Expression Of Sigma Receptor 1 In Rat Retina And Its Interaction With AMPA Receptors In Retinal Ganglion

Sigma receptor (aR) is a unique receptor family, which includes two subtypes: σR1 and aR2. aRl is widely distributed in the central nervous system (CNS), which has quite high expression levels in olfactory bulb and hypothalamus, but less in cortex and hippocampus. aRl is involved in modulating neuronal firing, neurotransmitter release and voltage-gated and ligand-gated ion channels. Moreover, aRl participates in a variety of physiological and pathological processes, including learning and memory, antipsychosis, drug abuse and neuroprotection. It has been reported that aRl is expressed in the mouse retinal neurons, but the identification of the aRl-positive cells located in the ganglion cell layer and inner nuclear layer has not been precisely determined. In the retina, many studies have demonstrated the efficacy of aRl ligands in the protection of retinal neurons. aRl ligands may protect cultured retinal ganglion cells (GCs) from glutamate-induced excitotoxicity and slow down the pathological course of diabetic retinopathy in mouse. However, little is known about transmission and modulation of visual signals by σR1 in the retina. A previous work in this laboratory shows that activation of σR1 suppresses NMDA receptor mediated currents in rat retinal GCs.In this work, using RT-PCR and Western blot techniques, we found that aRl mRNA and protein were present in rat retina. Immunofluorescence double labeling showed that σR1 was expressed in horizontal cells, most of GABAergic amacrine cells (ACs) (including dopaminergic and cholinergic ACs) and in some glycinergic ACs. In contrast, no aRl-immunoreactivity (IR) was detected in photoreceptors, several subtypes of bipolar cells (BCs) and glycinergic AⅡ ACs. In additon, aRl-IR was seen in almost all somata of the GCs retrogradely labeled by fluorogold. These results suggest that aRl may have neuromodulatory role in the retina.GCs are the only type of output neurons in the retina, which receive and integrate visual signals from BCs and ACs to visual cortex in manner of action potentials. Glutamate is considered to be the most important excitatory neurotransmitter in retina. Both ionotropic and metabotropic glutamate receptors are expressed in GCs. Excitatory signals from BCs to GCs are mainly mediated by AMPA receptors (AMPARs) that are located on GCs.Using whole-cell patch clamp techniques, we further investigated how activation of the aRl modulates AMPAR-mediated light-evoked excitatory postsynaptic currents (L-eEPSCs) of ON type GCs (ON GCs) in rat retinal slice preparations. AMPAR-mediated L-eEPSCs of GCs were pharmacologically isolated by adding the NMDA receptor antagonist MK-801, GABAA receptor antagonist bicuculline, glycine receptor antagonist strychnine, and voltage-gated sodium channel blocker TTX to bath Ringer’s. The currents could be completely blocked by the specific AMPAR antagonist NBQX or GYKI52466. We further showed that the aRl agonist (+)-SKF10047 (SKF) (50 μM) suppressed AMPAR-mediated L-eEPSCs from ON GCs and the effect could be reversed by the aRl antagonist BD1047, suggesting that SKF effect was mediated by aRl. The suppression of SKF on AMPAR-mediated L-eEPSCs may be attributed to two actions, one is that SKF directly suppresses AMPAR of ON GCs, another is that SKF inhibits the glutamate release from presynaptic BCs. In order to determine the effect of activation of aRl on glutamate release from BCs, we tested the effect of SKF on the paired-pulse ratio (PPR) of AMPAR-mediated electricity-evoked excitatory postsynaptic currents (E-eEPSCs). The results showed that SKF did not change the PPR of AMPAR-mediated E-eEPSCs of ON GCs, indicating that SKF did not affect the glutamate release probability of BCs.Signaling pathways mediating the suppression of SKF on AMPAR-mediated L-eEPSCs were further explored. Internal infusion of G protein inhibitor GDP-β-S eliminated the SKF-induced suppression on AMPAR-mediated L-eEPSCs of ON GCs, suggesting the involvement of G protein in the SKF effect. Moreover, internal application of the protein kinase G (PKG) agonist cGMP or PKG inhibitor KT5823 abolished the SKF effect on L-eEPSCs, indicating the SKF effect was mediated by cGMP-PKG pathway. Furthermore, calcium imaging revealed that SKF did not change [Ca2+]i in GCs. The SKF effect persisted when [Ca2+]i was chelated by BAPTA, suggesting no involvement of Ca2+ in the SKF effect. Neither the phosphatidylinostiol (PI)-phospholipase C (PLC) inhitor U73122 nor the protein kinase C (PKC) inhibitor BisIV/Go6976 abolished the effect of SKF. Similarly, protein kinase A (PKA) inhibitor KT5720 failed to block the SKF effect.In conclusion, our work demonstrates the expression of aRl in rat retina. We further shows that suppression of AMPAR-mediated L-eEPSCs by the activation of aRl may be mediated by a distinct cGMP-PKG signaling pathway.