Imre Kalló

Oestrogen Receptor β-Immunoreactivity in Gonadotropin Releasing Hormone-Expressing Neurones: Regulation by Oestrogen

Double‐label immunohistochemistry was employed to establish whether immunoreactivity for the β subtype of the oestrogen receptor (ERβ‐IR) is present in gonadotropin releasing hormone (GnRH)‐containing cells. In the immortalized GnRH cell line, GT1‐7, almost all nuclei were immunoreactive for ERβ. In the preoptic area of ovariectomized rats, more than one‐half of the GnRH neurones (52.0–63.5%) contained ERβ‐IR within the nucleus; a smaller proportion of these neurones (5–10%) displayed a particularly intense nuclear signal for ERβ. The presence of ERβ‐IR in the nuclei of GT1‐7 cells and GnRH neurones is consistent with recent reports of ERβ mRNA in these cells. Oestrogen treatment reduced the percentage of GnRH neurones with detectable ERβ‐IR. The range of signal intensity for ERβ and the incidence of the ERβ signal in GnRH neurones were comparable following double‐label immunohistochemistry involving either bright field or fluorescent techniques. These findings raise the possibility that ERβ receptors mediate direct effects of oestrogen on GnRH neurones.

Estrogen receptor-beta in oxytocin and vasopressin neurons of the rat and human hypothalamus: Immunocytochemical and in situ hybridization studies

Topographical distribution of estrogen receptor‐β (ER‐β)‐synthesizing oxytocin (OT) and vasopressin (VP) neurons was studied in the hypothalamic paraventricular and supraoptic nuclei (PVH; SO) of ovariectomized rats. In distinct subregions, 45–98% of OT neurons and 88–99% of VP neurons exhibited ER‐β immunoreactivity that was confined to cell nuclei. Neuronal populations differed markedly with respect to the intensity of the ER‐β signal. Magnocellular OT neurons in the PVH, SO, and accessory cell groups typically contained low levels of the ER‐β signal; in contrast, robust receptor labeling was displayed by OT cells in the ventral subdivision of medial parvicellular subnucleus and in the caudal PVH (dorsal subdivision of medial parvicellular subnucleus and lateral parvicellular subnucleus). Estrogen receptor‐β signal was generally more intense and present in higher proportions of magnocellular and parvicellular VP vs. OT neurons of similar topography. Immunocytochemical observations were confirmed via triple‐label in situ hybridization, an approach combining use of digoxigenin‐, fluorescein‐, and 35S‐labeled cRNA hybridization probes. Further, ER‐β mRNA was also detectable in corticotropin‐releasing hormone neurons in the parvicellular PVH. Finally, double‐label immunocytochemical analysis of human autopsy samples showed that subsets of OT and VP neurons also express ER‐β in the human. These neuroanatomical studies provide detailed information about the topographical distribution and cellular abundance of ER‐β within subsets of hypothalamic OT and VP neurons in the rat. The variable receptor content may indicate the differential responsiveness to estrogen in distinct OT and VP neuronal populations. In addition, a relevance of these findings to the human hypothalamus is suggested. J. Comp. Neurol. 473:315–333, 2004.

Oestrogen Receptor α and β Immunoreactive Cells in the Suprachiasmatic Nucleus of Mice: Distribution, Sex Differences and Regulation by Gonadal Hormones

Oestrogen regulates various aspects of circadian rhythm physiology. The presence of oestrogen receptors within the suprachiasmatic nucleus (SCN), the principal circadian oscillator, indicates that some actions of oestrogen on circadian functions may be exerted at that site. The present study analysed sex differences, topographic distribution, and neurochemical phenotype of neurones expressing the α and β subtypes of oestrogen receptors (ERα and ERβ) in the mouse SCN. We found that relatively few neurones in the SCN are immunoreactive (IR) for ERα (approximately 4.5% in females and 3% in males), but five‐ to six‐fold more SCN neurones express ERβ. ER‐IR neurones are primarily in the shell subdivision of the nucleus and show differences between the sexes, significantly greater numbers being found in females. Treatment of male or female gonadectomised mice with oestradiol benzoate for 24u2003h substantially reduced the number of ERβ‐IR neurones, but not ERα‐IR neurones. Double‐labelling immunocytochemical experiments to characterise the phenotype of the oestrogen‐receptive neurones showed the presence of the calcium‐binding proteins calretinin or calbindin D28K in approximately 12% and 10%, respectively, of ERα‐IR neurones. A higher proportion (approximately 38%) of ERβ‐IR neurones contains calbindin D28K; a few (approximately 2%) express calretinin or vasopressin. These double‐labelled cells appear primarily in the shell subdivision of the SCN. Neither vasoactive intestinal polypeptide‐ nor gastrin releasing peptide‐immunoreactivity was observed in ER‐IR neurones. These data indicate that the primary target cells for oestrogen are in the shell subdivision of the nucleus. The sexually differentiated expression and distribution of ERα and ERβ in various cell populations of the SCN suggest multiple modes of oestrogen signalling within this nucleus, which may modulate circadian functions.

