Orphanin FQ in the mediobasal hypothalamus facilitates sexual receptivity through the deactivation of medial preoptic nucleus mu-opioid receptors

Sexual receptivity, lordosis, can be induced by sequential estradiol and progesterone or extended exposure to high levels of estradiol in the female rat. In both cases estradiol initially inhibits lordosis through activation of β-endorphin (β-END) neurons of the arcuate nucleus of the hypothalamus (ARH) that activate μ-opioid receptors (MOP) in the medial preoptic nucleus (MPN). Subsequent progesterone or extended estradiol exposure deactivates MPN MOP to facilitate lordosis. Opioid receptor-like receptor-1 (ORL-1) is expressed in ARH and ventromedial hypothalamus (VMH). Infusions of its endogenous ligand, orphanin FQ (OFQ/N, aka nociceptin), into VMH–ARH region facilitate lordosis. Whether OFQ/N acts in ARH and/or VMH and whether OFQ/N is necessary for steroid facilitation of lordosis are unclear. In Exp I, OFQ/N infusions in VMH and ARH that facilitated lordosis also deactivated MPN MOP indicating that OFQ/N facilitation of lordosis requires deactivation of ascending ARH-MPN projections by directly inhibiting ARH β-END neurons and/or through inhibition of excitatory VMH–ARH pathways to proopiomelanocortin neurons. It is unclear whether OFQ/N activates the VMH output motor pathways directly or via the deactivation of MPN MOP. In Exp II we tested whether ORL-1 activation is necessary for estradiol-only or estradiol + progesterone lordosis facilitation. Blocking ORL-1 with UFP-101 inhibited estradiol-only lordosis and MPN MOP deactivation but had no effect on estradiol + progesterone facilitation of lordosis and MOP deactivation. In conclusion, steroid facilitation of lordosis inhibits ARH β-END neurons to deactivate MPN MOP, but estradiol-only and estradiol + progesterone treatments appear to use different neurotransmitter systems to inhibit ARH-MPN signaling.     

Orphanin FQ in the mediobasal hypothalamus facilitates sexual receptivity through the deactivation of medial preoptic nucleus mu-opioid receptors

Sexual receptivity, lordosis, can be induced by sequential estradiol and progesterone or extended exposure to high levels of estradiol in the female rat. In both cases estradiol initially inhibits lordosis through activation of β-endorphin (β-END) neurons of the arcuate nucleus of the hypothalamus (ARH) that activate μ-opioid receptors (MOP) in the medial preoptic nucleus (MPN). Subsequent progesterone or extended estradiol exposure deactivates MPN MOP to facilitate lordosis. Opioid receptor-like receptor-1 (ORL-1) is expressed in ARH and ventromedial hypothalamus (VMH). Infusions of its endogenous ligand, orphanin FQ (OFQ/N, aka nociceptin), into VMH–ARH region facilitate lordosis. Whether OFQ/N acts in ARH and/or VMH and whether OFQ/N is necessary for steroid facilitation of lordosis are unclear. In Exp I, OFQ/N infusions in VMH and ARH that facilitated lordosis also deactivated MPN MOP indicating that OFQ/N facilitation of lordosis requires deactivation of ascending ARH-MPN projections by directly inhibiting ARH β-END neurons and/or through inhibition of excitatory VMH–ARH pathways to proopiomelanocortin neurons. It is unclear whether OFQ/N activates the VMH output motor pathways directly or via the deactivation of MPN MOP. In Exp II we tested whether ORL-1 activation is necessary for estradiol-only or estradiol + progesterone lordosis facilitation. Blocking ORL-1 with UFP-101 inhibited estradiol-only lordosis and MPN MOP deactivation but had no effect on estradiol + progesterone facilitation of lordosis and MOP deactivation. In conclusion, steroid facilitation of lordosis inhibits ARH β-END neurons to deactivate MPN MOP, but estradiol-only and estradiol + progesterone treatments appear to use different neurotransmitter systems to inhibit ARH-MPN signaling.     

