Regulation of galanin receptor GalR1 mRNA expression by ovarian steroids in oestrogen receptor alpha-immunoreactive neurones: identification of distinct populations of neurones in the preoptic area

This study examined whether gonadal steroids are involved in regulating galanin receptor 1 (GalR1) mRNA expression in neurones that contain oestrogen receptor alpha (ERalpha), in three regions of the preoptic area (POA) known to be involved in the control of gonadotropin secretion. Double-labelling immunohistochemistry using an antibody against the ERalpha and in situ hybridization experiments using a 35S-labelled riboprobe specific for GalR1 mRNAs revealed that ERalpha is expressed in a large proportion of GalR1 mRNA-expressing neurones of the POA in the ovariectomized (OVX) female rat. Oestradiol (E2) and oestradiol plus progesterone (E2 + P) treatments of OVX rats significantly decreased the proportion of GalR1 mRNA/ERalpha immunoreactive (ERalpha-IR) neurones in the anteroventral periventricular nucleus (AVPV), medial preoptic nucleus (MPN) and medial preoptic area (MPO). The expression of GalR1 mRNA in ERalpha-IR neurones varied according the hormonal status of the female animals. In the AVPV, during the oestrous cycle, the hybridization signal significantly increased at oestrus. E2 and E2 + P treatments of OVX rats did not induced significant variation of levels of GalR1 mRNAs in ERalpha-IR neurones. In the MPN, E2 treatment of OVX rats resulted in significant increase in GalR1 mRNA expression in ERalpha-IR neurones. Similarly, levels of the GalR1 hybridization signal increased during afternoon of proestrus and oestrus. In the MPO, treatment of OVX rats with E2 + P significantly decreased GalR1 mRNA expression in ERalpha-IR neurones. The expression of GalR1 mRNA did not change during the oestrous cycle in this area. These findings suggest that the hypothalamic action of galanin on gonadotopin-releasing hormone (GnRH) secretion may pass through the specific population of GalR1/ERalpha-IR neurones of the MPN in mediating the oestrogen action on the GnRH system at the moment of the luteinizing hormone surge.     

Evidence for Oestrogen Receptor α-Immunoreactivity in Gonadotrophin-Releasing Hormone-Expressing Neurones

Having used the cingulate cortex to demonstrate the validity of our methods for detecting hitherto unrecognized oestrogen receptor alpha (ERalpha)-immunoreactive neurones, we have now employed immunoprecipitation and double-label immunohistochemistry to investigate whether the ERalpha protein is present in gonadotrophin-releasing hormone (GnRH)-containing cells. The immortalized GnRH cell line GT1-7 and GnRH neurones within the rat preoptic area were found to possess ERalpha-immunoreactivity (ERalpha-IR). These observations indicate that oestrogen may regulate the synthesis and release of GnRH by direct actions on GnRH neurones.     

Oestrogen Receptor-α-Immunoreactive Neurones Project to the Suprachiasmatic Nucleus of the Female Syrian Hamster

Ovarian steroid hormones regulate circadian period and phase, but classical receptors for these hormones are absent in the circadian pacemaker localized in the suprachiasmatic nucleus of the hypothalamus (SCN). In order to determine whether effects of oestrogen may be exerted through steroid-binding systems afferent to the SCN we have performed double label immunocytochemistry for oestrogen receptor-alpha(ER-alpha) and the retrograde tracer cholera toxin B subunit (CtB) after its application to the SCN. Most of the areas that contain ER-alpha-immunoreactive (ERalpha-ir) cells also contained cells afferent to the SCN. The percentage of neurones afferent to the SCN which show ERalpha-immunoreactivity varies between areas. As many as one-third of the neurones afferent to the SCN in some parts of the preoptic area and the corticomedial amygdala are ERalpha-ir. Very few of the afferent neurones from the septum and the central grey are ERalpha-ir, whereas an intermediate proportion of afferents from the bed nucleus of the stria terminalis and the arcuate nucleus are ERalpha-ir. Our retrograde tracing results were compared with results of anterograde tracing from some of the sites containing SCN afferents. Using a combined retrograde and anterograde tracing technique we tested the possibility that single ERalpha-ir neurones afferent to the SCN could receive reciprocal innervation by SCN efferents. Although we found SCN input to some SCN afferent neurones, we found no evidence of reciprocity between single ERalpha-ir cells and the SCN. Our results indicate the existence of oestrogen binding systems afferent to the SCN. These neuroanatomical pathways may mediate effects of gonadal steroid hormones on circadian rhythms.     

