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Maternal behaviors are essential for the survival of the young. Previous studies implicated the medial preoptic area (MPOA) as an important region for maternal behaviors, but details of the maternal circuit remain incompletely understood. Here we identify estrogen receptor alpha (Esr1)-expressing cells in the MPOA as key mediators of pup approach and retrieval. Reversible inactivation of MPOA Esr1+ cells impairs those behaviors, whereas optogenetic activation induces immediate pup retrieval. In vivo recordings demonstrate preferential activation of MPOA Esr1+ cells during maternal behaviors and changes in MPOA cell responses across reproductive states. Furthermore, channelrhodopsin-assisted circuit mapping reveals a strong inhibitory projection from MPOA Esr1+ cells to ventral tegmental area (VTA) non-dopaminergic cells. Pathway-specific manipulations reveal that this projection is essential for driving pup approach and retrieval and that VTA dopaminergic cells are reliably activated during those behaviors. Altogether, this study provides new insight into the neural circuit that generates maternal behaviors., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The neurovascular unit (NVU) can be conceptualized as a functional entity consisting of neurons, astrocytes, pericytes, and endothelial and smooth muscle cells that operate in concert to affect blood flow to a very circumscribed area. Although we are currently in a "golden era" of bioengineering, there are, as yet, no living NVUs-on-a-chip modules available and the development of a neural chip that would mimic NVUs is a seemingly lofty goal. The sexually dimorphic nucleus of the preoptic area (SDN-POA) is a tiny brain structure (between 0.001~0.007 mm 3 in rats) with an assessable biological function (i.e., male sexual behavior). The present effort was undertaken to determine whether there are identifiable NVUs in the SDN-POA by assessing its vasculature relative to its known neural components. First, a thorough and systematic review of thousands of histologic and immunofluorescent images from 201 weanling and adult rats was undertaken to define the characteristics of the vessels supplying the SDN-POA: its primary supply artery/arteriole and capillaries are physically inseparable from their neural elements. A subsequent immunofluorescent study targeting α-smooth muscle actin confirmed the identity of an artery/arteriole supplying the SDN-POA. In reality, the predominant components of the SDN-POA are calbindin D28k-positive neurons that are comingled with tyrosine hydroxylase-positive projections. Finally, a schematic of an SDN-POA NVU is proposed as a working model of the basic building block of the CNS. Such modules could serve the study of neurovascular mechanisms and potentially inform the development of next generation bioengineered neural transplants, i.e., the construct of an NVU neural chip.

Vasomotor symptoms (VMS; or hot flashes) plague millions of reproductive-aged men and women who have natural or iatrogenic loss of sex steroid production. Many affected individuals are left without treatment options because of contraindications to hormone replacement therapy and the lack of equally effective nonhormonal alternatives. Moreover, development of safer, more effective therapies has been stymied by the lack of an animal model that recapitulates the hot-flash phenomenon and enables direct testing of hypotheses regarding the pathophysiology underlying hot flashes. To address these problems, we developed a murine model for hot flashes and a comprehensive method for measuring autonomic and behavioral thermoregulation in mice. We designed and constructed an instrument called a thermocline that produces a thermal gradient along which mice behaviorally adapt to a thermal challenge to their core body temperature set point while their thermal preference over time is tracked and recorded. We tested and validated this murine model for VMS by administration of a TRPV1 agonist and a neurokinin B receptor agonist, capsaicin and senktide, respectively, to unrestrained mice and observed their autonomic and behavioral responses. Following both treatments, the mice exhibited a VMS-like response characterized by a drop in core body temperature and cold-seeking behavior on the thermocline. Senktide also caused a rise in tail skin temperature and increased Fos expression in the median preoptic area, a hypothalamic temperature control center. This dynamic model may be used to fully explore the cellular and molecular bases for VMS and to develop and test new therapeutic options., (Copyright © 2017 Endocrine Society.)

Aromatase (ARO) is a cytochrome P450 enzyme that accounts for local estrogen production in the brain. The goal of this study was to develop a microsomal based assay to sensitively and reliably detect the low levels of ARO activity in different brain regions. Enzyme activity was detected based on the conversion of testosterone to estradiol. Quantity of estradiol was measured using ultra performance liquid chromatography-mass spectrometry. Detection was linear over a range of 2.5-200pg/ml estradiol, and was reproducible with intra- and inter-assay coefficients of variation (CV) <15%. Estradiol production using isolated microsomes was linear with time up to 30min as well as linearly related to amount of microsome. Substrate concentration curves revealed enzymatic kinetics (hippocampus: Vmax and Km: 0.57pmol estradiol/h per mg microsome and 48.58nM; amygdala: Vmax and Km: 1.69pmol estradiol/h per mg microsome and 48.4nM; preoptic area: Vmax and Km: 0.96pmol estradiol/h per mg microsome and 44.31nM) with testosterone used at a saturating concentration of 400nM. Anastrozole treatment blocked ARO activity in hippocampal and ovarian microsomes, indicating that the assay is specific for ARO. Also, we showed that the distribution of the long form ARO mRNA (CYP19A1) in different regions of the brain is correlated with ARO activity, with highest levels in the amygdala, followed by preoptic area and hippocampus. In the frontal cortex, very little long form ARO mRNA, and little to no ARO activity, were detected. These findings demonstrate that the microsomal incubation (MIB) assay is a sensitive and reliable method for quantifying ARO activity in discrete brain regions., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

This study describes the distribution of galanin (Gal) and galanin receptor 2 (GalR2) in the pre-optic area (POA) of the female guinea pig. Frozen sections were undergone for a routine immunofluorescence labelling. Gal and GalR2 display immunoreactivity in all parts of the pre-optic area. Gal shows reactivity both in perikarya and fibres, whereas GalR2 was observed only in perikarya. Gal- and GalR2-immunoreactive (-ir) perikarya were the most numerous in the medial pre-optic area (MPA) with the highest reactivity in its dorsal part. In the median pre-optic nucleus (MPN) and periventricular pre-optic nucleus (PPN), only single Gal- and GalR2-ir neurons were observed. The highest density of Gal-ir fibres was revealed in the PPN and the lowest in the lateral pre-optic area (LPA). The results of this study indicate that the distribution pattern of Gal containing neurons overlaps well with the distribution pattern of GalR2-positive neurons, especially in the MPA. This may suggest GalR2-dependent activity in this brain region., (© 2014 Blackwell Verlag GmbH.)

The aim of this study was to investigate how acute insulin-induced hypoglycaemia (IIH) alters the activity of cells containing oestradiol receptor α (ERα) or somatostatin (SST) in the arcuate nucleus (ARC) and ventromedial nucleus (VMN), and ERα cells in the medial preoptic area (mPOA) of intact ewes. Follicular phases were synchronized with progesterone vaginal pessaries. Control animals were killed at 0 h or 31 h (n = 5 and 6, respectively) after progesterone withdrawal (PW; time zero). At 28 h, five other animals received insulin (INS; 4 iu/kg) and were subsequently killed at 31 h. Hypothalamic sections were immunostained for ERα or SST each with c-Fos, a marker of neuronal transcriptional activation. Insulin did not alter the percentage of activated ERα cells in the ARC; however, it appeared visually that two insulin-treated animals (INS responders, with no LH surge) had an increase in the VMN (from 32 to 78%) and a decrease in the mPOA (from 40 to 12%) compared to no increase in the two INS non-responders (with an LH surge). The percentage of activated SST cells in the ARC was greater in all four insulin-treated animals (from 10 to 60%), whereas it was visually estimated that activated SST cells in the VMN increased only in the two insulin responders (from 10 to 70%). From these results, we suggest that IIH stimulates SST activation in the ARC as part of the glucose-sensing mechanism but ERα activation is unaffected in this region. We present evidence to support a hypothesis that disruption of the GnRH/LH surge may occur in insulin responders via a mechanism that involves, at least in part, SST cell activation in the VMN along with decreased ERα cell activation in the mPOA., (© 2014 Blackwell Verlag GmbH.)

