Role of brain transmigrating neutrophils in depression-like behavior during systemic infection.

Peripheral inflammation induces transmigration of interleukin (IL)-1ß-expressing neutrophils to the brain. We investigated the possibility that this presents a new route of immune-to-brain communication by assessing their role in sickness behaviors relevant for mood disorders. Mice treated with lipopolysaccharide (LPS) developed despair-like behavior, and administration of an anti-polymorphonuclear antibody abolished LPS-induced despair-like and asocial behaviors, which correlated with the levels of IL-1ß expression in the brain. These behavioral changes were directly mediated by the energy-regulating hormone, leptin. Increasing the concentration of endogenous leptin during obesity exacerbated, whereas its neutralization using a specific antiserum attenuated sickness behaviors and importantly the neutrophil transmigrating process. Our results indicate a role for peripheral neutrophils in conveying inflammatory signals to the brain, which appears to be dependent on the energy status of the organism. This constitutes a novel mechanism of immune-to-brain communication relevant to mood disorders.

Changes in dendritic spine density on layer 2/3 pyramidal cells within the cingulate cortex of late pregnant and postpartum rats.

A rapid upregulation of astrocytic protein expression within area 2 of the cingulate cortex (Cg2) of the maternal rat occurs within 3h postpartum and persists throughout lactation. Previous studies have shown that similar changes in astrocytic proteins can signal changes in local synapses and dendritic spines. Thus, here we used the Golgi-Cox impregnation technique to compare spine density in layer 2 and 3 pyramidal cells of Cg2, the CA1 region of the hippocampus and the parietal cortex (ParCx) among metestrus, late pregnant (LP), 3-hour postpartum (3H PP) and 16-day postpartum rats (D16 PP). Rats in the 3H PP group had higher numbers of dendritic spines/10 µm on the apical dendrites of pyramidal neurons in both Cg2 and CA1 than the other groups, which did not differ. A similar pattern was observed in basilar dendrites but this failed to reach significance. In Cg2, Sholl analysis revealed that rats in the D16 PP group had a significantly greater extent of dendritic arborization in the basilar region than any other group. These data suggest that the changes in astrocytic proteins that occur in Cg2 in the postpartum period are associated with neuronal plasticity in pyramidal layers 2 and 3.

Steroid hormones and maternal experience interact to induce glial plasticity in the cingulate cortex.

Neocortical plasticity is not usually associated with changes in reproductive function. However, we have shown a six to 10-fold increase in the number of astrocytes labeled with glial fibrillary acidic protein (GFAP) and astrocytic basic fibroblast growth factor or FGF-2 (bFGF) in the cingulate cortex area 2 (Cg2) in postpartum rats, indicative of changes in connectivity in this area. In the present studies, we investigated the necessary and sufficient stimuli for these changes to occur. We show that 3 h of maternal experience combined with a hormonal treatment that mimics late pregnancy induces the astrocytic changes in Cg2 in virgin rats. The extent of these changes was similar to those of postpartum females. Sensitized virgin females did not show any astrocytic changes after 3 h of maternal behavior, suggesting that a similar amount of maternal experience alone is not sufficient to increase astrocytic bFGF- and GFAP-immunoreactivity in Cg2. Consistent with these data, eliminating early maternal experience by removing pups immediately postpartum abolishes the increased bFGF and GFAP protein expression in the cingulate cortex. These results suggest that maternal experience and hormonal state interact to produce astrocytic remodeling in the Cg2. The current results are consistent with a role for the cingulate cortex in maternal responsivity as suggested by early lesion studies in rats and more recent imaging studies in humans.

The slippery slope: prediction of successful weight maintenance in anorexia nervosa.

