Preoptic-anterior Hypothalamic Area Research Articles

Neuroendocrine aging in the female rat: the changing relationship of hypothalamic gonadotropin-releasing hormone neurons and N-methyl-D-aspartate receptors.

The reproductive axis undergoes alterations during aging, resulting in acyclicity and the loss of reproductive function. In the hypothalamus, changes intrinsic to GnRH neurons may play a critical role in this process, as may changes in inputs to GnRH neurons from neurotransmitters such as glutamate. We investigated the effects of age and reproductive status on neuroendocrine glutamatergic NMDA receptors (NRs), their regulation of GnRH neurons, and their expression on GnRH neurons, in female rats. First, we quantified NR subunit messenger RNAs (mRNAs) in preoptic area-anterior hypothalamus (POA-AH) and medial basal hypothalamus (MBH), the sites of GnRH perikarya and neuroterminals, respectively. In POA-AH, NR1 mRNA levels varied little with age or reproductive status. NR2a and NR2b mRNA levels decreased significantly between cycling and acyclic rats. In MBH, NR mRNAs all increased with aging, particularly in acyclic animals. Second, we tested the effects of N-methyl-D,L-aspartate (NMA) on GnRH mRNA levels in POA-AH of aging rats. NMA elevated GnRH mRNA levels in young rats, but decreased them in middle-aged rats. Third, we quantified expression of the NR1 subunit on GnRH perikarya in aging rats using double label immunocytochemistry. NR1 expression on GnRH cell bodies varied with age and reproductive status, with 30%, 19%, and 46% of GnRH somata double labeled with NR1 in young proestrous, middle-aged proestrous, and middle-aged persistent estrous rats, respectively. Thus, 1) the expression of hypothalamic NR subunit mRNAs correlates with reproductive status; 2) changes in NR subunit mRNA levels, if reflected by changes in protein levels, may result in alterations in the stoichiometry of the NR during aging, with possible physiological consequences; 3) the effects of NR activation on GnRH mRNA switches from stimulatory to inhibitory during reproductive aging; and 4) expression of the NR1 subunit on GnRH perikarya changes with reproductive status. These molecular, physiological, and cellular neuroendocrine changes are proposed to be involved in the transition to acyclicity in aging female rats.

Effects of female pheromones on gonadotropin-releasing hormone gene expression and luteinizing hormone release in male wild-type and oestrogen receptor-alpha knockout mice.

Pheromones are an important class of environmental cues that affect the hypothalamic-pituitary-gonadal axis in a variety of vertebrate species, including humans. When male mice contact female-soiled bedding, or urine, they display a reflexive luteinizing hormone (LH) surge within 30 min. Aside from the requirement that males have gonads to show this response, the physiological mechanisms that underlie this pituitary response are unknown. In this experiment, we asked if female pheromones acted at the level of gonadotropin-releasing hormone (GnRH) gene expression to affect this hormone response. In addition, we also examined the contribution of one of the oestrogen receptors (ERalpha) by studying this neuroendocrine reflex in wild-type and oestrogen receptor-alpha knockout (ERalphaKO) males. Both ERalphaKO and wild-type males showed the expected LH surge, 45 and 90 min after contact with female pheromones. Males housed in clean bedding or bedding soiled by another adult male did not display the LH elevation. Interestingly, this dramatic change in LH concentrations was not accompanied by any alterations in GnRH mRNA expression or levels of primary transcript in the preoptic area-anterior hypothalamus. The one exception to this was a significant increase in GnRH mRNA expression in tissue collected from wild-type males exposed to bedding from another male. This is particularly intriguing since LH was not elevated in these males. These data replicate and extend our previous finding that ERalphaKO males do exhibit an LH surge in response to female pheromones. Thus, this neuroendocrine response is regulated by a steroid receptor other than ERalpha and does not require alterations in GnRH mRNA expression.

