The Journal of Neurophysiology Vol. 80 No. 6 December 1998, pp. 3336-3340
Copyright ©1998 by the American Physiological Society

RAPID COMMUNICATION

Glycine Uptake Governs Glycine Site Occupancy at NMDA Receptors of Excitatory Synapses

Albert J. Berger, Stéphane Dieudonné, and Philippe Ascher

Laboratoire de Neurobiologie, École Normale Supérieure, 75005 Paris, France

	ABSTRACT

Abstract Introduction Methods Results Discussion References

Berger, Albert J., Stéphane Dieudonné, and Philippe Ascher. Glycine uptake governs glycine site occupancy at NMDA receptors of excitatory synapses. J. Neurophysiol. 80: 3336-3340, 1998. At central synapses occupation of glycine binding sites of N-methyl-D-aspartate receptors (NMDA-Rs) is a necessary prerequisite for the excitatory neurotransmitter glutamate to activate these receptors. There is conflicting evidence as to whether glycine binding sites normally are saturated. If they are not, then alterations in local glycine concentration could modulate excitatory synaptic transmission. By using an in vitro brain stem slice preparation we investigated whether the glycine site is saturated for synaptically activated NMDA-Rs in neonatal rat hypoglossal motoneurons. We found that the NMDA-R-mediated component of spontaneous miniature excitatory postsynaptic currents could be potentiated by exogenously applied glycine as well as by D-serine. The effects of glycine were observed only at concentrations (100 µM or more) two orders of magnitude above the apparent dissociation constant of glycine from NMDA receptors. In contrast, D-serine, a nontransported NMDA-R glycine site agonist, was effective in the low micromolar range, i.e., at concentrations similar to those found to be effective on isolated cells or on outside-out patches. We conclude that at these synapses the glycine concentration around synaptic NMDA-Rs is set below the concentration required to saturate their glycine site and is likely to be stabilized by a powerful glycine transport mechanism.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

Johnson and Ascher (1987) first demonstrated in brain neurons that exogenously applied glycine augmented the response of N-methyl-D-aspartate receptors (NMDA-Rs) to NMDA. The mechanism of interaction between glycine and NMDA-Rs was the subject of intensive investigation (Kemp and Leeson 1993; Thomson 1990), and it was generally agreed that occupation of the glycine binding site is a prerequisite for NMDA-R activation by glutamate (Kemp and Leeson 1993).

A key issue is whether glycine is a significant modulator of NMDA-R activity or is it always present at supramaximal amounts and therefore not capable of modulating NMDA-R-mediated excitatory synaptic activity. The uncertainty regarding the regulatory role for glycine arises because the glycine concentration in the synaptic cleft is unknown and because different NMDA-R isoforms have different affinities for glycine. The EC₅₀ for glycine is as low as 100-300 nM for NMDA-Rs in cultured central neurons and some heteromeric NMDA-Rs expressed in Xenopus oocytes and can reach a few micromolar for other expressed heteromeric NMDA-Rs (Johnson and Ascher 1992; Mayer et al. 1989; McBain and Mayer 1994; Thomson 1990). As for the extracellular glycine concentration, it was measured in "wide" extracellular spaces (sampled by microdialysis) and in cerebrospinal fluid. In both it was found to be in the low micromolar range (Kemp and Leeson 1993; Westergren et al. 1994) and thus could be saturating for some NMDA-Rs and not for others. However, the concentration of glycine in the synaptic cleft could be much lower if glycine transporters (Borowsky et al. 1993; Lukasiewicz and Roeder 1995; Smith et al. 1992; Zafra et al. 1995) were strategically placed around the cleft. In rat brain some mRNAs encoding glycine transporters are localized to regions that also contain high levels of NMDA-Rs and low levels of inhibitory glycine receptors. This suggests that the local glycine concentration may be tightly regulated by glycine transporters at NMDA-R-containing synapses (Smith et al. 1992).

