Muller Cells In The Salamander Retina Express Functional Ca2+-permeable AMPA Receptors

Abstract

Purpose: : We have previously demonstrated that D-serine is a necessary co-agonist for activation of NMDA receptors during synaptic transmission in the vertebrate retina. However, the source of D-serine and the mechanisms for D-serine release in the retina are still unknown. Several models suggest that functional AMPA-type glutamate receptors are necessary for D-serine release, and our laboratory has demonstrated that glial cells may be a prominent source of D-serine release in the retina. Reconciling these two ideas for D-serine release requires that glial cells express functional AMPA receptors. We tested this hypothesis by challenging isolated Muller glia with AMPA receptor agonists and antagonists while measuring changes in cytosolic free Ca²⁺ and transmembrane currents.

Methods: : Muller glia and neurons were acutely isolated from larval tiger salamander retinas by enzymatic digestion, loaded with fluorescent Ca²⁺-sensitive dyes, and plated in a perfusion chamber on the stage of a fluorescence-equipped inverted microscope. Intact Muller cells were selected for simultaneous whole-cell patch recording and imaging of intracellular Ca²⁺. Agonists and antagonists of AMPA receptors were applied by bath perfusion.

Results: : Application of AMPA alone (up to 100μM) did not evoke either Ca²⁺ responses or conductance changes in isolated Muller cells. However, AMPA application along with 50μM cyclothiazide, which prevents AMPA receptor desensitization, evoked large Ca²⁺ increases (3-11x resting Ca²⁺) and large inward currents from rest (E_rev~ +3 mV). In 85% of cells, a selective antagonist of Ca²⁺-permeable AMPA receptors, N-acetyl spermine, reduced Ca²⁺ mobilization and evoked current amplitudes by 40%.

Conclusions: : Our results indicate that isolated Muller glia express functional AMPA glutamate receptors, which appear to be in a desensitized state at rest. Sensitivity of the observed responses to N-acetyl spermine suggests that a large proportion of glial AMPA receptors are a Ca²⁺-permeable variant. These results are consistent with our earlier capillary electrophoretic analyses, which indicate that cyclothiazide is necessary to evoke AMPA-receptor mediated increases in extracellular D-serine levels in the mouse retina. Our results support the hypothesis that glial cells may be a major source of D-serine released during light-evoked activity in the vertebrate retina.

Keywords: Muller cells • neurotransmitters/neurotransmitter systems • excitatory amino acid receptors