RANTES: a new prostaglandin dependent endogenous pyrogen in the rat

Fever, a hallmark of disease, is a highly complex process initiated by the action of a number of endogenous pyrogens on the thermosensitive cells of the brain. We describe the activity of RANTES, a chemotactic cytokine, as intrinsically pyrogenic in the rat, when it is delivered directly to the thermosensitive region of the rat's anterior hypothalamic, pre-optic area (AH/POA). RANTES, microinjected into the AH/POA in a dose of 1, 5, 10, 15, 25 or 50 pg, produces an immediate and intense dose-related fever following injection. Increasing the dose to 100 pg did not result in a further increase in the febrile response. No significant change in body temperature was produced by heat-inactivated RANTES. The intrahypothalamic injection of antibodies against RANTES (2.0 μg, 15 min prior to RANTES) significantly blocked the fever induced by this chemokine. Pretreatment with ibuprofen blocked the fever induced by RANTES. In order of potency, the magnitude of the febrile response induced by RANTES was greater than that produced with equipotent doses of either macrophage inflammatory protein-1β or interleukin-6. The results thus demonstrate that RANTES is the most potent endopyrogen discovered thus far and exerts its action directly on pyrogen-sensitive cells of the AH/POA through a prostaglandin-dependent pathway.

Pyrogen sensing and signaling: old views and new concepts.

Fever is thought to be caused by endogenous pyrogenic cytokines, which are elaborated and released into the circulation by systemic mononuclear phagocytes that are activated by exogenous inflammatory agents and transported to the preoptic-anterior hypothalamic area (POA) of the brain, where they act. Prostaglandin (PG) E2 is thought to be an essential, proximal mediator in the POA, and induced by these cytokines. It seems unlikely, however, that these factors could directly account for early production of PGE2 following the intravenous administration of bacterial endotoxic lipopolysaccharides (LPS), because PGE2 is generated before the cytokines that induce it are detectable in the blood and the before cyclooxygenase-2, the synthase that they stimulate, is expressed. Hence other, more quickly evoked mediators are presumed to be involved in initiating the febrile response; moreover, their message may be conveyed to the brain by a neural rather than a humoral pathway. This article reviews current conceptions of pyrogen signalling from the periphery to the brain and presents new, developing hypotheses about the mechanism by which LPS initiates fever.

Photoperiod and temperature interact to affect the GnRH neuronal system of male prairie voles (Microtus ochrogaster).

Individuals of numerous species limit energy expenditure during winter by inhibiting reproduction and other nonessential functions. To time these adaptations appropriately with the annual cycle, animals rely on environmental cues that predict, well in advance, the onset of winter. The most commonly studied environmental factor that animals use to time reproduction is photoperiod. Rodents housed in short photoperiods in the laboratory or in naturally declining day lengths exhibit pronounced alterations in reproductive function concomitant with alterations in the hypothalamic gonadotropin-releasing hormone neuronal system. Because animals in their natural environment use factors in addition to photoperiod to time reproduction, the present study sought to determine the independent effects of photoperiod and temperature, as well as the interaction between these factors, on reproductive parameters and the GnRH neuronal system. Male prairie voles were housed in either long (LD 16:8) or short (LD 8:16) day lengths for 10 weeks. Animals in each photoperiod were further subdivided into groups housed in either mild (i.e., 20 degrees C) or low (i.e., 8 degrees C) temperatures. As shown with immunohistochemistry, voles that underwent gonadal regression in response to short photoperiods and long-day voles housed in low temperatures (and maintained large gonads) exhibit higher GnRH-immunoreactive (GnRH-ir) neuron numbers in the preoptic area/anterior hypothalamus (POA/AH) relative to all other groups. In addition, voles that underwent gonadal regression in response to both short days and low temperatures did not exhibit an increase in GnRH-ir neuron numbers compared to long-day, mild-temperature controls. These data suggest that photoperiod and temperature interact to influence reproductive function potentially by alterations of the GnRH neuronal system.

Forebrain peptides modulate sexually polymorphic vocal circuitry.