Hypertrophy of the ageing rat medial preoptic nucleus

The medial preoptic nucleus (MPN) plays an essential role in the coordination of behaviours and physiological responses necessary for reproduction. Since ageing is associated with a progressive deterioration of reproductive functions we have explored the possibility that changes in the structural organization of the MPN might be implicated in this process. Thus, we have estimated the volume of the MPN, and the total number and size of its neurons, using stereological methods, and quantitatively evaluated the dendritic trees of MPN neurons in Golgi-impregnated material. Male and female rats, aged 6, 24 and 30 months, were independently analysed. No cell loss was observed in aged rats of both sexes. However, the volume of the MPN and the somatic size of its neurons were remarkably enlarged in aged rats. No significant age-related changes in the size or shape of the dendritic trees or in dendritic spine density were found. To evaluate whether the changes observed in aged rats could be ascribed to an altered interaction between gonadal steroids and steroid-sensitive neurons, we have additionally estimated the to tal number of MPN neurons immunoreactive for the estrogen receptor-α. No significant age-related variations were detected. The age effects upon the MPN were more marked in females than in males and, consequently, the sexual dimorphisms in neuronal size and in the number of estrogen receptor-immunoreactive neurons were blunted in aged rats.     

Age-related changes in sexual function and steroid-hormone receptors in the medial preoptic area of male rats

Testosterone is the main circulating steroid hormone in males, and acts to facilitate sexual behavior via both reduction to dihydrotestosterone (DHT) and aromatization to estradiol. The mPOA is a key site involved in mediating actions of androgens and estrogens in the control of masculine sexual behavior, but the respective roles of these hormones is not fully understood. As males age they show impairments in sexual function, and a decreased facilitation of behavior by steroid hormones compared to younger animals. We hypothesized that an anatomical substrate for these behavioral changes is a decline in expression and/or activation of hormone receptor-sensitive cells in the mPOA. We tested this by quantifying and comparing numbers of AR- and ERα-containing cells, and Fos as a marker of activated neurons, in the mPOA of mature (4–5 months) and aged (12–13 months) male rats, assessed one hour after copulation to one ejaculation. Numbers of AR- and ERα cells did not change with age or after sex, but the percentage of AR- and ERα-cells that co-expressed Fos were significantly up-regulated by sex, independent of age. Age effects were found for the percentage of Fos cells that co-expressed ERα (up-regulated in the central mPOA) and the percentage of Fos cells co-expressing AR in the posterior mPOA. Interestingly, serum estradiol concentrations positively correlated with intromission latency in aged but not mature animals. These data show that the aging male brain continues to have high expression and activation of both AR and ERα in the mPOA with copulation, raising the possibility that differences in relationships between hormones, behavior, and neural activation may underlie some age-related impairments.     

Involvement of norepinephrine activity in the regulation of alpha 1 adrenergic receptors in the medial preoptic nucleus of estradiol-treated rats

To establish whether the diurnal decrease in the density of alpha 1 receptors observed in the medial preoptic nucleus (MPN) of estrogen (E2)-treated rats is related to the concomitant diurnal increase in norepinephrine (NE) turnover rates, we quantitated the density of [3H]-Prazosin binding to alpha 1 receptors after blockade of NE turnover with alpha-methyl-paratyrosine (alpha MPT). A series of preliminary studies was performed to rule out an interference of this drug with [3H]-Prazosin binding to alpha 1 adrenergic receptors in vitro and in vivo. Incubation of brain slices with alpha MPT produced a dose-dependent inhibition of [3H]-Prazosin binding to alpha 1 adrenergic receptors with an IC50 of approximately 6 mM. Scatchard analysis demonstrated that alpha MPT exhibited a simple competitive interaction with [3H]-Prazosin binding sites as shown by an increase in the apparent dissociation constant (Kd) of the ligand and no change in the number of alpha 1 receptors (Bmax). In contrast, preincubation of brain slices with alpha MPT and prior in vivo administration of alpha MPT did not affect [3H]-Prazosin binding to alpha 1 adrenergic receptors. Once we established that alpha MPT could be used to suppress NE turnover without interfering with the measurement of alpha 1 receptor densities, we repeatedly injected this drug to ovariectomized (OVX) and E2-implanted rats. The density of alpha 1 adrenergic receptors in MPN was quantitated autoradiographically. Blockade of NE turnover with alpha MPT only partially prevented the reduction in alpha 1 receptor density observed in the E2-treated rats, suggesting that the decrease in the level of [3H]-Prazosin binding sites cannot be completely ascribed to increased NE turnover rates.     