Identification of neurones in the female rat hypothalamus that express oestrogen receptor-alpha and vesicular glutamate transporter-2

Oestrogen exerts its effects in the brain by binding to and activating two members of the nuclear receptor family, oestrogen receptor (ER)-alpha and ER-beta. Evidence suggests that oestrogen-receptive neurones participate in the generation of reproductive behaviours and that they convey the oestrogen message to gonadotropin-releasing hormone (GnRH) neurones. The aim of the present study was to identify the neurochemical phenotype of a subset of oestrogen receptor-expressing neurones. To this aim, we focused on the glutamate neuronal system, which is one of the most important stimulators of GnRH synthesis and release. We used the presence of vesicular glutamate transporter-2 (VGLUT2) mRNA as a specific marker to identify glutamate neurones and employed dual in situ hybridization to localize ERalpha mRNA-(35S-labelling) and VGLUT2 mRNA-(digoxigenin-labelling) expressing neurones within the hypothalamus. The results show that the overall distribution of VGLUT2 mRNA and ERalpha mRNA are consistent with previous data in the literature. Dual-labelled neurones were localized in the ventrolateral part of the ventromedial nucleus where 81.3 +/- 3.4% of the ERalpha mRNA containing neurones expressed VGLUT2 mRNA, in the anteroventral periventricular nucleus (30% colocalization) and in the medial preoptic nucleus (19% colocalization). Only 4.4% of the ERalpha expressing neurones in the arcuate nucleus contained VGLUT2 mRNA. These findings reveal that certain subpopulations of oestrogen-receptive neurones are glutamatergic in select hypothalamic areas that are known to regulate reproductive behaviour and GnRH neurones in the female rat. Thus, the oestrogen signal could be propagated through glutamate neurones to distant sites and influence the activity of the postsynaptic neurones.     

Modulation of oestrogen receptor-beta mRNA expression in rat paraventricular and supraoptic nucleus neurones following adrenal steroid manipulation and hyperosmotic stimulation

Magnocellular neurosecretory neurones in the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei express oestrogen receptor beta (ERbeta) but not ERalpha. In the PVN, ERbeta is strongly expressed in the ventromedial parvocellular neurones projecting to the brainstem. We used quantitative in situ hybridization, with (35)S-labelled riboprobes, to study heterologous regulation by manipulating adrenal steroid hormones (72 h after adrenalectomy +/- corticosterone replacement; repeated stress: halothane inhalation, environmental cold, immobilization, each daily for 3 days) in male rats. Adrenalectomy increased ERbeta mRNA expression in the magnocellular PVN and SON, by 2.2 and 2.5-fold, respectively, with no effect in the ventromedial parvocellular PVN neurones. Corticosterone replacement partially prevented the increases in ERbeta mRNA expression in magnocellular PVN and SON neurones. Repeated stress over 72 h had no effect on ERbeta mRNA expression in the magnocellular PVN or SON, but increased expression 1.4-fold in the ventromedial parvocellular PVN neurones. Although consequences of hydromineral balance derangement after adrenalectomy may stimulate magnocellular neurones, strongly stimulating the neurones by giving intact male rats 2% saline to drink for 72 h decreased ERbeta mRNA expression in the magnocellular PVN and SON neurones by approximately 60%, and in the ventromedial parvocellular PVN neurones by 13%. Thus, ERbeta mRNA expression is negatively regulated by basal glucocorticoid secretion in magnocellular PVN and SON neurones, and positively regulated by stress in ventromedial parvocellular PVN neurones. However, ERbeta mRNA expression in magnocellular neurones is negatively linked to hyperosmotic stimulation of the neurones. The 6.25-fold variation in ERbeta mRNA expression in magnocellular neurones from salt-loading to adrenalectomy could alter their sensitivity to oestrogens. Consequently, regulation of oxytocin and vasopressin neurone activity via ERbeta is expected to vary according to their functional state and, in particular, on basal glucocorticoid actions.     