The peptide arginine-vasopressin (mammals) and its evolutionary precursor arginine-vasotocin (non-mammals) modulate reproductive physiology and numerous related social behaviours, as do oxytocin (mammals) and its homologues mesotocin and isotocin (fish). The distributions in the brain and/or the behavioural functions of these peptides often differ between the sexes, and between species with divergent social structures. Here we present neurophysiological evidence that males with vocal characteristics typical of females share a pattern of neuropeptide function with females rather than conspecific males. The plainfin midshipman fish (Porichthys notatus) has two male morphs with different reproductive tactics and vocalizations (a key species-typical behaviour which varies in its physical attributes and contextual usage, depending on the morph's social strategy). Forebrain-evoked, rhythmic vocal-motor activity that precisely mimics natural vocalizations was modulated by arginine-vasotocin, isotocin and their antagonists delivered to the preoptic area-anterior hypothalamus, a primary site for behavioural integration in all vertebrates. Peptides had different effects in males that acoustically court females (arginine-vasotocin-sensitive) than in females and sneak-spawning males (isotocin-sensitive), showing that the neuromodulatory mechanisms that establish reproduction-related behaviour can be dissociated from gonadal sex.

Blockade of angiotensin receptors in the anterior hypothalamic preoptic area lowers blood pressure in DOCA-salt hypertensive rats.

It has been established that deoxycorticosterone acetate (DOCA)-salt hypertensive rats have an overactive brain angiotensin-system. The purpose of the present study was to identify the brain sites showing enhanced angiotensin-system activity responsible for the pathogenesis of hypertension in DOCA-salt hypertensive rats. The angiotensin receptor antagonist, losartan, was injected into brain ventricles or into tissues around the rostral parts of the third ventricle in conscious DOCA-salt hypertensive rats. Losartan (1 microg) injection into the lateral ventricle or into the rostral parts of the third ventricle produced a depressor response, whereas the agent did not affect blood pressure when injected into the caudal parts of the third ventricle or into the fourth ventricle. Losartan (0.1 microg) injection into the anterior hypothalamic preoptic area, anterior (AHA) produced a depressor response. Angiotensin II (0.1-1 ng) injection into the AHA produced a pressor response in sham-operated and DOCA-salt hypertensive rats, and the pressor response to angiotensin II (1 ng) was greater in DOCA-salt hypertensive rats than that in sham-operated rats. Release of angiotensin peptides in the AHA was greater in DOCA-salt hypertensive rats than that in sham-operated rats. These findings suggest that the angiotensin-system in the AHA is enhanced, and that this enhancement is involved in the maintenance of hypertension in DOCA-salt hypertensive rats. Both increased pressor reactivity to angiotensin II and increased release of angiotensin peptides in the AHA appear to be related to this enhancement of the angiotensin-system in DOCA-salt hypertensive rats.

Perinatal developmental changes in expression of the neuropeptide genes preoptic regulatory factor-1 and factor-2, neuropeptide Y and GnRH in rat hypothalamus.

The preoptic regulatory factor genes, PORF-1 and PORF-2, are expressed in the rat brain in a regional-, age- and gender-dependent fashion. They are also expressed in the testis, where PORF-2 mRNA localizes to dividing germ cells while PORF-1 mRNA is associated with newly differentiated sperm. This suggests that PORF-1 and PORF-2 may play distinct roles in cell growth and differentiation. Moreover, the two preoptic regulatory factors are also highly expressed in the immature and mature rat hypothalamus, and their expression is modulated by gonadal hormones. Therefore, in the present study we investigated the expression of these two factors in neuroendocrine regions of the developing rat brain by addressing the following questions. First, are PORF-1 and PORF-2 mRNAs expressed during perinatal development in the preoptic area-anterior hypothalamus (POA-AH) and medial basal hypothalamus (MBH), and how do their levels vary? Second, are there gender differences in their expression? We also compared expression of the PORF mRNAs with those of neuropeptide Y (NPY) and gonadotropin-releasing hormone (GnRH), which play critical neuroendocrine roles, in these brain regions. PORF-1, PORF-2, and NPY mRNAs in the POA-AH and MBH, and GnRH mRNA in the POA-AH, were quantified by RNase protection assay at embryonic day (E) 18-19, and postnatal days (P) 0, 5, 10 and 15 in male and female rats. The results show that the four neuropeptide genes are regulated differentially during the perinatal-prepubertal period. PORF-1 mRNA shows age-related increases in expression from E18-E19 to P15 in POA-AH and MBH, without significant gender differences. In contrast, PORF-2 mRNA shows both age and gender differences in expression in these brain regions, with decreases occurring during the same time period in development. NPY mRNA increases similarly in males and females with age in POA-AH and MBH during this period. GnRH mRNA does not change during this period. Taken together with previous studies, the results suggest possible roles for PORF-1 and NPY in the pubertal process, since their expression is maximal from the prepubertal to the early pubertal period. The observation of highest levels of expression of PORF-2 in embryonic neuroendocrine tissues suggests a possible involvement of this neuropeptide in prenatal/neonatal developmental events.

