Quantitative analysis of neuronal nitric oxide synthase-immunoreactive neurons in the mouse hippocampus with optical disector

A detailed quantitative analysis of immunocytochemically identified nonprin cipal neurons containing neuronal nitric oxide synthase (nNOS) was performe d on the mouse hippocampus, with particular reference to the dorsoventral g radient. The present study applied two variations of a stereologic techniqu e, the optical disector-one that; used confocal laser-scanning microscope o ptical sections to examine colocalization of nNOS and glutamic acid decarbo xylase 67 (GAD67), and the other that used conventional thick sections to e xamine numerical densities (NDs) and cell sizes of nNOS-immunoreactive (IR) neurons. Colocalization analysis indicated that practically all nNOS-IR ne urons (97.6%) were GAD67-IR, whereas a part of the GAD67-IR neurons (about 30%) were nNOS-IR in the whole hippocampus at both dorsal and ventral level s. The percentages of CAD67-IR neurons containing nNOS were higher in the d entate gyrus (DG, about; 50%), and lower in the Ammon's horn (about 20%). L aminar analysis revealed that the majority of GAD67-IR neurons contained nN OS in the stratum lacunosum-moleculare of the CA3 region (about 60%) and in the molecular layer of the DG (about 80%). The NDs of nNOS-IR neurons in t he whole hippocampus showed a dorsoventral gradient, which increased from d orsal(1.6 x 10(3)/mm(3)) to ventral (2.2 x 10(3)/mm(3)) levels. The NDs wer e relatively higher in the principal cell layers, where about 40% of nNOS-I R neurons were situated both in the Ammon's horn and DG. The mean cell size s of nNOS-IR neurons showed no remarkable laminar differences or dorsoventr al gradient in the Ammon's horn, but; they were extensively larger in the h ilus of the DG than in other layers. These results indicate that nNOS-IR ne urons in the mouse hippocampus represent a subpopulation of gamma-aminobuty ric acid (GABA)ergic neurons and suggest that the laminar distributions of nNOS-IR neurons related to possible functional heterogeneity of GABAergic n eurons in each hippocampal layer. (C) 1999 Wiley-Liss, Inc.