Inner retinal neurons display differential responses to N‐methyl‐D‐aspartate receptor activation

Abstract

The N-methyl-D-aspartate (NMDA) responses of neurons from within the inner rabbit retina were mapped using a channel permeable cation, 1-amino-4-guanidobutane (agmatine, AGB). Serial sections were subsequently probed with immunoglobulins targeting AGB, glutamate, gamma-aminobutyric acid (GABA), and glycine to visualize the NMDA responses of neurochemical subpopulations of neurons. Most inner retinal subpopulations of neurons demonstrated an NMDA concentration-dependent increase in activation. This NMDA-induced activation displayed a distinct pattern, with the most sensitive class to least sensitive class ranking being GC > GABA cAC > GABA/Gly cAC > Gly cAC > GABA dAC (GC, ganglion cells; AC, amacrine cells; c, conventional; d, displaced; Gly, glycine). The variable NMDA response may reflect differences in NMDA receptor subunit disposition or differences in receptor density. In addition to the variable NMDA activation pattern, we found that virtually all ganglion cells (87%) showed NMDA-gated AGB entry, compared with only 58% of amacrine cells. We conclude that a large cohort of amacrine cells do not possess functional NMDA receptors. In addition to most ganglion cells being activated by NMDA, a large subpopulation displayed the highest sensitivity to NMDA application. The functional significance of this finding is that the ganglion cell population will be the first neuronal class to be susceptible to glutamate-induced neurotoxicity mediated through the NMDA receptor. The addition of betaxolol significantly reduced NMDA-mediated AGB entry into most neuronal groups (ganglion cells, GABA, and glycine amacrine cells), with the greatest effect being on ganglion cells. Betaxolol had no significant effect on NMDA-gated entry of AGB on the GABA/Gly amacrine cell population.

Metabolic and functional profiling of the normal rat retina

“…AGB does not alter amino acid neurochemistry, and there is no detectable endogenous AGB in the rabbit, rat, or mouse retina (Marc, 1999a,b;Kalloniatis et al, 2002;Sun et al, 2003;Sun and Kalloniatis, 2006). Similar to previous studies (Marc and Jones, 2002), we qualitatively analyzed sections from Ͼ10 separate retinal samples with and without AGB and found no obvious change in neurochemistry secondary to AGB injection.…”

“…Although amino acid content alone did not allow for the identification of separate theme classes for presumed cone photoreceptors and rod photoreceptors, the addition of an AGB signal (Fig. 1H, black square; Kalloniatis et al, 2002Kalloniatis et al, , 2004Sun et al, 2003), allowed us to separate cones from rods. All photoreceptor elements contained low levels of most amino acids, and it is the levels of glutamate and taurine that segregate them from each other (e.g., inner Fig.…”

“…The use of AGB to trace cation channel activity has been previously used in understanding excitatory drive in the retina and olfactory bulb (Marc, 1999a,b;Edwards and Michel, 2002;Sun et al, 2003;Kalloniatis et al, 2004;Marc et al, 2005;Sun and Kalloniatis, 2006;Acosta et al, 2007). The influx of AGB is proportional to the channel permeability and the driving force on the channel; it reports the excitation history experienced by a cell due to cationic fluxes through glutamate-gated channels.…”

“…Detailed methods regarding the preparation of the tissue for postembedding immunocytochemistry have been previously described (Marc et al, 1990(Marc et al, , 1995Kalloniatis and Fletcher, 1993;Sun et al, 2003). Resin blocks were serially sectioned at 200 nm using a Reichert-Jung Ultracut S microtome.…”

“…Functional profiles were derived from the immunocytochemical localization of AGB secondary to endogenous or exogenous excitatory drive (Marc, 1999a,b;Edwards and Michel, 2002;Sun et al, 2003;Kalloniatis et al, 2004;Marc et al, 2005;Sun and Kalloniatis, 2006). The advantage of using AGB is the ability to study population responses within a single preparation, while preserving structural integrity.…”