Metabotropic glutamate receptors and glutamate transporters shape transmission at the developing retinogeniculate synapse

Abstract

Over the first few postnatal weeks, extensive remodeling occurs at the developing murine retinogeniculate synapse, the connection between retinal ganglion cells (RGCs) and the visual thalamus. Although numerous studies have described the role of activity in the refinement of this connection, little is known about the mechanisms that regulate glutamate concentration at and around the synapse over development. Here we show that interactions between glutamate transporters and metabotropic glutamate receptors (mGluRs) dynamically control the peak and time course of the excitatory postsynaptic current (EPSC) at the immature synapse. Inhibiting glutamate transporters by bath application of TBOA (DL-threo-β-benzyloxyaspartic acid) prolonged the decay kinetics of both α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) and N-methyl-D-aspartate receptor (NMDAR) currents at all ages. Moreover, at the immature synapse, TBOA-induced increases in glutamate concentration led to the activation of group II/III mGluRs and a subsequent reduction in neurotransmitter release at RGC terminals. Inhibition of this negative-feedback mechanism resulted in a small but significant increase in peak NMDAR EPSCs during basal stimulation and a substantial increase in the peak with coapplication of TBOA. Activation of mGluRs also shaped the synaptic response during high-frequency trains of stimulation that mimic spontaneous RGC activity. At the mature synapse, however, the group II mGluRs and the group III mGluR7-mediated response are downregulated. Our results suggest that transporters reduce spillover of glutamate, shielding NMDARs and mGluRs from the neurotransmitter. Furthermore, mechanisms of glutamate clearance and release interact dynamically to control the glutamate transient at the developing retinogeniculate synapse.

Fig. 1.

Effects of dl-threo-β-benzyloxyaspartic acid (TBOA) at the immature retinogeniculate synapse. Excitatory postsynaptic currents (EPSCs) were measured before and during bath application of 50 μM TBOA. A and B: traces from representative experiments are shown for N-methyl-d-aspartate receptor (NMDAR)-mediated (holding potential V_h, +40 mV; A, left) and AMPA receptor (AMPAR)-mediated EPSCs (V_h, −70 mV; B, left) before (control; black line) and during bath application of 50 μM TBOA (gray line). Graphs show time course of NMDAR (A, right) and AMPAR EPSC amplitudes (B, right) before and during bath application of 50 μM TBOA, followed by their respective antagonists, 3-[(R)-2-carboxypiperazin-4-yl]-propyl-1-phosphonic acid (CPP) and 2-amino-5-phosphonopentanoic acid (APV; A, right) or 2,3-dihydro-6-nitro-7-sulfamoyl-benzol[f]quinozaline-2,3-dione (NBQX; B, right). C: summary graphs show the mean normalized amplitudes (±SE) of both NMDAR and AMPAR EPSCs (left) and the effects of TBOA on EPSC decay (T_1/2 or τ_s) shown as a percentage of control (right).*P < 0.01; **P < 0.001; #P < 0.05. D: effects of TBOA on the holding current (I_hold) in response to a +40-mV step. Representative traces (left) and time course (right) are shown before (control; thin line) and during bath application of TBOA (gray line) and NMDAR antagonists (dotted line). Recordings were performed at 35 ± 1°C.

Fig. 2.

Inhibition of group II/III metabotropic glutamate receptors (mGluRs) prevents TBOA-induced reduction in EPSC amplitude at the immature synapse. A: traces (left) and time course (right) of NMDA EPSC responses recorded at room temperature before (black) and during (dark gray, minute 1; open circles, minutes 2–3, light gray: minutes 3–5) bath application of 50 μM TBOA, followed by the NMDAR antagonist CPP. Stimulation frequency was 0.1 Hz. B: representative traces (left) and time course (right) of NMDAR EPSC responses in the presence of LY-341495 (LY; 50 μM) before (black line), during (solid gray line), and after (dashed gray line) bath application of 10 μM TBOA. C: NMDAR EPSC traces (left) and time course (right) before (black line) and during (gray line) bath application of LY. D: summary of data (means ± SE). Average T_1/2 is 87 ± 11 ms in LY vs. 185.4 ± 19.8 ms in LY and TBOA. #P < 0.05; **P < 0.01; ***P < 0.001 (n.s., not significant). Recordings in B and C were performed at 35 ± 1°C.

