Ontogeny of opiate receptors in the rat medial preoptic area: Critical periods in regional development

Opiate receptor labeling was examined throughout the early postnatal period using autoradiography to localize and quantify [³H]naloxone binding to μ-type opiate receptors in the medial preoptic area (MPOA). This region begins to exhibit sexual dimorphism of volume and dendritic growth shortly after birth. A distinct concentration of opiate receptor labeling appears on postnatal day 3 in females: this labeling is directly associated with the sexually dimorphic nucleus of the preoptic area (SDN-POA). SDN-POA labeling becomes denser through postnatal day 10 in females and the densely labeled area increases in size to encompass and surround the SDN-POA. These changes in opiate receptor labeling occur only in females, since males show relatively uniform labeling across the region throughout the early postnatal period.The critical time of formation of dense MPOA opiate receptor labeling may be related to endogenous MPOA opioid function and to the critical period of dendritic growth of SDN-POA neurons. The timing of these critical periods and their focus in the SDN-POA are coincident. The possible role of MPOA opiate receptors in modulating growth of MPOA neurons is discussed.     

Pre- and postnatal influence of testosterone propionate and diethylstilbestrol on differentiation of the sexually dimorphic nucleus of the preoptic area in male and female rats

The volume of the sexually dimorphic nucleus in the preoptic area (SDN-POA) of the rat brain is several fold larger in males than in females. When female rats were treated pre- and postnatally with testosterone propionate (TP) or with diethylstilbestrol (DES) they became anovulatory and their SDN-POA developed equivalent in size to that of normal males. Identical treatment of male rats resulted in deficient testicular development, but had no influence on SDN-POA volume. The results indicate that the gross morphological sex difference in SDN-POA volume can exclusively be controlled by the hormonal environment during the critical period of sexual brain differentiation, and that non-steroidal estrogens are just as effective as convertible androgens in stimulating SDN-POA differentiation.     

Sex Differences and the Roles of Sex Steroids in Apoptosis of Sexually Dimorphic Nuclei of the Preoptic Area in Postnatal Rats

The brain contains several sexually dimorphic nuclei that exhibit sex differences with respect to cell number. It is likely that the control of cell number by apoptotic cell death in the developing brain contributes to creating sex differences in cell number in sexually dimorphic nuclei, although the mechanisms responsible for this have not been determined completely. The milieu of sex steroids in the developing brain affects sexual differentiation in the brain. The preoptic region of rats has two sexually dimorphic nuclei. The sexually dimorphic nucleus of the preoptic area (SDN-POA) has more neurones in males, whereas the anteroventral periventricular nucleus (AVPV) has a higher cell density in females. Sex differences in apoptotic cell number arise in the SDN-POA and AVPV of rats in the early postnatal period, and an inverse correlation exists between sex differences in apoptotic cell number and the number of living cells in the mature period. The SDN-POA of postnatal male rats exhibits a higher expression of anti-apoptotic Bcl-2 and lower expression of pro-apoptotic Bax compared to that in females and, as a potential result, apoptotic cell death via caspase-3 activation more frequently occurs in the SDN-POA of females. The patterns of expression of Bcl-2 and Bax in the SDN-POA of postnatal female rats are changed to male-typical ones by treatment with oestrogen, which is normally synthesised from testicular androgen and affects the developing brain in males. In the AVPV of postnatal rats, apoptotic regulation also differs between the sexes, although Bcl-2 expression is increased and Bax expression and caspase-3 activity are decreased in females. The mechanisms of apoptosis possibly contributing to the creation of sex differences in cell number and the roles of sex steroids in apoptosis are discussed.     