Ultrastructural characterization of the sexually dimorphic medial preoptic nucleus of male Japanese quail

The medial preoptic nucleus is a sexually dimorphic structure whose cytoarchitecture, afferent and efferent connections, and functions have been previously described. No detailed ultrastructural study has, however, been perfomed to date. Here we describe the ultrastructural organization of this important preoptic structure of the male quail. Neuronal cell bodies of the medial preoptic nucleus generally show extensive development of protein-synthesis-related organelles (rough endoplasmic reticulum, polysomes), and of secretory structures (Golgi complexes, secretory vesicles, dense bodies). Previous morphometrical studies at the light-microscopical level have demonstrated the presence of a medial and a lateral neuronal population distinguished by the size of their cell bodies (the medial neurons are smaller than the lateral neurons). The present ultrastructural investigation confirms the difference in size, but no difference has been observed in the ultrastructural organization of the neurons. In both the medial and the lateral part, the nucleus is characterized by a large variety of cell bodies, including some that, on the basis of their ultrastructure, can be considered as putative peptidergic neurons. Close contacts are frequently observed between adjacent cell bodies that are normally arranged in clusters. Various types of synaptic endings are also present, suggesting a rich supply of nerve fibers. A few glial cells are scattered within the nucleus. In view of the crucial role of this region in regulating quail sexual behavior, the large heterogeneity of neurons and of afferent nervous fibers suggest that this region might have an important role in the integration of information arriving from different brain regions.     

Oxytocin in the medial preoptic area facilitates male sexual behavior in the rat

Oxytocin (OT) is a versatile neuropeptide that is involved in a variety of mammalian behaviors, and its role in reproductive function and behavior has been well established. The majority of pharmacological studies of the effects of OT on male sexual behavior have focused on the paraventricular nucleus (PVN), ventral tegmental area (VTA), hippocampus, and amygdala. Less attention has been given to the medial preoptic area (MPOA), a major integrative site for male sexual behavior. The present study investigated the effects of intra-MPOA administration of OT and (d(CH2)51, Tyr(Me)2, Thr4, Orn8, Tyr-NH29)-vasotocin, an OT antagonist (OTA), on copulation in the male rat. The relationship between OT receptor (OTR) binding levels in the MPOA and sexual efficiency was also explored. Microinjection of OT into the MPOA facilitated copulation in sexually experienced male rats, whereas similar injections of an OTA inhibited certain aspects of copulation but had no significant effect on locomotor activity in an open field. Contrary to expectation, sexually efficient males had lower levels of OTR binding in the rostral MPOA compared to inefficient animals. The present data suggest that OT activity in the MPOA is not necessary for the expression of male sexual behavior but is sufficient to facilitate copulatory behaviors and improve sexual efficiency in sexually experienced male rats. These data also suggest that OTR activity in the MPOA stimulates anogenital investigation, facilitates the initiation of copulation, and plays a role in the sensitization effect of the first ejaculation on subsequent ejaculations.     

Onset of the hormone-sensitive perinatal period for sexual differentiation of the sexually dimorphic nucleus of the preoptic area in female rats

The volume of the sexually dimorphic nucleus of the preoptic area (SDN-POA) of the rat brain is severalfold larger in males than in females. The volume of the SDN-POA can be influenced significantly by the hormonal milieu during the perinatal "critical period" of sexual differentiation of the brain. The purpose of the present study was to determine the onset of this period of sexual differentiation of the SDN-POA. Pregnant rats received no treatment or were injected subcutaneously with oil on day 17, 18, or 20, or testosterone (T;5 mg) on days 16-22 of gestation. On postnatal day 15, unilateral SDN-POA volumes from female offspring prenatally exposed to testosterone on day 16 or 17 were not different from values of control (untreated or oil-injected) offspring. Female offspring from mothers treated with testosterone on day 18, 19, or 20 of gestation showed a significant and similar increase in SDN-POA volume over values from control animals. SDN-POA volumes from female offspring exposed to testosterone on day 21 or 22, although larger than those of controls, were not different statistically. We conclude that with the specific paradigm used in this study SDN-POA development is insensitive prior to day 18 of gestation, the day on which the onset of the hormone-sensitive period occurs.     

