Abstract
Purpose: :
Electrophysiological studies indicate the presence of NaVs in ganglion cells, amacrine cells and, recently, in cone bipolar cells in the mammalian retina. Our purpose was to localize NaV α–subunit isoforms known to be expressed in the retina and to determine which, if any, of these subunits were localized to second order neurons.
Methods: :
Adult rat, mouse and rabbit eyes were enucleated immediately following euthanasia, paraformaldehyde fixed, embedded, cryosectioned and mounted on slides. Single and double immunolabeling using a pan–specific NaV α–subunit antibody (Pan–NaV) or NaV 1.1, 1.2, and 1.6 subunit–specific antibodies and a panel of cell–specific markers was examined, after an optional antigen retrieval step, by confocal microscopy. Ganzfeld ERGs were recorded from anesthetized and fully dark adapted Brown Norway rats using brief flashes (< 5ms, max=462 nm; –5.8 to 1.9 log sc td s) and after intravitreal injections of TTX (3 µM, to block Na+–dependent spikes), CNQX (200 µM, to block iGluR’s) or TTX+ CNQX. Dark–adapted cone signals were isolated by silencing rod signals with a transient background and probing for residual cone signals with test flashes.
Results: :
The Pan–NaV and isoform–specific antibodies showed expected labeling of ganglion cells, ganglion cell axons and optic nerve. In the INL, subsets of amacrine cells (e.g., AII cells) showed strong labeling for pan–NaV, consistent with previous physiological studies showing the presence of NaVs on amacrine cells. No clear NaV α–subunit labeling was identified in bipolar cell bodies. Surprisingly, Pan–NaV and NaV1.2 and 1.6 subunit, with weaker NaV1.1 labeling were found in horizontal (Hz) cells and their processes in all three species. In rodents, diffuse labeling for NaV1.1 distal to Hz cell processes in the OPL was seen. TTX did not alter the amplitude of the leading edge of the scotopic photoreceptor a–wave or the rod bipolar cell–driven b–wave. CNQX and CNQX + TTX reduced the maximum amplitude of the dark adapted b–wave. CNQX+TTX, but not CNQX alone, caused substantial reduction in amplitude of the cone–isolated b–wave, implicating cone bipolar cells as the loci for these TTX effects.
Conclusions: :
NaV labeling was found in ganglion cells, amacrine cells and Hz cells and their processes. NaV1.1 was diffusely distributed distal to Hz cell processes in the rodent OPL. ERG results indicate that NaVs in cone bipolar cells but not Hz cells contribute to cone driven b–wave response to brief flashes. The bipolar cell NaVs may be too sparsely distributed for reliable detection by immunolabeling, or they are the NaV1.1 isoforms found in the OPL.
Keywords: retina • ion channels • electrophysiology: non-clinical