Neonatal stimulation of 5-HT(2) receptors reduces androgen receptor expression in the rat anteroventral periventricular nucleus and sexually dimorphic preoptic area

Masculinization of the brain is dependent upon a perinatal surge in testosterone. It also requires a transient decrease in hypothalamic 5-HT concentration and turnover and an increase in androgen receptor (AR) expression during the second postnatal week. We have previously shown that increasing 5-HT activity over this period in male or androgenized female rats feminizes their adult behaviour and also feminizes the size of their anteroventral periventricular nucleus (AVPV) and sexually dimorphic nucleus of the preoptic area (SDN-POA). To investigate the role of 5-HT in sexual differentiation of the brain, 5-HT activity was raised over postnatal days 8-16 in male, female and androgenized female rats by daily administration of the 5-HT(2) receptor agonist (-)[2,5 dimethoxy-4-iodophenyl]-2-amino propane hydrochloride [(-)DOI]. By postnatal day 18, the size of the AVPV and SDN-POA was sexually dimorphic; their sizes were feminized by (-)DOI treatment. In the absence of (-)DOI treatment, there were significantly more AR-immunoreactive cells in the AVPV of males, and in the SDN-POA of males and androgenized females, than in those of females on postnatal day 18. (-)DOI treatment reduced the number of AR-immunoreactive cells in the AVPV and SDN-POA of males and androgenized females, but not of females, by postnatal day 18. These results suggest that 5-HT(2) receptor activation can influence sexual differentiation of the brain by controlling AR expression.     

Effects of prenatal stress on differentiation of the sexually dimorphic nucleus of the preoptic area (SDN-POA) of the rat brain

The present study was designed to determine the effects of prenatal malnutrition or environmental stress on the development of the sexually dimorphic nucleus of the preoptic area (SDN-POA). Pregnant rats were divided into a control group and two treatment groups (immobilization-illumination-heat or environmental stress, and nutritional stress). The two forms of stress were administered during the third trimester of gestation (days 14-20). Male and female offspring were sacrificed at birth, 20, and 60 days postnatally. The cross-sectional area of the SDN-POA was identified under light microscopy and was measured. The data confirm previous studies by showing a significant sex difference in the SDN-POA between control male and female rats. Prenatally stressed males sacrificed 20 and 60 days after birth showed SDN-POA areas 50% smaller than the nuclear areas of control males. The size of the SDN-POA of female offspring, however, was not significantly altered by prenatal treatments.     

The sexually dimorphic region of the preoptic area in rats contains denser opiate receptor binding sites in females

Quantitative autoradiographic analysis of opiate receptor binding using [³H]naloxone shows higher levels in the sexually dimorphic region of the medial preoptic area in female rats than in males. Opiate receptor density varies across the estrous cycle being densest in diestrous females. The sexually dimorphic nucleus of the preoptic are lies within the opiate receptor-rich region. Endogenous opiates in the medial preoptic region acting at opiate receptors which are of differential density in males and females could influence sex-specific behavior mediated by the region.     

Cerebellar projections to the lateral posterior-pulvinar thalamic complex in the cat

Quantitative autoradiographic analysis of opiate receptor binding using [3H]naloxone shows higher levels in the sexually dimorphic region of the medial preoptic area in female rats than in males. Opiate receptor density varies across the estrous cycle being densest in diestrous females. The sexually dimorphic nucleus of the preoptic area lies within the opiate receptor-rich region. Endogenous opiates in the medial preoptic region acting at opiate receptors which are of differential density in males and females could influence sex-specific behavior mediated by the region.     

Steroid autoradiography of the sexually dimorphic nucleus of the preoptic area

Autoradiography was performed to determine if the neurons of the sexually dimorphic nucleus of the preoptic area (SDN-POA) in the adult rat accumulate estradiol (E2), testosterone (T), and/or dihydrotestosterone (DHT). Three days prior to steroid administration, adult male and female Sprague-Dawley rats were gonadectomized and adrenalectomized. Animals were then given either [3H]T, [3H]E2, or [3H]DHT through an indwelling jugular cannula. One hour later, animals were decapitated and brain sections processed for thaw mount autoradiography. The autoradiograms which contained the SDN-POA and an adjacent area of the medial preoptic area (MPOA) were quantitatively analyzed using the 3 times background, 5 times background, and Poisson criteria for labeled cells. In general, cells in the SDN-POA and the MPOA accumulate T, E2, or DHT. For both sexes, there is a greater percentage of labeled cells in the SDN-POA than in the MPOA, and a greater percentage of labeled cells following E2 exposure than following T or DHT exposure. In addition, there is a sex difference (male greater than female) in the percentage of labeled cells following T exposure. In summary, these data indicate that adult SDN-POA neurons do accumulate gonadal steroids.     