BACKGROUND: Previous research has found that many patients with anorexia nervosa (AN) are unable to maintain normal weight after weight restoration. The objective of this study was to identify variables that predicted successful weight maintenance among weight-restored AN patients. METHOD: Ninety-three patients with AN treated at two sites (Toronto and New York) through in-patient or partial hospitalization achieved a minimally normal weight and were then randomly assigned to receive fluoxetine or placebo along with cognitive behavioral therapy (CBT) for 1 year. Clinical, demographic and psychometric variables were assessed after weight restoration prior to randomization and putative predictors of successful weight maintenance at 6 and 12 months were examined. RESULTS: The most powerful predictors of weight maintenance at 6 and 12 months following weight restoration were pre-randomization body mass index (BMI) and the rate of weight loss in the first 28 days following randomization. Higher BMI and lower rate of weight loss were associated with greater likelihood of maintaining a normal BMI at 6 and 12 months. An additional predictor of weight maintenance was site; patients in Toronto fared better than those in New York. CONCLUSIONS: This study found that the best predictors of weight maintenance in weight-restored AN patients over 6 and 12 months were the level of weight restoration at the conclusion of acute treatment and the avoidance of weight loss immediately following intensive treatment. These results suggest that outcome might be improved by achieving a higher BMI during structured treatment programs and on preventing weight loss immediately following discharge from such programs.

Resource partitioning during reproduction in the Norway rat.

Rat pups nursed by pregnant dams grow as fast as pups reared by dams that are not pregnant. Moreover, litters that were in utero during a lactation are as numerous at birth and grow as fast as pups developing in a nonlactating, pregnant mother. These litters continue to grow as fast as pups born to nonlactating dams whether or not the first litter remains after the birth of the second litter. When pregnant and lactating dams have a restricted food supply, some dams are capable of extending the duration of their pregnancies by over 2 weeks past that of nonlactating, pregnant dams. This facultative prolongation of pregnancy apparently allows females to carry normal litters to term.

Modulation of brain reward circuitry by leptin.

Leptin, a hormone secreted by fat cells, suppresses food intake and promotes weight loss. To assess the action of this hormone on brain reward circuitry, changes in the rewarding effect of lateral hypothalamic stimulation were measured after leptin administration. At five stimulation sites near the fornix, the effectiveness of the rewarding electrical stimulation was enhanced by chronic food restriction and attenuated by intracerebroventricular infusion of leptin. In contrast, the rewarding effect of stimulating neighboring sites was insensitive to chronic food restriction and was enhanced by leptin in three of four cases. These opposing effects of leptin may mirror complementary changes in the rewarding effects of feeding and of competing behaviors.

Fluctuations in astrocytic basic fibroblast growth factor in the cingulate cortex of cycling, ovariectomized and postpartum animals.

In the current studies we investigated the timing of onset and the conditions needed for the maintenance of the upregulation of basic fibroblast growth factor (bFGF) and glial fibrillary acidic protein (GFAP) in the cingulate cortex area 2 (Cg2) that occurs in postpartum animals. We have previously shown that this upregulation is present from day 4 to day 24, and is not seen late in pregnancy (days 21-22). In the current studies, we demonstrate that bFGF and GFAP are both upregulated in Cg2 as early as 3 h postpartum, and are maintained until at least day 24 postpartum in animals deprived of pup stimulation for 8 days prior to perfusion. bFGF and GFAP immunoreactivity returns to prepartum levels by 5-6 weeks post-weaning, and the typical postpartum increase is not further enhanced in multiparous rats. We also show that, although there are significant changes in levels of bFGF immunoreactivity across the phases of the estrous cycle, peak cycling levels remain much lower than those observed in lactating rats. Possible stimuli involved in the induction of bFGF and GFAP in Cg2, and the potential relevance of these changes to the maternal state are discussed.

Inhibition of nitric oxide synthase within the medial preoptic area impairs pup retrieval in lactating rats.

