Coagonist release modulates NMDA receptor subtype contributions at synaptic inputs to retinal ganglion cells

Abstract

NMDA receptors (NMDARs) are tetrameric protein complexes usually comprising two NR1 and two NR2 subunits. Different combinations of four potential NR2 subunits (NR2A-D) confer diversity in developmental expression, subsynaptic localization, and functional characteristics, including affinity for neurotransmitter. NR2B-containing NMDARs, for example, exhibit relatively high affinity both for glutamate and the coagonist glycine. Although multiple NMDAR subtypes can colocalize at individual synapses, particular subtypes often mediate inputs from distinct functional pathways. In retinal ganglion cells (RGCs), NMDARs contribute to synaptic responses elicited by light stimulus onset ("ON") and offset ("OFF"), but roles for particular NMDAR subtypes, and potential segregation between the ON and OFF pathways, have not been explored. Moreover, elements in the retinal circuitry release two different NMDAR coagonists, glycine and d-serine, but the effects of endogenous coagonist release on the relative contribution of different NMDAR subtypes are unclear. Here, we show that coagonist release within the retina modulates the relative contribution of different NMDARs in the ON pathway of the rat retina. By pharmacologically stimulating functional pathways independently in acute slices and recording synaptic responses in RGCs, we show that ON inputs, but not OFF inputs, are mediated in part by NMDARs exhibiting NR2B-like pharmacology. Furthermore, suppressing release of NMDAR coagonist reduces NMDAR activation at ON synapses and increases the relative contribution of these putative NR2B-containing receptors. These results demonstrate direct evidence for evoked coagonist release onto NMDARs and indicate that modulating coagonist release may regulate the relative activation of different NMDAR subtypes in the ON pathway.

Figure 1.

EPSCs elicited by light and pharmacological stimulation of ON and OFF bipolar cells. A–I, Responses from three different RGCs elicited by three kinds of stimulation: a 3 s, full-field light stimulus at an intensity of 10³–10⁴ photons/μm²/s (A, D, G); pressure application of CPPG (600 μm, 240 ms) in the OPL (B, E, H); and pressure application of KA (100 μm, 140 ms) in the OPL (C, F, I). Responses were recorded from ON (A–C), OFF (D–F), and ON–OFF (G–I) RGCs. Puff-evoked responses were blocked by the L-type Ca_v channel antagonist isradipine (10 μm, gray traces), indicating that they required synaptic transmission. V_hold = +40 mV for all EPSCs. J, Correlation between light responses (x-axis) and puff responses (y-axis). EPSC charge transfer (Q_EPSC) was calculated for 3 s windows following light onset and light offset and from the time of CPPG or KA puff stimulation until the EPSC amplitude returned to 5% of its peak value. x values represent ON light-evoked Q_EPSC as a fraction of total (ON + OFF) light-evoked Q_EPSC, while y values represent CPPG Q_EPSC as a fraction of total (CPPG + KA) Q_EPSC. Filled symbols denote individual cells (n = 14), the solid line shows linear regression, and the dashed line represents unity. K, Schematic of retinal circuit showing parallel ON and OFF pathways activated by CPPG and KA, respectively.

Figure 2.

RGC EPSCs are mediated primarily by NMDARs. A, CPPG-evoked (“ON”) EPSCs recorded from RGCs (V_hold = +40 mV) are blocked almost completely by the NMDAR antagonist R,S-CPP (10 μm, gray trace). B, ON EPSCs exhibited the J-shaped charge–voltage relationship that is characteristic of NMDARs; the small, CPP-insensitive component exhibited an ohmic (linear) conductance (gray symbols). For each cell, data points were normalized to the control Q_EPSC recorded at +40 mV. C, D, As in A and B, but EPSCs were elicited by KA. E, Electrically evoked EPSCs were recorded from the same cells as shown in C. F, Fraction of Q_EPSC remaining in the presence of CPP compared across stimulation modalities (KA puff, CPPG puff, electrical stimulation). n values are indicated in parentheses.

Figure 3.

CPPG-evoked ON EPSCs and KA-evoked OFF EPSCs recorded from ON–OFF RGCs. A, B, CPPG-evoked (A) and KA-evoked (B) EPSCs (V_hold = +40 mV) recorded from the same cell in the absence and presence of the NR2B NMDAR antagonist Ro 25-6981 (3 μm). C, Correlation between the fraction of ON and OFF Q_EPSC remaining in the presence of Ro 25-6981. The filled circle represents average ± SD (n = 6); open circles represent data from individual cells. The dashed line indicates equal inhibition of ON and OFF EPSCs. Ro 25-6981 reduced the ON component of the EPSCs more than the OFF component (p = 0.004, paired t test).

Figure 4.

