Distribution and neurochemical features of neuronal nitric oxide synthase-expressing interneurons in the rat dentate gyrus

Neuronal nitric oxide synthase (nNOS)-expressing interneurons are abundant in the dentate gyrus (DG) of rodents. In the present study, we immunohistochemically analyzed nNOS-positive cells in the rat DG by focusing on their GABAergicity, laminar distribution, and co-localization with calcium-binding proteins and neuropeptides. Experiments were conducted in adult male Sprague Dawley rats. Within the DG, nNOS-positive cells were found to reside in all three layers of DG; percentages of distribution in the molecular layer, granule cell layer and the hilus are 25.4%, 9.4% and 65.2%, respectively. Almost every nNOS-positive cell expressed glutamic acid decarboxylase 67 (GAD67) or glutamic acid decarboxylase 65 (GAD65). In the molecular layer, nearly two-thirds of GAD67-positive cells expressed nNOS. Percentages of nNOS-positive interneurons that expressed cholecystokinin, vasoactive intestinal polypeptide, parvalbumin, somatostatin, neuropeptide Y, and calretinin were approximately 0.8%, 1.8%, 9.2%, 10.3%, 13.8%, and 24.4%, respectively. In the molecular layer, the number of nNOS-positive cells far exceeded the sum total of cells positive for both nNOS and any of the above mentioned calcium-binding proteins or neuropeptides, indicating that a large proportion of nNOS-positive interneurons seldom express calcium-binding proteins or neuropeptides in this area. We conclude that nNOS expressing cells are an important neurochemically defined type of GABAergic interneuron in the rat DG showing a specific laminar-dependent distribution and expressing calcium-binding proteins and neuropeptides at different frequencies. In the molecular layer, most nNOS-positive interneurons do not express calcium-binding proteins or neuropeptides; they could be the missing pieces in the GABAergic interneuron jigsaw puzzle of this DG layer.

► nNOS-positive cells reside in all three layers of rat DG.
► Every nNOS-positive cells are GABAergic.
► nNOS-positive cells constitute a large proportion of GABAergic interneurons.
► nNOS-positive interneurons express individual calcium-binding proteins and neuropeptides at different frequencies.
► In the molecular layer, nNOS-positive cells seldom express calcium-binding proteins and neuropeptides.