Prenatal morphine exposure alters estrogen regulation of kappa receptors in the cortex and POA of adult female rats but has no effects on these receptors in adult male rats

The binding characteristics of kappa receptors were assessed in the frontal cortex (CX), striatum, hypothalamus, preoptic area (POA), cerebellum, and ventral tegmental area of adult male and female rats exposed prenatally to morphine or saline. Prenatal morphine exposure altered estrogen regulation of kappa receptors in the CX and POA of females, but had no effects on kappa receptors in any of the examined brain regions in male rats.     

Prenatal morphine exposure alters estrogen regulation of κ receptors in the cortex and POA of adult female rats but has no effects on these receptors in adult male rats

The binding characteristics of κ receptors were assessed in the frontal cortex (CX), striatum, hypothalamus, preoptic area (POA), cerebellum, and ventral tegmental area of adult male and female rats exposed prenatally to morphine or saline. Prenatal morphine exposure altered estrogen regulation of κ receptors in the CX and POA of females, but had no effects on κ receptors in any of the examined brain regions in male rats.     

Prenatal exposure to morphine alters brain μ opioid receptor characteristics in rats

Prenatal morphine exposure alters neither the binding capacity nor the affinity of ligand binding to μ opioid receptors of adult male brains. However, males have significantly higherB_max in the hypothalamus than ovariectomized females. In females, prenatal exposure to morphine reduces theB_max of μ opioid receptors 25% in the hypothalamus and preoptic area. Estrogen treatment increases theB_max of μ opioid receptors in the striatum of all ovariectomized females but in the hypothalamus only of morphine-exposed females, thereby eliminating the sex difference observed in control animals.     

Prenatal exposure to morphine alters brain μ opioid receptor characteristics in rats

Prenatal morphine exposure alters neither the binding capacity nor the affinity of ligand binding to μ opioid receptors of adult male brains. However, males have significantly higherB_max in the hypothalamus than ovariectomized females. In females, prenatal exposure to morphine reduces theB_max of μ opioid receptors 25% in the hypothalamus and preoptic area. Estrogen treatment increases theB_max of μ opioid receptors in the striatum of all ovariectomized females but in the hypothalamus only of morphine-exposed females, thereby eliminating the sex difference observed in control animals.     

Changes in prodynorphin and POMC gene expression in several brain regions of rat fetuses prenatally exposed to Delta(9)-tetrahydrocannabinol

Recently, we demonstrated that prenatal Delta(9)-tetrahydrocannabinol (Delta(9)-THC) exposure alters proenkephalin mRNA levels in several brain regions of rat fetuses. In the present study, we analyzed mRNA levels of the two other opioid peptide precursors, prodynorphin and pro-opiomelanocortin (POMC), in several brain nuclei of rat fetuses which were exposed daily to Delta(9)-THC from day 5 of gestation. Prenatal Delta(9)-THC exposure altered POMC and prodynorphin mRNA levels in most of the brain areas studied at different fetal ages, but the effects were sex-dependent. Thus, POMC mRNA levels increased in Delta(9)-THC-exposed females, but decreased in Delta(9)-THC-exposed males at GD21 in the arcuate nucleus, cerebral cortex and habenular nuclei. POMC mRNA levels also increased in the arcuate nucleus and cerebral cortex of Delta(9)-THC-exposed fetuses at GD18. Prodynorphin mRNA levels were not altered by the prenatal Delta(9)-THC exposure in the striatum, cerebral cortex, hippocampus and hypothalamic structures of fetuses at GD16 and GD18, but a sexually dimorphic response was observed at GD21. Thus, prodynorphin mRNA levels increased in the cerebral cortex, hippocampus and paraventricular hypothalamic nucleus of Delta(9)-THC-exposed females, whereas no changes were observed in Delta(9)-THC-exposed males. In summary, Delta(9)-THC exposure altered the prenatal development of POMC and prodynorphin mRNA levels in several brain structures. Changes in POMC were similar to those reported previously for proenkephalin, increases in females but decreases in males, whereas changes in prodynorphin were only observed in females.     