Central suppression of nitric oxide (NO) production by administering 250 microg of Nitro-superw--subL-Argenine Methyl Ether (L-NAME), an inhibitor of NO synthase, into the 3rd ventricle disrupts both pup retrieval and maternal aggression in postpartum rats. In these studies, the authors examined the ability of varying doses of L-NAME to produce these effects on maternal behavior. Doses of L-NAME that were shown to be ineffective when injected into the 3rd ventricle were administered bilaterally into the medial preoptic area (MPOA) of rats on Day 4 postpartum. To assess the specificity of L-NAME's effect within the MPOA, the authors bilaterally injected Nitro-superw--subD-Argenine Methyl Ether (D-NAME), an inactive isomer of L-NAME, into the MPOA. When administered intracerebroventricularly, the 2 highest doses of L-NAME used, 250 microg and 200 microg, disrupted retrieval behavior and maternal aggression. Bilateral injections of L-NAME into the MPOA at doses of 20 microg and 40 microg/side also disrupted pup retrieval, and D-NAME injections into the MPOA had no effect on the maternal behaviors measured. All rats in these experiments showed normal maternal behavior 24 hr after drug administration. These results suggest that NO acts within the MPOA to facilitate retrieval behavior.

Role of voltage-dependent calcium channel long-term potentiation (LTP) and NMDA LTP in spatial memory.

This experiment explores the role of two forms of long-term potentiation (LTP) in behavioral memory. NMDA and/or voltage-dependent calcium channels (VDCCs) were antagonized pharmacologically at levels that block nmdaLTP and vdccLTP, respectively, in rats learning an eight-arm radial maze task. Animals were trained twice a day for 11 d under the systemic influence of MK-801, verapamil, both drugs, or saline. During acquisition, the mixed drug group displayed significantly more working memory errors and reference memory errors than all other groups. The mixed drug group was markedly impaired on the first daily trial but improved dramatically on their second daily trial. After a 7 d delay, saline and MK-801 animals maintained their predelay level of performance. The performance of the verapamil groups declined significantly over the delay. These results demonstrate that: (1) vdccLTP is necessary for the retention of information over a 7 d period, (2) the blockade of both forms of LTP prevents the retention of information over a 21 hr period, and (3) blockade of both forms of LTP does not prevent the storing of information over a short period of time (3 hr).

Differential effects of reproductive and hormonal state on basic fibroblast growth factor and glial fibrillary acid protein immunoreactivity in the hypothalamus and cingulate cortex of female rats.

Morphological changes in astrocytes occur in a number of brain regions including the hypothalamus and hippocampal regions as a function of hormonal and reproductive state. Because basic fibroblast growth factor has been shown to play an important role in morphological changes in astrocytes, we investigated whether basic fibroblast growth factor immunoreactivity would also be influenced by reproductive state and circulating gonadal steroids. To do this we compared astrocytic basic fibroblast growth factor and glial fibrillary acid protein immunoreactivity in hypothalamic nuclei and the cingulate cortex, area 2 among groups of cycling, late pregnant and lactating rats as well as in ovariectomized and ovariectomized hormone-replaced females. Significant differences in both basic fibroblast growth factor and glial fibrillary acid protein immunoreactivity were observed across groups in the supraoptic nucleus, parvocellular paraventricular nucleus, medial preoptic area of the hypothalamus and cingulate cortex 2. The pattern of change in basic fibroblast growth factor and glial fibrillary acid protein immunoreactivity varied across regions both in direction and magnitude. For example, although in the supraoptic nucleus ovariectomized rats had lower levels of basic fibroblast growth factor-ir than cycling females, this pattern was reversed within cingulate cortex. Overall the results of this study suggest that reproductive and hormonal states are associated with robust changes in basic fibroblast growth factor and glial fibrillary acid protein immunoreactivity in a number of brain areas but that the changes observed vary in magnitude as well as direction from one brain region to another.

Suppression of LH secretion in food-restricted lactating females: effects of ovariectomy and bromocryptine treatment.