In the absence of direct evaluations of either the NMDA-R glycine EC₅₀ or the concentration of glycine in the synaptic cleft, attempts to determine whether the resting level of glycine is saturating mostly examined if adding exogenous glycine potentiates either the response to exogenous NMDA or the NMDA component of synaptic currents. Results were variable because both an absence of potentiation (Fletcher et al. 1989; Kemp et al. 1988) and potentiation (Thomson et al. 1989; Watanabe et al. 1992; Wilcox et al. 1996) were described. Addition of D-serine, which is a potent agonist at the glycine binding site (Schell et al. 1995; Thomson 1990) but is not taken up by glycine transporters (Supplisson and Bergman 1997) mostly produced a potentiation of NMDA synaptic currents (Daw et al. 1993; Watanabe et al. 1992). The interpretation of these observations is difficult. Absence of potentiation by glycine could indicate saturation of the glycine site but could also result from the presence of very powerful uptake systems that protect the synaptic cleft from a rise in glycine concentration. Conversely, the observation that there is a potentiation by glycine or D-serine in a slice certainly indicates that the glycine site is not saturated.

In light of recent work demonstrating that glycine transporters can lower the local glycine concentration by many orders of magnitude in restricted spaces (Supplisson and Bergman 1997), we reevaluated the hypothesis made by many previous authors (Kemp and Leeson 1993; Kleckner and Dingledine 1988; McBain and Mayer 1994; Smith et al. 1992; Supplisson and Bergman,1997; Wilcox et al. 1996) that in the synaptic cleft transporters lower the glycine concentration far below that of the surrounding extrasynaptic space. We compared the concentrations of glycine and D-serine producing a given potentiation of the NMDA component of spontaneous mEPSCs in rat hypoglossal motoneurons. D-Serine at 1-30 µM produced a potentiation. Glycine produced a potentiation only when its concentration was raised to the 100 µM range. Because the EC₅₀s for the two compounds are very similar (Monahan et al. 1989; Snell et al. 1988; Thomson 1990; Watson et al. 1990), the discrepancy suggests that the glycine sites are not saturated and are isolated from the exogenous glycine by a powerful uptake system.

	METHODS

Abstract Introduction Methods Results Discussion References

Brain stem slice preparation

Experiments were performed on hypoglossal motoneurons recorded in brain stem slices of rats aged 6-18 days. The brain stem slice preparation was essentially that described by Umemiya and Berger (1994), with the exception that transverse brain stem slices were cut at a thickness of 250-300 µm instead of 120-130 µm. Slices were incubated at 34°C for ~1 h and then maintained at room temperature while submerged in a bicarbonate-buffered solution bubbled with carbogen (95% O₂-5% CO₂); this same solution was also used for cutting (at 0°C) and recording (at 20-25°C).

Recording

Hypoglossal motoneurons were viewed with a fixed-stage upright microscope (Zeiss) equipped with infrared differential interference contrast optics and a video camera. Incubation and recording chambers were continuously perfused with carbogen gassed bicarbonate-buffered solution containing (in mM) 125 NaCl, 2.5 KCl, 1 MgCl₂, 2 CaCl₂, 1.25 NaH₂PO₄, 26 NaHCO₃, and 25 glucose. When recording spontaneous miniature excitatory postsynaptic currents (mEPSCs) at negative holding potentials, MgCl₂ was excluded from the perfusate. During recording of mEPSCs, the following inhibitors were added to the external solution (in µM): 0.5-1 tetrodotoxin, 1 strychnine, and 10 bicuculline. In some experiments we blocked the NMDA component of the mEPSCs with 50 µM aminophosphonovaleric acid (APV) and the non-NMDA component with 5 µM 6-nitro-7-sulfamoylbenzo[f]quinoxaline-2,3-dione (NBQX).

Patch electrodes were fabricated from borosilicate glass capillaries on a two-stage puller to a DC resistance of 4-5 M and filled with a solution containing (in mM) 100 cesium gluconate, 20 tetraethylammonium chloride, 10 N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid (HEPES), 4 NaCl, 10 ethylene glycol-bis(-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 5 lidocaine N-ethyl bromide (QX-314), 1 CaCl₂, 4 ATP-Mg (pH adjusted to 7.2-7.3). After establishment of whole cell recording conditions access resistance was maintained at <10 M and generally compensated by ~95%. No correction was made for the liquid junction potential.