NBQX reduces NMDAR EPSCs by blocking input from the rod pathway. A, CPPG-evoked EPSCs were reduced by NBQX (5 μm). B, The NBQX-sensitive component of the EPSC exhibited a J-shaped voltage dependence, indicating that it was mediated primarily by NMDARs. C, NBQX does not block NMDARs, as it did not affect responses in RGCs to exogenous NMDA (100 μm; V_hold = +40 mV). D, NBQX did not effect CPPG responses in RBCs, indicating that it did not act on mGluR6. E, NBQX blocked CPPG-evoked EPSCs in AII amacrine cells. F, NBQX strongly reduced CPPG responses in ON CBCs. G, As in F, but in a wild-type (WT) mouse ON CBC. H, NBQX did not affect CPPG responses in ON CBCs from Cx36−/− mice. I, NBQX reduced CPPG-evoked EPSCs in RGCs from WT mice. J, NBQX also reduced CPPG-evoked EPSCs in Cx36−/− mice, but to a lesser extent than in WT. K, Summarized effects of NBQX on CPPG-evoked responses in ON CBCs and RGCs. n values are indicated in parentheses.

Figure 5.

Synaptic release of coagonist contributes to NMDAR activation. A, d-Serine (100 μm) did not affect CPPG-evoked EPSCs, indicating that the NMDAR coagonist site is saturated during evoked responses. B, d-Serine potentiates the EPSC in the presence of NBQX, indicating that NBQX relieves saturation of the coagonist site. C, NBQX's reduction of CPPG-evoked EPSCs was less in the presence of d-serine. D, NBQX reduced EPSCs in WT mice in the presence of d-serine. E, NBQX exerted no effect on EPSCs in the presence of d-serine in Cx36−/− mice. F, Addition of NMDA (100 μm, red trace) to the superfusion solution increased the holding current and reduced CPPG-evoked EPSC amplitude (V_hold = +40 mV). Subsequent addition of d-serine (100 μm, blue trace) further increased the holding current and eliminated the EPSC. The gray trace indicates NMDA and d-serine washed out. The dashed line indicates zero holding current.

Figure 6.

Synaptic release of coagonist modulates relative contribution of different NMDAR subtypes to the EPSC. V_hold = +40 mV in all experiments. A, CPPG-evoked EPSCs were reduced by the NR2B NMDAR antagonist Ro 25-6981 (3 μm). B, Ro 25-6981 exerted similar effects in the presence of d-serine (100 μm). C, In the presence of NBQX, Ro 25-6981 blocked a larger fraction of the CPPG-evoked EPSC. D, The increased effect of Ro 25-6981 was eliminated in the presence of the NMDAR coagonist d-serine. E, Ro 25-6981 reduced CPPG-evoked EPSCs to similar extents in 2.5 mm and 1.0 mm Ca²⁺_o. Recordings are from the same cell in the continuous presence of 5 μm NBQX and 100 μm d-serine. F, Effects of Ro 25-6981 on EPSCs under different pharmacological conditions. n values are indicated in parentheses.

Figure 7.

d-Serine and glycine play complementary roles in activating NMDARs. A, Immunoreactivity (IR) for d-serine (DS) in P17 rat retina. DS IR was evident in the inner plexiform layer (IPL), the ganglion cell layer (GCL), the inner nuclear layer (INL) and the outer plexiform layer (OPL; A1) and was colocalized with glutamine synthetase (GS), a Müller cell marker (A2). ELM, External limiting membrane; ILM, internal limiting membrane. DS IR was eliminated by preadsorption of DS (A3) but not l-serine (A4). B, Serine racemase (SR) IR in P17 rat retina. SR IR was evident primarily in the INL and GCL (B1), but no SR IR was detected following preadsorption with SR antigen peptide (B2). C, As in A but in P22 retina. DS IR was weaker in the IPL and stronger in the ELM and ILM compared with P17. D, As in B but in P22 retina. Scale bar in A1 (50 μm) applies to A–D. E, Degradation of d-serine with DAAO (100 μg/ml) reduced CPPG-evoked EPSCs. F, Exogenous NMDA increased holding current and decreased CPPG-evoked EPSCs (red trace, V_hold = +40 mV). In the presence of NMDA, DAAO reduced the holding current but not the EPSC (blue trace). The gray trace indicates both NMDA and DAAO washed out. The dashed line indicates zero holding current. G, Ro 25-6981 reduced CPPG-evoked EPSCs in the presence of DAAO.

Figure 8.

Multiple NMDARs and coagonists in the inner retina. Simplified schematic illustrating distinct NMDAR subtypes at ON and OFF synapses onto RGCs (GC). ON synapses contain NR2B and non-NR2B NMDARs, and OFF synapses express primarily non-NR2B NMDARs (see Fig. 3). d-Serine (gray), perhaps released diffusely from Müller glial cells, may contribute primarily to ambient coagonist site occupancy. Glycine (violet), likely released from amacrine cells, may increase coagonist site occupancy during evoked responses.