Erik Hrabovszky

Estrogen Receptor-β Immunoreactivity in Luteinizing Hormone-Releasing Hormone Neurons of the Rat Brain

Abstract Feedback regulation of luteinizing hormone-releasing hormone (LHRH) neurons by estradiol plays important roles in the neuroendocrine control of reproduction. Recently, we found that the majority of LHRH neurons in the rat contain estrogen receptor-β (ER-β) mRNA, whereas, they seemed to lack ER-α mRNA expression. In addition, we observed nuclear uptake of 125I-estrogen by a subset of these cells. These data suggest that ER-β is the chief receptor isoform mediating direct estrogen effects upon LHRH neurons. To verify the translation of ER-β protein within LHRH cells, the present studies applied dual-label immunocytochemistry (ICC) to free-floating sections obtained from the preoptic area of rats. The improved ICC method using the silver-gold intensification of nickel-diaminobenzidine chromogen, enabled the observation of nuclear ER-β-immunoreactivity in the majority of LHRH cells. The incidence of ER-β expression was similarly high in LHRH neurons of ovariectomized female (87.8 ± 2.3%, mean ± SEM),...

Detection of Estrogen Receptor-β Messenger Ribonucleic Acid and 125I-Estrogen Binding Sites in Luteinizing Hormone-Releasing Hormone Neurons of the Rat Brain

Luteinizing hormone-releasing hormone (LHRH) neurons of the forebrain play a pivotal role in the neuroendocrine control of reproduction. Although serum estrogen levels influence many aspects of LHRH neuronal activity in the female, earlier studies were unable to detect estrogen receptors (ERs) within LHRH neurons, thus shaping a consensus view that the effects of estradiol on the LHRH neuronal system are mediated by interneurons and/or the glial matrix. The present studies used dual-label in situ hybridization histochemistry (ISHH) and combined LHRH-immunocytochemistry/125I-estrogen binding to readdress the estrogen-receptivity of LHRH neurons in the female rat. In ISHH experiments we found that the majority of LHRH neurons exhibited hybridization signal for the “β” form of ER (ER-β). The degree of colocalization was similar in topographically distinct populations of LHRH neurons and was not significantly altered by estradiol (67.2±1.8 % in ovariectomized and 73.8±4.2 % in ovariectomized and estradiol-tre...

The kisspeptin system of the human hypothalamus: sexual dimorphism and relationship with gonadotropin-releasing hormone and neurokinin B neurons.

Kisspeptin signaling via the kisspeptin receptor G‐protein‐coupled receptor‐54 plays a fundamental role in the onset of puberty and the regulation of mammalian reproduction. In this immunocytochemical study we addressed the (i) topography, (ii) sexual dimorphism, (iii) relationship to gonadotropin‐releasing hormone (GnRH) neurons and (iv) neurokinin B content of kisspeptin‐immunoreactive hypothalamic neurons in human autopsy samples. In females, kisspeptin‐immunoreactive axons formed a dense periventricular plexus and profusely innervated capillary vessels in the infundibular stalk. Most immunolabeled somata occurred in the infundibular nucleus. Many cells were also embedded in the periventricular fiber plexus. Rostrally, they formed a prominent periventricular cell mass (magnocellular paraventricular nucleus). Robust sex differences were noticed in that fibers and somata were significantly less numerous in male individuals. In dual‐immunolabeled specimens, fine kisspeptin‐immunoreactive axon varicosities formed axo‐somatic, axo‐dendritic and axo‐axonal contacts with GnRH neurons. Dual‐immunofluorescent studies established that 77% of kisspeptin‐immunoreactive cells in the infundibular nucleus synthesize the tachykinin peptide neurokinin B, which is known to play crucial role in human fertility; 56 and 17% of kisspeptin fibers in the infundibular and periventricular nuclei, respectively, contained neurokinin B immunoreactivity. Site‐specific co‐localization patterns implied that kisspeptin neurons in the infundibular nucleus and elsewhere contributed differentially to these plexuses. This study describes the distribution and robust sexual dimorphism of kisspeptin‐immunoreactive elements in human hypothalami, reveals neuronal contacts between kisspeptin‐immunoreactive fibers and GnRH cells, and demonstrates co‐synthesis of kisspeptins and neurokinin B in the infundibular nucleus. The neuroanatomical information will contribute to our understanding of central mechanisms whereby kisspeptins regulate human fertility.

