Metabolic and functional profiling of the normal rat retina

Abstract

We established a metabolic and functional profile map of the normal rat retina, given the premise that: 1) amino acid neurochemistry reflects metabolic integrity and cellular identity, and 2) the permeation of a cation channel probe, agmatine (1-amino-4-guanidobutane, AGB), reflects cation channel functionality. The purpose was to provide a unique method of simultaneously assessing the metabolic and functional characteristics of the normal retina, upon which a comparison can be made to disease models. Quantitative pattern recognition analysis of overlapping amino acid and AGB expression profiles was used to provide a statistically robust classification of all neural elements according to their metabolic and functional characteristics. This classification was spatially complete and with single-cell resolution. The resulting classification demonstrated 28 statistically separable theme classes dominated by characteristic glutamate, GABA, glycine, and/or taurine profiles, with each of the neuronal theme classes containing further subtypes. The inclusion of a functional parameter (AGB mapping) in the classification process nearly doubled the number of neural elements that could be ascribed a neurochemical/cation profile, compared to when amino acid labeling was used alone. Strong endogenous glutamate gated AGB labeling was observed in horizontal cells, rod bipolar cells, cholinergic amacrine cells, and AII amacrine cells. The resulting amino acid and AGB profile matrix constitutes a nomogram for assessing cellular responses to experimental challenges in models of ocular disease.

Functional and neurochemical development in the normal and degenerating mouse retina

“…His assumption was based on the known location of cholinergic cell somata and distinct bistratified dendritic location in the inner plexiform layer. Subsequent development of techniques to combine AGB and macromolecular markers (Marc et al, 2005) confirmed that Marc's linking proposition was indeed correct (Sun et al, 2007b). We acknowledge that unless other cell markers are used (e.g., macromolecular markers) or pattern recognition is undertaken, some errors will occur in cell assignment in borders or locations where the cells coexist.…”

“…Similar to other rodent retinas (Fletcher andKalloniatis, 1996, 1997a,b), the mouse retina displays distinct cellular location during development, identified using pattern recognition of amino acid labeling patterns (Chua, 2008;Kalloniatis et al, 2012). Soma size, dendritic disposition, and labeling intensity are also supportive components in cellular classification (Sun et al, 2007b). The use of all or some of these principles allows the identification of core cell classes, e.g., bipolar cells, amacrine cells, ganglion cells.…”

Metabolic and functional profiling of the ischemic/reperfused rat retina

“…Briefly, a 3-L intravitreal injection of AGB (final vitreal concentration of 10 mM) was administered 30 minutes prior to enucleation. Details of the tissue preparation following enucleation have also been described in Sun et al (2007b). Briefly, tissue was collected Ϸ1-2 mm from the optic nerve head, fixed in 2.5% glutaraldehyde / 1% paraformaldehyde, resin-embedded, and processed for postembedding silver-intensified immunocytochemistry (Kalloniatis and Fletcher, 1993;Marc, 1999a,b;Sun et al, 2003).…”

“…Procedures for the in vivo injection of AGB have been previously described (Sun et al, 2007b). Briefly, a 3-L intravitreal injection of AGB (final vitreal concentration of 10 mM) was administered 30 minutes prior to enucleation.…”

“…Quantitative pattern recognition analysis of overlapping amino acid and AGB immunoreactivity patterns was used to provide a statistically robust classification of all retinal elements within the reperfused retina. Our previous work has characterized the metabolic and functional profiles of all neurons and glia in the normal rat retina (Sun et al, 2007b). In this study, a number of key issues were addressed: 1) the duration of the amino acid changes following ischemia/reperfusion, and whether there is recovery of the amino acid expression profiles; 2) the identification of cell types with a particular amino acid or cation channel characteristic that are susceptible to ischemic insult; and 3) whether the changes in cation channel function parallel the change in metabolic state (as reflected by amino acid labeling).…”