Nitric oxide (NO) acts in the medial preoptic area (mPOA) of the hypothalamus to facilitate the expression of male sexual behavior and has also been widely implicated in mechanisms of experience, learning, and memory. Using immunohistochemistry for Fos, as a marker for neural activity, and nitric oxide synthase (NOS), the enzyme that catalyzes the production of nitric oxide (NO), we examined whether sexual activity and sexual experience influence Fos co-expression in NOS-containing neurons in the mPOA of male rats. Consistent with previous findings, results indicate that mating increased activity in the mPOA, and that sexual experience facilitated the expression of sexual behaviors, together with increased mating-induced Fos and NOS in the mPOA. Results also indicate that mating increased co-expression of Fos in NOS-containing neurons, and that this increase was highest in animals undergoing their first sexual encounter, indicating that initial sexual experience increases NO production in the mPOA of male rats., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

The aim of this study was to investigate the medial preoptic nucleus (MPN) of the anterior hypothalamus by resonance Raman spectroscopy (514.5 nm) to determine if it is possible to enhance the Raman scattering of hemoproteins in fresh brain tissue slices. The resonance effect was compared with near-infrared Raman spectra. Two groups of male Sprague Dawley rats were studied, one control group on a normal diet and one group on a low-iron diet to evoke iron deficiency. Each group consisted of four rats, 38-41 days old. The diets lasted for 11, 12, and 15 days. The MPN regions of brain tissue slices were analyzed by monitoring raw and pre-processed mean data, by cluster analysis, and by deriving difference spectra from pre-processed mean spectra. Cluster analysis of the resonance Raman spectra could identify different hemoprotein groups, namely, hemoglobin (Hb) and neuroglobin (Ngb). Spectra from randomly distributed spots revealed high Hb content, whereas Ngb was evenly distributed in the MPN. The different spectra showed a decrease of the Ngb and lipid content for the animals on the low-iron diet. The Ngb decrease was approximately 20%. The data show that resonance Raman spectroscopy is well suited to study hemoproteins in fresh brain tissue.

Many temperate zone songbird species exhibit marked seasonal variation in song quality as well as in the motivation to sing. Two brain systems are known to mediate such annual variation in song quality and motivation: (1) the song control system (SCS), and (2) the social behavior network (SBN), respectively. How these two circuits interact to produce changes in singing behavior is not well understood. The opioid enkephalin is expressed in both the SCS and SBN and may function to modulate song quality in a socially relevant manner. Using immunocytochemistry, we examined variation in enkephalin immunoreactivity (ENK-ir) in male European starlings (Sturnus vulgaris) that were in breeding conditions (i.e. photostimulated) or non-breeding conditions (i.e. photorefractory). We also included a group of castrated photostimulated males to investigate the relationship between gonadal steroids and ENK-ir. ENK-ir in the preoptic area (POA) and lateral septum (LS) was greater in photostimulated intact birds as compared to photorefractory males, but not in other regions within the SBN. There was a significant difference in ENK-ir in two forebrain song nuclei, HVC and the lateral nucleus of the anterior medial nidopallium (lMAN), with lower expression in photostimulated intact as compared to photorefractory birds. ENK-ir did not change across breeding conditions in the Nucleus Interface (NIf). After accounting for the volumetric change in HVC and lMAN, the pattern of ENK-ir remained greater in photorefractory compared to intact photostimulated starlings. We propose that the observed regulation of ENK-ir in the POA and LS may be related to seasonal changes in the motivation to engage in singing behavior, while the change in ENK-ir in the song system are associated with the quality of the song produced. Thus seasonal changes in a single neuromodulatory system can have very different functional effects based on the neuroanatomical specificity of its expression., (Copyright © 2012. Published by Elsevier B.V.)

Kisspeptin has been thought to play pivotal roles in the control of both pulse and surge modes of gonadotropin-releasing hormone (GnRH) secretion. To clarify loci of kisspeptin action on GnRH neurons, the present study examined the morphology of the kisspeptin system and the associations between kisspeptin and GnRH systems in gonadally intact and castrated male goats. Kisspeptin-immunoreactive (ir) and Kiss1-positive neurons were found in the medial preoptic area of intact but not castrated goats. Kisspeptin-ir cell bodies and fibers in the arcuate nucleus (ARC) and median eminence (ME) were fewer in intact male goats compared with castrated animals. Apposition of kisspeptin-ir fibers on GnRH-ir cell bodies was very rare in both intact and castrated goats, whereas the intimate association of kisspeptin-ir fibers with GnRH-ir nerve terminals was observed in the ME of castrated animals. Neurokinin B immunoreactivity colocalized not only in kisspeptin-ir cell bodies in the ARC but also in kisspeptin-ir fibers in the ME, suggesting that a majority of kisspeptin-ir fibers projecting to the ME originates from the ARC. A dual immunoelectron microscopic examination revealed that nerve terminals containing kisspeptin-ir vesicles made direct contact with GnRH-ir nerve terminals at the ME of castrated goats. There was no evidence for the existence of the typical synaptic structure between kisspeptin- and GnRH-ir fibers. The present results suggest that the ARC kisspeptin neurons act on GnRH neurons at the ME to control (possibly the pulse mode of) GnRH secretion in males., (Copyright © 2011 S. Karger AG, Basel.)

The posterodorsal preoptic nucleus (PdPN), lateral part of the posterodorsal medial amygdala (MeApd) and medial part of the medial preoptic nucleus (MPNm) are activated at ejaculation in male gerbils as assessed by Fos expression. We sought to immunocytochemically visualize substance P (SP), cholecystokinin (CCK), oxytocin, vasopressin and tyrosine hydroxylase (TH), a catecholaminergic marker, in the mating-activated cells, but the need for colchicine precluded behavioral testing. Instead, we detailed distributions of cells containing these molecules in the medial amygdala, caudal preoptic area and caudal bed nuclei of the stria terminalis (BST) and quantified their densities in the PdPN, MPNm and lateral MeApd for comparison to densities previously assessed for mating-activated efferents from these sites. TH cells were as dense in the PdPN and lateral MeApd as activated efferents to the anteroventral periventricular nucleus. In the lateral MeApd, TH cells were grouped where cells activated at ejaculation are clustered and where CCK cells form a ball. Lateral MeApd CCK cells and PdPN SP cells were as dense as activated efferents to the principal BST. Oxytocinergic PdPN cells and SP cells in the MPNm were as dense as mating-activated efferents to the lateral MeApd. If some oxytocin cells in the PdPN project to the neurohypophysis, as in rats, they could be a source of the oxytocin secreted at ejaculation. Since gerbils are monogamous and biparental, it was also interesting that, unlike monogamous prairie voles, they had few TH cells in the MeApd or dorsal BST, resembling promiscuous rats, hamsters and meadow voles., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Atrazine (ATRA) is the most commonly applied herbicide in the United States and is detected frequently in drinking water at significant levels. Following oral exposure, metabolism of ATRA generates diaminochlorotriazine (DACT), an electrophilic molecule capable of forming covalent protein adducts. At high doses, both ATRA and DACT can disrupt the preovulatory luteinizing hormone (LH) surge in rats, thereby altering normal reproductive function. This research was designed to identify DACT protein adducts formed in three distinct brain regions of ATRA-exposed rats, including the preoptic area (POA), medial basal hypothalamus (MBH), and cortex (CTX). Proteins with DACT adducts were identified following 2-dimensional electrophoresis (2-DE), immunodetection, and MALDI-TOF mass spectrometry analysis. Western blots from exposed animals revealed over 30 DACT-modified spots that were absent in controls. Protein spots were matched to concurrently run 2-DE gels stained with Sypro Ruby, excised, and in-gel digested with trypsin., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

The magnocellular division of the medial Preoptic nucleus (MPN mag) plays a critical role in the regulation of male sexual behavior in the hamster. Results from previous studies indicated that the number of neurons in the MPN mag is greater in males than females but failed to find significant differences in the volume of the nucleus suggesting that other elements in the nucleus may be greater in the female. The results of the present study, using NeuN to identify neurons, are in line with this hypothesis. The data show that (1) neurons in the MPN mag display two distinct phenotypes, those with a single nucleolus and those with multiple nucleoli; (2) the percentage of each phenotype is sex specific, differing over the course of development and (3) there is no sex difference in the number of glial cells at any age. Sex differences in the numbers of each type are correlated with developmental milestones and suggest that morphological changes are influenced by changes in circulating gonadal steroids during development., (2010 Elsevier B.V. All rights reserved.)

