Biophysical Basis of the in vivo ERG of the Mouse: Rod, RPE and Müller glial Cell Contributions To Dark and to Light-driven Changes in Sub-Retinal Space o

Abstract

The subretinal space (SRS) is a contiguous extracellular volume bounded by the external limiting membrane (ELM) and the retinal pigment epithelium (RPE). Approximately 90% of mouse rod photoreceptor dark current circulates in the SRS, and rod photoresponses materially alter SRS concentrations of permeant ions, especially K+. Following intravitreal injection of drugs that block glutamatergic synapses, and drugs that inhibit Kir4.1 and Kir7.1 channels expressed in Muller and RPE cells respectively, we measured electroretinograms (ERGs) in response to light flashes that completely suppress rod dark current, and compared ERG c-waves with predictions of a biophysical model of extracellular current sources generated by rod, Muller and RPE cells. SRS K+ fluxes in the dark steady-state were dominated by rods - NKX (-6.6 mM s-1), Kv2.1 channels (+5.4 mM s-1), NCKX (0.3 mM s-1) and HCN1 channel fluxes (1.0 mM s-1) - with much smaller contributions from Kir4.1 and Kir7.1 channels and trans-ELM and trans-RPE fluxes combining to zero net flux at resting SRS Ko of 5 mM (re 3.5 mM [K+] in blood plasma). In response to rod-saturating stimulation, Ko was predicted to decline to [~]3.5 mM with a half-time of [~] 0.5 s, driven mainly by a hyperpolarization-dependent decline in Kv2.1 current. The kinetics and amplitude of the c-wave are explained by the decline in SRS Ko, which negatively shifts the Nernst potential of apical RPE Kir7.1 channels, increasing outward current that sinks through the paracellular resistance to basal Bestrophin and CFTR chloride channels causing a trans-epithelial potential of [~] 2 mV.