Fig. 3.

mGluR agonists modulate synaptic currents at the immature synapse. Representative traces (left) and time course (right) of AMPAR EPSC responses to pairs of pulses before (thin line) and during (thick line) bath application of the group II mGluR agonist (2R,4R)-4-aminopyrrolidine-2,4,-dicarboxylate (APDC; 30 μM; A), group III agonist l-(+)-2-amino-4- phosphonobutyric acid (l-AP4; 500 μM; B), and group I agonist (S)-3,5-dihydroxyphenylglycine (DHPG; 25 μM; C). Peak amplitude of first (EPSC₁; circles) and second AMPAR EPSC (EPSC₂; squares) and paired-pulse ratio (PPR; triangles) are plotted. D: summary data of peak EPSCs (top) and PPR (bottom) shown as a percentage of control in the presence of various concentrations of mGluR agonists. #P < 0.05; *P < 0.01. Recordings were performed at 25 ± 1°C.

Fig. 4.

Effects of TBOA on EPSCs at the mature retinogeniculate synapse. A: representative traces (left) and time course (middle) of NMDAR EPSCs recorded from a mature (postnatal day 28) relay neuron before (black line), during (solid gray line), and after (dashed gray line) bath application of 10 μM TBOA; summary data (means ± SE) shown as a percentage of control (right). B: representative traces (left) and time course (middle) of AMPAR EPSCs recorded before (black line), during (dark gray line), and after (dashed gray line) application of TBOA, followed by receptor antagonist; summary data (means ± SE) shown as a percentage of control (right). C: I_hold in response to a +40-mV step. Average traces (left) and time course (middle) are shown before (black line), during (dark gray line), and after (dashed gray line) application of TBOA; summary I_hold data (means ± SE) shown as a percentage of control. Recordings were performed at 35 ± 1°C. #P < 0.05; *P < 0.01. **P < 0.001.

Fig. 5.

Downregulation of mGluRs at the mature retinogeniculate synapse. A: representative traces (left) and time course (right) of pairs of AMPAR EPSCs before (gray) and during (black) application of 50 μM l-AP4. Peak EPSC₁ (circles), EPSC₂ (squares), and PPR (triangles) are plotted. B: summary data (means ± SE) shown as a percentage of control in response to 4 concentrations of l-AP4 (10, 30, 50, and 500 μM). C: summary data (means ± SE) for different mGluR agonists: 10 μM DHPG (EPSC₁: 93 ± 3% of control, n = 6, P = 0.16; PPR: 100 ± 3% of control, n = 6, P = 0.68) and 30 μM APDC (EPSC₁: 94 ± 5% of control, n = 4, P = 0.24; PPR: 110 ± 5% of control, n = 4, P = 0.11). #P < 0.05. Recordings were performed at 25 ± 1°C.

Fig. 6.

mGluRs are activated during physiologically relevant trains. A–C, left: representative traces of NMDAR EPSCs in response to trains of 5 stimuli at 10 (A), 20 (B), and 50 Hz (C) shown before (black) and during (gray) bath application of 50 μM LY. Middle: NMDAR responses normalized to EPSC₁ before (black) and during (gray) application of 50 μM LY. Right: summary of the relative changes in EPSC amplitudes during trains of stimuli at frequencies before (black) and during (gray) application of LY (means ± SE). Peak amplitudes in a train are normalized to EPSC₁. Synaptic depression in response to trains of stimuli was significantly increased in the presence of LY for 10, 20, and 50 Hz (P << 0.001; 2-way ANOVA). Recordings were performed at 35 ± 1°C.