Comparative distribution of estrogen receptor alpha and beta immunoreactivities in the forebrain and the midbrain of the female guinea pig

Estrogen plays an important role in regulating gonadotropin secretion and reproductive behavior. The estrogen receptor alpha (ERalpha) was believed to be the only receptor which mediated the actions of the hormone until the identification of a novel ER called ERbeta. In the present study, the map of ERalpha immunoreactive (IR) neurons was compared with the distribution pattern of ERbeta-IR neurons in the forebrain and midbrain of ovariectomized guinea pigs using immunocytochemistry. The immunoreactivities appeared to be mainly nuclear in their subcellular distribution. Both ERalpha- and ERbeta-like immunoreactivities were highly expressed in the bed nucleus of the stria terminalis and the ventrolateral hypothalamic nucleus but were found to be differentially expressed in discrete subregions of the amygdaloid complex. A large number of intensely labeled ERalpha cells were observed throughout the rostrocaudal extent of the preoptic region, whereas only a few ERbeta-IR neurons were found in the periventricular preoptic nucleus bordering the third ventricle or scattered in the medial preoptic area. In contrast, only ERalpha-immunoreactivity was seen in the septum, and in the magnocellular supraoptic, paraventricular, arcuate, and premammillary nuclei. In the midbrain, neurons containing ERalpha were observed throughout the rostrocaudal extent of the gray matter, whereas ERbeta was only detected within the dorsal raphe nucleus. These observations provide evidence of a distinct neuroanatomical pattern for the two subtypes of the ER which may have different roles in regulating behavior and the neuroendocrine mechanisms of reproduction. Species similarities and differences in the distributions of ERalpha and ERbeta immunoreactivities are discussed.     

Suprachiasmatic nucleus in the mouse: retinal innervation, intrinsic organization and efferent projections

The suprachiasmatic nucleus (SCN) is the principal circadian pacemaker of the mammalian circadian timing system. The SCN is composed of two anatomically and functionally distinct subdivisions, designated core and shell, which can be distinguished on the basis of their chemoarchitecture and connections in the rat. In the present study, we examine the intrinsic organization and the afferent and efferent connections of the mouse SCN using immunocytochemistry and ocular injections of cholera toxin. Neurons of the SCN shell contain GABA, calbindin (CALB), arginine vasopressin (AVP), angiotensin II (AII) and met-enkephalin (mENK), and receive input from galanin (GAL) and vasoactive intestinal polypeptide (VIP) immunoreactive fibers. Neurons of the SCN core synthesize GABA, CALB, VIP, calretinin (CALR), gastrin releasing peptide (GRP), and neurotensin (NT), and receive input from the retina and from fibers that contain neuropeptide Y (NPY) and 5-hydroxytryptamine (5HT). Fibers projecting from SCN neurons that are immunoreactive for AVP and VIP exhibit a characteristic morphology, and project to the lateral septum, a series of medial hypothalamic areas extending from the preoptic to the posterior hypothalamic area and to the paraventricular thalamic nucleus. The organization of the mouse SCN, and its connections, are similar to that in other mammalian species.     