Postnatal treatment of rats with the beta 2-adrenergic agonist salbutamol influences the volume of the sexually dimorphic nucleus in the preoptic area

Sexual differentiation of the brain seems to be influenced by postnatal interaction of gonadal steroids with neurotransmitter systems, in particular the adrenergic system. Stimulation or inhibition of adrenergic receptors during early postnatal development had previously been shown to influence steroid-induced sexual differentiation of rat brain function. In the present study newborn male and female rats were treated daily for 5 days with salbutamol, a specific beta 2-receptor agonist, or with alprenolol, a beta-receptor antagonist and the volume of the sexually dimorphic nucleus of the preoptic area (SDN-POA) was examined in adulthood. This nucleus, one of the most striking sex differences in brain anatomy, is several-fold larger in male than in female rats. Postnatal treatment with salbutamol increased SDN-POA volume in female and in male rats. The effect was particularly striking in males, because any previous pre- and/or postnatal treatment of male rats with large amounts of gonadal steroids had been unable to increase the volume of the SDN-POA above normal. The beta-receptor antagonist alprenolol had no effect on SDN-POA differentiation. The results indicate that beta 2-adrenergic stimulation influences development and differentiation of the SDN-POA.     

Effects of discrete lesions of the sexually dimorphic nucleus of the preoptic area or other medial preoptic regions on the sexual behavior of male rats

The sexually dimorphic nucleus of the rat medial preoptic area (SDN-POA) has a volume five times larger in the adult male compared with that of the adult female. In the present study, the effects of discrete electrolytic destruction of the SDN-POA or other specific medial preoptic (MPOA) regions on masculine sexual behavior were determined in adult, sexually experienced male rats. Small lesions encompassing the SDN-POA had no effect on the maintenance of copulatory behavior. Lesions of similar size placed within the ventral or anterio-dorsal MPOA also did not consistently affect the display of masculine sexual behavior. However, animals that received small lesions within their dorsal MPOA showed a substantial, long-term decrease in number of mounts, intromissions, and ejaculations compared to these parameters in sham-lesioned control rats, thus indicating a lesion-induced disruption of those neural mechanisms mediating these behaviors. Collectively these data suggest that the SDN-POA is not critical for a full expression of male sexual behavior and that the dorsal MPOA may be more important than other MPOA regions for copulatory behavior.     

Neurogenesis of the sexually dimorphic nucleus of the preoptic area in the rat

The sexually dimorphic nucleus of the preoptic area (SDN-POA) of Sprague-Dawley rats is larger in volume in the male and hormone-dependent early in postnatal life. In the present study, we compared for each sex the time course of neuroblast proliferation which forms SDN-POA or adjacent medial preoptic area (MPOA) neurons. Additionally, we investigated whether there is a temporal gradient of production of neurons in relation to their final position within the SDN-POA. On day 14, 15, 16, 17, or 18 postfertilization (pf) pregnant rats were given a single injection of ³H-thymidine (*thy). At 30 postnatal days of age the pups were sacrificed and brain sections were prepared and processed for autoradiography. Three sections of the SDN-POA and an adjacent area just lateral to it in the MPOA wer also analyzed. In the MPOA and the SDN-POA the percentage (%) of labeled neurons decreases as the day of injection of *thy approaches the end of gestation, but the time period in which neuroblast divisions occurred is markedly different for the SDN-POA as compared to that for the MPOA. DNA synthesis occurs as late as day 18pf for neurons which form the SDN-POA but ceases on day 16pf for those destined for the MPOA. There is a sex difference in neuronal production on both day 14 and 17pf for neurons destined for the SDN-POA. After injection on day 14pf the % labeled neurons is larger in the female than in the male but after injection on day 17pf this is reversed. There are also significant sex differences as well as a temporal gradient associated with the % labeled neurons in the SDN-POA in relation to their final anterior-posterior position. In addition, this study confirms our previous results which justify labeling the SDN-POA a nucleus, since neuronal density in this region in the male and female is significantly greater than that in the surrounding MPOA. These data illustrate that the specific neurons which comprise the SDN-POA in both the male and female are being produced as late as day 18pf, whereas neurons located in the MPOA but not in the SDN-POA have all been born by day 16pf. Neuroblast division which produces the neurons of the SDN-POA may begin earlier and terminate sooner in the female than in the male. These differences in neuronal production may partially account for the sexual dimorphism seen in the volume and neuronal number of the SDN-POA of the adult rat.     