Release of orphanin FQ/nociceptin in the medial preoptic nucleus and ventromedial nucleus of the hypothalamus facilitates lordosis

Opioid regulation of reproduction has been widely studied. However, the role of opioid receptor-like 1 receptor (NOP; also referred to as ORL-1 and OP4) and its endogenous ligand orphanin FQ/nociceptin (OFQ/N) have received less attention despite their extensive distribution throughout nuclei of the limbic-hypothalamic system, a circuit that regulates reproductive behavior in the female rat. Significantly, the expression of both receptor and ligand is regulated in a number of these nuclei by estradiol and progesterone. Activation of NOP in the ventromedial nucleus of the hypothalamus (VMH) of estradiol-primed nonreceptive female rats facilitates lordosis. NOPs are also expressed in the medial preoptic nucleus (MPN), however, their roles in reproductive behavior have not been studied. The present experiments examined the role of NOP in the regulation of lordosis in the MPN and tested whether endogenous OFQ/N in the MPN and VMH mediates reproductive behavior. Activation of NOP by microinfusion of OFQ/N in the MPN facilitated lordosis in estradiol-primed sexually nonreceptive female rats. Passive immunoneutralization of OFQ/N in either the MPN or the VMH reduced lordosis in estradiol-primed females, but had no effect on lordosis in estradiol+progesterone-primed sexually receptive rats. These studies suggest that OFQ/N has a central role in estradiol-only induced sexual receptivity, and that progesterone appears to involve additional circuits that mediate estradiol+progesterone sexual receptivity.     

Preliminary observations on the fine structure of the medial preoptic nucleus in Coturnix japonica

The organization of the sexually dimorphic medial preoptic nucleus of the Japanese quail was studied at ultrastructural level. The region was characterized by clusters of parvocellular neurons showing a rich supply of axo-somatic synapses and a peculiar distribution of synthetic (rough endoplasmic reticulum) and secretory (Golgi complexes) structures. Further analyses are required to relate these features with sex, hormonal status and sexual behaviour of quail.     

Selective contributions of the medial preoptic nucleus to testosterone-dependant regulation of the paraventricular nucleus of the hypothalamus and the HPA axis

Previous data have consistently demonstrated an inhibitory effect of androgens on stress-induced hypothalamic-pituitary-adrenal (HPA) responses. Several brain regions may influence androgen-mediated inhibition of the HPA axis, including the medial preoptic area. To test the role of the medial preoptic nucleus (MPN) specifically, we examined in high- and low-testosterone-replaced gonadectomized rats bearing discrete bilateral lesions of the MPN basal and stress-induced indexes of HPA function, and the relative levels of corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP) mRNA in the amygdala. High testosterone replacement decreased plasma adrenocorticotropin hormone (ACTH) and paraventricular nucleus (PVN) Fos responses to restraint exposure in sham- but not in MPN-lesioned animals. AVP-, but not CRH-immunoreactivity staining in the external zone of the median eminence was increased by testosterone in sham animals, and MPN lesions blocked this increment in AVP. A similar interaction between MPN lesions and testosterone occurred on AVP mRNA levels in the medial nucleus of the amygdala. These findings support an involvement of MPN projections in mediating the AVP response to testosterone in both the medial parvocellular PVN and medial amygdala. We conclude that the MPN forms part of an integral circuit that mediates the central effects of gonadal status on neuroendocrine and central stress responses.     

The LHRH neuronal system in female rats: relation to the medial preoptic nucleus

Previously we have found that small lesions confined to the medial preoptic nucleus (MPN) or the suprachiasmatic nucleus (SCN) blocked the cyclic release of gonadotropins in the female rat, inducing a persistent estrous state. Since the MPN is located just caudal to the organum vasculosum of the lamina terminalis (OVLT) where LHRH cell bodies are most concentrated, we applied an immunocytochemical technique to examine the possibility that the lesions had simply disrupted LHRH neurons or fibers. Using a new anti-LHRH provided by Dr. V. D. Ramirez, we found that the distribution pattern of immunoreactive LHRH cell bodies and fibers was similar to that previously reported, although the staining was more intense and extensive with low background. There was no concentration of LHRH cell bodies and fibers in the MPN or SCN and, in fact, these nuclei generally showed a lower density of stained elements than did surrounding tissue. In persistent estrous animals with lesions confined to the MPN there was no detectable reduction of stained fibers in the median eminence. These results, along with the results of other workers, suggest that persistent estrus following lesions of the MPN or SCN is not due to reduction of LHRH neurons or fibers. Rather, they support the hypothesis that these nuclei are critical for triggering the ovulatory release of LHRH.     

