BRAIN RESEARCH ELSEVIER Brain Research714 (1996) 135-144 Research report Localisation of NADPH diaphorase activity and NOS immunoreactivity in astroglia in normal adult rat brain P.L.A. Gabbott *, S.J. Bacon University Departmentof Pharmacology, MansfieldRoad, Oxford, OXI 3QT, UK Accepted21 November1995 Abstract This study demonstrates the, co-localisation of NADPH diaphorase activity and GFAP immunoreactivity in non-neuronal cells in weakly fixed brain sections from normal adult rats. The presence of GFAP immunoreactivity in these cells indicates that they are astroglia. In addition, cells possessing the morphological characteristics of astroglia were weakly immunoreactive for the endothelial isoform of nitric oxide synthase (eNOS) - these cells also co-localised NADPH diaphorase activity. Furthermore, cells immunoreactive for eNOS displayed GFAP imnmnoreactive processes. This cytochemical evidence strongly suggests that resting astroglia are potential sources of nitric oxide - a powerful modulator of cell activity. Keywords: ConstitutiveNOS; Nitric:oxide; GFAP; Weak aldehydefixation 1. Introduction Nitric oxide (NO) is a recently described transcellular messenger that can strongly influence cell function in the nervous system [13,19,25,27]. Cells with the ability to synthesise NO, possess the enzyme nitric oxide synthase (NOS) which catalyses the formation of NO and citrulline from L-arginine [25]. Several NOS isoforms have been described and localised within distinct cell classes in the brain [12,13,25-27]. Constitutive calcium/calmodulin de- pendent NOS isoforms are present in neurons (bNOS), endothelial cells (eNOS) aJad some resting glial cells [1,12,13,27]. The inducible calcium-independent NOS iso- forms occur in activated astroglia and microglia, and in blood-borne macrophages (mNOS) [13,17,21,22]. The identified NOS isoforms have highly conserved consensus sequences for NADPH binding sites [3,4,8]. These sites exhibit NADPH diaphoretic reductase activity that can be detected in aldehyde fixed brain tissue using enzyme histochemistry [6-8,13,25-27]. Different types of NOS containing cells in the brain can therefore be identi- fied via NADPH diaphorase histochemistry or via immunocytochemistry using antisera directed against the * Corresponding author. Fax: (44) (1865) 271853; e-mail: paul.gab- bott@pharm.ox.ac.uk. 0006-8993/96/$15.00 © 1996 ElsevierScienceB.V. All rights reserved SSDI 0006- 8993(95)01509-4 various NOS isoforms [27]. The isoforms of NOS in neurons, endothelial cells and macrophages have been well investigated (see review by Vincent [27]). By contrast, although pharmacological and biochemical data indicate that astroglia in vitro are 'sources and targets' of NO (see review by Murphy et al. [13]), there is little morphological evidence demonstrating the in situ presence of NOS in astroglia in normal brain tissue [13,22]. The aim of this study was to provide supportive mor- phological evidence for the localisation of NADPH di- aphorase activity and NOS immunoreactivity in resting astroglial cells in the rat brain. 2. Materials and methods 2.1. Tissue fixation Material from twelve young adult Sprague Dawley rats (200-300 g) of both sexes were used in this study. These rats had been reared normally and had not undergone any previous surgical procedures. The animals were deeply anaesthetised with equithesin [10] (0.3 ml/100 g) and transcardially perfused, initially with 0.9% physiological saline followed by one of the following aldehyde solu- tions: Fixative (i) 4% paraformaldehyde alone; fixative (ii) 0.2% glutaraldehyde and 2% paraformaldehyde; or fixative