Purpose: To investigate the influence of angiotensin II (Ang II) AT1 receptors blockade on central fatigue induced by brain content of serotonin (5-HT) and dopamine (DA) during exercise., Methods: Losartan (Los) was intracerebroventricularly injected in rats before running until fatigue (n = 6 per group). At fatigue, brains were quickly removed for measurement of 5-HT, 5-hydroxyindoleacetic acid (5-HIAA), DA, and 3,4-dihydroxyphenylacetic acid by high-pressure liquid chromatography in the preoptic area, hypothalamus, hippocampus, and frontal cortex., Results: Intracerebroventricular injection of Los increased 5-HT content in the preoptic area and hypothalamus. Such results correlated positively with body heating rate and inversely with time to fatigue. On the other hand, time to fatigue was directly correlated with the diminished concentration of 5-HT in the hippocampus of Los rats. Although the levels of DA were not affected by Los treatment during exercise in any of the brain areas studied, a higher 5-HT/DA ratio was seen in the hypothalamus of Los animals. This higher hypothalamic 5-HT/DA ratio correlated positively with body heating rate and negatively with time to fatigue., Conclusions: Our results show that central fatigue due to hyperthermia and increased body heating rate induced by central Ang II AT1 receptor blockade in exercising rats is related with higher 5-HT content in the preoptic area and hypothalamus as well as with decreased levels of this neurotransmitter in the hippocampus. Furthermore, the interaction between 5-HT and DA within the hypothalamus seems to contribute to hyperthermia and premature central fatigue after angiotensinergic inhibition.

Lactation is an energy-demanding process characterized by massive food and water consumption, cessation of the reproductive cycle and induction of maternal behavior. During lactation, melanin-concentrating hormone (MCH) mRNA and peptide expression are increased in the medial preoptic area (MPO) and in the anterior paraventricular nucleus of the hypothalamus. Here we show that MCH neurons in the MPO coexpress the GABA synthesizing enzyme GAD-67 mRNA. We also show that MCH neurons in the MPO of female rats are innervated by neuropeptides that control energy homeostasis including agouti-related protein (AgRP), alpha-melanocyte stimulating hormone (alphaMSH) and cocaine- and amphetamine-regulated transcript (CART). Most of these inputs originate from the arcuate nucleus neurons. Additionally, using injections of retrograde tracers we found that CART neurons in the ventral premammillary nucleus also innervate the MPO. We then assessed the projections of the female MPO using injections of anterograde tracers. The MPO densely innervates hypothalamic nuclei related to reproductive control including the anteroventral periventricular nucleus, the ventrolateral subdivision of the ventromedial nucleus (VMHvl) and the ventral premammillary nucleus (PMV). We found that the density of MCH-ir fibers is increased in the VMHvl and PMV during lactation. Our findings suggest that the expression of MCH in the MPO may be induced by changing levels of neuropeptides involved in metabolic control. These MCH/GABA neurons may, in turn, participate in the suppression of cyclic reproductive function and/or sexual behavior during lactation through projections to reproductive control sites.

Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) play key roles in vertebrate gametogenesis and steroidogenesis. They are mainly synthesized in the pituitary gland. While investigating the ontogeny of FSH and LH cells in the cichlid fish Cichlasoma dimerus by immunohistochemistry (IHC), we unexpectedly found immunoreactive neurons in the preoptic area, sending their projections through different brain areas and neurohypophysis. Our previous work using Western blot and IHC techniques applied to the adult brain confirmed these findings. To further demonstrate the extrapituitary expression of these hormones, we performed RT-PCR detecting sequences coding for beta-FSH and beta-LH subunits in the C. dimerus pituitary and brain (preoptic-hypothalamic area). The expression of these transcripts in both organs was consistent with their peptide expression showing a high sequence homology when compared with other phylogenetically related fish. An individual pituitary in vitro culture system was utilized to study the possible modulatory effect of brain-derived gonadotropins on pituitary hormone secretion. Pituitary explants were cultured with different concentrations of LH or FSH, and the culture media were analyzed by Western blot. Exogenous LH produced a dose-dependent increase in pituitary beta-LH, beta-FSH and somatolactin (SL) releases. No effect was observed on growth hormone (GH). The effect on prolactin (PRL) was not consistent among treatments. Exogenous FSH produced an inhibition in beta-LH release, dose-dependent increases in beta-FSH and SL releases, and no effect on PRL and GH releases. These findings support the concept of regulation of pituitary trophic hormones by brain-derived gonadotropins.

We studied the expression of sGnRH mRNA in the neurons of the nucleus preopticus (NPO) of the Indian major carp, Cirrhinus cirrhosus, and their correlation with the reproductive status of the fish. Non-radioisotopic in situ hybridization histochemistry protocol employing biotinylated-oligonucleotide probes complementary to salmon GnRH, cichlid GnRH I, catfish GnRH, chicken GnRH II (from cichlid and catfish), and mammalian GnRH, were applied to the sections through the POA of the female Indian major carp Cirrhinus cirrhosus. Incubation with the probe complimentary to salmon GnRH (sGnRH) mRNA from salmon, produced distinct hybridization signal in the cytosol of several neurosecretory neurons of the magnocellular and parvocellular subdivisions of the NPO of the fish collected during February-April (preparatory phase) and May-June (prespawning phase). However, no signal was detected in the NPO of fish collected during July-August (spawning phase). Application of other antisense probes, or sense probe for salmon GnRH mRNA, produced no signal. We suggest that NPO neurons in C. cirrhosus may express sGnRH mRNA, produce GnRH peptide, and play a role in regulation of pituitary-ovary axis.

One of the challenges of gene targeting is to achieve regulated transgene expression in specific target cells. The hypogonadal (hpg) mice are genetically deficient in hypothalamic gonadotropin-releasing hormone (GnRH) production due to a deletion in the GnRH gene, resulting in hypogonadotropic hypogonadism. Here we show an improvement in reproductive parameters of adult female homozygous hpg mice by direct infusion into the hypothalamic preoptic area (POA) of a herpes simplex virus (HSV)-based amplicon vector containing a 13.5 kb genomic fragment encoding the GnRH gene together with its cognate promoter and regulatory elements. Following vector injection, GnRH-expressing neurons were detected in the POA, and pituitary and plasma gonadotropin levels as well as ovarian and uterine weights increased. In addition, a subset of injected hpg mice demonstrated cyclic estrous changes, consistent with regulated control of GnRH production. Administration of kisspeptin-10 resulted in an increase in plasma luteinizing hormone levels, further supporting appropriate regulation of the introduced GnRH transgene. These findings indicate that delivery of the GnRH gene resulted in selective neuronal expression of GnRH and regulated hypothalamic GnRH release. To our knowledge, this is the first example of the correct targeting of a gene under its cognate promoter to neurons resulting in selective and regulated synthesis of a biologically active peptide, and thus may have a wide range of applications in the treatment of human disorders.

Seasonal anoestrus in the ewe results from enhanced oestrogen negative feedback. Recent data have implicated the ventromedial preoptic area (vmPOA) as an important site of oestrogen action. This study addressed whether NO acts within the vmPOA to inhibit LH during seasonal anoestrus. In Experiment 1, microimplants containing Nomega-nitro-l-arginine methyl ester (l-NAME, NOS inhibitor), S-methyl thiocitrulline (SMTC, neural NOS (nNOS) inhibitor) or empty implants (control) were administered during mid-anoestrus to the vmPOA. l-NAME, but not SMTC, significantly increased LH pulse frequency. For Experiment 2, ewes in late anoestrus were administered 7-nitroindazole (7NI; nNOS inhibitor), l-NAME, SMTC, or empty implants. 7NI, but not l-NAME or SMTC, increased LH pulse frequency. In Experiment 3, the effects of microimplants and microinjections of l-NAME were compared in mid-anoestrus. Microinjections of l-NAME (300 nl at 10 microg/microl) increased LH pulse frequency, but microimplants did not. In late anoestrus, similar microinjections were ineffective. Taken together, the results of Experiments 1-3 suggested that NO inhibition may be stronger during the middle than at the end of seasonal anoestrus. To test this hypothesis, ewes in Experiment 4 received microinjection of l-NAME or vehicle thrice during the non-breeding season; none of the treatments increased LH pulse frequency. These results indicate that NO plays a role in the vmPOA in suppressing LH secretion during seasonal anoestrus because NOS inhibitors were consistently stimulatory when LH pulse frequency was low. However, the inconsistent and modest effects of these inhibitors suggest that NO actions in this area cannot completely account for the effects of inhibitory photoperiod.

