The sex hormone-dependent development of opiate receptors in the rat medial preoptic area

The opiate receptor content of the sexually dimorphic medial preoptic area (MPOA) was examined in newborn and 5-day-old (D6) male and female rats. A significant increase of [³H]naloxone binding was observed in and around the sexually dimorphic nucleus of the preoptic area (SDN-POA) in D6 female rats, relative to newborn females. Opiate receptor labeling did not increase over this period in males, nor was labeling different between males and females at birth. This dramatic alteration of MPOA opiate receptor content was observed to occur in either sex in the absence of testosterone postnatally; that is, neonatally-castrated males exhibited the same increase of labeling by D6 as did normal females. Conversely, daily postnatal testosterone treatment of females from birth to D6 resulted in the development of male-like MPOA opiate receptor pattern. The sex hormone-dependence of MPOA opiate receptor development is discussed in relation to the sex hormone-dependent ontogeny of SDN-POA structure. The overlap of critical periods for the development of these structural and chemical sexual dimorphisms suggests a role for endogenous opioids in modulating MPOA development.     

μ-Opiate receptor binding in the medial preoptic area is cyclical and sexually dimorphic

The density and distribution of μ- and κ-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 κ-receptor labeling was observed across sex or estrous stage, selective μ-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 μ-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 opiod peptides (e.g. β-endorphin) is cyclically regulated in females, which may explain the gonadal streoid-dependent effects of MPOA β-endorphin on lordosis and luteinizing hormone secretion in females.     

Effects of testosterone on a selected neuronal population within the preoptic sexually dimorphic nucleus of the Japanese quail

The effects of testosterone on the volume and cytoarchitecture of the sexually dimorphic nucleus of the preoptic area (POM) were investigated in male and female Japanese quail. It was confirmed that castration decreases the POM volume in males and that, in gonadectomized birds of both sexes, testosterone increases this volume to values similar to those observed in intact sexually mature males. This suggests that the sex difference in POM volume results from a differential activation by T so that this brain morphological characteristic is not truly differentiated in the organizational sense. This conclusion was extended here by demonstrating that males exposed to a photoperiod simulating long days and that are known to have high plasma levels of testosterone have a larger POM than short-day males that have inactive testes. Detailed morphometric studies of POM neurons revealed a structural heterogeneity within the nucleus. A population of large neurons (cross-sectional area larger than 70-80 microns2) was well represented in the dorsolateral but was almost absent in the medial part of POM. This lateral population of neurons was sensitive to variations of testosterone levels in males but not in females. The cross-sectional area, diameter, and perimeter of the dorsolateral neurons were significantly increased in males exposed to high testosterone levels (intact birds exposed to long days or castrated birds treated with the steroid). These changes were not observed in the medial part of the nucleus. Interestingly, the size of the dorsolateral neurons was not affected by testosterone treatments in females. These results suggest that the swelling of neurons in the lateral POM of males might be responsible for the increase in total volume of the nucleus, which is observed in physiological situations associated with a high testosteronemia. In addition, the sensitivity to testosterone of the dorsolateral neurons in the POM appears to be sexually differentiated. This differential response to testosterone might represent a truly dimorphic feature in the organizational sense and additional studies manipulating the early steroid environment should be performed to test this possibility.     

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.     

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.     

Sex difference in dendritic development of the sexually dimorphic nucleus of the preoptic area in the rat

Sex differences in the growth and dendritic development of neurons in the sexually dimorphic nucleus of the preoptic area were examined with quantitative Golgi techniques during early postnatal life in rats. Neuronal size and dendritic extent were found to increase more in males than in females during the first 10 postnatal days, while the numbers of primary and terminal dendrites were similar in the two sexes. The onset of greater dendritic growth in males occurs just after the volume of the nucleus begins to exhibit sexual dimorphism, between 24 and 26 days after fertilization. Growth of dendrites in this region may be related to the presence of sex hormones during the critical period of sexual brain differentiation.     

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.     

