Glycine receptors in a population of adult mammalian cones

Abstract

Glycinergic interplexiform cells provide a feedback signal from the inner retina to the outer retina. To determine if cones receive such a signal, glycine was applied on cultured porcine cone photoreceptors recorded with the patch clamp technique. A minor population of cone photoreceptors was found to generate large currents in response to puff application of glycine. These currents reversed close to the calculated equilibrium potential for chloride ions. These glycine-elicited currents were sensitive to strychnine but not to picrotoxin consistent with the expression of alpha-beta-heteromeric glycine receptors. Glycine receptors were also activated by taurine and beta-alanine. The glycine receptor antibody mAb4a labelled a minority of the cone photoreceptors identified by an antibody specific for cone arrestin. Finally, expression of the beta subunit of the glycine receptor was demonstrated by single cell RT-PCR in a similar proportion (approximately 13%) of cone photoreceptors freshly isolated by lectin-panning. The identity of cone photoreceptors was assessed by their specific expression of the cone arrestin mRNA. The population of cone photoreceptors expressing the glycine receptor was not correlated to a specific colour-sensitive subtype as demonstrated by single cell RT-PCR experiments using primers for S opsin, cone arrestin and glycine receptor beta subunit. This glycine receptor expression in a minority of cones defines a new cone population suggesting an unexpected role for glycine in the visual information processing in the outer retina.

Figure 1. Glycine responses in cultured and freshly dissociated photoreceptors of the adult pig retina

A–B, current–voltage relationships of cultured rod (A) and cone (B) photoreceptors when stepping the membrane potential from −120 mV to +50 mV in 10 mV increments. C–E, effect of bath-perfusion of glycine (1 mm) in a representative cultured rod (C) and in two cultured cones (D and E) voltage-clamped at −70 mV. Note that some cultured cones responded to glycine whereas rod never generated a current (n = 20). F and G, response of a freshly isolated cone photoreceptor submitted to a puff-application of 1 mm glycine (CP: cone pedicle; Ax: axon; CB: cell body; IS: inner segment; OS: outer segment).

Figure 2. Cl⁻ dependence of glycine-elicited responses in cultured cones

A, glycine-elicited currents in two cones with different calculated equilibrium potentials for Cl⁻ (left: 0 mV; right: −30 mV). Cells were voltage clamped at potentials ranging from −70 mV to 40 mV in 10 mV increments and glycine (1 mm) was puff-applied for 50 ms. B, current–voltage relation of the averaged glycine-elicited responses in cone photoreceptors with different calculated equilibrium potentials for Cl⁻ (▪, E_Cl= 0 mV, n = 3; •, E_Cl=−30 mV; n = 7).

Figure 3. Pharmacology of glycine receptors in cultured cone photoreceptors

A and B, strychnine (1 μm) completely suppressed the glycine-elicited current (A) whereas picrotoxin (100 μm) did not affect the response (B). C and D, β-alanine- and taurine-elicited currents were partly suppressed by bath application of the GABA_A and GABA_C receptor antagonists, SR95531 (100 μm) and TPMPA (50 μm), the remaining currents being totally inhibited by strychnine. E, both β-alanine (6 mm) and taurine (5 mm) completely suppressed the glycine-elicited response while inducing a steady state current. In this experiment, SR95531 (100 μm) and TPMPA (50 μm) were continuously bath-applied. F, measurements of glycine-elicited responses and the holding current following during β-alanine and taurine bath applications. Note that the holding current increased during agonist applications and reflecting the steady state current generated by these amino acids. Amino acids were puff-applied on cultured cones voltage clamped at −70 mV (glycine in A and B: 1 mm, 50 ms; β-alanine in C: 6 mm, 3 s; taurine in D: 5 mm, 3 s). In E, glycine (1 mm, 3 s) was puff-applied while β-alanine (6 mm) and taurine (5 mm) were bath-perfused.

Figure 4. Single-cell RT-PCR analysis of the glycine receptor β subunit expression in different subpopulations of freshly purified cones photoreceptors

A 100 bp DNA standard marker ladder is shown on lanes M. Lanes 1 and 2 were negative controls: RT-PCR on PCR buffer and patch clamp buffers (intrapipette buffer + perfusing solution) and samples without reverse transcriptase, respectively. Lanes 3–7 illustrate amplified DNA products corresponding to cone arrestin mRNA (top), S-opsin mRNA (middle) and β-subunit mRNA (bottom) from 5 independent freshly isolated cells. DNA products were resolved in a 1.2% agarose gel and visualized with ethidium bromide.

Figure 5. Glycine receptor immunolabelling in pig retinal sections and cultured photoreceptors on a glial cell feeder layer

A and B, fluorescence images showing the mAb4a immunoreactivity (red) in a vertical section of the pig retina also labelled for the cone arrestin (green). Retinal layers are visible on the superimposed Nomarski image (A). Arrows in B indicate an OPL location of glycine receptors in the pig retina. C–E, epifluorescence images focused on the OPL showing mAb4a-immunoreactive puncta (red) in some cone photoreceptor synaptic endings as revealed with the cone arrestin antibody (green). Arrows in C and E point out puncta of glycine receptors on a cone terminal. F–H, glycine receptor immunolabelling in cultured photoreceptors on a glial feeder cell layer. Very few cone photoreceptors that were identified with the cone arrestin antibody (green) were double-labelled with the mAb4a antibody (red) in vitro. Scale bars represent 10 μm in (A and B), 5 μm (C–H).