AMPA/Kainate Receptors Modulate Release Of The NMDA Receptor Co–agonist D–serine From Müller Glia

Abstract

Abstract: : Purpose: Recent evidence indicates that glial cells in the CNS synthesize D–serine, an endogenous co–agonist at the "glycine site" of NMDA receptors. We have previously shown that Müller glia express serine racemase, which catalyzes synthesis of D–serine, and that D–serine modulates NMDA receptor–mediated currents in intact retina. In this study we used microdialysis coupled to capillary electrophoresis to determine whether AMPA/KA receptors modulate extracellular D–serine concentration in intact retinas. The second purpose of this study was to determine whether AMPA/KA receptors could stimulate release of D–serine from Müller glia by elevating internal Ca²⁺ in these cells. Methods: Capillary electrophoresis (CE): Intact retinas from tiger salamander (Ambystoma tigrinum) were acutely isolated and placed under constant perfusion in a low–volume chamber. Amino acid efflux was analyzed online by CE with microdialysis and laser–induced fluorescence detection of derivatized D– and L–amino acids. Kainic acid (KA, 25–100 µM) and cyclothiazide (CTZ, 25–50 µM) were added by bath perfusion. Electropherograms were collected continuously and compared against standard curves to determine identity and concentrations of amino acids in the dialysate. Calcium imaging: Salamander Müller cells were enzymatically dissociated, loaded with Fura–2, and viewed in a chamber under constant perfusion. KA (.1–100 µM), CTZ (25 µM), and AMPA (.1–100 µM) were added by bath perfusion. Peak ratiometric values reflecting [Ca²⁺]were continuously measured. Results: Stimulation of intact retinas with KA (up to 100 µM) in the presence of CTZ (50 µM) led to dose–dependent, transient increases in extracellular D–serine concentration (2– to 7–fold). Increases in GABA, glutamate, and taurine were also reliably detected with this method. In isolated Müller cells, KA or AMPA application (15–30 sec) led to transient increases of [Ca²⁺], which usually took the form of a "wave" that progressed from the distal tuft to proximal endfoot. These responses were dose dependent (EC 50 for KA ∼ 3 µM; AMPA ∼5 µM). In the presence of CTZ, responses to KA in the major regions of Müller cells were amplified by 29–1400%. Conclusions: Amphibian Müller cells express receptors with pharmacological characteristics of AMPA/KA receptors, including sensitivity to cyclothiazide, and may be capable of modulating local neuronal activity by release of D–serine. Our ongoing experiments are using CE/LIF to determine directly whether a purified preparation of Müller cells releases D–serine in response to AMPA/KA receptor activation.

Keywords: Muller cells • neurotransmitters/neurotransmitter systems • calcium