Atrial natriuretic peptide-synthesizing neurons in the hypothalamic paraventricular nucleus constitute the major sources of ANP in the three lobes of the pituitary gland. Complete transection of the pituitary stalk eliminated 93% of ANP from the intermediate lobe, 47 and 77% from the anterior and the posterior lobes, respectively. Meantime, increased levels of immunoreactive ANP were measured in the median eminence, due to the accumulation of the peptide in the transected axons centrally to the transected stalk and in the paraventricular nucleus. It is likely that ANP neurons in the paraventricular nucleus innervate the pituitary, but those in the periventricular (median) preoptic nucleus and the organum vasculosum laminae terminalis may not contribute to the ANP innervation of the pituitary gland.

Hot flushes and night sweats, referred to as vasomotor symptoms (VMS), are presumed to be a result of declining hormone levels and are the principal menopausal symptoms for which women seek medical treatment. To date, estrogens and/or some progestins are the most effective therapeutics for alleviating VMS; however, these therapies may not be appropriate for all women. Therefore, nonhormonal therapies are being evaluated. The present study investigated a new reuptake inhibitor, desvenlafaxine succinate (DVS), in animal models of temperature dysfunction. Both models used are based on measuring changes in tail-skin temperature (TST) in ovariectomized (OVX) rats. The first relies on naloxone-induced withdrawal in morphine-dependent (MD) OVX rats, resulting in an acute rise in TST. The second depends on an OVX-induced loss of TST decreases during the dark phase as measured by telemetry. An initial evaluation demonstrated abatement of the rise in TST with long-term administration of ethinyl estradiol or with a single oral dose of DVS (130 mg/kg) in the MD model. Further evaluation showed that orally administered DVS acutely and dose dependently (10-100 mg/kg) abated a naloxone-induced rise in TST of MD rats and alleviated OVX-induced temperature dysfunction in the telemetry model. Oral administration of DVS to OVX rats caused significant increases in serotonin and norepinephrine levels in the preoptic area of the hypothalamus, a key region of the brain involved in temperature regulation. These preclinical studies provide evidence that DVS directly impacts thermoregulatory dysfunction in OVX rats and may have utility in alleviating VMS associated with menopause.

Objectives: The current research was conducted to determine whether hyperpolarization-activated cyclic nucleotide-gated (HCN1-4) channels are expressed in gonadotropin-releasing hormone (GnRH) neurons in the female rat hypothalamus and immortalized GnRH neurons (GT1-7 cells)., Methods: Double-label fluorescence immunohistochemistry was used to colocalize HCN1-4 channels and GnRH in GnRH neurons in the female rat hypothalamus. Reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting, and immunocytochemistry were used to analyze HCN channel gene expression in GT1-7 cells., Results: Double-label fluorescence immunohistochemistry showed that 43% of hypothalamic GnRH neurons immunostained for HCN2 and 90% of GnRH neurons immunostained for HCN3. RT-PCR and Western blot showed expression of all four HCN channel subunits in GT1-7 cells. Double-label immunocytochemistry showed cytoplasmic immunostaining of HCN2 and HCN3 in GT1-7 cells., Conclusions: This study demonstrates for the first time that HCN channels are expressed in GnRH neurons in the rat hypothalamus and GT1-7 cells. Our research supports the hypothesis that HCN channels may be involved in electrical bursting activity and pulsatile GnRH secretion in endogenous GnRH neurons and GT1-7 cells.

The innate immune system of mammals is able to detect bacteria when they infect local tissue or enter the blood stream, and initiate an immediate immune response. Prostaglandin (PG) E2 is considered as the most important link between the peripheral immune system and the brain. Due to four PGE2 receptors (EP receptors) and their differential expression in various areas of the hypothalamus and brain stem, PGE2 mediates different components of the acute phase reaction. A fever model is discussed in which the preoptic area contains the mechanisms for both hyperthermic and hypothermic responses and EP receptors in the median preoptic area (MnPO) modulate the thermogenic system. The neuron-specific modulation of EP receptors in the MnPO can be critically tested by using Cre-recombinase-mediated DNA recombination in genetically engineered mice. A concept for mice with conditional expression of EP3R and EP4R to investigate the different roles of those receptors in lipopolysaccharide (LPS)-induced fever is presented.

The median preoptic nucleus (MnPN) of the hypothalamus contains sleep-active neurones, and sleep-related Fos-immunoreactivity (IR) in this nucleus is primarily expressed in GABAergic cells. The MnPN also contains cells responsive to hypertonic saline and to angiotensin-II (Ang-II). To clarify functional relationships between MnPN neurones involved in the regulation of sleep and body fluid homeostasis, we examined c-fos expression in the MnPN after administration of hypertonic saline and Ang-II in both spontaneously sleeping and sleep-deprived rats. Systemic administration of hypertonic saline and intracerebroventricular (i.c.v.) injection of Ang-II increased Fos-IR in both spontaneously sleeping and sleep-deprived rats, compared to control animals. To determine if the population of MnPN neurones activated in response to osmotic and hormonal stimuli is similar to or different from neurones activated during sleep, we quantified Fos-IR in MnPN GABAergic neurones in spontaneously sleeping hypertonic saline- and Ang-II-treated rats versus respective control rats. Fos-IR evoked by these treatments occurred primarily (80-85%) in non-GABAergic neurones. Findings of the present study provide evidence that separate populations of MnPN neurones are involved in the regulation of sleep and body fluid homeostasis.

The objectives of this study were to characterize rainbow trout (Oncorhynchus mykiss) corticotropin-releasing factor (CRF)-binding protein (CRF-BP) cDNA and to examine the variations in CRF-BP and CRF mRNA levels in response to different intensities of stress. Trout were physically disturbed by a single or three consecutive periods of chasing until exhaustion followed by 2 h of recovery. The pituitary CRF-BP and preoptic area CRF1 mRNA contents were significantly increased only after repeated chasing events. Physical disturbance increased plasma cortisol levels with the largest change occurring in the group of trout that were exposed to repeated chasing events. Trout were also individually isolated in 120 l tanks or confined to 1.5 l boxes for 4, 24 or 72 h. CRF-BP mRNA levels in confined fish were greater than those of isolated fish at 72 h although there were no differences compared with the control group. CRF1 mRNA levels in the preoptic area were greater and remained elevated for a longer period in confined compared with isolated trout. Isolation led to a transient increase in plasma cortisol levels, but the higher cortisol values developed in the confined fish suggest that this treatment was more stressful than isolation. These results demonstrate that the intensity and duration of stress are important factors regulating CRF and CRF-BP mRNA levels in rainbow trout. We hypothesize that pituitary CRF-BP is involved in regulating the activity of the stress axis, possibly by reducing access to CRF1 receptors in the corticotropes.

Neonatal handling induces anovulatory estrous cycles and decreases sexual receptivity in female rats. The synchronous secretion of hormones from the gonads (estradiol (E2) and progesterone (P)), pituitary (luteinizing (LH) and follicle-stimulating (FSH) hormones) and hypothalamus (LH-releasing hormone (LHRH)) are essential for the reproductive functions in female rats. The present study aimed to describe the plasma levels of E2 and P throughout the estrous cycle and LH, FSH and prolactin (PRL) in the afternoon of the proestrus, and the LHRH content in the medial preoptic area (MPOA), median eminence (ME) and medial septal area (MSA) in the proestrus, in the neonatal handled rats. Wistar pup rats were handled for 1 min during the first 10 days after delivery (neonatal handled group) or left undisturbed (nonhandled group). When they reached adulthood, blood samples were collected through a jugular cannula and the MPOA, ME and MSA were microdissected. Plasma levels of the hormones and the content of LHRH were determined by RIA. The number of oocytes counted in the morning of the estrus day in the handled rats was significantly lower than in the nonhandled ones. Neonatal handling reduces E2 levels only on the proestrus day while P levels decreased in metestrus and estrus. Handled females also showed reduced plasma levels of LH, FSH and PRL in the afternoon of the proestrus. The LHRH content in the MPOA was significantly higher than in the nonhandled group. The reduced secretion of E2, LH, FSH and LHRH on the proestrus day may explain the anovulatory estrous cycle in neonatal handled rats. The reduced secretion of PRL in the proestrus may be related to the decreased sexual receptiveness in handled females. In conclusion, early-life environmental stimulation can induce long-lasting effects on the hypothalamus-pituitary-gonad axis.

