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.

Blocking nitric oxide (NO) production, by 3rd ventricle administration of a nitric oxide synthase (NOS) inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME; 250 microg/5 microl, postpartum [pp]) decreased milk ejections in Day 10 pp rats. On Day 4 pp, L-NAME treatment eliminated pup retrieval and at both stages of lactation suppressed maternal aggression. Fewer rats treated with L-NAME on Day 10 pp retrieved 4-day-old pups than controls, although all nursed older litters. Following exposure to a mobile intruder, Fos expression was lower in the medial preoptic area and the bed nucleus of the stria terminalis in L-NAME-treated rats than in controls but was lower in the medial amygdala only following exposure to an anaesthetized intruder. Thus, the elevated levels of NO observed in lactation may contribute to the mechanism(s) that mediate maternal behavior and aggression.

We investigated the contribution of prolactin and oxytocin to the increase in staining for NADPH-d and oxytocin mRNA in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) observed at the end of pregnancy, or following a steroid-priming regimen that mimics the hormonal profile of late pregnant females. Ovariectomized rats received chronic implants of silastic capsules containing oestrogen and progesterone followed by progesterone removal. In experiment 1, oxytocin antagonist (OTA) was administered to rats to investigate whether intranuclear oxytocin release was necessary for NADPH-d staining. In experiments 2a and b, rats received concurrent treatment with bromocryptine (0.5 mg/day) to suppress endogenous prolactin release, and either systemic prolactin (0.5 mg once daily), or prolactin (2 micro g/ micro l), or vehicle infused twice a day into the third ventricle, or chronic oxytocin infusion (24 ng/day) for 3 days following progesterone removal. Brains were then processed for NADPH-d histochemistry. In experiment 3, the interaction of prolactin and oxytocin on oxytocin mRNA within the SON and PVN was examined. NADPH-d staining in the SON and PVN was reduced by the highest dose of the OTA, and by bromocryptine treatment. Central prolactin and oxytocin replacement completely restored NADPH-d staining in bromocryptine-treated rats. Finally, both bromocryptine and the OTA suppressed oxytocin mRNA expression and prolactin replacement restored expression levels to that of controls. Together, these data suggest that the increased capacity to produce nitric oxide in the SON and PVN during late pregnancy is dependent on prolactin stimulating oxytocin gene mRNA and hence intranuclear oxytocin release.

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