The distribution and origin of the calretinin-containing innervation of the nucleus accumbens of the rat

The nucleus accumbens of the rat consists of several subregions that can be distinguished on the basis of histochemical markers. For example, the calcium-binding protein calbindin D28k is a useful marker of the core compartment of the nucleus accumbens. Calretinin, another calcium-binding protein, is found in a dense fibre plexus in the accumbal shell and septal pole regions. The source of the accumbal calretinin innervation is not known. We examined the distribution of calretinin in the nucleus accumbens and used tract-tracing and lesion methods to determine the source of this calretinin innervation. Intense calretinin immunoreactivity was present in the medial shell, but the density of calretinin axons diminished sharply in the ventrolateral shell. Regions of dense calretinin immunostaining and those areas with calbindin-like immunoreactive cell bodies were generally segregated in the nucleus accumbens, although some overlap in the transition region between the core and shell was seen. Small clusters of calretinin-immunoreactive fibres were seen in the core, where they were restricted to calbindin-negative patches. Injections of the anterograde tracer biotinylated dextran amine into the paraventricular thalamic nucleus labelled fibres in calretinin-rich regions of the accumbens. Conversely, injections of Fluoro-gold into the accumbal shell retrogradely labelled numerous cells in the paraventricular thalamic nucleus that were calretinin-immunoreactive. Electrolytic lesions of the paraventricular thalamic nucleus reduced calretinin levels in the shell by approximately 80%. These data indicate that the calretinin innervation of the nucleus accumbens is derived primarily from the thalamic paraventricular nucleus, and marks accumbal territories that are largely complementary to those defined by calbindin.     

Expression of estrogen receptor -alpha and -beta immunoreactivity in the cultured neonatal suprachiasmatic nucleus: with special attention to GABAergic neurons.

This study investigated the expression patterns of estrogen receptor -alpha (ERalpha) and -beta (ERbeta) in cultured cells of the suprachiasmatic nucleus (SCN) in neonatal rats by combined application of cell culture and double-label immunocytochemistry. The results revealed that the immunoreactivity for either ERalpha or ERbeta (with predominance of ERbeta) was localized in not only neurons but also astrocytes. The co-expression of both ERalpha and ERbeta in the same individual cell was also demonstrated by the double-label immunocytochemistry. The observations also provide a direct evidence for the differential expression of ER subtypes within GABAergic SCN neurons in vitro and suggest that estrogen's effect on the SCN may be mediated at least in part by its ER-containing GABAergic neurons.     

Oestrogen receptor beta-immunoreactive neurones in the ovine hypothalamus: distribution and colocalisation with gonadotropin-releasing hormone

Oestrogen powerfully affects the secretion of gonadotropin-releasing hormone (GnRH) from the brain in all species investigated, including sheep. Until recently, it was hypothesised that such regulation occurs indirectly because few or no GnRH neurones were found to express oestrogen receptor (ER) alpha. The discovery of a second oestrogen receptor, ERbeta, and its subsequent localisation in numerous GnRH neurones in the rat, led to a reconsideration of this hypothesis. However, colocalisation of immunoreactive ERbeta protein in GnRH neurones has only been demonstrated in the rat, raising the possibility that such putative direct regulation of GnRH neurones by oestrogen may be peculiar to this species. We have previously shown that steroid receptors in the sheep brain are acutely sensitive to fixation and the full complement of immunoreactive cells can only be visualised after antigen retrieval. The aims of this study were therefore to map immunocytochemically the distribution of ERbeta neurones in the ewe brain, and to determine which proportion of GnRH neurones express ERbeta. Brain sections (20 microm) from four ewes killed in anestrus were subjected to high temperature antigen retrieval and immunocytochemistry. Numerous ERbeta-immunoreactive cells were located throughout the hypothalamus and, following dual-label immunocytochemistry, over 50% of the GnRH neurones were found to express immunoreactive ERbeta. The functional significance of these ERbeta-expressing GnRH neurones in the ovine brain remains to be determined.     

