_A receptor-mediated currents, glycine responses are much smaller (Fig. 2 and Fig. 5), and we found no evidence for intrahippocampal glycinergic synaptic transmission (Fig. 6). Our experiments do not, however, rule out an input from an as yet unidentified extrahippocampal glycinergic pathway. An alternative function for hippocampal glycine receptors may lie in the modulation of excitability in neuronal circuits. Although activation of non-synaptic glycine receptors may not change resting membrane potential, a tonic background chloride current could act to shunt synaptic signals, changing the amplitude and kinetics of fast synaptic potentials. The fact that whole-cell glycinergic responses were largest in CA3 pyramidal cells, which represent a central relay synapse in the hippocampal synaptic network, suggests that non-synaptic glycine receptors may play an important role in regulating hippocampal information processing.

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Acknowledgements

We thank L. Heeb, L. Rietschin, S. Giger, H. Kasper, R. Dürr and R. Schöb for excellent technical assistance and P. Benquet, K. Fischer, C. Gee and M. Scanziani for valuable discussions and critical reading of the manuscript. This work was supported by the Swiss National Science Foundation (U.G.), and the Ministry of Education, Science, and Culture of Japan (M.M.).

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