In this study and the accompanying article (Folgueira et al., 2004a), the fluorescent carbocyanine dye 1,1'-dioctadecyl 3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) was used in fixed tissue to comprehensively analyze the connections of the different regions of the telencephalic lobes and the preoptic region of the rainbow trout. Here, we analyze the connections of the dorsal area (D; pallium) of the telencephalon, and the preoptic region, as well as the telencephalic connections of several structures in the diencephalon and brainstem of juvenile trout. The dorsal plus dorsolateral pallial zone of D (Dd+Dl-d) receives afferents from contralateral Dd+Dl-d, the ventral area of the telencephalon, preoptic nucleus, suprachiasmatic nucleus, medial thalamus, preglomerular complex, anterior and lateral tuberal nuclei, posterior tuberal nucleus, posterior hypothalamic lobe, superior raphe nucleus, and the rhombencephalic central gray and reticular formation, and projects to the central zone of D (Dc), medial thalamus, and some caudomedial hypothalamic regions. The medial zone of D (Dm) maintains reciprocal connections with the preglomerular complex and also receives afferents from the preoptic nucleus, suprachiasmatic nucleus, anterior tuberal nucleus, preglomerular tertiary gustatory nucleus, posterior tubercle, superior raphe nucleus, locus coeruleus, and the rhombencephalic central gray, and reticular formation. Dc receives fibers mainly from Dd+Dl-d, preoptic nucleus, preglomerular complex, and torus semicircularis and projects to several extratelencephalic centers, including the paracommissural nucleus, optic tectum, torus semicircularis, thalamus, preglomerular complex, posterior tubercle nuclei, and inferior hypothalamic lobes. The posterior zone of D (Dp) is mainly connected with the olfactory bulbs, the ventral and supracommissural nuclei of the ventral area (subpallium), the preoptic nucleus, and the preglomerular complex and projects to wide hypothalamic and posterior tubercular regions. The preoptic nucleus projects to the olfactory bulb, to most regions of the telencephalic lobes, and to several diencephalic and brainstem structures. These results reveal complex and specialized connectional patterns in the rainbow trout dorsal telencephalon and preoptic region. Most of these connections have not been described previously in salmonids. These connections indicate that the salmonid telencephalon is involved in multisensorial processing and modulation of brain activity., (2004 Wiley-Liss, Inc.)

Although many studies has been shown that serotonin (5-HT) in the preoptic area and anterior hypothalamus (PO/AH) is important for regulating body temperature (Tb), the exact role is not established yet due to conflicting results probably related to experimental techniques or conditions such as the use of anesthesia. The purpose of present study was to clarify the role of 5-HT in the PO/AH using the combined methods of telemetry, microdialysis and high performance liquid chromatography (HPLC), with a special emphasis on the regulation of Tb in freely moving rats. Firstly, we measured changes in Tb and levels of extracellular 5-HT and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) in the PO/AH during cold (5 degrees C) and heat (35 degrees C) exposure. We also perfused fluoxetine (5-HT re-uptake inhibitor) and 8-hydroxy-2-(Di-n-propylamino)tetralin (8-OH-DPAT: 5-HT1A agonist) into the PO/AH. During both exposures, although Tb changed significantly, no significant changes were noted in extracellular levels of 5-HT and 5-HIAA in the PO/AH. In addition, although perfusion of fluoxetine or 8-OH-DPAT into the PO/AH increased or decreased extracellular 5-HT and 5-HIAA levels in the PO/AH respectively, but Tb did not change at all. Our results suggest that 5-HT in the PO/AH may not mediate acute changes in thermoregulation.

It is generally assumed that the inhibitory neurotransmitter GABA and the stimulatory neurotransmitter glutamate are released from different neurons in adults. However, this tenet has made it difficult to explain how the same afferent signals can cause opposite changes in GABA and glutamate release. Such reciprocal release is a central mechanism in the neural control of many physiological processes including activation of gonadotropin-releasing hormone (GnRH) neurons, the neural signal for ovulation. Activation of GnRH neurons requires simultaneous suppression of GABA and stimulation of glutamate release, each of which occurs in response to a daily photoperiodic signal, but only in the presence of estradiol (E2). In rodents, E2 and photoperiodic signals converge in the anteroventral periventricular nucleus (AVPV), but it is unclear how these signals differentially regulate GABA and glutamate secretion. We now report that nearly all neurons in the AVPV of female rats express both vesicular glutamate transporter 2 (VGLUT2), a marker of hypothalamic glutamatergic neurons, as well as glutamic acid decarboxylase and vesicular GABA transporter (VGAT), markers of GABAergic neurons. These dual-phenotype neurons are the main targets of E2 in the region and are more than twice as numerous in females as in males. Moreover, dual-phenotype synaptic terminals contact GnRH neurons, and at the time of the surge, VGAT-containing vesicles decrease and VGLUT2-containing vesicles increase in these terminals. Thus, we propose a new model for ovulation that includes dual-phenotype GABA/glutamate neurons as central transducers of hormonal and neural signals to GnRH neurons.

In this study, we employed triple fluorescent labelling to reveal the distribution of the direct serotonergic neurons within Barrington's nucleus (BN) that supply branching collateral input to the medial preoptic area (MPA) and to the lumbo-sacral spinal cord (LSC). Immunocytochemical detection of the monoclonal antibody raised against serotonin was used for identification of the neurons. The projections were defined by injections of two retrograde tracers: fluoro gold and rhodamine in the MPA and LSC, respectively. The aim of this study is to identify the direct projections to BN and MPA and/or LSC. The present study confirms findings of others describing BN-LSC projections and extends previous findings by demonstrating an single or collateralized fibers with MPA, and serotonergic immunoreactive fibers.

The aim of the present study was investigate, in young rats, the effects of malnutrition on astrocyte distribution of two hypothalamic regions, the circadian pacemaker suprachiasmatic nucleus (SCN) and the medial preoptic area (MPA). Control rats were born from mothers fed on commercial diet since gestation and malnourished rats from mothers fed on multideficient diet, from the beginning of gestation (GLA group) or from the onset of lactation (LA group). After weaning, pups received ad libitum the same diet as their mothers, and were maintained under a 12/12 h light/dark cycle. The animals were analyzed either at 30-33, or 60-63 days of life. Brain coronal sections (50 microm) were processed to visualize glial fibrillary acidic protein (GFAP) immunoreactivity. Compared to control rats, both malnourished groups of 30 and 60 days exhibited a reduced number of GFAP-immunoreactive astrocytes in the SCN. The total GFAP-immunoreactive area in the SCN of the GLA group differed from the control group at both age ranges analyzed. The GFAP expression as measured by the relative optical density (ROD) exhibited a 50-60% reduction in the MPA in both malnourished groups, compared to controls. The results suggest that malnutrition early in life leads to alterations in gliogenesis or glial cell proliferation in both nuclei, being these alterations greater in the MPA. Compensatory plasticity mechanisms in the GFAP-expression seem to be developed in the astrocyte differentiation process in the SCN, especially when the malnutrition is installed from the lactation.

Adrenocorticotropic hormone (ACTH) takes a central role in the hypothalamo-pituitary-interrenal axis (HPI axis), which is activated during stress. ACTH is produced by the corticotrope cells of the pituitary pars distalis (PD) and is under control of factors from the nucleus preopticus (NPO). The distribution of ACTH in the hypothalamo-pituitary system in common carp (Cyprinus carpio L.) was assessed by immunohistochemistry. ACTH and beta-endorphin immunoreactivity was observed in the ACTH cells in the PD and in the NPO. Nerve fibers, originating from the NPO and projecting to the pituitary gland, contain beta-endorphin, but not ACTH, and these fibers either control the pituitary pars intermedia (PI) through beta-endorphin or release it to the blood. The release of pituitary ACTH (studied in a superfusion setup) must in vivo be under predominant inhibitory control of dopamine. Release of ACTH is stimulated by corticotropin-releasing hormone, but only when ACTH cells experience dopaminergic inhibition. The expression of the precursor pro-opiomelanocortin in (POMC) NPO, PD and PI was studied in an acute restraint stress paradigm by real-time quantitative polymerase chain reaction (RQ-PCR). POMC gene expression is upregulated in these three key tissues of the hypothalamo-pituitary complex, revealing a hitherto unforeseen complex role for POMC-derived peptides in the regulation of responses to stress.