It has been shown that restricting food in lactating rats for the first 2 weeks postpartum at a level of 60% of the ad-libitum daily ration increases the length of lactational dioestrus by about 7 days but little is known about correlated changes in hormone levels. In the first experiment we report changes in LH, prolactin (PRL) and ACTH secretion in food-restricted and ad-libitum fed lactating rats at various stages of lactation. Our results demonstrate that food restriction during the first 2 weeks of lactation did not affect PRL or ACTH secretion, but decreased plasma LH levels despite comparable GnRH receptor density between food-restricted and ad-libitum fed females. In the second experiments we investigated a possible causal relationship between the increased secretion of progesterone seen in food-restricted females and the suppression of plasma LH levels, by determining the effects of bromocryptine treatment and ovariectomy on LH secretion in both ad-libitum fed and food-restricted lactating females. LH suppression in food-restricted lactating females was not affected by ovariectomy or bromocryptine treatment, although the latter treatment significantly increased GnRH receptor number. These data suggest that factors other than ovarian steroids, PRL or increased adrenocortical activity modulate LH secretion and the length of lactational dioestrus in food-restricted lactating females.

Neuroendocrine basis of thermally regulated maternal responses to young in the rat.

Over the first two weeks postpartum there is a decline in the amount of time that the rat dam spends with her young, resulting from a decrease in the duration of each nest bout. The duration of each nest bout is limited by the rate of rise of maternal temperature when she huddles with her litter. This pattern of mother-young contact is dependent on the dam's hormonal status, because adrenalectomised dams fail to show the expected decline in mother-young contact over time. Ovariectomy, on the other hand, does not have any effect on this behavior. Replacement therapy with glucocorticoids or placing the dam in a warm (25 degrees C) ambience reinstates the normal pattern of mother-litter contact in adrenalectomised-ovariectomised dams. These data suggest that the elevated level of serum glucocorticoids in lactating dams affects maternal behavior by increasing maternal heat load, thereby making the dam vulnerable to an acute rise in temperature when huddling with her young. Prolactin suppression also results in an increase in contact time between mother and young, but only in the second week postpartum. The effects of prolactin suppression are reversed by progesterone replacement or placing prolactin-suppressed females in a warm ambience. However, progesterone is ineffective in restoring the normal pattern of mother-litter contact in adrenalectomised females. These findings suggest that progesterone raises the thermal set point, thereby permitting the thermogenic effects of glucocorticoids to occur.

Effect of nitric oxide synthase inhibition on fos expression in the hypothalamus of female rats following central oxytocin and systemic urethane administration.

Three experiments were carried out to investigate the pattern of neuronal activation induced by central oxytocin administration and its modulation by nitric oxide (NO). First, we compared the induction of Fos-like immunoreactivity (lir) in the supraoptic (SON) and paraventricular (PVN) nuclei and medial preoptic area (MPOA) after central oxytocin administration between nonlactating and lactating rats. Next, we investigated whether NO modulated Fos induction following central oxytocin administration using a nitric oxide synthase (NOS) inhibitor, N omega-nitro-L-arginine methyl ester (L-NAME). Finally, to determine whether the effects of NOS inhibition on Fos induction would generalize to stimuli other than oxytocin, we compared Fos-lir in the SON and PVN of lactating and nonlactating rats following L-NAME and urethane administration. In the first two experiments, oxytocin (50 ng in 2 microl) or vehicle was administered into the third ventricle. L-NAME (50 mg/kg) was given by an intraperitoneal (i.p.) injection 30 min before oxytocin administration (experiment 2) or an i.p. injection of urethane (1.4 g/kg) (experiment 3). In all experiments, lactating rats were tested on day 12 or 13 postpartum and nonlactating females at least 11 days after surgery or the start of the experiment. Central oxytocin infusion induced Fos expression in the SON and PVN in lactating and nonlactating rats and in the MPOA and bed nucleus of the stria terminalis in lactating rats. Overall, lactating rats that received L-NAME and oxytocin had a greater number of cells showing Fos-lir in both the SON and PVN. Conversely, L-NAME administration reduced Fos-lir in the SON and PVN in oxytocin-stimulated nonlactating rats. In urethane-treated rats, L-NAME administration did not change Fos-lir in lactating rats but reduced Fos-lir in nonlactating rats. These data suggest that the role of NO in modulating the activity of neurones in discrete nuclei in the hypothalamus varies across reproductive state and with the stimulus presented.