Motoneurons were voltage-clamped with an Axopatch 200B amplifier (Axon Instruments). The membrane current was filtered at 2 kHz and recorded on a digital tape recorder (Biologic) for subsequent data analysis. This recorded signal was later low-pass filtered (1 kHz) and sampled at 5 kHz (PClamp by Axon Instruments) to acquire 120 s of continuously digitized data for identification and analysis of the mEPSCs. mEPSCs were detected and analyzed by a computer program kindly provided by Dr. Pierre Vincent. Event detection was done automatically and occurred when the recorded current showed a rapid excursion from the baseline and reached a preset current threshold. Detected events were viewed individually, and each was accepted for further analysis only if during the first 100 ms of its time course there was no indication of contamination by other synaptic events. Each 120 s of data contained ~50-250 spontaneous mEPSCs. These events were averaged, and the averages were used for subsequent quantitative analyses.

Analysis

The spontaneous mEPSCs were composed of the two components, a fast non-NMDA and a slower NMDA component (O'Brien et al. 1997). The non-NMDA-R-mediated mEPSC could be isolated by perfusing the slice with APV. We found that the non-NMDA-R-mediated component decayed to 5% of its peak value 9.9 ± 0.5 ms after its onset (n = 8 cells recorded in the presence of APV). In control conditions, the NMDA component decayed to ~10% of its maximal value after 100 ms. Thus, to quantify the NMDA component, we integrated the average mEPSC waveforms (acquired in the absence of APV) for a 90-ms time period beginning 10 ms after the start of the mEPSC. This integral was used as an index of the NMDA-R-mediated charge transfer (e.g., see Figs. 1A2 and 2A2).

View larger version (36K): [in this window] [in a new window]   FIG. 1. The N-methyl-D-aspartate (NMDA) component of spontaneous miniature excitatory postsynaptic currents (mEPSCs) increases on application of exogenous glycine. A1: mEPSCs recorded in the control conditions (top 3 traces) and in the presence of increasing concentrations of glycine and finally after application of 50 µM of aminophosphonovaleric acid (APV) to block the NMDA component of the mEPSC. All traces are from the same motoneuron at VH = 70 mV. Increasing concentrations of glycine lead to an increasing NMDA component. A2: average mEPSCs under the 4 conditions and from the same motoneuron shown in A1; averages are derived from 231, 188, 170, and 80 events in control, 300 µM and 1 mM glycine, and in 50 µM APV, respectively. B: average NMDA component as a function of increasing concentrations of extracellular glycine. The NMDA component was measured as charge transfer between 10 and 100 ms after mEPSC onset and plotted here as a percentage of its magnitude during control conditions. Bar heights are means ± SE, and each data point is derived from average mEPSCs in 3 motoneurons.

View larger version (43K): [in this window] [in a new window]   FIG. 2. The NMDA component of spontaneous mEPSCs increases with application of D-serine. A1: mEPSCs recorded in the control conditions (top 3 traces) and in the presence of increasing concentrations of D-serine and finally after application of 50 µM of APV to block the NMDA component of the mEPSC. All traces are from the same motoneuron at VH = 80 mV. Increasing concentrations of D-serine lead to an increasing NMDA component as shown by the increasing decay times of the mEPSCs. A2: average mEPSCs under the 4 conditions and from the same motoneuron shown in A1; averages are derived from 146, 95, 110, and 110 events in control, 3 and 10 µM D-serine, and in 50 µM APV, respectively. B: average charge transfer (10-100 ms) of the NMDA component with increasing concentrations of extracellular D-serine, as a percentage of its magnitude during control conditions. Bar heights are means ± SE, and each data point is derived from average mEPSCs in 3 motoneurons.

In all cases the results are presented as means ± SE. Significance was accepted if P < 0.05 for paired or unpaired t-tests as appropriate.

	RESULTS

Abstract Introduction Methods Results Discussion References

Whole cell recordings of spontaneous mEPSCs were made from 27 cells (Fig. 1A). Figure 1A shows that application of 50 µM APV (n = 8 neurons) abolished the slowly decaying phase of the mEPSC, indicating that this latter portion of the mEPSC was due solely to activation of NMDA-Rs. After APV application, the remaining fast component of the mEPSC was abolished by application of 5 µM NBQX (data not shown). These data indicate that mEPSCs recorded in hypoglossal motoneurons result from activation of both NMDA and non-NMDA glutamatergic receptors (see also (O'Brien et al. 1997).