The aim of the present work was to study the potential involvement of hypothalamic galanin system in the anorectic mechanism of fluoxetine in obese Zucker rats. Male obese Zucker (fa/fa) rats were administered fluoxetine (10 mg/kg; i.p.) daily for two weeks. The control group was given 0.9% NaCl solution. Significant decreases in food intake, final body weight and total body fat were observed after fluoxetine treatment. Although fluoxetine-treated rats showed a decrease in urine elimination, this effect was not enough to compensate decreased water intake, leading to dehydration, as showed by decreased body water content. Chronic fluoxetine administration increased the numbers of galanin positively immunostained neural cells in medial and lateral preoptic areas, lateral hypothalamic area and paraventricular nucleus (rostral and magnocellular regions), without changes in dorsomedial, ventromedial, supraoptic, suprachiasmatic and arcuate nuclei. Taken into account that galanin stimulates appetite, these results could represent rather a compensatory response against reduced food intake than a direct anorectic mechanism. Changes in the magnocellular region of the hypothalamic paraventricular nucleus suggest a role for galanin neural circuits at this level in fluoxetine-induced hydro-osmotic impairment.

Unilateral microdialysis-perfusion of the preoptic area with 50 microM muscimol decreased the sleep period of rats to less than 3% of the baseline value over a 90 min period before death (p = 0.018 by Wilcoxon signed-rank test). These rats showed the expression of FOS in 36% of the orexin neurons located in the perifornical/lateral hypothalamic areas on the side ipsilateral to the perfusion site, but in only 3% of the orexin neurons on the side contralateral to it (p = 0.018 by Wilcoxon signed-rank test). These results suggest that subpopulations of the preoptic neurons give an inhibitory tone to the activities of the orexin neurons in the perifornical/lateral hypothalamic areas., (Copyright 2004 Lippincott Williams and Wilkins)

Estrous behavior induced by progesterone (P) treatment of estradiol-primed rats is followed by a period in which females do not respond behaviorally to a second administration of P [sequential inhibition (SI)]. SI is thought to involve P-dependent down-regulation of hypothalamic P receptor (PR) content. This study tested the hypothesis that the 26S proteasome participates in the regulation of SI and brain PR content in female rats. Ovariectomized, estrogen-primed (estradiol benzoate, 2 microg s.c.) adult rats were injected with P (1 mg s.c.) alone or P with the proteasome inhibitors Z-Ile-Glu (OBu(1))-Ala-Leu-H (PSI, 300 microg/100 g s.c.) or N alpha-tosyl-lysyl chloromethyl ketone (TLCK, 200 microg i.p.) administered 48 h after estradiol priming. Sexual behavior was assessed in all animals 4 h later. These two agents inhibit 26S proteasome-mediated protein degradation by different mechanisms. To explore SI, the animals received a second P injection 24 h after the first, and a second sexual behavior test was performed 4 h later. After this test, brains were excised, and proteins were extracted from the preoptic area and the hypothalamus and processed for semiquantitative immunoblotting. In the first sexual behavior test (facilitation test), all animals treated with estradiol + P exhibited intense lordosis behavior. In the second sexual behavior test (inhibition test), both lordosis and proceptivity were significantly reduced in response to the second administration of P (SI). The magnitude of SI was significantly attenuated by the administration of either PSI or TLCK concurrently with the first P injection. The first P injection reduced PR content in the hypothalamus but not in the preoptic area. In contrast, PSI and TLCK significantly increased PR content in both structures. Our results suggest that PR degradation by the 26S proteasome participates in the expression of P-induced SI in female rats.

Neurones in the median preoptic nucleus (MnPN) and the ventrolateral preoptic area (vlPOA) express immunoreactivity for c-Fos protein following sustained sleep, and display elevated discharge rates during both non-REM and REM sleep compared to waking. We evaluated the hypothesis that MnPN and vlPOA sleep-active neurones are GABAergic by combining staining for c-Fos protein with staining for glutamic acid decarboxylase (GAD). In a group of six rats exhibiting spontaneous total sleep times averaging 82.2 +/- 5.1% of the 2 h immediately prior to death, >75% of MnPN neurones that were Fos-immunoreactive (IR) were also GAD-IR. Similar results were obtained in the vlPOA. In a group of 11 rats exhibiting spontaneous sleep times ranging from 20 to 92%, the number of Fos + GAD-IR neurones in MnPN and vlPOA was positively correlated with total sleep time. Compared to control animals, Fos + GAD-IR cell counts in the MnPN were significantly elevated in rats that were sleep deprived for 24 h and permitted 2 h of recovery sleep. These findings demonstrate that a majority of MnPN and vlPOA neurones that express Fos-IR during sustained spontaneous sleep are GABAergic. They also demonstrate that sleep deprivation is associated with increased activation of GABAergic neurones in the MnPN and vlPOA.

Pupfishes in the Death Valley region of California and Nevada comprise a monophyletic group of populations that became isolated in remote streams and springs over the past 20,000 years. These aquatic habitats show considerable ecological diversity, and allopatric populations have evolved differences in morphology and behavior. Here we investigated whether the divergence of pupfish populations in Death Valley might be associated with changes in arginine vasotocin (AVT). We used immunocytochemistry to compare the expression of AVT in the brain of Amargosa pupfish (Cyprinodon nevadensis) from two Death Valley populations: (1) the Amargosa River-a highly variable desert stream containing the Cyprinodon nevadensis amargosae subspecies, and (2) Big Spring-a comparatively stable springhead and outflow inhabited by Cyprinodon nevadensis mionectes. These particular populations have been isolated from each other for only 400-4000 years. In both populations AVT-immunoreactive somata localized to parvocellular and magnocellular neurons in the preoptic area, with AVT-immunoreactive fibers extending ventrolaterally to innervate the pituitary. We found that both parvocellular and magnocellular AVT-immunoreactive neurons were significantly larger in males and females from the Amargosa River population than in same sex pupfish from Big Spring. Our findings suggest that the divergent ecological conditions of these two habitats have brought about changes in AVT pathways in the brain.

The objectives of this study were to characterize rainbow trout (Oncorhynchus mykiss) corticotropin-releasing factor (CRF) and neuropeptide Y (NPY) cDNAs and to determine their mRNA levels in response to social stress. Standard cloning techniques were used to obtain cDNAs, sequences for trout NPY and two CRF isoforms. At the predicted amino acid level, our NPY sequence differs from the trout amino acid sequence reported by. A phylogenetic analysis suggests that the two CRF isoforms result from a gene duplication that occurred in a common ancestor of salmonids. A tissue distribution demonstrated that the mRNAs of both CRF isoforms are predominantly present in the preoptic area of the trout brain, whereas NPY mRNA is more abundant in the telencephalon. Pairs of sized-matched juvenile female trout were allowed to interact for 72 h and social ranks were assigned on the basis of behavioural observations. Mean plasma cortisol levels were 13-fold higher in subordinate than in dominant trout. As measured by ribonuclease protection assay, CRF1 and NPY mRNA levels were respectively 51 and 32% higher in the preoptic area of subordinate trout; in addition, CRF1 and NPY mRNA levels were positively correlated (R2=0.44). These results suggest that subordinate rainbow trout chronically maintain high levels of CRF mRNA during social stress and that NPY may be involved in the control of the stress axis in trout.

The present study was undertaken to investigate the effects of pre- and early postnatal social stress on the functioning of hormonal, autonomic and behavioural systems, by studying the distribution of sex hormone receptors in limbic brain systems. Dams had either lived in groups with a constant composition (= stable social environment) or in groups with changing compositions, i.e. every third day, two females from different groups were exchanged (= unstable social environment). The subjects were male offspring of dams who had either lived in a stable social environment during pregnancy and lactation (= control males) or in an unstable social environment during this period of life (= early stressed males). From days 20-80, the spontaneous behaviour of control males and early stressed males was recorded in their home cages. Five control males and five early stressed males were killed at 75 days, and five control males and five early stressed males at 120 days. Blood samples were taken to determine serum concentrations of cortisol, testosterone, dehydroepiandrosterone, dehydroepiandrosterone sulphate and oestrogen. The adrenals were prepared to determine tyrosine hydroxylase activities and the brains were used to investigate the distribution of sex-hormone receptors in specific hypothalamic and hippocampal brain areas. Early stressed males showed a behavioural infantilization that was accompanied by significantly decreased adrenal tyrosine hydroxylase activities and dehydroepiandrosterone levels. Furthermore, early stressed males showed a down-regulation of androgen receptors in the medial preoptic area and the nucleus arcuatus of the hypothalamus, as well as of oestrogen receptor alpha in the hippocampus compared to control males. Thus, the present study provides clear evidence that early social stress induces changes in endocrine, autonomic and limbic brain function, which is mirrored by changes in male social behaviour.