GABAergic influence on the development of the sexually dimorphic nucleus of male and female rats.

A large number of estrogen-sensitive neurons in rat hypothalamus use gamma-aminobutyric acid (GABA) as a neurotransmitter. As estrogens influence the size of the sexually dimorphic nucleus (SDN) of the preoptic area it was tested whether perinatal treatment of rats with the GABA-agonistic drug muscimol can induce similar changes as estrogens. Male and female rats were treated perinatally with muscimol or with the vehicle only and the vols. of the SDN of the preoptic area were determined morphometrically following maturation of the animals. The SDN vols. in treated males were significantly smaller (80.3%) compared to controls. There was no striking effect in females. Our data suggest an influence of the GABAergic system on the development of the sexually dimorphic nucleus of the preoptic area (SDN-POA). The muscimol treatment, however, did not mimic the effects of perinatal estrogen treatment. Since muscimol is a GABAA receptor stimulating drug it appears that this receptor subtype is not involved in the estrogen-induced changes in size of the SDN.     

Pre- or postnatal testosterone and flutamide effects on sexually dimorphic nuclei of the rat hypothalamus

Utilizing the sexually dimorphic nature of hypothalamic nuclei, a determination of the effects of pre- or postnatal flutamide and testosterone treatments were examined in male and female rats. Statistical analysis compared treatments, sex, and time of injection in terms of the sexually dimorphic nucleus of the preoptic area (SDN-POA) and the anteroventral periventricular nucleus (AVPV) volumes and lengths. The present findings establish that pre- or postnatal hormonal environments are crucial in influencing sexual morphology on the developing brain.     

Mu-opiate receptor binding in the medial preoptic area is cyclical and sexually dimorphic

The density and distribution of mu- and kappa-opiate receptors in the medial preoptic area (MPOA) of male and female rats across the estrous cycle was examined using quantitative in vitro autoradiography of [3H]D-Ala2,MePhe4,Gly-ol5-enkephalin (DAGO), [3H]naloxone and [3H]bremazocine binding. While no difference in kappa-receptor labeling was observed across sex or estrous stage, selective mu-receptor labeling with [3H]DAGO revealed a significant variation of density and distribution in the MPOA across the estrous cycle and between sexes. A dense concentration of mu-receptors located in the central, sexually dimorphic portion of the MPOA was observed during metestrus and diestrus in females, but not during proestrus nor in males. This region appeared to be the same as that labeled similarly using [3H]naloxone. These results suggest that a regional substrate for functional activation by endogenous opioid peptides (e.g. beta-endorphin) is cyclically regulated in females, which may explain the gonadal steroid-dependent effects of MPOA beta-endorphin on lordosis and luteinizing hormone secretion in females.     