Topographic organization of suprachiasmatic nucleus projection neurons

The mammalian circadian pacemaker, the hypothalamic suprachiasmatic nucleus (SCN), has two subdivisions. The core is located above the optic chiasm, receives primary and secondary visual afferents, and contains neurons producing vasoactive intestinal polypeptide and gastrin-releasing peptide. The shell largely surrounds the core, receives input from nonvisual sources and contains neurons producing arginine vasopressin and calretinin. In this study, we tested the hypothesis that SCN efferent projections are topographically organized with respect to the subdivision of origin. Injections of retrograde tracers were placed in major sites of efferent termination, described from prior studies that used anterograde tracers (Watts and Swanson, [1987] J. Comp. Neurol. 258:230-252; Watts et al. [1987] J. Comp. Neurol. 258:204-229). After retrograde tracer injections in the medial preoptic area, dorsomedial and paraventricular hypothalamic nuclei, bed nucleus of stria terminalis, paraventricular thalamic nucleus, zona incerta, and medial subparaventricular zone, retrogradely labeled SCN cells are clustered in the shell with few labeled neurons in the core. After injections centered in the lateral subparaventricular zone, peri-suprachiasmatic region, lateral septum, or ventral tuberal area, the majority of neuronal label is in the core with moderate to sparse neuronal label in the shell. Both subdivisions are labeled after injections in the paratenial thalamic nucleus. The same pattern of retrograde labeling is found with four tracers, cholera toxin-beta subunit, Fluoro-Gold, the Bartha strain of pseudorabies virus, and biotinylated dextran amine. These data extend our understanding of the significance of the division of the SCN into shell and core by demonstrating that the subdivisions differ in the pattern of projections. Together with prior observations that the subdivisions differ with respect to afferents, local connections, and neuroactive substances, the present study provides an anatomic basis for discrete control of circadian function by the SCN core and shell. In this novel view, the nature of the signal conveyed to areas receiving core or shell projections varies as a function of the subdivision from which innervation is derived.     

Transgenic approach reveals expression of the VPAC2 receptor in phenotypically defined neurons in the mouse suprachiasmatic nucleus and in its efferent target sites

Circadian rhythms in mammals depend on the properties of cells in the suprachiasmatic nucleus (SCN). The retino-recipient core of the mouse SCN is characterized by vasoactive intestinal peptide (VIP) neurons. Expression within the SCN of VPAC2, a VIP receptor, is required for circadian rhythmicity. Using transgenic mice with beta-galactosidase as a marker for VPAC2, we have phenotyped VPAC2-expressing cells within the SCN and investigated expression of the VPAC2 marker at sites previously shown to receive VIP-containing SCN efferents. In situ hybridization and immunohistochemistry demonstrated identical distributions for VPAC2 mRNA and beta-galactosidase and coexpression of the two signals in the SCN. Double-label confocal immunofluorescence identified beta-galactosidase in 32% of the VIP and 31% of the calretinin neurons in the SCN core. Of the arginine-vasopressin neurons that characterize the SCN shell, 45% expressed beta-galactosidase. In contrast, this marker was not apparent in astrocytes within the SCN core or shell. Cell bodies containing beta-galactosidase were detected at sites reportedly receiving VIP-containing SCN efferents, including the subparaventricular zone and lateral septum and the anteroventral periventricular, preoptic suprachiasmatic, medial preoptic and paraventricular hypothalamic nuclei. The detection of a marker for VPAC2 expression in the SCN in almost one-third of the VIP and calretinin core neurons and nearly half of the arginine-vasopressin shell neurons and also in cell bodies at sites receiving VIP-immunoreactive projections from the SCN indicates that VPAC2 may contribute to autoregulation and/or coupling within the SCN core and to the control of the SCN shell and sites distal to this nucleus.     

Effects of organisational oestradiol on adult immunoreactive oestrogen receptors (alpha and beta) in the male mouse brain