A sex comparison of serotonin immunoreactivity and content in the ferret preoptic area/anterior hypothalamus

Previous studies with rats raised the possibility that sexually dimorphic features of the medial preoptic area/anterior hypothalamus (POA/AH) may result, in part, from a sex difference in the serotonergic innervation of this region. We asked whether a similar phenomenon may occur in a carnivore, the ferret. A sexually dimorphic male nucleus of the dorsal POA/AH (Mn-POA/AH) has previously been characterized in Nissl-stained sections of the male ferret forebrain; this nucleus is absent in females. A nondimorphic ventral nucleus of the POA/AH is found in both sexes. In the present study numerous serotonin (5-HT) immunoreactive (ir) fibers were observed in the dorsal POA/AH of gonadectomized adult ferrets of both sexes. By contrast, in both sexes the ventral nucleus of the POA/AH had many fewer 5-HTir fibers. A similar difference in the distribution of immunoreactivity between dorsal and ventral POA/AH was observed for tyrosine hydroxylase (TH) localized in cell bodies and in nerve fibers and for H222ir estrogen receptors localized in cell nuclei. Likewise, in both sexes the content of 5-HT and dopamine (DA), measured by high pressure liquid chromatography with electrochemical detection, were significantly higher in the dorsal than the ventral POA/AH, thereby corroborating observed regional differences in 5-HTir and THir fibers, respectively. The present findings provide no support for the notion that sexually dimorphic cytoarchitectonic features of the dorsal POA/AH in ferrets are associated with a sex difference in the serotonergic innervation of this region.     

Effects of neonatal rat Borna disease virus (BDV) infection on the postnatal development of the brain monoaminergic systems

Effects of neonatal Borna disease virus infection (BDV) on the postnatal development of brain monoaminergic systems in rats were studied. Tissue content of norepinephrine (NE), dopamine (DA) and its metabolite, 3,4-dihydroxyphenol acetic acid (DOPAC), and serotonin (5-HT) and its metabolite, 5-hydroxyindole-3-acetic acid (5-HIAA) were assayed by means of HPLC-EC in frontal cortex, cerebellum, hippocampus, hypothalamus and striatum of neonatally BDV-infected and sham-inoculated male Lewis rats of 8, 14, 21, 60 and 90 days of age. Both NE and 5-HT concentrations were significantly affected by neonatal BDV infection. The cortical and cerebellar levels of NE and 5-HT were significantly greater in BDV-infected rats than control animals at postnatal days (PND) 60 and 90. Tissue content of NE in hippocampus was unaffected. In hippocampus, neonatally BDV-infected rats had lower 5-HT levels at PND 8 and significantly elevated levels at PND 21 and onwards. Neither striatal levels of 5-HT nor hypothalamic levels of 5-HT and NE were affected by neonatal BDV infection, suggesting that the monoamine systems in the prenatally maturing brain regions are less sensitive to effects of neonatal viral infection. 5-HIAA/5-HT ratio was not altered in BDV-infected rats indicating no changes in the 5-HT turnover in the brain regions damaged by the virus. Neither DA nor DOPAC/DA ratio was affected by neonatal BDV infection in any of the brain regions examined. The present data demonstrate significant and specific alterations in monoaminergic systems in neonatally BDV-infected rats. This pattern of changes is consistent with the previously reported behavioral abnormalities resulting from neonatal BDV infection.     

Prenatal exposure to morphine differentially alters gonadal hormone regulation of delta-opioid receptor binding in male and female rats

The present study tested the hypothesis that exposure to morphine on gestation days 11-18 differentially alters delta-opioid receptors in the brain of adult male and female rats. In Experiment 1, the binding characteristics of delta-opioid receptors were examined in membrane homogenates from six brain regions, including the hypothalamus (HYP), preoptic area, frontal cortex (CX), ventral tegmental area, striatum (STR) and cerebellum of adult male and female rats. In Experiment 2, the density of delta-opioid receptors was assessed in the CX and STR using receptor autoradiography. Prenatal morphine exposure has no effects on delta-opioid receptors in the brain of gonadally intact, adult male rats regardless of methodology. However, when male rats were gonadectomized in Experiment 2, morphine-exposed males have fewer delta-opioid receptors than controls in the CX but not in the STR. These reductions in cortical delta-opioid receptors are restored by testosterone replacement, demonstrating that prenatal morphine exposure alters testosterone regulation in the CX of male rats. In ovariectomized (OVX) female rats, prenatal morphine exposure increases the density of delta-opioid receptors in the frontal CX. Interestingly, this up-regulation of delta-opioid receptors is not present when the CX is investigated by autoradiography. Moreover, progesterone given alone or in combination with estrogen reduces the density of delta-opioid receptors in the CX and STR of both saline- and morphine-exposed, OVX females. Thus, mid to late gestational morphine exposure differentially alters the influence of adult gonadal hormones on delta-opioid receptors in the CX, decreasing the sensitivity in females and increasing it in males. This is also the first report to demonstrate that gonadal hormones regulate delta receptor densities in brain regions other than the HYP of OVX 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.     