Expression of VIP and/or PACAP receptor mRNA in peptide synthesizing cells within the suprachiasmatic nucleus of the rat and in its efferent target sites

The suprachiasmatic nucleus (SCN) contains the predominant circadian pacemaker in mammals. Considerable evidence indicates that VPAC2 and PAC1, receptors for vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase‐activating peptide (PACAP), play critical roles in maintaining and entraining circadian rhythms. Retinal projections to the rat SCN contain PACAP and terminate mostly in the ventral SCN, the site of VIP neurons. The incidence of VPAC2 and PAC1 mRNAs within distinct neuronal populations of the rat SCN has been determined using double‐label in situ hybridization. VPAC2 mRNA was detected in almost all arginine‐vasopressin (AVP) neurons of the dorsomedial SCN and in 41% of the VIP neurons; somatostatin (SST) neurons, predominantly in dorsomedial and intermediate regions, showed a decreased incidence (23%). PAC1 mRNA was present in nearly half of the VIP and SST neurons (45% and 40%, respectively) and in one‐third of the AVP neurons (32%). Cells expressing VPAC2 mRNA also were detected in diencephalic areas that receive VIP‐immunoreactive SCN efferents, such as the peri‐suprachiasmatic region, lateral subparaventricular zone, parvocellular hypothalamic paraventricular subdivisions, dorsomedial hypothalamic nucleus, and anterior thalamic paraventricular and paratenial nuclei. The extensive distribution of PAC1 mRNA within the SCN suggests that actions of PACAP are not restricted to the predominantly retinorecipient region. The presence of VPAC2 mRNA in nearly half the VIP neurons, in almost all the AVP neurons, and at sites receiving VIP‐immunoreactive SCN efferents suggests that the SCN VIP neurons are coupled and/or autoregulated and also influence the AVP‐containing dorsomedial SCN and distal sites via VPAC2. J. Comp. Neurol. 475:19–35, 2004.

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 β‐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 β‐galactosidase and coexpression of the two signals in the SCN. Double‐label confocal immunofluorescence identified β‐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 β‐galactosidase. In contrast, this marker was not apparent in astrocytes within the SCN core or shell. Cell bodies containing β‐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.

Cellular expression of V1a vasopressin receptor mRNA in the female rat preoptic area: effects of oestrogen.

The preovulatory luteinizing hormone (LH) surge in female rats is dependent upon signals from the suprachiasmatic nucleus (SCN), the site of a dominant circadian pacemaker. Various lines of evidence indicate that arginine‐vasopressin (AVP)‐containing projections from the SCN to the preoptic area (POA) contribute to the production of the surge of LH‐releasing hormone (LHRH). These actions may be mediated by V1a because the transcript for this AVP receptor subtype is predominant within the POA of the female rat. In this study, in situ hybridization histochemistry was used to examine V1a mRNA expression, either by itself or together with LHRH or glutamic acid decarboxylase 65 (GAD65) mRNA, within the POA of ovariectomized rats in the presence or absence of oestrogen. V1a mRNA was found in cells across the rostro‐caudal axis of the POA; some were in close proximity to cells expressing LHRH mRNA. Coexpression of V1a and LHRH mRNAs was detected only very rarely. By contrast, cells with V1a mRNA commonly displayed GAD65 mRNA. The density of V1a mRNA‐expressing cells was particularly high within the anteroventral periventricular nucleus; at this site, V1a mRNA expression was elevated following oestrogen treatment. The present results indicate that V1a‐mediated AVP actions may influence LHRH release via cells in the immediate vicinity of LHRH neurones and/or via oestrogen‐regulated cells in the anteroventral periventricular nucleus, which is a site that lacks LHRH neurones but plays an essential role in initiating the preovulatory LH surge.

Ageing and the diurnal expression of mRNAs for vasoactive intestinal peptide and for the VPAC2 and PAC1 receptors in the suprachiasmatic nucleus of male rats

Ageing alters fundamental aspects of circadian rhythmicity in mammals; the effects include reduced rhythm amplitude and alterations in period length and in entrainment to the light/dark cycle. Such changes may reflect disruptions in cellular function within the suprachiasmatic nucleus (SCN), the site of the predominant circadian pacemaker. In the SCN, vasoactive intestinal peptide (VIP)‐synthesizing neurones receive various inputs, including retinohypothalamic projections containing pituitary adenylate cyclase activating peptide (PACAP). SCN VIP cells establish connections with local neurones and send efferents beyond the nucleus. Considerable evidence implicates VIP and PACAP in circadian rhythm maintenance and/or entrainment to photic Zeitgebers. These actions involve members of a distinct family of receptors; mRNAs for two such receptors, VPAC2 and PAC1, are present in the SCN. This study used isotopic in situ hybridization to examine the effects of ageing on expression of mRNAs for VIP, VPAC2 and PAC1 in the SCN of male rats under a 12u2003:u200312u2003h light/dark cycle. Analysis of film autoradiographs from young adult (2–3u2003months) or aged (19–20u2003months) rats, at eight time points across the light/dark cycle, showed loss of diurnal rhythmicity and reduced levels for VIP mRNA in the aged group. A diurnal rhythm of VPAC2 receptor mRNA was present in both groups, but its levels were reduced in the aged rats. There were no differences between the two groups for PAC1 receptor mRNA expression. The present results indicate that ageing reduces VIP and VPAC2 receptor mRNA and eliminates diurnal expression of VIP mRNA within the SCN of aged male rats.