Stereological evaluation and Golgi study of the sexual dimorphisms in the volume,cell numbers,and cell size in the medial preoptic nucleus of the rat

The medial preoptic nucleus (MPN) and the sexually dimorphic nucleus of the preoptic area (SDN-POA) stand out as prominent sexually dimorphic cell groups of the rat brain. However, quantitative data on sex-related differences in these nuclei in the adult rat are confined to their volume. We have used stereological methods and Golgi-impregnated material to examine whether, in young adult rats, the sexual dimorphism in the volume of the MPN, including its divisions, and of the SDN-POA, reflect similar differences in the number and size of their neurons. We found that the total number of neurons in all MPN divisions is higher and the mean somatic volume larger in males than in females. In addition, the total dendritic length of MPN neurons is greater, but the dendritic spine density is smaller, in males than in females. Likewise, in the SDN-POA the total number and size of its neurons is greater in males than in females. The sex differences in all quantitative parameters evaluated accounted for the larger volume of the MPN and SDN-POA in males relative to females. In addition, the MPN neuropil also displays sex-related differences in its volume, and these differences closely match those detected for the volume of each MPN division. It deserves to be emphasised that the numerical density of neurons was the only parameter found to be significantly higher in females than in males in all MPN divisions and in the SDN-POA. Our results show that the MPN and the SDN-POA display sex differences in the volume, total number of neurons, and size of neuronal cell bodies and dendritic trees. Furthermore, they also indicate that the neuropil is critical for the establishment of sexual dimorphism in the size of the MPN.     

Conjugated estradiol increases female sexual receptivity in response to oxytocin infused into the medial preoptic area and medial basal hypothalamus

The ovarian steroid estradiol (E) has been found to increase both receptor affinity and release of the neuropeptide oxytocin (OT) in plasma membrane preparations. Therefore, we hypothesized that E conjugated to bovine serum albumin at position 6 (E-6-BSA) would increase behavioral responsiveness to OT. Preliminary results showed that 200 ng/microl of E-6-BSA increased sexual receptivity slightly, but not significantly. Therefore, this dose was used as a subthreshold dose to test whether it would increase sexual responsiveness when infused in combination with 100 ng/microl OT. After recovery from cannula implantation surgery animals were injected with 0.5 microg E benzoate daily for 3 days before testing. On the fourth day, after a baseline preinfusion test rats were infused bilaterally with E-6-BSA alone or with OT, OT with BSA, or conjugated progesterone, luteinizing hormone-releasing hormone equimolar to OT alone, or with E-6-BSA or conjugated progesterone alone. When infused into either the medial preoptic area-anterior hypothalamus or the medial basal hypothalamus the combination of OT and E-6-BSA significantly increased sexual receptivity over receptivity after artificial cerebrospinal fluid control infusions. Neither bilateral infusions of OT in combination with conjugated progesterone nor E-6-BSA in combination with luteinizing hormone-releasing hormone enhanced sexual receptivity. Results presented here strongly support the conclusion that some of the effects that E has in sensitizing brain systems to the facilitating effects of OT occur at the membrane level in the medial preoptic area-anterior hypothalamus and medial basal hypothalamus.     

Estradiol acts in the medial preoptic area, arcuate nucleus, and dorsal raphe nucleus to reduce food intake in ovariectomized rats

Estradiol (E2) exerts an inhibitory effect on food intake in a variety of species. While compelling evidence indicates that central, rather than peripheral, estrogen receptors (ERs) mediate this effect, the exact brain regions involved have yet to be conclusively identified. In order to identify brain regions that are sufficient for E2's anorectic effect, food intake was monitored for 48 h following acute, unilateral, microinfusions of vehicle and two doses (0.25 and 2.5 μg) of a water-soluble form of E2 in multiple brain regions within the hypothalamus and midbrain of ovariectomized rats. Dose-related decreases in 24-h food intake were observed following E2 administration in the medial preoptic area (MPOA), arcuate nucleus (ARC), and dorsal raphe nucleus (DRN). Within the former two brain areas, the larger dose of E2 also decreased 4-h food intake. Food intake was not influenced, however, by similar E2 administration in the paraventricular nucleus, lateral hypothalamus, or ventromedial nucleus. These data suggest that E2-responsive neurons within the MPOA, ARC, and DRN participate in the estrogenic control of food intake and provide specific brain areas for future investigations of the cellular mechanism underlying estradiol's anorexigenic effect.     