The organization of neural inputs to the medial preoptic nucleus of the rat

There is general agreement that the medial preoptic nucleus (MPN) receives projections from widespread regions of the brain, although there are significant discrepancies in the literature with regard to certain specific inputs. Therefore, we have reexamined the inputs to this nucleus with both retrograde and anterograde axonal transport techniques. First, injections of the retrograde tracers true blue, SITS, or wheat germ agglutinin were made into the region of the MPN and the distribution of retrogradely labeled cells was charted. Then, autoradiographic material was used to confirm the results of the retrograde studies, to identify the route taken by fibers projecting to the MPN, and to describe the distribution of projections with respect to the three cytoarchitectonic subdivisions of the nucleus. The results indicate that the MPN receives inputs from widely distributed areas in both the forebrain and brainstem, and that these inputs appear to be distributed topographically within the three cytoarchitectonic subdivisions of the nucleus. Direct inputs to the MPN arise from all major areas of the hypothalamus (except for the median and magnocellular preoptic nuclei, the supraoptic and suprachiasmatic nuclei, and the medial and lateral mammillary nuclei). Projections from nuclei within the periventricular zone of the hypothalamus end primarily in the medial part of the MPN, while inputs from the lateral zone are mainly confined to the lateral part of the nucleus, as are projections from the nuclei within the medial zone, except for those from the anterior and ventromedial nuclei, which appear to be more widespread. The MPN receives major inputs from limbic regions including the amygdala, ventral subiculum, and ventral lateral septal nucleus, all of which end preferentially in the lateral part of the MPN. In contrast, the projection from the encapsulated part of the bed nucleus of the stria terminalis appears to end preferentially in the central part of the MPN and in immediately adjacent regions of the medial subdivision. In addition, the MPN may receive relatively sparse inputs from infralimbic and insular cortical areas, the nucleus accumbens, and the substantia innominata. Finally, ascending serotoninergic projections from the raphe nuclei appear to terminate principally in the lateral part of the MPN, whereas inputs from regions containing noradrenergic cell groups are chiefly distributed to the central and medial parts of the nucleus. Other brainstem regions that appear to provide modest inputs include the ventral tegmental area, central tegmental field, periaqueductal gray, pedunculopontine nucleus, and the peripeduncular nucleus.(ABSTRACT TRUNCATED AT 400 WORDS)     

Estrogen/progesterone treatment in adulthood affects the size of several components of the medial preoptic area in the male rat

The results of preliminary studies suggested that steroid and/or propylthiouracil (PTU) treatment of adult gonadectomized (Gxd) male rats significantly reduced the volume of the sexually dimorphic nucleus of the preoptic area (SDN-POA). Therefore, we designed a study to examine this effect in detail. Groups of adult rats were sham Gxd (intact) or Gxd, then treated with multiple injections of oil (males and females), or estrogen and progesterone (males). Gonadectomized estrogen/progesterone-treated males had a significantly smaller SDN-POA volume, smaller volume of the medial division of the medial preoptic nucleus (MPNm), smaller volume of the anteroventral MPNm (MPNav), and larger volume of the anteroventral periventricular nucleus (AVPv). The volume of the central division of the medial preoptic nucleus (MPNc) or of the suprachiasmatic nucleus was not affected. There were no differences between Gxd estrogen/progesterone-treated males vs the group that received PTU as well, indicating that the PTU treatment was unnecessary. The reduced volume of the SDN-POA was due to a reduced volume of the MPNav and of the portion of the SDN-POA located within the MPNm-exclusive of the MPNav and MPNc. In conclusion, estrogen/progesterone treatment in adulthood caused significant changes in the volume of several medial preoptic structures in two separate groups of Gxd males. Because the steroids produced no significant effects in intact males, testicular hormones appear to "protect" these structures from the effects of the estrogen/progesterone treatment.     

Ovarian steroids alter dopamine receptor populations in the medial preoptic area of female rats: implications for sexual motivation,desire, and behaviour

Dopamine (DA) transmission in the medial preoptic area (mPOA) plays a critical role in the control of appetitive sexual behaviour in the female rat. We have shown previously that a DA D1 receptor (D1R)‐mediated excitatory state appears to occur in females primed with estradiol benzoate (EB) and progesterone (P), whereas a DA D2 receptor (D2R)‐mediated inhibitory state appears to occur in females primed only with EB. The present experiment employed three techniques to better understand what changes occur to DA receptors (DARs) in the mPOA under different hormonal profiles. Ovariectomized females were randomly assigned to one of three steroid treatment groups: EB + P (10 and 500 μg, respectively), EB + Oil, or the control (Oil + Oil), with hormone injections administered at 48 and 4 h prior to euthanizing. First, the number of neurons in the mPOA that contained D1R or D2R was assessed using immunohistochemistry. Second, the mPOA and two control areas (the prelimbic cortex and caudate putamen) were analysed for DAR protein levels using western blot, and DAR functional binding levels using autoradiography. Ovarian steroid hormones affected the two DAR subtypes in opposite ways in the mPOA. All three techniques supported previous behavioural findings that females primed with EB have a lower D1R : D2R ratio, and thus a D2R‐mediated system, and females primed with EB + P have a higher D1R : D2R ratio, and thus a D1R‐mediated system. This provides strong evidence for a DA‐driven pathway of female sexual motivation, desire, and behaviour that is modified by different hormone priming regimens.     