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, 30.79u2003±u20031.63% 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 (46.95u2003±u20031.88%) 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.

Distribution of type 1 cannabinoid receptor (CB1)-immunoreactive axons in the mouse hypothalamus

Type 1 cannabinoid receptor (CB1) is the principal receptor for endocannabinoids in the brain; it mainly occurs in preterminal/terminal axons and mediates retrograde neuronal signaling mechanisms. A large body of physiological and electrophysiological evidence indicates the critical role of CB1 in the regulation of hypothalamic functions. Conversely, the distribution of CB1‐containing axons in the hypothalamus is essentially unknown. Therefore, we have analyzed the distribution and the ultrastructural characteristics of the CB1‐immunoreactive (IR) axons in the mouse hypothalamus by using an antiserum against the C‐terminal 31 amino acids of the mouse CB1. We found that CB1‐IR axons innervated densely the majority of hypothalamic nuclei, except for the suprachiasmatic and lateral mammillary nuclei, in which only scattered CB1‐IR fibers occurred. CB1‐IR innervation of the arcuate, ventromedial, dorsomedial, and paraventricular nuclei and the external zone of the median eminence corroborated the important role of CB1 in the regulation of energy homeostasis and neuroendocrine functions. Ultrastructural studies to characterize the phenotype of CB1‐IR fibers established that most CB1 immunoreactivity appeared in the preterminal and terminal portions of axons. The CB1‐IR boutons formed axospinous, axodendritic, and axosomatic synapses. Analysis of labeled synapses in the paraventricular and arcuate nuclei detected approximately equal numbers of symmetric and asymmetric specializations. In conclusion, the study revealed the dense and differential CB1‐IR innervation of most hypothalamic nuclei and the median eminence of the mouse brain. At the ultrastructural level, CB1‐IR axons established communication with hypothalamic neurons via symmetric and asymmetric synapses indicating the occurrence of retrograde signaling by endocannabinoids in hypothalamic neuronal networks. J. Comp. Neurol. 503:270–279, 2007.

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.

The amygdala as a neurobiological target for ghrelin in rats: neuroanatomical, electrophysiological and behavioral evidence.

Here, we sought to demonstrate that the orexigenic circulating hormone, ghrelin, is able to exert neurobiological effects (including those linked to feeding control) at the level of the amygdala, involving neuroanatomical, electrophysiological and behavioural studies. We found that ghrelin receptors (GHS-R) are densely expressed in several subnuclei of the amygdala, notably in ventrolateral (LaVL) and ventromedial (LaVM) parts of the lateral amygdaloid nucleus. Using whole-cell patch clamp electrophysiology to record from cells in the lateral amygdaloid nucleus, we found that ghrelin reduced the frequency of mEPSCs recorded from large pyramidal-like neurons, an effect that could be blocked by co-application of a ghrelin receptor antagonist. In ad libitum fed rats, intra-amygdala administration of ghrelin produced a large orexigenic response that lasted throughout the 4 hr of testing. Conversely, in hungry, fasted rats ghrelin receptor blockade in the amygdala significantly reduced food intake. Finally, we investigated a possible interaction between ghrelins effects on feeding control and emotional reactivity exerted at the level of the amygdala. In rats allowed to feed during a 1-hour period between ghrelin injection and anxiety testing (elevated plus maze and open field), intra-amygdala ghrelin had no effect on anxiety-like behavior. By contrast, if the rats were not given access to food during this 1-hour period, a decrease in anxiety-like behavior was observed in both tests. Collectively, these data indicate that the amygdala is a valid target brain area for ghrelin where its neurobiological effects are important for food intake and for the suppression of emotional (anxiety-like) behaviors if food is not available.

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.