Changes in NADPH-d staining in the paraventricular and supraoptic nuclei during pregnancy and lactation in rats: role of ovarian steroids and oxytocin.

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.

Food restriction during lactation results in prolonged hyposensitivity to the positive-feedback effects of oestradiol.

Food restriction prolongs lactational infertility in rats. Here, we investigated whether an attenuated response to the positive-feedback effects of oestrogen on luteinizing hormone release contributed to this effect. The ability of oestrogen to induce surges in luteinizing hormone in ad libitum fed and food-restricted dams at different times of lactation was compared. The results showed that, on day 20 postpartum, ad libitum fed dams showed luteinizing hormone surges after oestrogen treatment, but food-restricted dams did not. Ovariectomy or RU486 treatment restored the ability of oestrogen to induce luteinizing hormone surges in food-restricted dams, and chronic progesterone exposure reduced oestrogen-induced surges of luteinizing hormone in ad libitum fed ovariectomized dams. Food restriction also resulted in a reduced ability of oestrogen to induce progesterone receptor immunoreactivity, but did not reduce the number of oestrogen receptors (ERalpha) in the anteroventralperiventricular area. As with the surge in luteinizing hormone, the effects of food restriction on oestrogen induction of progesterone receptors were mediated by progesterone. Together, these results suggest that the ability of food restriction to extend the length of lactational diestrus is mediated, in part, by a decrease in sensitivity to the positive-feedback effects of oestrogen. This results from high circulating concentrations of progesterone which apparently reduce the ability of oestrogen to induce progesterone receptor expression.

Chronic neuropeptide Y Y5 receptor stimulation suppresses reproduction in virgin female and lactating rats.

Continuous infusion of neuropeptide Y (NPY) disrupts cyclicity and delays the onset of puberty in female rats indicating that NPY can suppress reproduction. Central application of NPY also reliably increases food intake in rats. States with heavy demands on energy resources where reproduction is also inhibited, such as lactation, are similarly accompanied by elevations in central NPY expression. In previous studies, we have shown that, compared to lactating rats fed ad libitum, food-restricted lactating rats exhibit a longer period of lactational diestrus that is correlated with increased central NPY expression. These studies link NPY to the inhibition of reproduction that is mediated by low availability of energy resources. Here, we examine the effect of chronic 7-day infusion of the mixed Y1/Y4/Y5 agonist (Leu31, Pro34) NPY and selective agonists to the Y2 (NPY13-36) and Y5 (D-Trp32 NPY and D-Trp34 NPY) receptors on food intake and the oestrous cycle of virgin female rats. We also investigated the effect of chronic infusion from day 8-15 postpartum (pp) of D-Trp32 NPY and D-Trp34 NPY on food-intake and the length of lactational diestrus in lactating rats fed ad libitum. In virgin females, infusion of (Leu31, Pro34) NPY and both the Y5 agonists lengthened the period between consecutive oestrus days while the Y2 agonist NPY13-36 was without effect. Selective Y5 receptor activation alone caused an increase in food intake in virgin females. In lactating females, D-Trp32 NPY extended the length of lactational diestrus, while D-Trp34 NPY had no effect on this parameter. These data suggest that Y5 receptor activation suppresses the reproductive axis in both virgin and lactating rats and that Y5 receptor activation enhances food-intake in virgin females.

Y1 receptor activation is involved in the effect of exogenous neuropeptide Y on pup growth and the early termination of lactational diestrus in the postpartum rat.