Evidence for extensive distribution of oestrogen receptor alpha-immunoreactivity in the cerebral cortex of adult rats

The presence of oestrogen receptor alpha (ERα)‐immunoreactivity (IR) in the cerebral cortex of adult rats has been investigated. Previous studies have reported a negligible presence of ERα or ERα mRNA in this region of the adult rat brain. We have used immunoprecipitation and immunohistochemistry, with various antibodies and fixatives, to detect this protein in the cingulate cortex. When the tissue was fixed using paraformaldehyde alone only faint ERα–IR was observed at this site. In contrast, following fixation either with acrolein (with or without paraformaldehyde) or with a mixture of paraformaldehyde and glutaraldehyde there was extensive ERα–IR throughout layers II to VI; this was absent when the antibodies were preincubated with the peptide fragment used in their production. The presence of ERα–IR in the nonfixed cingulate cortex of adult rats was confirmed by immunoprecipitation.

Oestrogen receptor-β and neurohypophysial hormones: Functional interaction and neuroanatomical localisation

Oestrogens affect fluid balance, influencing both ingestive behaviour and renal excretion. The renal effects are partly due to altered release of vasopressin and oxytocin. This study was designed to explore the role of oestrogen receptor-beta (ERbeta) in neurohypophysial hormonal function. Following dietary administration, soya isoflavones reach the brain in sufficient concentration to activate ERbeta, but not oestrogen receptor-alpha (ERalpha). ERbeta function was therefore manipulated by feeding rat diets differing in soya isoflavone content. Fluid balance and neurohypophysial hormone release were measured in male rats maintained for 14 days on a soya isoflavone-free diet or one containing 150 microg/g genistein+daidzein. Food and water intake, body weight, urine flow, osmolality and sodium concentrations were determined daily. After 14 days, plasma and urine osmolality and sodium, vasopressin and oxytocin concentrations were determined. There was no significant difference in weight gain between the two groups or in their excretion of sodium and water or plasma sodium and plasma oxytocin. However, plasma vasopressin was significantly lower in the iso-free group. Double-label immunocytochemistry was used to assess colocalisation of ERbeta with the neurohypophysial hormones in male rats. Cell nuclei showing ERbeta immunoreactivity were abundant in the posterior magnocellular paraventricular nucleus (PVNpm) and in the supraoptic nucleus (SON). Vasopressin-immunoreactive neurones were similarly distributed, forming the core of the PVNpm and the ventral portion of the SON; majority were positive for ERbeta. Cells with oxytocin immunoreactivity were located mainly at the periphery of the PVNpm and in the dorsal SON; only approximately a quarter of these cells showed ERbeta immunoreactivity. Thus, the difference in the effects of the soya diet on vasopressin and oxytocin release may be related to the ERbeta-activating properties of this diet and to the preponderance of this receptor in vasopressin as opposed to oxytocin cells.

Neonatal stimulation of 5-HT2 receptors reduces androgen receptor expression in the rat anteroventral periventricular nucleus and sexually dimorphic preoptic area

Masculinization of the brain is dependent upon a perinatal surge in testosterone. It also requires a transient decrease in hypothalamic 5‐HT concentration and turnover and an increase in androgen receptor (AR) expression during the second postnatal week. We have previously shown that increasing 5‐HT activity over this period in male or androgenized female rats feminizes their adult behaviour and also feminizes the size of their anteroventral periventricular nucleus (AVPV) and sexually dimorphic nucleus of the preoptic area (SDN‐POA). To investigate the role of 5‐HT in sexual differentiation of the brain, 5‐HT activity was raised over postnatal daysu20038–16 in male, female and androgenized female rats by daily administration of the 5‐HT2 receptor agonist (–)[2,5 dimethoxy‐4‐iodophenyl]‐2‐amino propane hydrochloride [(–)DOI]. By postnatal dayu200318, the size of the AVPV and SDN‐POA was sexually dimorphic; their sizes were feminized by (–)DOI treatment. In the absence of (–)DOI treatment, there were significantly more AR‐immunoreactive cells in the AVPV of males, and in the SDN‐POA of males and androgenized females, than in those of females on postnatal dayu200318. (–)DOI treatment reduced the number of AR‐immunoreactive cells in the AVPV and SDN‐POA of males and androgenized females, but not of females, by postnatal dayu200318. These results suggest that 5‐HT2 receptor activation can influence sexual differentiation of the brain by controlling AR expression.