Effect of nociceptin and [Phe1Ψ (CH2–NH) Gly2]-nociceptin-(1–13)-NH2 on tonic activity of rat hypothalamic neurons

The effect of nociceptin, an endogenous ligand for a unique member of the cloned opioid receptor family ORL1-receptor, on tonic activity of neurons in the preoptic area/anterior hypothalamus (PO/AH) has been examined in rat brain slices using extracellular recordings. Nociceptin (1, 10 and 100 nM) decreased dose-dependently tonic activity of PO/AH neurons. This effect was not significantly different from the effect of [Phe1Ψ (CH2–NH) Gly2]-nociceptin-(1–13)-NH2 (1, 10 and 100 nM), recently proposed as a selective antagonist of the nociceptin receptor. Thus, [Phe1Ψ (CH2–NH) Gly2]-nociceptin-(1–13)-NH2 appears to be an agonist rather than an antagonist of nociceptin (ORL1) receptor in rat PO/AH neurons. However, there was neither antagonism nor additive synergism when nociceptin and [Phe1Ψ (CH2–NH) Gly2]-nociceptin-(1–13)-NH2 were applied simultaneously at equimolar concentrations. The effect of nociceptin on tonic activity of rat PO/AH neurons was not blocked by selective μ-, κ- and δ-opioid receptor antagonists (d-Phe-Cys-Tyr-d-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP), nor-binaltorphimine and naltrindol, respectively) at 10 times higher concentrations than nociceptin. These data suggest that the effect of nociceptin on tonic activity of PO/AH neurons is not due to an action on μ-, κ-, or δ-opioid receptors but results from a specific effect on the ORL1-receptor.

Volumetric analysis of sexually dimorphic limbic nuclei in normal and sex-reversed whiptail lizards

Sex differences in the size of key limbic nuclei have been found in many species. In some of these species, steroid hormones have been implicated in both the development and the maintenance of the sex difference. However, the possible role of sex-specific genes has not been examined, in part due to lack of an appropriate model system. In this study we measured the size of the ventromedial hypothalamus and preoptic area–anterior hypothalamus in normal female whiptail lizards and in genetic female whiptails that had been sex-reversed by treatment early in development with the aromatase inhibitor fadrozole. We found no difference in the size of these two nuclei between females and the sex-reversed animals. These results suggest that either the sex-reversing treatment itself interfered with the masculinization process, or that a male genome is required to produce a male-like limbic phenotype.

The preoptic area/anterior hypothalamus of different strains of mice: sex differences and development

While sex differences in neural morphology in the preoptic area/anterior hypothalamus (POA/AH) have been demonstrated in many species, their existence in mice have been controversial. Given the increased use of transgenic and gene-disrupted mice, we characterized sex differences using Nissl stains, and the immunocytochemical location of estrogen receptor-α (ER-α) and galanin in the POA/AH of two widely used strains, C57BL/6 and 129SvEv, and a mixed strain (C57BL/6×129Sv); the wild-type littermates of steroidogenic factor-1 (SF-1) gene-disrupted mice. Cell grouping was not a reliable marker of sex. In adults, cells located beneath the anterior commissure (AC) were reliably larger in females than males in 129SvEv, but not in the other strains. Caudally, cells in a group medial to the medial extension of the bed nucleus of the stria terminalis (BST) were significantly larger in males than females in C57BL/6J and SF-1 gene-disrupted wild-types. Cell groups discernible by embryonic day (E) 18 were not sexually dimorphic for cell size in C57BL/6J mice at E18 or postnatal day (P) 4. The pattern of distribution of cells containing ER-α was similar among the strains, reduced in the group medial to the BST; a pattern established by P0. Galanin-containing cells and fibers were seen from E15 to adulthood ventral to the AC. Caudally, a smaller group ventromedial to the BST was found only in 129SvEv adults. Sex differences in neural morphology which develop within the POA/AH depend upon multiple factors, particularly including genetic background.

