Protein MAP1B was recently reported to link GABA(C) receptors to the cytoskeleton at neuronal synapses. This interaction was demonstrated in the mammalian retina, where GABA(C) receptors were thought to be exclusively expressed in bipolar cells. Our previous studies on cultured photoreceptors suggested however the presence of GABA(C) receptors in cones. To further investigate GABA(C) receptor expression in cones, we measured GABA responses in mammalian photoreceptors in situ, and we examined the distribution of the receptor and that of protein MAP1B in the mammalian outer retina. Photoreceptors were recorded from flat-mounted retinas of retinal degeneration mice at an age when the retina becomes cone-dominated after rod cell death. GABA(A) and GABA(C)-gated currents were produced only in cones but not rods. Recording freshly dissociated retinal cells from wild-type C57 mice confirmed the presence of GABA(A) and GABA(C) receptors in cones. Immunohistochemical labeling of mouse and rat retinal sections localized GABA(C) receptors to cone terminals that were identified by peanut agglutinin lectin staining. As expected from previous studies on bipolar cells, the punctate immunostaining was not restricted to cone terminals in the outer plexiform layer. MAP1B immunolabeling was obtained in rat and pig retinas and was similarly found in cone terminals identified by the peanut agglutinin lectin staining. These results provide physiological and histological evidence that cones receive a GABA feedback in the mammalian retina and are consistent with the notion that protein MAP1B links GABA(C) receptors to the cytoskeleton at postsynaptic sites.

In different animal models, photoreceptor degeneration was correlated to an abnormal increase in cGMP concentration. The cGMP-induced photoreceptor toxicity was demonstrated by applying the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine on retinal explants. To assess the role of cGMP-gated channels in this cGMP toxicity, the Ca(2+) channel blockers verapamil and L- and D-diltiazem, which block cGMP-gated channels with different efficacies, were applied to in vitro animal models of photoreceptor degeneration. These models included: (i) adult rat retinal explants incubated with zaprinast, a more specific inhibitor of the rod phosphodiesterase than 3-isobutyl-1-methylxanthine and (ii) rd mouse retinal explants. Photoreceptor apoptosis was assessed by terminal dUTP nick end labelling and caspase 3 activation. Effects of the blockers on the synaptic rod Ca(2+) channels were measured by patch-clamp recording. In the zaprinast-induced photoreceptor degeneration model, both diltiazem isomers rescued photoreceptors whereas verapamil had no influence. Their neuroprotective efficacy was correlated to their inhibition of cGMP-gated channels (l-diltiazem>d-diltiazem>verapamil=0). In contrast, all three Ca(2+) channel blockers suppressed rod Ca(2+) channel currents similarly. This suppression of the currents by the diltiazem isomers was very weak (16.5%) at the neuroprotective concentration (10 microm). In rd retinal explants, both diltiazem isomers also slowed down rod degeneration in contrast to verapamil. L-diltiazem exhibited this effect at concentrations ranging from 1 to 20 microm. This study further supports the photoreceptor neuroprotection by diltiazem particularly in the rd mouse retina, whereas the absence of neuroprotection by verapamil further suggests the role of cGMP-gated channel activation in the induction of photoreceptor degeneration.

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