Addition to the perfusate of 30 µM glycine, a concentration that is saturating for all NMDA-Rs analyzed in isolated cells or in outside-out patches, did not produce a significant change of the NMDA component of the mEPSCs (Fig. 1B). However, a significant increase was detected when the concentration of exogenous glycine was raised to 100 µM. This increase was observed at both negative (Fig. 1) and positive holding potentials (data not shown).

The mean potentiating effect of 100 µM glycine on the NMDA component of the average mEPSC was ~40%, and additional increases occurred when the concentration was raised to 300 µM and to 1 mM (Fig. 1B). These results indicate that at synaptically activated NMDA-Rs the glycine binding site is not saturated. Further, the fact that high concentrations of exogenously applied glycine are required to enhance NMDA-R-mediated mEPSCs suggests that the glycine concentration in the region of synaptically activated NMDA-Rs is tightly regulated by active glycine transport.

D-Serine is an agonist at the glycine site with an EC₅₀ similar to that of glycine when measured in the same system (Monahan et al. 1989; Snell et al. 1988; Thomson 1990; Watson et al. 1990). However, D-serine is not transported by glycine transporters (Supplisson and Bergman 1997) and therefore should equilibrate rapidly between the perfusion solution and the synaptic cleft. We therefore measured the NMDA component of the mEPSCs after addition of various concentrations of D-serine (Fig. 2). We observed that D-serine caused a concentration-dependent increase in the NMDA component of the mEPSCs. The augmentation occurred, however, at much lower concentrations than with glycine. A mean potentiation of 52 ± 16% was detected with 1 µM D-serine, and somewhat larger increases were detected with 10 and 30 µM D-serine (Fig. 2B).

To confirm the specificity of D-serine for the glycine binding site of the NMDA-R we tested whether its isomer L-serine could enhance the NMDA component of mEPSCs. The average EC₅₀ for L-serine at the glycine binding site of the NMDA-R is more than an order of magnitude greater than that for D-serine (Thomson 1990). Application of L-serine (1 µM) resulted in an NMDA component that was 97 ± 7% (n = 5 HMs) of its control value. The responses to L- and D-serine were significantly different (P < 0.01).

	DISCUSSION

Abstract Introduction Methods Results Discussion References

Application of either exogenous glycine or D-serine (but not L-serine) enhanced the NMDA component of mEPSCs recorded in rat hypoglossal motoneurons. We suggest that this potentiation occurs because glycine in the synaptic cleft is present at subsaturating concentrations. Saturation can be produced by bath application of either very high concentrations of glycine or nontransported glycine agonists of the NMDA-R (such as D-serine). That the effective concentration of glycine for enhancement of NMDA-R-mediated mEPSCs is 100 times higher than that of D-serine demonstrates that the glycine concentration in the synaptic cleft of glutamatergic synapses is regulated by a powerful glycine uptake system. Various glycine transporters (GLYT1a, GLYT1b, and GLYT2 subtypes) are present within the rodent brain stem, and in particular within the hypoglossal motor nucleus (Borowsky et al. 1993; Guastella et al. 1992; Jursky and Nelson 1996; Smith et al. 1992; Zafra et al. 1995). This supports the idea that active transport by glycine transporters could lower the local glycine concentration.

