Density and neurochemical profiles of neuronal nitric oxide synthase-expressing interneuron in the mouse basolateral...

Neuronal nitric oxide synthase (nNOS)-expressing interneurons reside in the basolateral nucleus of the amygdala (BLA) of rodents. In the present study, we immunohistochemically analyzed nNOS-positive cells in the mouse BLA by focusing on their density, γ-Aminobutyric acid (GABA)ergicity, and co-localization with calcium-binding proteins and neuropeptides. The density of nNOS-containing neurons was analyzed with unbiased stereology. Experiments were conducted in both adult wild-type C57BL/6 and glutamic acid decarboxylase-green fluorescence protein (GAD₆₇-GFP) knock-in mice, in which GFP is expressed in GABAergic neurons under the control of the endogenous GAD₆₇ gene promoter. In the BLA, the density of nNOS-positive cells was 3.92×10³cells/mm³. Immunofluorescence revealed that nNOS-containing neurons constituted almost 26.93±2.36% of the GAD₆₇-GFP neurons. Almost every nNOS-positive cell expressed glutamic acid decarboxylase 65 (GAD₆₅). Proportions of nNOS-positive interneurons that expressed calbindin, calretinin, parvalbumin, somatostatin and neuropeptide Y were approximately 5.20%, 15.63%, 26.50%, 87.50% and 88.00%, respectively; but exhibited no co-localization with vasoactive intestinal polypeptide. By contrast, percentages of calbindin, calretinin, parvalbumin, somatostatin and neuropeptide Y-positive cells that expressed nNOS were approximately 1.93%, 7.25%, 25.25%, 80.25% and 87.50%, respectively. Together, these findings suggest that nNOS-expressing cells are a discrete interneuronal subpopulation in the mouse BLA and may play a functional role in the inhibitory circuitry of this brain region.