Steroid hormones act on developing neural circuits that regulate the hypothalamic-pituitary-gonadal axis and are involved in hormone-sensitive behaviours. To test the hypothesis that developmental exposure to oestradiol (E(2)) organises the quantity of adult oestrogen receptors (ERalpha and ERbeta), we used male mice with a targeted mutation of the aromatase enzyme gene (ArKO) and their wild-type (WT) littermates. These mice are unable to aromatise testosterone to E(2), but still express both ERalpha and beta. To evaluate adult responsiveness to E(2), gonadectomised males were implanted with Silastic capsules containing E(2), or an empty implant, 5 days prior to sacrifice. Immunoreactivity for ERalpha and ERbeta was quantified in the caudal ventromedial nucleus (VMN) and the medial preoptic area (POA). Regardless of genotype, adult treatment with E(2) reduced ERalpha-immunoreactive (ir) and ERbeta-ir cell numbers in the POA, as well as ERbeta-ir, but not ERalpha-ir, cell numbers in the VMN. Genotype, and thus endogenous exposure to E(2), produced opposite effects on ER expression in the two brain areas. In the VMN, ArKO males had more ERalpha-ir and ERbeta-ir cells than did WT males. In the POA, ArKO males had fewer ERalpha-ir and ERbeta-ir cells than did WT males. Thus, numbers of immunoreactive neurones containing both ERs in the adult ArKO male were enhanced in the POA, but decreased in the VMN, and most likely these patterns were established during the developmental critical period. Furthermore, although both ERalpha and beta-ir cell numbers are altered by the disruption of the aromatase gene, ERbeta is altered in a more robust and region-specific manner.     

Intraspecific variation in estrogen receptor alpha and the expression of male sociosexual behavior in two populations of prairie voles

Estrogen (E) regulates a variety of male sociosexual behaviors. We hypothesize that there is a relationship between the distribution of estrogen receptor alpha (ERalpha) and the degree of male social behavior. To test this hypothesis, ERalpha immunoreactivity (IR) was compared in prairie voles (Microtus ochrogaster) from Illinois (IL), which are highly social, and Kansas (KN), which are less social. The expression of androgen receptors (AR) in males also was compared between populations. The expression of ERalpha and AR were compared in brains from KN and IL males and females using immunocytochemistry (ICC). There were significant intrapopulational differences, with males expressing less ERalpha-IR than females in the medial preoptic area, ventromedial nucleus, ventrolateral portion of the hypothalamus, and bed nucleus of the stria terminalis (BST). IL males also displayed less ERalpha-IR in the medial amygdala (MeA) than IL females. While IL males expressed significantly less ERalpha-IR in the BST and MeA than KN males, there was no difference in AR-IR. Differences in the pattern of ERalpha-IR between KN and IL males were behaviorally relevant, as low levels of testosterone (T) were more effective in restoring sexual activity in castrated KN males than IL males. The lack of difference in AR combined with lower expression of ERalpha-IR in IL males suggests that behavioral differences in response to T are associated with aromatization of T to E and that reduced sensitivity to E may facilitate prosocial behavior in males.     

Immunocytochemical Evidence for Oestrogen Receptors within GABA Neurones Located in the Perinuclear Zone of the Supraoptic Nucleus and GABAA Receptor β2/β3 Subunits on Supraoptic Oxytocin Neurones

The mechanisms by which oestrogen modulates the biosynthetic and secretory activity of magnocellular oxytocin neurones are poorly understood. Using an antibody directed against the oestrogen receptor (ER), the distribution of ER-immunoreactive (-IR) cells in relation to the supraoptic nucleus (SON) was examined. Although no ER-IR cells were detected within the SON, a small population of immunoreactive cells separate from those in the preoptic area was identified in the perinuclear zone of the SON. Double-labelling experiments with an antibody specific for glutamic acid decarboxylase (GAD), the neuronal enzyme producing gamma aminobutyric acid (GABA), revealed that approximately 60% of perinuclear zone ER-IR cells contained GAD. A further set of immunocytochemistry experiments using an antibody raised against the β₂. and β₃ sub-units of the GABA_A receptor revealed immunoreactivity in the SON. Double-labelling experiments demonstrated that both oxytocin-IR and non-oxytocin-IR neurones in the SON were immunoreactive for β₂ and/or β₃ sub-units of the GABA_A receptor. These studies have identified ERs within a GABAergic neural population in the perinuclear zone of the SON and shown that magnocellular oxytocin neurones in the SON possess GABA_A receptors comprised of β₂ and/or β₃ sub-units. In conjunction with previous evidence that the perinuclear zone GABA neurones are an important source of GABA terminals in the SON, these results provide a morphological basis for the hypothesis that perinuclear zone GABA neurones may be part of a steroid-sensitive neural circuitry transmitting oestrogen input to oxytocin neurones in the SON.     