Radioligand assays for oestradiol and progesterone conjugated to protein reveal evidence for a common membrane binding site in the medial preoptic area-anterior hypothalamus and differential modulation by cholera toxin and GTPgammaS.

In this study membrane oestradiol (E) binding sites in the medial preoptic area-anterior hypothalamus (MPOA-AH) of ovariectomized (OVX) rats were characterized using standard radioligand binding techniques employing E conjugated to bovine serum albumin (BSA) at position 6 and radiolabeled with 125I (E-6-[125I-BSA]). In previous studies binding of a radioactive conjugate of progesterone (P) and BSA (P-3-[125I-BSA]) was examined using the same membrane preparation. E-6-[125I-BSA] binding was linear across a tissue concentration range of 0.005-0.02 mg protein/0.1 ml of membrane suspension. An association T1/2 of 9.5 min and a dissociation T1/2 of 52.1 min for E-6-[125I-BSA] were derived from kinetic experiments. Competition binding experiments revealed high (Ki=0.63+/-(0.50 nM) and low (Ki=161.5(96.5 nM) affinity binding sites for E-6-[125I-BSA], demonstrating different binding parameters than shown in our previous work for P-3-[125I-BSA] binding. Further studies on MPOA-AH membranes treated with cholera toxin (CTX) and GTPgammaS suggested that E-6-BSA binding sites are associated with G proteins. E-6-[125I-BSA] binding demonstrated both high-and low-affinity sites. GTPgammaS added to the assay reduced both E-6-[125I-BSA] and P-3-[125I-BSA] binding suggesting that G proteins are associated with both binding sites. Extensive analysis of both E-6-[125I-BSA] and P-3-[125I-BSA] binding sites demonstrated a reciprocal relationship such that high-affinity E-6-[125I-BSA] binding sites exhibit low affinity for P-3-[125I-BSA] and low-affinity E-6-[125I-BSA] binding sites exhibit high affinity for P-3-[125I-BSA]. Preincubating membranes with CTX or GTPgammaS reduced high-affinity E-6-[125I-BSA] binding and enhanced high-affinity P-3-[125I-BSA] binding. These results suggest that, in the MPOA-AH, membrane steroid binding sites exist in two interconvertible conformations that preferentially bind either E-6-BSA or P-3-BSA, depending on their association with a G protein. Additional studies with free steroids revealed that: (1) oestrogens (17beta-oestradiol, diethylstilbestrol) as well as synthetic oestrogen antagonists tamoxifen and ICI 182 780 displaced P-3-[125I-BSA] further suggesting a relationship between membrane binding sites for E and P-3-[125I-BSA] binding sites; and (2) treatment of OVX rats with E decreased displacement by P-3-BSA and increased displacement by ICI 182,780 and tamoxifen suggesting these antagonists affect membrane P-3-[125I-BSA] binding sites after in-vivo E treatment. The membrane binding sites for E and P demonstrate interrelationships not demonstrated by their nuclear receptors.

Perinatal Changes in Hypothalamic N-Methyl-D-Aspartate Receptors and Their Relationship to Gonadotropin-Releasing Hormone Neurons