Influences of cholecystokinin antiserum on the dopamine,norepinephrine and serotonin contents of different brain regions in rats

The effects of intraventricular administration of cholecystokinin antiserum on the dopamine (DA), norepinephrine (NE) and serotonin (5-HT) contents of the hypothalamus, mesencephalon, amygdala, septum, striatum and cerebral cortex were tested 24 hr following injection in rats.Cholecystokinin antiserum decreased the DA and NE contents of the hypothalamus, mesencephalon, amygdala and septum, while it increased the DA and decreased the NE content of the striatum. In the cerebral cortex the NE content was decreased. The 5-HT contents decreased in the mesencephalon, amygdala and septum, and increased in the striatum.     

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.     

Influence of prenatal ethanol exposure on hormonal responses to clonidine and naloxone in prepubescent male and female rats

The expression of sexually dimorphic behavior has been found to be altered in adult animals following prenatal alcohol exposure. The present study examined whether such exposure would alter the sexually dimorphic response of luteinizing hormone (LH) to clonidine and naloxone observed in normal prepubescent animals. Both LH and corticosterone (CS) were measured in 16 day old male and female rats 30 min after injection of naloxone (2 mg/kg) or clonidine (0.1 mg/kg). Prenatal alcohol exposure did not influence the LH response to either drug in females. An LH response to clonidine in normal males did not occur, but it was present in the males exposed to alcohol in utero and in the pair-fed controls. Prenatal alcohol exposure influenced the CS response to both drugs. CS levels were depressed in the naloxone-treated males prenatally exposed to alcohol compared to their saline-injected counterparts. The CS levels of other groups following naloxone administration were unchanged compared to saline injection. Normal animals of both sexes exhibited an elevation in CS levels following clonidine. However, this stimulatory effect of clonidine on CS release was absent in both female and male animals prenatally exposed to alcohol. The results of this study indicate that prenatal alcohol exposure may alter noradrenergic and opioid modulation of corticosterone and possibly of LH in young animals.     

Prenatal Androgens Defeminize Activation of GnRH Neurons in Response to Estradiol Stimulation

To determine if prenatal androgens prevent activation of GnRH neurons in response to estradiol stimulation, Fos colocalization with GnRH was compared in the brains of normal female lambs, normal males, and androgenized females in response to a surge-inducing dose of estradiol. Blood samples were collected every 1–2  h for 6  h before estradiol treatment up to the time of sacrifice at 17–19  h post-treatment. Following perfusion, 60  μm coronal brain sections were immunostained for Fos (1:1000, Santa Cruz Biochemicals) and GnRH (1:40,000, LR-1) using NiCl-enhanced and unenhanced DAB, respectively. Although LH secretion increased in females before sacrifice, no increase was observed in males or androgenized females. Despite differences in LH secretion, the number and distribution of GnRH neurons was not sexually dimorphic. Moreover, Fos immunostaining was visible throughout steroid-responsive limbic regions in all three groups of lambs. However, the colocalization of Fos with GnRH was highly sexually dimorphic. In females perfused after the peak of the LH surge, 65.7% of GnRH neurons in the preoptic area, anterior hypothalamus, and mediobasal hypothalamus expressed Fos, whereas only 1.7% of GnRH neurons were Fos-positive in males and androgenized females. These findings indicate that sex differences in the activation of GnRH neurons in response to estradiol are determined prenatally through the actions of testosterone.     

Prenatal exposure to methadone affects central cholinergic neuronal activity in the weanling rat.

The effect of prenatal exposure to methadone via maternal osmotic minipumps was studied on brain regional acetylcholine (ACh) turnover and dopamine (DA), norepinephrine (NE), serotonin (5-hydroxytryptamine, 5-HT) and their metabolites in 21-day-old female and male rats. ACh content was not affected in any region studied. However, the turnover rate of ACh (TRAch) was increased significantly in the striata and parietal cortices of both sexes. Two gender-specific changes were observed: a profound decrease in hypothalamic TRACh in the females and an increase in hippocampal TRACh in the males. No changes were observed in TRACh in the medulla-pons or the frontal cortex of either sex. The reduction in TRACh was accompanied by a threefold increase in DA content in the hypothalamus of the methadone-exposed females. No other changes were observed in DA, NE, or 5-HT, save for increased 5-HT content in the medulla-pons of the male methadone-exposed rats. Thus, prenatal methadone exposure produces several lingering changes in cholinergic function, many of which were not apparent in the immediate postnatal period. Although striatal ACh content was no longer reduced in methadone-exposed rats, striatal cholinergic function remains disrupted. It remains to be proven whether these differences are a direct effect of methadone exposure or are a consequence of neonatal withdrawal.     