Relationship between sexual behavior and sexually dimorphic structures in the anterior hypothalamus in control and prenatally stressed male rats

The present study was designed to examine the effects of prenatal stress on the morphological development of sexually dimorphic structures in the anterior hypothalamus in male rats and to determine if there is a relationship between morphologic development of the brain and copulatory behavior in individual animals. Dams in the stress group were subjected to treatments of heat-light restraint during the third trimester of gestation (day 14 to parturition) three times daily for 45-min periods. At 90 days of age, prenatally stressed and control male offspring were tested during the dark cycle for spontaneous male sexual behavior. Volumes of the sexually dimorphic nucleus of the preoptic area (SDN-POA) and the anteroventral periventricular nucleus (AVPV) were measured. Comparisons were made between copulatory behavior and hypothalamic nuclear volumes. SDN-POA volumes were significantly reduced (feminized; males have a larger SDN-POA than females) in prenatally stressed males that did not copulate, whereas, SDN-POA volumes in prenatally stressed males that copulated were not altered. The few control males that did not copulate (sexually non-active) also had significantly reduced SDN-POA volumes compared to the control males that did copulate (sexually active). The volume of the AVPV was significantly increased (feminized; males have a smaller AVPV than females) in prenatally stressed males that were sexually non-active compared to AVPV volumes in sexually active males. The results obtained in this study provide a strong positive relationship between sexual behavior and the morphology of the two sexually dimorphic structures measured.     

Hormonal modification of the number of total and late-arising neurons in the central part of the medial preoptic nucleus of the rat

The sexually dimorphic nucleus of the preoptic area (SDN-POA) is larger in volume in males, is responsive to steroids developmentally, and contains a subpopulation of late-arising neurons that can be specifically labeled with 3H-thymidine on embryonic day 18 (E18). The cytoarchitecture of this region has been described, and one component, the central part of the medial preoptic nucleus (MPNc), shows considerable overlap with the SDN-POA. One goal of the present study was to relate the two by determining if testosterone propionate (TP) exposure perinatally increases MPNc volume and neuronal number, and by characterizing the distribution of the late arising neurons of the SDN-POA with respect to the MPNc. A second goal was to determine if these late-arising neurons are a representative, hormone-sensitive population. Finally, TP exposure was delayed past the time of the endogenous testosterone surge in males and after the neurons have become postmitotic, to determine if female brain structure could still be sex-reversed under these conditions. Pregnant rats were injected on E18 with 3H-thymidine. Daily injections of 2.0 mg TP were given to the mothers starting on either E16 or E20 and continued through birth. The pups were injected daily with 100 micrograms TP from birth through postnatal day 10. Control rats, from mothers given oil from E16 until birth, were injected with oil from birth through postnatal day 10. Rats were sacrificed at 30 days of age and their brains processed for autoradiography.(ABSTRACT TRUNCATED AT 250 WORDS)     

Perinatal androgenization prevents age-related neuron loss in the sexually dimorphic nucleus of the preoptic area in female rats

To investigate the effect of perinatal testosterone exposure, which simulates the endogenous testosterone peak, on neuron loss during aging, nuclear morphology was evaluated in male and female rats as well as in female rats treated with testosterone perinatally followed by ovariectomy (TE/Ovx). Additionally, neuronal apoptosis, which occurred primarily at postnatal day 8 (PND8), was identified by in situ TUNEL staining. Neuronal density, nuclear volume, total neuronal number and pyknotic ratio were estimated after HE stain at PND8, middle age and old age. The results showed that age-related decrease in neuronal nuclear volume and total neuron number in the sexually dimorphic nucleus of the preoptic area (SDN-POA) of female rats was significantly diminished by TE/Ovx. The pyknotic ratio in the SDN-POA of female rats at PND8 was significantly higher than that of males, and neuronal death was reversed by testosterone exposure, while no significant difference of pyknotic ratios was observed among male, female and TE/Ovx female rats at both middle and old age. Moreover, the high apoptotic incidence of female rats at PND8 was significantly diminished by testosterone exposure. These results suggest that neuron loss in the SDN-POA during aging may be predominantly determined by perinatal testosterone through modulation of postnatal neuronal apoptosis.     