During the neonatal period, the brain is subject to profound alterations in neuronal circuitry due to high levels of synaptogenesis and gliogenesis. In neuroendocrine regions such as the preoptic area-anterior hypothalamus (POA-AH), the site of GnRH perikarya, these changes could affect the maturation of GnRH neurons. Because the GnRH system is developmentally regulated by glutamatergic neurons, we hypothesized that changes in the N-methyl-d-aspartate (NMDA) receptor system begin early in postnatal development, before the onset of puberty, thereby playing a role in establishing the appropriate environment for the subsequent maturation of GnRH neurons. To this end, we determined developmental changes in NMDA receptors, alterations in GnRH gene expression, and the regulation of GnRH neurons by the NMDA receptor system in developing male and female rats. In Exp I, NMDA receptor subunit (NR) 1 mRNA levels in the POA-AH were found to increase significantly (∼5-fold) from E18 through P10 in both males and females. NR2b mRNA increased significantly between P0 and P5 in both males and females. In contrast, NR2a subunit mRNA, which was in very low abundance in both males and females, increased only in males between P10 and P15. In Exp II we determined that GnRH gene expression changes differentially in developing male and female rats, with increases from P0 to P5 in males, and decreases from P5 to P10 in females. This latter effect in females is attributed to a change in GnRH gene transcription because GnRH primary transcript RNA levels paralleled changes in GnRH mRNA levels. In Exp III, we tested effects of treatment with an NMDA receptor analog on GnRH mRNA levels and found that only P5 and P10 male rats responded to NMDA receptor activation with an increase in GnRH mRNA levels, via a posttranscriptional mechanism. This greater responsiveness of males to NMDA receptor stimulation may be due to differences in the composition and levels of NMDA receptor subunits. Exp IV examined the localization of NR1 in the POA-AH during neonatal development. No GnRH neurons were immunopositive for NR1, indicating that effects of glutamate on GnRH neurons are mediated by interneurons or other glutamate receptor subunits or types. Taken together, these data indicate that glutamatergic inputs to the POA-AH change dramatically during the early postnatal period, before puberty and before the GnRH system is fully responsive to glutamate, consistent with the hypothesis that the maturation of inputs to GnRH neurons, and the establishment of the proper neurotransmitter“ milieu” enabling the activation of GnRH neurons, occurs before the onset of puberty.

Perinatal changes in hypothalamic N-methyl-D-aspartate receptors and their relationship to gonadotropin-releasing hormone neurons.

During the neonatal period, the brain is subject to profound alterations in neuronal circuitry due to high levels of synaptogenesis and gliogenesis. In neuroendocrine regions such as the preoptic area-anterior hypothalamus (POA-AH), the site of GnRH perikarya, these changes could affect the maturation of GnRH neurons. Because the GnRH system is developmentally regulated by glutamatergic neurons, we hypothesized that changes in the N-methyl-D-aspartate (NMDA) receptor system begin early in postnatal development, before the onset of puberty, thereby playing a role in establishing the appropriate environment for the subsequent maturation of GnRH neurons. To this end, we determined developmental changes in NMDA receptors, alterations in GnRH gene expression, and the regulation of GnRH neurons by the NMDA receptor system in developing male and female rats. In Exp I, NMDA receptor subunit (NR) 1 mRNA levels in the POA-AH were found to increase significantly (approximately 5-fold) from E18 through P10 in both males and females. NR2b mRNA increased significantly between P0 and P5 in both males and females. In contrast, NR2a subunit mRNA, which was in very low abundance in both males and females, increased only in males between P10 and P15. In Exp II we determined that GnRH gene expression changes differentially in developing male and female rats, with increases from P0 to P5 in males, and decreases from P5 to P10 in females. This latter effect in females is attributed to a change in GnRH gene transcription because GnRH primary transcript RNA levels paralleled changes in GnRH mRNA levels. In Exp III, we tested effects of treatment with an NMDA receptor analog on GnRH mRNA levels and found that only P5 and P10 male rats responded to NMDA receptor activation with an increase in GnRH mRNA levels, via a posttranscriptional mechanism. This greater responsiveness of males to NMDA receptor stimulation may be due to differences in the composition and levels of NMDA receptor subunits. Exp IV examined the localization of NR1 in the POA-AH during neonatal development. No GnRH neurons were immunopositive for NR1, indicating that effects of glutamate on GnRH neurons are mediated by interneurons or other glutamate receptor subunits or types. Taken together, these data indicate that glutamatergic inputs to the POA-AH change dramatically during the early postnatal period, before puberty and before the GnRH system is fully responsive to glutamate, consistent with the hypothesis that the maturation of inputs to GnRH neurons, and the establishment of the proper neurotransmitter "milieu" enabling the activation of GnRH neurons, occurs before the onset of puberty.

Mechanisms for the regulation of gonadotropin-releasing hormone gene expression in the developing mouse.