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.     

Sexually dimorphic activation of galanin neurones in the ferret's dorsomedial preoptic area/anterior hypothalamus after mating

Male ferrets in breeding condition possess three times as many galanin-immunoreactive (IR) neurones as oestrous females in the sexually dimorphic dorsomedial preoptic area/anterior hypothalamus (dmPOA/AH). Using Fos-IR as a marker of activation, we investigated whether mating with intromission differentially activates this sexually dimorphic group of galanin-IR neurones in male and female ferrets. Male ferrets that intromitted had a significantly greater percentage of galanin-IR neurones in the dmPOA/AH that were colabelled with nuclear Fos-IR than oestrous females that received an intromission. Intromissive stimulation augmented Fos-IR in an equal percentage of galanin-IR neurones in both sexes in the medial amygdala (MA) and bed nucleus of the stria terminalis (BNST). Peripheral anosmia induced by bilateral occlusion of males' nares did not reduce the mating-induced activation of galanin-IR neurones in the dmPOA/AH, and there was a significant correlation among individual males between intromission duration and the percentage of dmPOA/AH galanin-IR neurones colabelled with Fos-IR. Exposure of castrated, testosterone propionate-treated male ferrets to either soiled bedding or to volatile odours from oestrous females failed to induce nuclear Fos-IR in galanin-IR neurones located in the dmPOA/AH, BNST or MA, suggesting that the mating-induced activation of galanin-IR forebrain neurones in male ferrets depends more on genital-somatosensory than on olfactory inputs. The observed sex dimorphism in the mating-induced activation of galanin-IR neurones in the dmPOA/AH raises the possibility that these neurones perform a mating-dependent function that occurs only in males.     

Afferent connections of the sexually dimorphic area of the hypothalamus of male and female gerbils

We studied neural inputs to the sexually dimorphic area (SDA) of the gerbil hypothalamus by injecting wheat-germ agglutinin-horseradish peroxidase into its medial or lateral components in males and females. To confirm the topography of SDA afferents, we injected Phaseolus vulgaris-leucoagglutinin into areas where retrograde labeling from the medial and lateral SDA differed. Both methods indicated that the medial SDA received stronger inputs from the medial part of the bed nucleus of the stria terminalis, the ventral part of the lateral septal nucleus, the medial amygdaloid nucleus, and the amygdalohippocampal area, than the lateral SDA does. In contrast, the rostrodorsal part of the lateral septum, the lateral part of the bed nucleus of the stria terminalis, the anterior and posterior hypothalamic areas, and the dorsomedial hypothalamic nucleus project more heavily to the lateral than to the medial SDA. In addition, retrograde labeling suggested that the ventral part of the premammillary nucleus projects more strongly to the medial than to the lateral SDA, whereas the infralimbic area of the cortex and the lateral preoptic area project more strongly to the lateral than to the medial SDA. The densities of cells in the bed nucleus of the stria terminalis and medial amygdaloid nucleus that could be retrogradely labeled from the medial SDA were greater in males than in females. This was not true of labeling in the arcuate nucleus or in the ventral part of the lateral septal nucleus. Since the medial SDA receives strong inputs from areas with many steroid-accumulating cells, it could respond to steroids directly and via these afferents. In contrast, hormonal effects on the lateral SDA are more likely to occur locally.     

Estradiol binding in the sexually dimorphic nucleus of the preoptic/anterior hypothalamic area of adult male ferrets and in the equivalent region of females

A large number of estradiol-concentrating cells were visualized by autoradiography in a subpopulation of large neurons located in and around the sexually dimorphic male nucleus of the preoptic/anterior hypothalamic area (MN-POA/AH) of castrated male ferrets and in a comparable dorsal portion of the POA/AH of ovariectomized females. Considerably fewer estradiol-labelled cells were seen in the non-dimorphic ventral POA/AH nucleus of both sexes. Estrogen binding in cells in or around the MN-POA/AH may contribute to the formation of this sexually dimorphic nucleus in fetal males and may mediate specific estrogen-dependent behavioral functions in adulthood.     