Retrograde endocannabinoid signaling reduces GABAergic synaptic transmission to gonadotropin-releasing hormone neurons

Cannabinoids suppress fertility via reducing hypothalamic GnRH output. γ-Aminobutyric acid (GABA)A receptor (GABAA-R)-mediated transmission is a major input to GnRH cells that can be excitatory. We hypothesized that cannabinoids act via inhibiting GABAergic input. We performed loose-patch electrophysiological studies of acute slices from adult male GnRH-green fluorescent protein transgenic mice. Bath application of type 1 cannabinoid receptor (CB1) agonist WIN55,212 decreased GnRH neuron firing rate. This action was detectable in presence of the glutamate receptor antagonist kynurenic acid but disappeared when bicuculline was also present, indicating GABAA-R involvement. In immunocytochemical experiments, CB1-immunoreactive axons formed contacts with GnRH neurons and a subset established symmetric synapses characteristic of GABAergic neurotransmission. Functional studies were continued with whole-cell patch-clamp electrophysiology in presence of tetrodotoxin. WIN55,212 decreased the frequency of GABAA-R-mediated miniature postsynaptic currents (mPSCs) (reflecting spontaneous vesicle fusion), which was prevented with the CB1 antagonist AM251, indicating collectively that activation of presynaptic CB1 inhibits GABA release. AM251 alone increased mPSC frequency, providing evidence that endocannabinoids tonically inhibit GABAA-R drive onto GnRH neurons. Increased mPSC frequency was absent when diacylglycerol lipase was blocked intracellularly with tetrahydrolipstatin, showing that tonic inhibition is caused by 2-arachidonoylglycerol production of GnRH neurons. CdCl2 in extracellular solution can maintain both action potentials and spontaneous vesicle fusion. Under these conditions, when endocannabinoid-mediated blockade of spontaneous vesicle fusion was blocked with AM251, GnRH neuron firing increased, revealing an endogenous endocannabinoid brake on GnRH neuron firing. Retrograde endocannabinoid signaling may represent an important mechanism under physiological and pathological conditions whereby GnRH neurons regulate their excitatory GABAergic inputs.

Co-localisation of kisspeptin with galanin or neurokinin B in afferents to mouse GnRH neurones.

The gonadotrophin‐releasing hormone (GnRH) secreting neurones, which form the final common pathway for the central regulation of reproduction, are directly targeted by kisspeptin (KP) via the G protein‐coupled receptor, GPR54. In these multiple labelling studies, we used ovariectomised mice treated with 17β‐oestradiol (OVXu2003+u2003E2) or vehicle (OVXu2003+u2003oil) to determine: (i) the ultrastructural characteristics of KP‐immunoreactive (IR) afferents to GnRH neurones; (ii) their galanin or neurokinin B (NKB) content; and (iii) the co‐expression of galanin or NKB with KP in the two major subpopulations of KP neurones located in the rostral periventricular area of the third ventricle (RP3V) and the arcuate nucleus (Arc). Electron microscopic investigation of the neuronal juxtapositions revealed axosomatic and axodendritic synapses; these showed symmetrical or asymmetrical characteristics, suggesting a phenotypic diversity of KP afferents. Heterogeneity of afferents was also demonstrated by differential co‐expression of neuropeptides; in OVXu2003+u2003E2 mice, KP afferents to GnRH neurones showed galanin‐immunoreactivity with an incidence of 22.50u2003±u20032.41% and NKB‐immunoreactivity with an incidence of 5.61u2003±u20032.57%. In OVXu2003+u2003oil animals, galanin‐immunoreactivity in the KP afferents showed a major reduction, appearing in only 5.78u2003±u20031.57%. Analysis for co‐localisation of galanin or NKB with KP was extended to the perikaryal level in animal models, which showed the highest KP incidence; these were OVXu2003+u2003E2 females for the RP3V and OVXu2003+u2003oil females for the ARC. In the RP3V of colchicine‐treated OVXu2003+u2003E2 animals, 87.84u2003±u20032.65% of KP‐IR neurones were galanin positive. In the Arc of the colchicine‐treated OVXu2003+u2003oil animals, galanin immunoreactivity was detected in only 12.50u2003±u20031.92% of the KP expressing neurones. By contrast, the incidence of co‐localisation with NKB in the Arc of those animals was 98.09u2003±u20031.30%. In situ hybridisation histochemistry of sections from OVXu2003+u2003E2 animals identified galanin message in more than a third of the KP neurones in the RP3V (38.67u2003±u200311.57%) and in the Arc (42.50u2003±u200312.52%). These data suggest that GnRH neurones are innervated by chemically heterogeneous KP cell populations, with a small proportion deriving from the Arc group. The presence of galanin within KP axons innervating GnRH neurones and the oestrogen‐dependent regulation of that presence add a new dimension to the roles played by galanin in the central regulation of reproduction.