The release of GnRH peptide from neuroterminals in the median eminence increases during postnatal development. We were interested in determining the biosynthetic component contributing to the regulation of GnRH decapeptide levels, and ascertaining the molecular mechanism for these changes. Male and female C57bl/6 mice, from embryonic day (E)16 through postnatal day (P)60, were killed, and the preoptic area-anterior hypothalamus was dissected out. Cytoplasmic and nuclear RNA were extracted separately. Levels of GnRH messenger RNA (mRNA) and primary transcript were quantitated in individual preoptic area-anterior hypothalamus cytoplasmic and nuclear fractions, respectively, by ribonuclease protection assays. Serum LH levels were assayed by RIA. GnRH mRNA levels in the cytoplasm increased gradually and significantly during postnatal development in both males and females, reaching a peak at P55 in females and P40 in males. GnRH primary transcript levels in the nucleus, an index of GnRH gene transcription, changed in a completely different manner developmentally, and they differed between male and female mice. GnRH primary transcript levels in males were quite low until P5, when they underwent an increase of approximately 4-fold, between P5 and P7. They continued to increase through P15, at which time they reached adult levels. In females, GnRH primary transcript levels were high at E16, decreased to a nadir at P5, and then underwent an increase of approximately 5-fold to P7, which were comparable with adult levels. The large and sexually dimorphic changes in GnRH primary transcript between E16 and P7, in the absence of similar changes in GnRH mRNA, suggest that differential mechanisms, such as gene transcription and mRNA stability, play a role in determining levels of GnRH mRNA at different stages of development.

Mechanisms for the Regulation of Gonadotropin-Releasing Hormone Gene Expression in the Developing Mouse

The release of GnRH peptide from neuroterminals in the median eminence increases during postnatal development. We were interested in determining the biosynthetic component contributing to the regulation of GnRH decapeptide levels, and ascertaining the molecular mechanism for these changes. Male and female C57bl/6 mice, from embryonic day (E)16 through postnatal day (P)60, were killed, and the preoptic area-anterior hypothalamus was dissected out. Cytoplasmic and nuclear RNA were extracted separately. Levels of GnRH messenger RNA (mRNA) and primary transcript were quantitated in individual preoptic area-anterior hypothalamus cytoplasmic and nuclear fractions, respectively, by ribonuclease protection assays. Serum LH levels were assayed by RIA. GnRH mRNA levels in the cytoplasm increased gradually and significantly during postnatal development in both males and females, reaching a peak at P55 in females and P40 in males. GnRH primary transcript levels in the nucleus, an index of GnRH gene transcription, changed in a completely different manner developmentally, and they differed between male and female mice. GnRH primary transcript levels in males were quite low until P5, when they underwent an increase of approximately 4-fold, between P5 and P7. They continued to increase through P15, at which time they reached adult levels. In females, GnRH primary transcript levels were high at E16, decreased to a nadir at P5, and then underwent an increase of approximately 5-fold to P7, which were comparable with adult levels. The large and sexually dimorphic changes in GnRH primary transcript between E16 and P7, in the absence of similar changes in GnRH mRNA, suggest that differential mechanisms, such as gene transcription and mRNA stability, play a role in determining levels of GnRH mRNA at different stages of development.

Androgen receptor messenger ribonucleic acid in brains and pituitaries of male rhesus monkeys: studies on distribution, hormonal control, and relationship to luteinizing hormone secretion.

Because the distribution and hormonal regulation of the androgen receptor (AR) mRNA in brains and pituitaries of adult rhesus monkeys have not been studied, we cloned and sequenced a 329-base pair segment of the 5' coding region of the rhesus AR cDNA. Monkey AR cDNA was 99% identical with the human sequence and 96% homologous with the rat sequence. Using a ribonuclease protection assay, we studied the distribution and regulation of AR mRNA in brains and anterior pituitary glands of three groups of male rhesus monkeys: intact (n = 3), castrated (Cx, n = 4), and Cx treated with testosterone (n = 6). Serum testosterone levels of Cx males treated with testosterone differed significantly (p < 0.05) in the morning but not in the evening hours from those in intact controls. Serum LH concentrations were significantly suppressed (p < 0.05) in both morning and evening serum samples of testosterone-treated males compared to intact controls. We found the highest concentrations of AR mRNA in the medial basal hypothalamus, the bed nucleus of the stria terminalis, the medial preoptic area-anterior hypothalamus, and the lateral dorsomedial hypothalamus. Intermediate amounts were found in the septum and amygdala. Low amounts were found in the hippocampus, cingulate cortex, parietal cortex, and cerebellum. The anterior pituitary gland also contained a large amount of AR mRNA. Surprisingly, neither Cx for 3 wk nor Cx plus testosterone replacement for 3 wk significantly affected AR mRNA in any brain area or in the pituitary gland. The present study demonstrates that the effectiveness of testosterone as a regulator of LH secretion in male monkeys is not related to changes of AR mRNA in the brain or pituitary gland. It appears that AR mRNA in the monkey brain and pituitary gland is not regulated at the transcriptional level by androgen.