Double oestrogen receptor alpha and beta knockout mice reveal differences in neural oestrogen-mediated progestin receptor induction and female sexual behaviour

To test the hypothesis that oestrogen receptor alpha (ERalpha) and ERbeta act together to mediate the actions of oestrogen in the ventromedial hypothalamus (VMH), we used mice with single or double knockout mutations of the ERalpha and ERbeta genes. Ovariectomized mice were implanted with 17beta-oestradiol and killed 5 days later. Oestradiol treatment promoted progestin receptor (PR)-immunoreactivity (-ir) in the VMH of all genotypes, but was maximal in brains of wild-type and ERbetaKO females. Analysis of specific VMH subregions revealed that PR-ir induction was limited to the caudal VMH in ERalphaKO and ERalphabetaKO mice. In the rostral VMH, oestradiol only induced PR-ir in wild-type and ERbetaKO mice, and the number of PR-ir neurones in this region was greater in ERbetaKO than wild-type females. Next, we tested the ability of a dopamine agonist and progesterone to facilitate sexual behaviour in females lacking functional ERalpha, ERbeta, or both receptors. Ovariectomized mice were implanted with oestradiol, and tested for sexual behaviour three times after administration of the dopamine agonist, apomorphine, followed by two tests concurrent with progesterone treatment and a final test with just apomorphine treatment. ERalphaKO and ERalphabetaKO females failed to display lordosis under any testing conditions, while ERbetaKO females exhibited lordosis behaviour equal to that of wild-type females. Our data show that a subpopulation of PR-ir neurones is induced by oestradiol in the caudal VMH of female mice lacking both ERalpha and ERbeta genes. We hypothesize that this action of oestradiol is either mediated by a novel ER or by the mutant portion of the AF2 subregion of the ERalpha gene present in ERalphaKO brain. However, despite the presence of PR in VMH, females lacking a functional ERalpha gene do not display sexual behaviour, via either ligand-dependent or -independent activation.     

Evidence for Suprachiasmatic Vasopressin Neurones Innervating Kisspeptin Neurones in the Rostral Periventricular Area of the Mouse Brain: Regulation by Oestrogen

In rodents, a circadian signal from the suprachiasmatic nucleus (SCN) is essential for the pro‐oestrous surge of gonadotrophin‐releasing hormone (GnRH), which, in turn, induces luteinising hormone (LH) surge and ovulation. We hypothesised that kisspeptin (KP) neurones in the anteroventral periventricular and periventricular preoptic nuclei (AVPV/PeN) form part of the communication pathway between the SCN and GnRH neurones. In anterograde track tracing studies, we first identified vasopressin (VP)‐containing axons of SCN origin in apposition to KP‐immunoreactive (IR) neurones. Studies to quantify this input relied on the observation that VP‐synthesising neurones in the SCN differ from other VP systems in their lack of galanin expression. In ovariectomised mice, 30.79 ± 1.63% of KP‐IR perikarya and proximal dendrites within the AVPV/PeN received galanin‐negative VP‐IR varicosities. Oestrogen‐treatment significantly increased the number of KP‐IR neurones, with their percentage apposed by galanin‐negative VP‐IR varicosities (46.95 ± 1.88%) and the number of VP‐IR appositions on individual KP‐IR neurones. At the ultrastructural level, the VP‐IR terminals formed symmetric synapses with KP‐IR neurones, which was in accordance with the morphology of inhibitory synapses established by SCN neurones. By contrast to VP, vasoactive intestinal polypeptide (VIP), which is synthesised by a distinct subset of SCN neurones, occurred only rarely in axons apposed to KP‐IR neurones. Altogether, our results are consistent with the hypothesis that KP neurones located in the mouse AVPV/PeN receive circadian information from the SCN via a vasopressinergic monosynaptic pathway, which is enhanced by oestrogen.     

