Journal of Neuroendocrinology, 1999, Vol. 11, 53–61 Changes in NADPH-d Staining in the Paraventricular and Supraoptic Nuclei During Pregnancy and Lactation in Rats: Role of Ovarian Steroids and Oxytocin N. Popeski, S. Amir and B. Woodside Centre for Studies in Behavioural Neurobiology, Psychology Department, Concordia University, Montre ´ al, Que ´ bec, Canada. Key words: nitric oxide, reproduction, hypothalamus, neuroendocrine regulation. Abstract Staining for nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d), a histochemical marker for nitric oxide synthase (NOS), is increased in the supraoptic (SON) and paraventricular (PVN) nuclei in late pregnant rats. To determine whether increases in staining were evident at other times during pregnancy and lactation the number of cells that stained for NADPH-d in the SON and PVN in rats on days 4, 12, 16, and 22 of pregnancy and on days 4, 12, and 20 of lactation was compared to that in virgin females. In a second experiment the influence of ovarian hormones on NADPH-d staining was assessed by comparing staining in the SON and PVN among ovariectomized animals exposed to either a steroid hormone replacement schedule that mimics late pregnancy (oestrogen and progesterone with progesterone removal), oestrogen alone, oestrogen and progesterone, or cholesterol alone. In the last experiment of this series staining was compared among ovariectomized animals given either oestrogen or cholesterol priming accompanied by oxytocin (OT) or vehicle infusion into the third ventricle for 7 days. The number of cells showing dense staining for NADPH-d in both the SON and PVN increased on days 12 and 22 of pregnancy and 4 and 12 of lactation compared to that observed in virgins. NADPH-d staining in these areas was also increased by both the steroid treatment that mimicked late pregnancy and chronic central OT infusion in oestrogen-primed animals. These data suggest that NADPH-d staining in the SON and PVN is increased at times when oxytocinergic cells are known to be active and that the hormonal state associated with late pregnancy is sufficient to increase NADPH-d staining. Nitric oxide (NO) is a gaseous messenger molecule that was mone (ACTH ) following immune stress (12) but has been found to suppress the expression of Fos in response to first identified as an endothelium derived relaxing factor (EDRF) (1). Subsequently NO has been implicated in a immobilization stress (13). Similar treatment increases oxyto- cin (OT ) release from the pituitary under both basal and variety of physiological and pathophysiological processes in both the peripheral and central nervous systems (2, 3) stimulating conditions suggesting that NO suppresses OT release (14, 15). including excitotoxicity (4), neuroplasticity (5–7) and cardio- vascular regulation (8). In addition, recent evidence suggests NO is synthesized by a number of isozymes of nitric oxide synthase (NOS), which catalyse the oxidation of the amino- that NO plays an important role in neuroendocrine function both at the hypothalamic and pituitary level (9 ). For example, acid -arginine to NO and citrulline (16). Nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d), a NO has been shown to mediate NMDA-induced gonadotro- pin releasing hormone release (10) and the facilitatory effect histochemical marker for NOS (17), as well as NOS immuno- cytochemistry (18) and in-situ hybridization of NOS mRNA of neuropeptide Y on the oestrogen-induced surge of lutein- izing hormone (11). NO also has diverse effects on the have been used to label cells that produce NO. Consistent with its role in modulating the hypothalamo-pituitary- hypothalamic-pituitary-adrenal axis (HPA). Blocking NO production enhances the release of adrenocorticotropic hor- gonadal and HPA axes NOS has been found in numerous Correspondence to: Barbara Woodside, Department of Psychology, Concordia University, 7141 Sherbrooke Street West, Montre ´al, Que ´bec H4B 1R6, Canada. © 1999 Blackwell Science Ltd