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/μl 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/μl OT. After recovery from cannula implantation surgery animals were injected with 0.5 μg 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.

Large Somal Size Is Associated with the Expression of Galanin but Not with Neuronal Birthdate in the Sexually Dimorphic Male Nucleus of Ferret’s Preoptic Area/Anterior Hypothalamus

Using Nissl and Golgi stains, a sexually dimorphic male nucleus (MN) comprised of a cluster of large cells with large dendritic arbors has been identified in the dorsal preoptic area/anterior hypothalamus (POA/AH) of male ferrets. The MN-POA/AH is formed only in males by the action of estradiol derived from the neural aromatization of testosterone during the last quarter of a 41-day gestation. The ferret’s dorsal POA/AH is also characterized by a sex difference in the expression of the neuropeptide galanin which first arises in males around embryonic day (e) 34. We asked whether the male-typical phenotype of large somal size is related to birthdate and/or the capacity of dorsal POA/AH neurons to express galanin. In experiment 1 we labeled cohorts of cells born on E20, E24, or E28 by injecting the amniotic sacs of individual fetuses with the thymidine analogue bromodeoxyuridine (BrdU). On postnatal day 20, BrdU-immunoreactive cells were visualized immunohistochemically, counterstained with cresyl violet, and their somal sizes were measured. BrdU-immunoreactive cells were significantly larger in the males’ MN-POA/AH than in a comparable region of females, regardless of when they were born between E20 and E28. In experiment 2 galanin-immunoreactive cells in the dorsal POA/AH of adult ferrets were visualized immunohistochemically, and their somal sizes were measured. Somal areas of galanin-immunoreactive cells were significantly larger in the MN-POA/AH of intact, breeding, or castrated and testosterone-treated males than in the corresponding area of females. Our results suggest that cells in the males’ MN-POA/AH are more likely to be larger than cells in females’ corresponding region, regardless of birthdate. Finally, in adulthood the male-typical phenotype of large Nissl-stained somal areas of MN-POA/AH cells may, in part, reflect their increased galanin expression.

Sleep–waking discharge patterns of ventrolateral preoptic/anterior hypothalamic neurons in rats

Numerous lesion, stimulation and recording studies in experimental animals demonstrate the importance of neurons within the preoptic/anterior hypothalamic area (POA) in the regulation of sleep induction and sleep maintenance. Recently, a discrete cluster of cells in the ventrolateral POA (vlPOA) of rats was found to exhibit elevated c- fos gene expression during sleep, indicating that these neurons are strongly activated during nonREM and/or REM sleep stages. We examined neuronal discharge during wakefulness and sleep throughout the dorsal to ventral extent of the lateral POA in rats, using chronic microwire technique. We found that neurons with elevated discharge rates during sleep, compared to waking, were localized to the vlPOA. As a group, vlPOA neurons displayed elevated discharge rates during both nonREM and REM sleep. Discharge of vlPOA neurons reflected the depth of sleep, i.e., discharge rates increased significantly from light to deep nonREM sleep. During recovery sleep following 12–14 h of sleep deprivation, vlPOA neurons displayed increased sleep-related discharge, compared to baseline sleep. Neurons in the vlPOA displaying increased neuronal discharge during sleep were located in the same area where neurons exhibit increased c- fos gene expression during sleep. Such neurons are likely components of a rostral hypothalamic mechanism that regulates sleep onset and sleep maintenance.