Mapping glutamate responses in immunocytochemically identified neurons of the mouse retina

Abstract

The mammalian retina contains as many as 50-60 unique cell types, many of which have been identified using various neurochemical markers. Retinal neurons express N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxyl-5-methylisoxazole-4-propionic acid (AMPA), and kainic acid (KA) receptor subunits in various mixtures, densities, and spatial distributions. Ionotropic glutamatergic drive in retinal neurons can be mapped using a cation channel permeant guanidinium analog called agmatine (1-amino-4-guanidobutane; AGB). This alternative approach to physiologically characterize neurons in the retina was introduced by Marc (1999, J Comp Neurol 407:47-64, 407:65-76), and allows the simultaneous mapping of responses of glutamate receptor-gated channels from an entire population of neurons. Unlike previous AGB studies, we colocalized AGB with various macromolecular markers using direct and indirect immunofluorescence to characterize the glutamate agonist sensitivities of specific cell types. Activation with NMDA, AMPA, and KA resulted in AGB entry into neurons in a dose-dependent manner and was consistent with previous receptor subunit localization studies. Consistent with the various morphological phenotypes encompassed by the calbindin and calretinin immunoreactive cells, we observed various functional phenotypes revealed by AGB labeling. Not all calbindin or calretinin immunoreactive cells showed ligand-evoked AGB permeation. A small proportion either did not possess functional glutamate receptors, required higher activation thresholds, or express functional channels impermeable to AGB. AMPA and KA activation of bipolar cells resulted in AGB permeation into the hyperpolarizing variety only. We also studied the glutamate ligand-gating properties of 3[alpha1-3]-fucosyl-N-acetyl-lactosamine (CD15) immunoreactive cells and show functional responses consistent with receptor subunit gene expression patterns. CD15-immunoreactive bipolar cells only responded to AMPA but not KA. The CD15 immunoreactive amacrine cells demonstrated an identical selectivity to AMPA activation, but were also responsive to NMDA. Finally, localization of AGB secondary to glutamate receptor activation was visualized with a permanent reaction product.

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.…”

“…There was no crossreactivity of AGB against arginine, glutamate, and other amino acids used in this study (Marc, 1999a;Marc and Jones, 2002) (Chemicon datasheet for AB1568-2000T). Normal retinae probed with anti-AGB antibodies do not demonstrate endogenous AGB labeling (Marc, 1999a,b;Kalloniatis et al, 2004;Sun and Kalloniatis, 2006;Marc et al, 2007), with AGB immunoreactivity detected in the retina only when AGB was included in the incubation medium or via intravitreal injection. Amino acid antibodies have been used in numerous other retinal studies and show an identical labeling pattern (Marc et al, 1990(Marc et al, , 1995(Marc et al, , 2007Kalloniatis and Fletcher, 1993;Marc, 1999a;Marc and Jones, 2002;Kalloniatis et al, 2002Kalloniatis et al, , 2004.…”

“…These methods are as previously described (Sun and Kalloniatis, 2006). Briefly, for indirect immunofluorescence, retinae were fixed in 4% paraformaldehyde/0.01% glutaraldehyde, mounted on freezing medium (Reichert, Nussloch, Germany), sectioned vertically at 16 m on a cryostat, and collected on coated slides.…”

“…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.…”

Early remodeling of müller cells in the rd/rd mouse model of retinal dystrophy

“…Similar calculations comparing total rd/rd and control mice populations resulted in a sampling error of <2%. As per previous studies, only GSpositive cells that had distinct soma labeling above background levels were selected (Sun and Kalloniatis, 2006;Acosta et al, 2007).…”