​(Fig.13A).13A). nNOS neurons were scattered and intermingled with GFAPIP cells; in either experimental case no cells contained both nNOS and GFAP (Fig. ​(Fig.13).13). These experiments indicated that: (1) NO-producing intracallosal cells are neurons; (2) glial cells do not contain nNOS. Figure 13 Confocal laser scanning photomicrographs showing the lack of colocalization (C) of GFAP+ (A, red fluorescence) and nNOSIP neurons Inhibitors,research,lifescience,medical (B, green fluorescence) in the rat corpus callosum. Calibration bar: in C for A–C 25 μm. Discussion The results of the present experiments can be summarized as follows: Double-labeling experiments disclosed that nNOS-positive

cells did not contain GFAP, indicating that they may be considered as neurons. The rat cc contains numerous NO-producing Inhibitors,research,lifescience,medical neurons. NADPH-d+/nNOSIP neurons show a lateromedial gradient, that is, they are more numerous in lateral than in medial cc regions. NADPH-d+/nNOSIP neurons are morphologically heterogeneous. Many NADPH-d+ neurons are closely associated with intracallosal blood vessels. The fluorescence experiments indicated that NO-producing cells in the rat cc are neurons. In both brains studied by this method, nNOSIP neurons never contained GFAP Inhibitors,research,lifescience,medical and callosal glial cells did not contain nNOS. The presence of nNOS in glial cells in normal nervous tissue is debated.

Previous studies have shown a very small proportion of nNOSIP glial cells in the rat visual cortex (Lüth 1997) and guinea pig optic nerve (Qi and Guy 1996). Our study is in line with work performed in the rat intermediolateral cell column, where double-labeling experiments Inhibitors,research,lifescience,medical demonstrated the lack of nNOS/GFAP colocalization (Blottner and Baumgarten 1992). NO-producing neurons in the rat cc The free radical NO is involved in many aspects of normal CNS functioning Inhibitors,research,lifescience,medical (Vincent 1994). NO is synthesized from l-arginine by three different isoforms of the NOS enzyme: nNOS, endothelial NOS (eNOS) and inducible NOS (iNOS) (Garthwaite 1991; Moncada et al. 1991; Knott and Bossy-Wetzel 2009). Neuronal

NOS is found in CNS and peripheral nervous system neurons; NO-producing neurons GPX6 can therefore be visualized by immunocytochemical nNOS detection. Moreover, nNOS activity can be evaluated using a histochemical stain for the enzyme NADPH-d, which is considered a neuronal NOS (Hope et al. 1991). We therefore used immunocytochemistry and histochemistry to investigate the presence, distribution, and morphology of NO-producing neurons in the rat cc. Our findings partially confirm those obtained in monkey (Rockland and Nayyar 2012), but in addition, they provide a detailed description of the LY2157299 number and distribution of NO-producing neurons. Special attention was devoted to defining cc boundaries, especially in the most lateral stereotaxic planes, where it is harder to discriminate the cc dorsal border from overlying white matter.