Abstract
Voltage-gated sodium channels (Na(v) channels) in retinal neurons are known to contribute to the mammalian flash electroretinogram (ERG) via activity of third-order retinal neurons, i.e. amacrine and ganglion cells. This study investigated the effects of tetrodotoxin (TTX) blockade of Na(v) channels on the b-wave, an ERG wave that originates mainly from activity of second-order retinal neurons. ERGs were recorded from anaesthetized Brown Norway rats in response to brief full-field flashes presented over a range of stimulus energies, under dark-adapted conditions and in the presence of steady mesopic and photopic backgrounds. Recordings were made before and after intravitreal injection of TTX (approximately 3 microm) alone, 3-6 weeks after optic nerve transection (ONTx) to induce ganglion cell degeneration, or in combination with an ionotropic glutamate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 200 microm) to block light-evoked activity of inner retinal, horizontal and OFF bipolar cells, or with the glutamate agonist N-methyl-D-aspartate (NMDA, 100-200 microm) to reduce light-evoked inner retinal activity. TTX reduced ERG amplitudes measured at fixed times corresponding to b-wave time to peak. Effects of TTX were seen under all background conditions, but were greatest for mesopic backgrounds. In dark-adapted retina, b-wave amplitudes were reduced only when very low stimulus energies affecting the inner retina, or very high stimulus energies were used. Loss of ganglion cells following ONTx did not affect b-wave amplitudes, and injection of TTX in eyes with ONTx reduced b-wave amplitudes by the same amount for each background condition as occurred when ganglion cells were intact, thereby eliminating a ganglion cell role in the TTX effects. Isolation of cone-driven responses by presenting test flashes after cessation of a rod-saturating conditioning flash indicated that the TTX effects were primarily on cone circuits contributing to the mixed rod-cone ERG. NMDA significantly reduced only the additional effects of TTX on the mixed rod-cone ERG observed under mesopic conditions, implicating inner retinal involvement in those effects. After pharmacological blockade with CNQX, TTX still reduced b-wave amplitudes in cone-isolated ERGs indicating Na(v) channels in ON cone bipolar cells themselves augment b-wave amplitude and sensitivity. This augmentation was largest under dark-adapted conditions, and decreased with increasing background illumination, indicating effects of background illumination on Na(v) channel function. These findings indicate that activation of Na(v) channels in ON cone bipolar cells affects the b-wave of the rat ERG and must be considered when analysing results of ERG studies of retinal function.