Temporal and spatial expression patterns of canonical clock genes and clock-controlled genes in the suprachiasmatic nucleus

In mammals, the suprachiasmatic nuclei (SCN) of the hypothalamus control endogenous circadian rhythms and entrainment to the environment. A core SCN region of calbindin (CalB)-containing cells is retinorecipient and the cells therein lack rhythmic expression of clock genes and electrical activity. The core is surrounded by a 'shell' of rhythmic oscillator cells. In the present experiments, we studied the spatial arrangement of oscillator cells by examining the spatial and temporal patterns of expression of the canonical clock genes Per1, Per2 and vasopressin mRNA, a clock-controlled gene. Surprisingly, in the SCN shell, the dorsomedial cells were the first to rhythmically express both Per1 and VP mRNA, with gene expression then spreading very slowly through much of the nucleus for the next 12 h then receding to baseline levels. Following a light pulse, Per expression increased after 1 h in the core SCN and after 1.5 h in the shell. Although expression in the shell occurred earlier in light-pulsed animals than in those housed in constant darkness, it still followed the same spatial and temporal expression pattern as was observed in constant darkness. The results suggest that not only is the SCN organized into light-responsive and rhythmic regions but also that the rhythmic region of the SCN itself has an ordered arrangement of SCN oscillator cells.     

Calcium-binding proteins in the circadian centers of the common marmoset (Callithrix jacchus) and the rock cavy (Kerodon rupestris) brains

The hypothalamic suprachiasmatic nucleus (SCN) and the thalamic intergeniculate leaflet (IGL) are considered to be the main centers of the mammalian circadian timing system. In primates, the IGL is included as part of the pregeniculate nucleus (PGN), a cell group located mediodorsally to the dorsal lateral geniculate nucleus. This work was carried out to comparatively evaluate the immunohistochemical expression of the calcium-binding proteins calbindin D-28k (CB), parvalbumin (PV), and calretinin (CR) into the circadian brain districts of the common marmoset and the rock cavy. In both species, although no fibers, terminals or perikarya showed PV-immunoreaction (IR) into the SCN, CB-IR perikarya labeling was detected throughout the SCN rostrocaudal extent, seeming to delimit its cytoarchitectonic borders. CR-IR perikarya and neuropil were noticed into the ventral and dorsal portions of the SCN, lacking immunoreactivity in the central core of the marmoset and filling the entire nucleus in the rock cavy. The PGN of the marmoset presented a significant number of CB-, PV-, and CR-IR perikarya throughout the nucleus. The IGL of the rocky cavy exhibited a prominent CB- and CR-IR neuropil, showing similarity to the pattern found in other rodents.By comparing with literature data from other mammals, the results of the present study suggest that CB, PV, and CR are differentially distributed into the SCN and IGL among species. They may act either in concert or in a complementary manner in the SCN and IGL, so as to participate in specific aspects of the circadian regulation.     

Estrogen receptor alpha and vasopressin in the paraventricular nucleus of the hypothalamus in Peromyscus

The purpose of this study was to determine the presence of estrogen receptor alpha (ERalpha) and the relationship between neurons that express ERalpha and produce vasopressin (AVP) in the paraventricular nucleus of the hypothalamus (PVN) in new world mice of the genus Peromyscus. Brains were collected from male and female Peromyscus californicus, Peromyscus leucopus, Peromyscus maniculatus, and Peromyscus polionotus, and double labeled for the expression of ERalpha and AVP immunoreactivity (IR). The number of cells expressing ERalpha-IR and AVP-IR was determined in the medial and posterior region of the PVN. The results indicate that Peromyscus is the first taxonomic group reported to have ERalpha widely distributed in the PVN, occurring in both medial and posterior regions of the PVN. While estrogen can regulate the production of AVP, AVP and ERalpha were rarely colocalized. There was, however, a significant inverse relationship between the number of cells that expressed ERalpha-IR and the number expressing AVP-IR. There were no sex differences in the expression of ERalpha-IR or AVP-IR.