Projections of the sexually dimorphic calcitonin gene-related peptide neurons of the preoptic area determined by retrograde tracing in the female rat

The medial preoptic area of the rat exhibits morphologic sex differences and is implicated in the control of sexually dimorphic behavior and function. Neurons expressing calcitonin gene-related peptide (CGRP) within the anteroventral periventricular (AVPV) and medial preoptic nucleus (MPN) of the medial preoptic area exhibit female-dominant sex differences in number through organizational and activational effects of gonadal steroids. The present study used retrograde tracing experiments to establish the projections of the AVPV and MPN CGRP neurons in the female rat. After the intraperitoneal administration of Fluoro-Gold to female rats (n = 5), we were unable to detect retrograde tracer in any CGRP-immunoreactive cells of the hypothalamus. Intracerebral injections of 50- to 100-nl volumes of Fluoro-Gold into the mediobasal hypothalamus resulted in up to 70% of CGRP neurons in the AVPV and MPN containing retrograde tracer. Similar large volume tracer depositions in the lateral septum, periaqueductal gray, two likely CGRP projection sites, resulted in no labeling of preoptic CGRP neurons. Experiments using small volume (30-nl) injections of Fluoro-Gold and green fluorescent microspheres at multiple sites in the mediobasal hypothalamus (n = 18) revealed that approximately 60% of AVPV and 30% of MPN neurons expressing CGRP were projecting to the region of the tuberal and ventral premammillary nuclei, with a minor projection to the dorsomedial nucleus. These findings demonstrate a major projection of the preoptic CGRP neurons to the posterior hypothalamus in the female rat and support further a functional role for these neurons in the sexually dimorphic regulation of reproductive functioning.     

Estrogen configures sexual dimorphism in the preoptic area of C57BL/6J and ddN strains of mice

Immunohistochemistry using a calbindin D28k antibody revealed a marked sex difference in neuronal distribution in the central portion of the medial preoptic area in C57BL/6J and ddN strains of mice when the animals were sacrificed on D65 (D1 = the day of birth). Male mice had a distinct ellipsoidal cell aggregate, whereas females lacked such a structure. This sex difference was not observed in Nissl‐stained sections. Co‐localization of calbindin D28k and the neuron‐specific nuclear protein NeuN confrmed that the cells in the aggregate were neurons. The aggregates were larger in males than in females in both strains. When observed on D65, males orchidectomized on D1 had smaller aggregates. However, daily injections of 2 μg estradiol benzoate through D1–D5 as well as a single injection of 100 μg testosterone propionate on D1 enlarged the aggregates in females, but a single injection of 100 μg dihydrotestosterone on D1 had no effect on the female phenotype. Similar endocrine manipulations had no effects in adult animals of both sexes. Thus, the calbindin‐immunoreactive cell aggregates in the preoptic area of C57BL/6J and ddN mice are homologous to the sexually dimorphic nucleus of the rat preoptic area in terms of the morphology and sex steroid‐dependent organization. J. Comp. Neurol. 518:3618–3629, 2010. © 2010 Wiley‐Liss, Inc.     

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.     

Sexual differentiation and hormonal control of the sexually dimorphic medial preoptic nucleus in the quail

We recently identified a sexually dimorphic nucleus in the preoptic region of the Japanese quail, the medial preoptic nucleus (POM), which is significantly larger in males than in females. In the present study, we investigated the hormonal control of this morphological neuroanatomical difference and the possible relationships between the sexual dimorphism in POM volume and in copulatory behavior. Treatments which are known to affect sexual behavior were thus applied to different groups of birds and the POM volume was then measured. In one experiment, male and female quails were either gonadectomized, gonadectomized and treated with testosterone or left intact. The larger size of the POM in males was confirmed and treatments significantly affected the nucleus size which was decreased by gonadectomy and restored by testosterone treatment in both sexes to a level similar to that seen in intact males. In two other experiments, eggs were injected with estradiol benzoate on day 9 of incubation and the POM volume was measured in adulthood either in intact birds or in gonadectomized birds receiving a replacement therapy with testosterone. Despite the fact that estradiol benzoate treatment completely suppressed copulatory behavior, it did not affect the volume of the POM or slightly increased it. These data thus show that the POM volume is controlled by testosterone levels in adulthood and could thus be an interesting model for the study of the effects of steroids on the 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.