The anteroventral periventricular nucleus (AVPV) of the preoptic area has been implicated in the induction of spontaneous ovulation. In the AVPV, we found a striking sex difference in the distribution of estrogen receptor (ER) positive cells. In females, a significantly larger number of ER mRNA-positive cells were visualized than in males using in situ hybridization in the most medial part of the AVPV next to the ependymal lining of the third ventricle. In males, the labeled cells were dispersed into more lateral region. Immunohistochemistry revealed a similar sexual dimorphism in the ER protein. The dimorphism persisted from Day 7 to Day 60. Orchidectomy of male neonates or estrogen treatment of female pups had reversed the brain phenotype when examined on Day 14. No gross sex difference was detected in the pattern of ER expression in the medial preoptic nucleus and the bed nucleus of the stria terminals. Estrogen receptor immunoreactive cells co-localization in 83% of ER mRNA positive cells in the AVPV of adult females. Infusion of an ER antisense oligonucleotide into the third ventricle resulted in a significantly longer period of successive vaginal estrus and 50% reduction in the number of ER-immunoreactive cells in the AVPV. These findings suggest an important role of ER AVPV in the female-typical estrogen-dependent induction of the luteinizing hormone surge.

Many neuropeptide systems subserving sex-typical behavior are dependent on sex steroids for both their organization early in life and activation during maturity. The arginine vasopressin/vasotocin (AVP/AVT) system is strongly androgen dependent in many species and critically mediates responses to sociosexual stimuli. The bluehead wrasse is a teleost fish that exhibits a female-to-male sex change in response to social cues, and neither the development nor the maintenance of male-typical behavior depends on the presence of gonads. To examine social and gonadal inputs on the AVP/AVT system in the preoptic area (POA) of the hypothalamus, we conducted three field experiments. In the first experiment, we found that AVT mRNA abundance is higher in sex-changing females that attain social dominance and display dominant male behavior than in subordinate females, regardless of whether the dominant females were intact or ovariectomized. However, AVT-immunoreactive (IR) soma size in the gigantocellular POA (gPOA), but not in the magnocellular or parvocellular POA, increased only when females were displaying both dominant male behavior and had developed testes. In the second experiment, castration of dominant terminal-phase males had no effect on AVT mRNA abundance or any behavior we measured but did increase gPOA AVT-IR soma size compared with sham-operated controls. In the third experiment, 11-ketotestosterone implants in socially subordinate, ovariectomized females had no effect on either AVT mRNA abundance or AVT-IR soma size compared with controls. These results demonstrate that the AVT neural phenotype in the bluehead wrasse can be strongly influenced by social status, and that these social influences can be manifested independent of gonads.

The posterodorsal medial amygdala (MeApd), the posterodorsal preoptic nucleus (PdPN), and the medial cell group of the sexually dimorphic preoptic area (mSDA) contain cells that are activated specifically at ejaculation as assessed by Fos expression. The mSDA also expresses Fos early in the mating context. Because little is known about the neurotransmitters of these activated cells, the possibility that they use gamma-aminobutyric acid (GABA) or glutamate was assessed. Putative glutamatergic cells were visualized with immunocytochemistry (ICC) for glutamate and its neuron-specific transporter. Their distributions were compared with those of GABAergic cells visualized with ICC for the 67-kDa form of glutamic acid decarboxylase (GAD(67)) and in situ hybridization for GAD(67) messenger RNA (mRNA). Colocalization of Fos and GAD(67) mRNA in recently mated males indicated that half of the activated cells in the PdPN, mSDA, and lateral MeApd are GABAergic. Colocalization of Fos and glutamate suggested that a quarter of the activated mSDA and lateral MeApd cells are glutamatergic. The PdPN does not appear to have glutamatergic cells. In the lateral MeApd, the percentage of activated cells that are GABAergic (45%) matches the percentage that project to the principal part of the bed nucleus of the stria terminalis (BST; 43%), and the percentage likely to be glutamatergic (27%) matches the percentage projecting to the mSDA (27%). The latter could help to trigger ejaculation. The distribution of GABAergic and putative glutamatergic cells in the caudal preoptic area, caudal BST, and medial amygdala of male gerbils is also described., (Copyright 2003 Wiley-Liss, Inc.)

Corticotropin-releasing hormone (CRH) is implicated in the suppression of pulsatile luteinizing hormone (LH) secretion by a variety of stressful stimuli; 17beta-oestradiol (E2) has been shown to modulate this inhibitory response. The present study in ovariectomized (OVX) rats was designed to investigate the effect of E2 and progesterone (P4) on hypoglycaemic stress-induced changes in pulsatile LH secretion and on the associated changes in both central and peripheral components of the hypothalamic-pituitary-adrenal axis. E2 enhanced the hypoglycaemic stress-induced suppression of LH pulses; P4 in addition to E2 further potentiated the inhibitory response. The rise in plasma corticosterone following insulin-induced hypoglycaemia (IIH) was highest in the E2 + P4 group. Nevertheless, when such levels were achieved by administration of corticosterone, the occurrence of LH pulses was completely unaffected, irrespective of ovarian steroid milieu. E2 and E2 + P4 up-regulated basal CRH mRNA expression in the paraventricular nucleus (PVN) as measured by in situ hybridization; this signal was also increased in the medial preoptic nucleus (MPN) following E2. IIH resulted in a rise in CRH mRNA in the PVN, but not in the MPN; this rise may reflect a more significant role for the PVN in the present context. Changes in neuropeptide mRNA expression may signal changes in neuronal activity; nevertheless, the profound differences in LH pulse suppression in OVX, E2 and E2 + P4 rats following IIH were not reflected in the concurrent changes in CRH mRNA in the PVN. The results suggest that while corticosterone has no acute effect on LH pulses in the rat, the up-regulation by ovarian steroids of basal CRH mRNA in the PVN and/or MPN may contribute to the central regulation of these pulses in response to stress.

The seasonal pattern of breeding in sheep offers an opportunity to examine plasticity of neuronal inputs to gonadotropin-releasing hormone (GnRH) neurones. We used conventional fluorescence microscopy and confocal microscopy to compare the extent of input to GnRH neurones from various neuropeptide/neurotransmitter systems in ewes during the breeding and anestrous seasons. Using double-labelling immunohistochemistry, we counted close appositions between GnRH cells and varicosities that were immunoreactive for either glutamic acid decarboxylase (GAD; for gamma-amino butyric acid-GABA-neurones), dopamine beta hydroxylase (DBH; for noradrenergic neurones), vesicular glutamate transporter-1 (VGluT-1, for glutamatergic neurones), neuropeptide Y (NPY) and tyrosine hydroxylase (TH; for dopaminergic/noradrenergic neurones). The percentage of GnRH cells displaying close appositions to GABA-ergic varicosities was higher (P < 0.02) in anestrus than in the breeding season. The percentage of GnRH cells receiving input from varicosities that were positive for TH, DBH and VGluT-1 was similar in both seasons. Approximately 26-49% of GnRH neurones were seen to receive inputs from NPY, TH, GABAergic or noradrenergic neurones, while a larger number of GnRH cells (72-75%) received input from glutamatergic neurones. Conventional microscopy consistently overestimated the number of close contacts on GnRH neurones compared to confocal microscopy. For TH-immunoreactive varicosities in the preoptic area, only 16-35% were also immunoreactive for DBH, suggesting that the remainder are dopaminergic. Approximately half of the noradrenergic inputs in the preoptic area were also immunoreactive for NPY. In conclusion, we present numerical data on the consensus between light and confocal microscopy and the level of input of various neuronal systems to GnRH cells; the data indicate a seasonal change in the GABAergic input to GnRH neurones.

