Morphometric multivariate analysis of GABAergic neurons containing calretinin and neuronal nitric oxide synthase in the mouse hippocampus

Abstract

Several studies reported the morphology of calretinin-positive (CR+) neurons and nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) labeled or neuronal nitric oxide synthase-positive (nNOS+) neurons in the rodent hippocampus, where these neurons showed similar morphological features. In addition, a previous study reported the frequent colocalization of CR and NADPH-d in the rat hippocampus. In this study, we aimed to examine whether CR+ neurons and nNOS+ neurons belong to a same morphological subpopulation of GABAergic neurons in the mouse hippocampus. Neurons were immunocytochemically classified into three groups, i.e., CR+/nNOS−, CR−/nNOS+ and CR+/nNOS+ groups. The present morphometric analysis was performed in the mouse Ammon’s horn, because CR+/nNOS+ neurons were rarely found in the mouse dentate gyrus. We selected three morphometric parameters, i.e., soma area, soma form factor (FF) and number of primary dendrites. Dunnett’s post-hoc analysis revealed that soma area, soma FF and number of primary dendrites were significantly larger in CR−/nNOS+ group than in CR+/nNOS− and CR+/nNOS+ groups. The morphometric data of CR+/nNOS+ group were quite similar to those of CR+/nNOS− group. The morphometric multivariate logistic regression analysis revealed that these three morphometric parameters were independent significant variables to discriminate between CR+/nNOS− and CR−/nNOS+ groups, and the majority of CR+/nNOS− and CR−/nNOS+ groups were correctly classified from the morphometric features. The present results clearly indicate that CR+/nNOS− neurons and CR−/nNOS+ neurons belong to different morphological subpopulations, and lead us to speculate that they might play different functional roles in the hippocampal circuit. The further application of morphometric multivariate analysis would be valuable to understand the functional roles of chemically defined neurons in the various brain regions.