Gonadal Steroid Action and Brain Sex Differentiation in the Rat

Gonadal steroids that establish sexually dimorphic characteristics of brain morphology and physiology act at a particular stage of ontogeny. Testosterone secreted by the testes during late gestational and neonatal periods causes significant brain sexual dimorphism in the rat. This results in both sex-specific behaviour and endocrinology in adults. Sexual differentiation may be due to neurogenesis, migration or survival. Each mechanism appears to be uniquely regulated in a site-specific manner. Thus, the volume of an aggregate of neurones in the rat medial preoptic area (POA), termed the sexually dimorphic nucleus of the POA (SDN-POA), is larger in males than in females. The anteroventral periventricular nucleus (AVPV) is packed with neurones containing oestrogen receptor (ER)β in female rats but, in males, ERβ-positive neurones scatter into the more lateral portion of the POA. POA neurones are born up to embryonic days 16–17 and not after parturition. Therefore, neurogenesis is unlikely to contribute to the larger SDN-POA in males. DNA microarray analysis for oestrogen-responsive genes and western blotting demonstrated site-specific regulation of apoptosis- and migration-related genes in the SDN-POA and AVPV.     

Termination of the hormone-sensitive period for differentiation of the sexually dimorphic nucleus of the preoptic area in male and female rats

The volume of the sexually dimorphic nucleus of the preoptic area (SDN-POA) in the rat brain is several-fold larger in males than in females. The volume of the SDN-POA can be influenced significantly by the hormone milieu during early postnatal life. The purpose of the present study was to identify when termination of the sensitive period occurs during which exogenous androgen administration influences SDN-POA volume in males gonadectomized on the first day of postnatal life (fales) or intact females. Analysis of the SDN-POA in fales showed that testosterone propionate (TP, 500 micrograms) treatment on days 2, 3, 4, or 5, significantly increased its volume over values from oil-treated fales. In contrast, TP treatment in fales on days 6, 7, or 8, failed to increase SDN-POA volume. A similar pattern was observed in females treated with TP. Females treated with TP (500 micrograms) on days 2, 3, 4, or 5, showed a significant increase in SDN-POA volume compared to the values from oil-injected animals, while the same TP treatment in females on days 6, 7, or 8, resulted in no such enhancement. The absolute and relative change in SDN-POA volume following postnatal androgen treatment is greater in males than in females. We conclude that (1) SDN-POA development is sensitive to hormone action through postnatal day 5 and then abruptly becomes insensitive to this dosage of TP, and (2) although the temporal pattern of the response is similar in males and females, androgen exposure postnatally results in a consistently greater increase in the male SDN-POA volume than in the female's. This greater response may be due to exposure prenatally to endogenous androgen in males.     

Intrauterine proximity to male fetuses affects the morphology of the sexually dimorphic nucleus of the preoptic area in the adult rat brain

Previous studies on polytocous rodents have revealed that the fetal intrauterine position influences its later anatomy, physiology, reproductive performance and behavior. To investigate whether the position of a fetus in the uterus modifies the development of the brain, we examined whether the structure of the sexually dimorphic nucleus of the preoptic area (SDN-POA) of rat brains accorded to their intrauterine positions. Brain sections of adult rats gestated between two male fetuses (2M) and between two female fetuses (2F) in the uterus were analysed for their immunoreactivity to calbindin-D28k, which is a marker of the SDN-POA. The SDN-POA volume of the 2M adult males was greater than that of the 2F adult males, whereas the SDN-POA volume of the 2M and 2F adult females showed no significant difference. This result indicated that contiguous male fetuses have a masculinizing effect on the SDN-POA volume of the male. To further examine whether the increment of SDN-POA volume in adulthood was due to exposure to elevated steroid hormones during fetal life, concentrations of testosterone and 17beta-estradiol in the brain were measured with 2M and 2F fetuses during gestation, respectively. On gestation day 21, the concentrations of testosterone and 17beta-estradiol in the brain were significantly higher in the 2M male rats as compared with the 2F male rats. The results suggested that there was a relationship between the fetal intrauterine position, hormone transfer from adjacent fetuses and the SDN-POA volume in adult rat brains.