The serotonin system has been implicated in the modulation of endocrine and behavioral components of reproduction. In this study, we examined endogenous hypothalamic indolamines during sexual differentiation and long-term effects of exogenous steroids during this time. In Experiment 1, Japanese quail were studied during the last half of embryonic development and early post-hatch. Samples were taken at embryonic day 10 (E10), E12, E14, E16, hatch (day 0), and days 3 and 5, post-hatch. Hypothalamic indolamines, including serotonin (5-HT) and its metabolite, 5-hydroxy indole acetic acid (5-HIAA) were measured by HPLC-EC detection. Females had relatively higher hypothalamic 5-HT at E14 than males, with both sexes showing increasing levels thereafter. By day 5, post-hatch, hypothalamic 5-HT content was higher in males than in females. When turnover was estimated by comparing relative concentrations of 5-HT to 5-HIAA, males were significantly higher at E12 and E14 than females. These data suggest that there are stage specific changes in the serotonin system, as well as sexually dimorphic patterns in the ontogeny and activity of this system. In Experiment 2, we investigated the effects of embryonic steroid hormone treatment on the serotonin system and on male sexual behavior. Birds were treated with either estradiol benzoate (EB), testosterone propionate (TP) or sesame oil (vehicle control) at selected embryonic days (E10, E12, E14, E16, 0, D3, and D5). At 4 weeks post-hatch, birds were transferred to short photoperiod (16D:8L) for 3 weeks to prevent photostimulated reproductive development. At 7 weeks of age, males were implanted with a 20mm silastic capsule filled with testosterone and sexual behavior was tested 1 week later. Brains were collected from both males and females, and preoptic area (POA) indolamines were measured. Steroid treatment at E10 or E12 resulted in the loss of male sexual behavior. Moreover, males treated with EB or TP on E12 also had increased POA 5-HT content as adults, compared to control males. Females treated with EB on either E10 or E 12 also had higher POA 5-HT content than control or TP treated females. These data provide evidence for sexual dimorphism in the hypothalamic 5-HT system at specific stages during embryonic development. Moreover, males were sensitive to exogenous EB and TP on E12, whereas females appeared to be affected by EB only and appeared to be sensitive to steroid effects over a longer period of time in development. Moreover, exogenous steroids at E12 in males also correlated with impaired sexual behavioral. These data suggest that long-term effects of embryonic steroid exposure may be mediated in part through effects on the serotonin neurotransmitter system.

Effects of N-methyl-D-aspartate (NMDA) receptor (NMDAR) activation on neuroendocrine function can be modulated by the steroid hormone milieu. For example, the hypothalamic GnRH neurons, the primary cells regulating reproductive function, are stimulated by NMDAR agonists, and this is greatly potentiated by estrogen. We hypothesized that the actions of glutamate and estrogen may converge at target cells in the brain in which the NMDA and estrogen receptors (ERs) are coexpressed. To this end, we used quantitative stereological techniques to determine the colocalization of the obligatory NMDAR subunit, NR1, and the ERalpha, in the anteroventral periventricular nucleus and the medial preoptic nucleus, two critical regions for reproductive physiology and behavior. We observed extensive colocalization of ERalpha and NR1 in these brain regions (approximately 80%). In the anteroventral periventricular nucleus, treatment of ovariectomized rats with estrogen up-regulated the coexpression, whereas in the medial preoptic nucleus, estrogen had no effect, demonstrating a regional specificity to the estrogen sensitivity. The number of ERalpha cells that did not express NR1 was not altered by estrogen treatment in either brain region. Thus, we speculate that the extensive colocalization of ERalpha and the NMDAR provides an anatomical level at which estrogen and glutamate can act at target cells, and potentially synergize, to influence neuroendocrine and autonomic functions.

Gamma-aminobutyric acid (GABA), acting through GABA(A) receptors (GABA(A)R), is hypothesized to suppress reproduction by inhibiting GnRH secretion, but GABA actions directly on GnRH neurons are not well established. In green fluorescent protein-identified adult mouse GnRH neurons in brain slices, gramicidin-perforated-patch-clamp experiments revealed the reversal potential (E(GABA)) for current through GABA(A)Rs was depolarized relative to the resting potential. Furthermore, rapid GABA application elicited action potentials in GnRH neurons but not controls. The consequence of GABA(A)R activation depends on intracellular chloride levels, which are maintained by homeostatic mechanisms. Membrane proteins that typically extrude chloride (KCC-2 cotransporter, CLC-2 channel) were absent from the GT1-7 immortalized GnRH cell line and GnRH neurons in situ or were not localized to the proper cell compartment for function. In contrast, GT1-7 cells and some GnRH neurons expressed the chloride-accumulating cotransporter, NKCC-1. Patch-clamp experiments showed that blockade of NKCC hyperpolarized E(GABA) by lowering intracellular chloride. Regardless of reproductive state, rapid GABA application excited GnRH neurons. In contrast, bath application of the GABA(A)R agonist muscimol transiently increased then suppressed firing; suppression persisted 4-15 min. Rapid activation of GABA(A)R thus excites GnRH neurons whereas prolonged activation reduces excitability, suggesting the physiological consequence of synaptic activation of GABA(A)R in GnRH neurons is excitation.

Excitatory amino acids (EAAs), particularly glutamate, have been implicated in the control of luteinizing hormone (LH) secretion through facilitation of gonadotropin-releasing hormone release. The effects of EAAs are mediated by means of ionotropic glutamate receptors, which are divided into N-methyl-D-aspartate (NMDA) and non-NMDA (kainate and AMPA) subtypes. Moreover, ovarian steroids are responsible for inducing the preovulatory surge of LH and are involved in the actions of EAAs on LH release. Progesterone is directly involved in the potentiating effect of ovarian steroids on the stimulating effect of AMPA neurotransmission on gonadotropin secretion. To broaden our understanding of the role of hypothalamic AMPA receptors in the steroid-induced LH surge, we determined the cellular localization of AMPA receptors in the hypothalamus of guinea pigs by using antibodies that recognize the GluR1, GluR2, GluR2/3, or GluR4 subunits, and then we examined the neuroanatomic relationships between these receptors and the progesterone receptor (PR). Different patterns of immunostaining within the preoptic area and hypothalamus were evident with the antibodies to the four subunits with marked contrasts between moderate staining for GluR1, intensely stained structures for GluR2 and GluR2/3, and little specific staining for GluR4. Immunoreactive (IR) neurons were visualized in many regions, including the two regions known to contain a dense population of estradiol-induced PR-IR cells: the preoptic periventricular and ventrolateral hypothalamic nuclei. Approximately 60% of GluR1-IR and 39% of GluR2-IR cells in the preoptic region possessed PR, whereas 46% of GluR1-IR and 54% of GluR2-IR cells in the ventrolateral nucleus expressed PR. These neuroanatomic results suggest that the coordinated actions of progesterone and glutamatergic inputs on mammalian reproductive functions are integrated at the cellular level., (Copyright 2002 Wiley-Liss, Inc.)

Recent evidence suggests that the dynorphin-kappa receptor opioid system acts to mediate the inhibitory effect of progesterone (P) on GnRH pulse frequency during the luteal phase of the ovine estrous cycle. It is known that progesterone receptors (PRs) are required for the actions of P on GnRH secretion. Therefore, if P acts directly on dynorphin (DYN) neurons, then these neurons should contain PRs. To test this hypothesis, we used a dual-label immunoperoxidase procedure to visualize PRs and DYN in the preoptic area (POA) and hypothalamus of ovary-intact ewes killed during the luteal phase of the estrous cycle. The PR was colocalized in more than 90% of parvicellular DYN neurons in the POA, anterior hypothalamus (AHA), and arcuate nucleus (ARC). By contrast, none of magnocellular DYN cells of the paraventricular and supraoptic nuclei coexpressed immunoreactive PRs. The high percentage of colocalization of PRs in parvicellular DYN cells of the POA, AHA, and ARC suggests that these cells are prime targets of P. In addition, DYN cells in the ARC, but not the POA or AHA, were found to receive synaptic inputs from DYN-positive axon terminals. This observation raises the possibility that an ultrashort feedback loop controls the release of DYN from ARC neurons.

C57BL/6J and DBA/2J inbred mice differ in aspects of maternal behavior and in the morphology of the medial preoptic nucleus (MPO), suggesting a possible association. DBA/2J mice have a compact subnucleus in the MPO, the MPOpc, that is sexually dimorphic and absent in C57BL/6J mice. To determine whether MPOpc cells are activated by maternal behavior, FOS and FOSB immunohistochemistry was performed on brain sections of C57BL/6J and DBA/2J mothers following the return of their pups after a separation of 2 days. In both light and dark phases of the daily cycle, stimulation of DBA/2J mothers evoked an increase in FOS- and FOSB-immunoreactivity in the MPOpc. Stimulated C57BL/6J mice, which lack the MPOpc, did not show an increase in cellular activity in the corresponding MPO region. Cells immediately lateral to the MPOpc were activated by pup stimulation, in both strains. These results suggest that MPOpc cells are active during maternal behavior, and that strain differences in maternal behavior are related to anatomical differences in the MPO.