The maximal potentiation observed with the highest concentrations of glycine (Fig. 1) and of D-serine (Fig. 2) was ~100%. If one assumes that under these conditions saturation of the glycine site was attained then one can conclude that in the absence of exogenous glycine the synaptic glycine concentration is close to the value of the EC₅₀ of the glycine site. Previously, by using this same preparation, it was shown that in the absence of exogenous glycine, application of the glycine site blocker (L-689,560) abolished the NMDA component of mEPSCs (O'Brien et al. 1997). Our current results do not allow us to determine the EC₅₀ for glycine quantitatively. Glycine affinity is known to depend on the subunit composition of the NMDA receptors (Mori and Mishina 1995). Recombinant receptors assembled from NR1 and NR2A subunits (or from their mouse equivalents 1 and 1) expressed in Xenopus oocytes have "low" glycine affinities (EC₅₀ = 2-3 µM), whereas receptors assembled from NR1 and NR2B have "high" glycine affinities (EC₅₀ = 0.1-0.3 µM) (McBain and Mayer 1994; Mori and Mishina 1995). Neurons in the mouse hypoglossal motor nucleus express the 1, 1, and 2 NMDA-R subunit mRNAs (Watanabe et al. 1994). Similarly, rats in the age range used in our experiments (P6-P18) express both NR2A and NR2B subunits in the hypoglossal nucleus (J. H. Singer and A. J. Berger, unpublished data). The subunit composition of NMDA synaptic receptors therefore remains uncertain. However, an indication of this composition may be derived from the kinetics of the NMDA synaptic currents. In neuronal cultures the time course of the decay of synaptic currents is similar to that of the decay of the patch response to a short pulse of glutamate (Lester et al. 1990), which itself is closely correlated with the affinity of glutamate. Thus the fact that NR1-NR2A has a lower affinity for glutamate than NR1-NR2B (McBain and Mayer 1994; Mori and Mishina 1995) is reflected by the faster decay of the response to a short pulse of glutamate for NR1-NR2A than for NR1-NR2B (Monyer et al. 1994; Vicini et al. 1998). In our experiments the decay of the NMDA components of mEPSCs was undoubtedly fast because 90 ms after the peak the current was reduced to <10% of its peak value (a single exponential fit to these first 90 ms gave a time constant in the range of 22-70 ms). Such a fast decay was seen only in recombinant receptors assembled from NR1 and NR2A subunits (Vicini et al. 1998). This suggests that synaptic NMDA receptors of motoneurons associate these two subunits and therefore not only have a relatively low affinity for glutamate but also a low affinity for glycine, with EC₅₀s in the low micromolar range.

Even if we cannot evaluate more precisely the glycine concentration in the synaptic cleft, the conclusion that this concentration is close to the EC₅₀ for the synaptic NMDA receptor glycine sites suggests that the NMDA component is sensitive to both increases and decreases in the extracellular glycine concentration. The system is thus set at the most efficient point for making the glycine concentration a physiological signal. The set point does not result primarily from an exchange of glycine between the slice and the surrounding solution because it is unchanged when glycine is increased from 0 to 30 µM in the bath solution. It is therefore a characteristic of the overall mechanisms of glycine homeostasis in the slice: biochemically regulated intracellular glycine concentration, efflux of glycine from neurons, and glia and reuptake by the various transporters.

Although transporters are not likely to be the only molecular components determining the value of the resting extracellular glycine level, they are crucial components in the protection against external sources of glycine such as blood or cerebrospinal fluid. They are also important in setting the spatial extent, amplitude, and duration of local perturbations such as those that may be induced in physiological conditions by glycine released from glycine containing nerve fibers. The hypoglossal motor nucleus is one of several brain stem structures that contain the highest density of glycinergic nerve fibers (Rampon et al. 1996). Glycinergic nerve terminals synapsing onto hypoglossal motoneurons show a high level of spontaneous activity in the slice (Umemiya and Berger 1995). This significant glycinergic input in fact precluded us from studying evoked NMDA receptor-mediated EPSCs because it was not possible to obtain a pure evoked glutamatergic input uncontaminated by synaptically released glycine. The striking glycine input suggests that glycine may normally modulate NMDA-Rs by "spillover" from glycinergic inhibitory synapses to glutamatergic synapses before being taken up by glycine transport mechanisms. On the basis of indirect evidence, it was proposed that in the retina glycinergic amacrine cells may modulate NMDA-R-mediated excitatory synaptic inputs to retinal ganglion cells (Lukasiewicz and Roeder 1995). A similar situation was proposed in spinal cord (Fern et al. 1996) and may be valid for brain stem structures.

	ACKNOWLEDGEMENTS

  We thank B. Barbour and J. Singer for comments on the manuscript.

  This work was supported by the Center National de la Recherche Scientifique (URA 1857), the Association Française contre les Myopathies, the Ministère de l'Education Nationale (Département des Affaires Internationales), the Université Pierre et Marie Curie and a National Institutes of Health Grant NS-14857.

	FOOTNOTES

  Address for reprint requests: A. J. Berger, Dept. of Physiology and Biophysics, School of Medicine, University of Washington, Box 357290, Seattle, WA 98195.

  Received 15 July 1998; accepted in final form 14 September 1998.

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