Distinct types of nitric oxide-producing neurons in the developing and adult mouse claustrum

We studied at the light and electron microscopic levels the nitric oxide-producing neurons in the mouse claustrum. Nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry and neuronal nitric oxide synthase (nNOS) immunohistochemical staining were used to reveal putative nitrergic neurons. We also analyzed colocalization of nNOS with the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) as well as the ontogenesis of the nNOS-immunoreactive neurons, providing evidence for different populations of nitrergic neurons in the mouse claustrum. The general staining pattern was similar for the histochemical and the immunohistochemical methods, resulting in neuron and neuropil staining throughout the whole claustrum. We described two populations of nitric oxide-producing neurons in the mouse claustrum on the basis of a different level of nNOS expression. Densely nNOS-stained neurons were mostly GABA immunoreactive, displayed ultrastructural features typically seen in aspiny neurons, and may originate in the subpallium; they were first seen in the claustrum at embryonic stage 17.5 and probably represent local inhibitory interneurons. Densely stained cells were found from rostral to caudal levels throughout the dorsal claustrum and the endopiriform nucleus. Lightly nNOS-stained neurons, on the other hand, were more numerous than densely stained ones, especially in the dorsal claustrum. These claustral lightly stained cells, barely observed in the NADPH-diaphorase reacted sections, were mostly non-GABAergic, and appeared earlier during ontogenesis than densely stained cells (at embryonic stages 15.5-16.5). We suggest that these neurons are probably projection neurons.