The effect of chronic administration of exogenous neuropeptide Y (NPY) and specific NPY receptor agonists and antagonists on reproductive function was examined in lactating rats. As previously demonstrated in our laboratory, chronic (7-day) intracerebroventricular (i.c.v.) NPY infusion (6 microg/day) from days 8-15 postpartum (pp) caused a significant decrease in milk production and an early termination of lactational diestrus. Similar application of the mixed Y1/Y4/Y5 receptor agonist (Leu31, Pro34) NPY (at 3, 6 and 9 microg/day) reproduced the effect of chronic NPY infusion on milk production in a dose-independent manner. Consistent with this effect, the potent Y1 antagonist/Y4 agonist, 1229U91, given concomitantly with NPY eliminated the decline in milk production. The Y2 receptor agonist, NPY13-36, had no effect on milk production at any of the doses used. Length of lactational diestrus was reduced following administration of the Y2 agonist at 18 microg/day but not at 9 microg or 27 microg/day whereas (Leu31, Pro34) NPY infusion had no effect on this parameter at any of the doses used. However, the group that was treated with NPY plus 1229U91 exhibited the usual length of lactational diestrus, indicating that there is at least some Y1 involvement in the effects of NPY on lactational infertility. To test the possibility that the effects of NPY infusion are mediated through changes in circulating prolactin and progesterone, plasma concentrations of these hormones were measured on day 15 pp in NPY-, (Leu31, Pro34) NPY- and vehicle-treated females. NPY-infused females had lower plasma prolactin concentrations than vehicle-infused dams but progesterone concentrations were similar across groups. Overall, these data indicate that chronic exogenous NPY-infusion in lactating females disrupts milk production and shortens lactational diestrus, most likely through reducing prolactin secretion, and that this effect is mediated via Y1 receptor activity.

Pups presence eliminates the stress hyporesponsiveness of early lactating females to a psychological stress representing a threat to the pups.

Blunted neuroendocrine responses to stress are reported in lactating females after exposure to various stressors. However, many of the stimuli used in these studies have little ethological relevance for maternal protection of the litter in a threatening environment. The question that arises is whether the relevance of the stressor to the infant is critical in the 'gating' of the neuroendocrine response. We hypothesized that the presence of pups with their mothers at the time of exposure to an intruder or a predator odour is an effective way to increase the emotional salience of the psychological stressor, thus eliminating the stress hyporesponsiveness in lactating females. We first compared neuroendocrine responses [corticotropin-releasing factor (CRF) mRNA in the paraventricular nucleus of the hypothalamus (PVN) and central nucleus of the amygdala (CeA), plasma adrenocorticotropic hormone (ACTH) and corticosterone] between early (EL, PPD3-5), late (LL, PPD 15) lactating and virgin (V) females to a male intruder in the home cage. We next investigated the effect of pups' presence at the time of stressor exposure on the magnitude of the hormonal response to a male intruder in the home cage or to a predator odour (fox urine) in a novel environment. In the male intruder paradigm, levels of CRF mRNA expression in the PVN and CeA were lower in LL compared to EL or V females and plasma ACTH and corticosterone secretion was not as elevated in LL compared to EL females. Aggression towards the intruder was high in EL females in the presence of their pups and a positive correlation was found with the integrated ACTH response. Aggression rapidly declined after pup separation (2.5 h or 48 h) or in LL nursing females. In EL females, the presence of the pups with their mothers (EL + pups) at the time of stress significantly increased plasma ACTH and corticosterone responses to either male intruder or predator odour compared to EL females without their pups for 2.5 h or 48 h (EL - pups). Plasma ACTH response to fox urine in EL + pups females was comparable to that of virgin females, suggesting that increasing the salience of emotionally relevant stimuli by keeping the pups present in the cage could eliminate the hyporesponsiveness detected for EL females without their pups. These studies indicate the critical role of the pups in modulating the maternal response to stressors that represent a threat for the litter. We hypothesize that the amygdala, because of its ability to process olfactory stimuli and stimuli with affective properties, might play an essential role in 'gating' the neuroendocrine response to stress during lactation.

Prolactin and oxytocin interaction in the paraventricular and supraoptic nuclei: effects on oxytocin mRNA and nitric oxide synthase.

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.