Sexually dimorphic expression of hypothalamic estrogen receptors α and β and Kiss1 in neonatal male and female rats

Release of gonadotropins in adult rodents is sex specific and dependent upon kisspeptin (Kiss1) neurons. This crucial pathway within the hypothalamic-pituitary-gonadal (HPG) axis is profoundly influenced by neonatal estrogens, which induce a male-like phenotype. Classically, estrogen activity is mediated via the estrogen receptors α and β (ERα and ERβ), but the relative roles each plays in organizing the sex-specific ontogeny of kisspeptin signaling pathways remain unresolved. Thus, the present study used in situ hybridization histochemistry (ISHH) to map the temporal and sexually dimorphic neonatal mRNA expression profiles of ERα, ERβ, and Kiss1 in the anterioventral periventricular nucleus (AVPV), medial preoptic area (MPOA), ventromedial nucleus (VMN), and arcuate nucleus (ARC), all regions critical for kisspeptin regulation of gonadotropin secretion. In general, females had higher levels of ERα, in all regions examined, a sex difference that persisted until postnatal day (PND) 19 except in the ARC. Males had significantly more ERβ expression in the AVPV at birth, but this sex difference was lost and then re-emerged on PND 19, with females having more than males. VMN ERβ levels were higher in females until PND 19. Kiss1 was not detectable until PND 11 in the anterior hypothalamus, but expression levels were equivalent at birth in the ARC. By PND 2, ARC ERα and Kiss1 levels were abundant, sexually dimorphic (higher in females), and, respectively, showed a U- and a bell-shaped pattern with age. Sex differences in ARC Kiss1 expression provide evidence that Kiss1 may play a role in the sexual dimorphic organization of the neonatal brain. These detailed profiles of neonatal Kiss1 and ERs mRNA levels will help elucidate the relative roles each plays in the sex-specific, estrogen-dependent organization of gonadotropin signaling pathways.     

Sexually dimorphic hormonal regulation of the gap junction protein, CX43, in rats and altered female reproductive function in CX43+/- mice

Astrocytic gap junctional communication is important in steroid hormone regulation of reproductive processes at the level of the hypothalamus, including estrous cyclicity and sexual behavior. We examined the effects of estradiol and progesterone on the abundance of the gap junctional protein, connexin 43 (CX43), which is highly expressed in astrocytes. Gonadectomized rats received hormone treatments that induce maximal sexual behavior and gonadotropin surges in females (estrogen for 48 h followed by progesterone, estrogen alone or progesterone alone). Control animals received vehicle (oil) injections. In the female rat preoptic area (POA), containing the gonadotropin-releasing hormone (GnRH) cell bodies, treatment with estrogen, progesterone or estrogen + progesterone significantly increased CX43 protein levels in immunoblots. In contrast, estrogen + progesterone significantly decreased CX43 levels in the male rat POA. This sexually dimorphic hormonal regulation of CX43 was not evident in the hypothalamus, which contains primarily GnRH nerve terminals. Treatment with estrogen + progesterone significantly decreased CX43 levels in both the male and female hypothalamus. To examine the role of CX43 in female reproductive function, we studied heterozygous female CX43 (CX43+/-) mice. Most mutant mice did not show normal estrous cycles. In addition, when compared to wild type females, CX43+/- mice had reduced lordosis behavior. These data suggest that hypothalamic CX43 expression is regulated by steroid hormones in a brain-region-specific and sexually dimorphic manner. Therefore, gap junctional communication in the POA and hypothalamus may be a factor regulating the estrous cycle and sexual behavior in female rodents.     

Prenatal morphine exposure differentially alters TH-immunoreactivity in the stress-sensitive brain circuitry of adult male and female rats

Previously, we demonstrated that exposure to morphine during gestation increases hypothalamic norepinephrine (NE) content and turnover rate in adult male rats and decreases these measures in adult females. To investigate the basis of these alterations, the present study examined the effects of prenatal exposure to morphine on tyrosine hydroxylase immunoreactivity (TH-IR) in the brains of adult male and female progeny. In male rats, prenatal morphine exposure significantly increased the density of TH-IR in cells and fibers in the caudal paraventricular nucleus of the hypothalamus (PVN) and locus coeruleus (LC), but had no effects in the lateral hypothalamus (LH). In female rats that were ovariectomized (OVX), prenatal morphine exposure significantly decreased the density of TH-IR in cells and fibers in the LC. Interestingly, an injection of estrogen in OVX control females reduced the mean optical density of TH-IR in the LC, but it was ineffective in drug-exposed females in the same brain region. Estrogen injections also reduced the mean optical density of TH-IR in the LH but not in the PVN of females, regardless of prenatal drug exposure. Thus, the present study suggests that prenatal morphine exposure induces long-term, sex-specific alterations in TH-IR in the PVN and LC of adult progeny.