Angiotensin and NMDA receptors in the median preoptic nucleus mediate hemodynamic response patterns to stress

The brain renin-angiotensin system plays an important role in the regulation of arterial pressure in response to stress, in part due to activation of AT1 receptors in the hypothalamic median preoptic nucleus (MnPO) by endogenous angiotensin II (ANG II). N-methyl-d-aspartate (NMDA) receptors are also involved in the angiotensinergic signaling pathway through the MnPO. We investigated whether AT1 and NMDA receptors in the MnPO are responsible for variable hemodynamic response patterns to stress. Cocaine or startle with cold water evoked a pressor response in Sprague-Dawley rats due, in some rats [vascular responders (VR)], to a large increase in systemic vascular resistance (SVR) and, in other rats [mixed responders (MR)], to small increases in SVR and cardiac output (CO). Microinjection of the GABAA agonist muscimol into the MnPO to block synaptic transmission attenuated the cocaine- or stress-induced increase in SVR and the decrease in CO seen in VR without altering either response in MR. Likewise, administration of either an AT1 receptor antagonist, losartan, or an NMDA receptor antagonist, MK-801, attenuated the increase in SVR and the decrease in CO in VR in response to either cocaine (losartan and MK-801) or startle with cold water (losartan) without altering either response in MR. We propose that the MnPO is responsible for greater SVR responses in VR and that AT1 and NMDA receptors play an important role in greater SVR responses in VR. These data provide additional support for the critical role of the MnPO in cardiovascular responses to stress.     

Mu opioid receptors in the medial preoptic area govern social play behavior in adolescent male rats

Neural systems underlying important behaviors are usually highly conserved across species. The medial preoptic area (MPOA) has been demonstrated to play a crucial role in reward associated with affiliative, nonsexual, social communication in songbirds. However, the role of MPOA in affiliative, rewarding social behaviors (eg, social play behavior) in mammals remains largely unknown. Here we applied our insights from songbirds to rats to determine whether opioids in the MPOA govern social play behavior in rats. Using an immediate early gene (ie, Egr1, early growth response 1) expression approach, we identified increased numbers of Egr1‐labeled cells in the MPOA after social play in adolescent male rats. We also demonstrated that cells expressing mu opioid receptors (MORs, gene name Oprm1) in the MPOA displayed increased Egr1 expression when adolescent rats were engaged in social play using double immunofluorescence labeling of MOR and Egr1. Furthermore, using short hairpin RNA‐mediated gene silencing we revealed that knockdown of Oprm1 in the MPOA reduced the number of total play bouts and the frequency of pouncing. Last, RNA sequencing differential gene expression analysis identified genes involved in neuronal signaling with altered expression after Oprm1 knockdown, and identified Egr1 as potentially a key modulator for Oprm1 in the regulation of social play behavior. Altogether, these results show that the MPOA is involved in social play behavior in adolescent male rats and support the hypothesis that the MPOA is part of a conserved neural circuit across vertebrates in which opioids act to govern affiliative, intrinsically rewarded social behaviors.     

Inactivation of the medial preoptic area or the bed nucleus of the stria terminalis differentially disrupts maternal behavior in sheep

The present study was designed to investigate the role of the medial preoptic area (MPOA) and the bed nucleus of the stria terminalis (BNST) in the onset and maintenance of maternal behavior in sheep. In a first experiment, the MPOA or BNST were transiently inactivated during the first 2 h postpartum in primiparous ewes with the use of the anaesthetic lidocaine. MPOA inactivation greatly impaired the display of maternal behavior whereas inactivation of BNST or of adjacent sites (septum or diagonal band of Broca) or infusion of cerebrospinal fluid did not. In a separation/reunion lamb test (S/R) performed at 2 h postpartum, ewes with MPOA inactivation exhibited little reaction after separation of their lambs and did not show any motivation to reunite with them. Ewes with BNST inactivation showed intermediate performances in the S/R test. Moreover, in control ewes that were maternal for the first 2 h postpartum, MPOA or BNST inactivation performed in the following 12 h induced deficits in the S/R test, indicating that the MPOA and to a lesser extent BNST are also involved in the maintenance of maternal behavior. A second experiment showed that, in multiparous ewes, MPOA inactivation at parturition induced less deficit in the display of maternal behavior and in the S/R test than in primiparous mothers. These findings indicate that the MPOA and, to some extent, the BNST are functionally involved in the initiation and in the maintenance of maternal behavior in sheep, but this involvement is influenced by maternal experience.