Arcuate Nucleus Of Female Rats Research Articles

ERαΔ4, an ERα splice variant missing exon4, interacts with caveolin-3 and mGluR2/3.

The two isoforms of the nuclear estrogen receptor, ERα and ERβ are widely expressed in the central nervous system. Although they were first described as nuclear receptors, both isoforms have also been found at the cell membrane where they mediate cell signaling. Surface biotinylation studies using neuronal and glial primary cultures label an alternatively spliced form of ERα. The 52kDa protein, ERαΔ4, is missing exon 4 and is highly expressed in membrane fractions derived from cultured cells. In vivo, both full-length (66kDa) ERα and ERαΔ4 are present in membrane fractions. In response to estradiol, full-length ERα and ERαΔ4 are initially trafficked to the membrane, and then internalized in parallel. Previous studies determined that only the full-length ERα associates with metabotropic glutamate receptor-1a (mGluR1a), initiating cellular signaling. The role of ERαΔ4, remained to be elucidated. Here, we report ERαΔ4 trafficking, association with mGluR2/3, and downstream signaling in female rat arcuate nucleus (ARH). Caveolin (CAV) proteins are needed for ER transport to the cell membrane, and using co-immunoprecipitation CAV-3 was shown to associate with ERαΔ4. CAV-3 was necessary for ERαΔ4 trafficking to the membrane: in the ARH, microinjection of CAV-3 siRNA reduced CAV-3 and ERαΔ4a in membrane fractions by 50%, and 60%, respectively. Moreover, co-immunoprecipitation revealed that ERαΔ4 associated with inhibitory mGluRs, mGluR2/3. Estrogen benzoate (EB) treatment (5μg; s.c.; every 4days; three cycles) reduced levels of cAMP, an effect attenuated by antagonizing mGluR2/3. Following EB treatment, membrane levels of ERαΔ4 and mGluR2/3 were reduced implying ligand-induced internalization. These results implicate ERαΔ4 in an estradiol-induced inhibitory cell signaling in the ARH.

Distinct dynorphin expression patterns with low- and high-dose estrogen treatment in the arcuate nucleus of female rats.

Kisspeptin (KISS1; encoded by Kiss1) neurons in the arcuate nucleus (ARC) coexpress tachykinin 3 (TAC3; also known as neurokinin B) and dynorphin A (PDYN). Accordingly, they are termed KNDy neurons and considered to be crucial in generating pulsatile release of gonadotropin-releasing hormone. Accumulating evidence suggests that Kiss1 and Tac3 are negatively regulated by estrogen. However, it has not been fully determined whether and how estrogen modulates Pdyn and PDYN. Here, we examined the expression of Pdyn mRNA and PDYN by in situ hybridization and immunohistochemistry, respectively, in the ARC of female rats after ovariectomy (OVX) and OVX plus low- or high-dose beta-estradiol (E2) replacement. We also investigated the effect of E2 on expression of Kiss1, KISS1, Tac3, and TAC3. Furthermore, colocalization of PDYN and estrogen receptor alpha (ESR1) was determined. Subsequently, we found that low-dose E2 treatment had no effect on Pdyn mRNA-expressing cells, but increased PDYN-immunoreactive (ir) cell numbers. In contrast, high-dose E2 treatment resulted in prominent reductions in both Pdyn mRNA-expressing and PDYN-ir cell numbers. Changes induced by low or high doses of E2 were similarly observed in the expression of Kiss1, KISS1, Tac3, and TAC3. The majority of PDYN-ir neurons coexpressed ESR1 in all groups. Our results indicate that E2 regulates the expression of PDYN, as well as KISS1 and TAC3, with regulation by E2 differing according to its levels.

Regulation of NPY and α-MSH expression by estradiol in the arcuate nucleus of Wistar female rats: a stereological study

Objectives: Feeding behavior in both animals and humans is modulated by estrogens, as shown by the increased adiposity observed in women and rats upon the drop of estradiol levels at menopause. Estradiol action on food intake is mediated through its cognate receptors within several hypothalamic nuclei, namely the arcuate nucleus (ARN). The ARN contains two neuronal populations expressing peptides that exert opposing effects on the central control of feeding: the orexigenic neuropeptide Y (NPY) and the anorexigenic α-melanocyte-stimulating hormone (α-MSH).Methods: To understand the role played by estradiol in the modulation of food intake, we have used an animal model of cyclic 17β-estradiol benzoate (EB) administration and stereological methods to estimate the total number of neurons immunoreactive for NPY and α-MSH in the ARN of ovariectomized rats.Results: Present results show that the experimentally induced EB cyclicity prompted a decrease in food consumption and in body weight. Data also show that ovariectomy induced an increase in NPY expression and a decrease in α-MSH expression in the ARN that were reverted by EB administration. Conversely, EB blocked the expression of NPY and increased the synthesis of α-MSH in ARN neurons, without affecting the overall sum of NPY and α-MSH neurons.Discussion: These results suggest that estradiol affects food intake and, consequently, body weight gain, through an overriding mechanism superimposed in the physiological balance between both peptides in the ARN of female rats.

Effect of Chronic Estradiol‐17β (E2) exposure on the expression of genes related to inflammation in the Arcuate Nucleus of female rats: Effects of aging.

The use of estrogen is widespread among young women as a contraceptive and in older women for hormone replacement therapy. Although estrogen is known to have several beneficial effects on the brain, these effects are limited to non‐hypothalamic regions. Earlier studies have demonstrated inflammatory changes in the arcuate nucleus (AN) of the hypothalamus after E2 treatment. We hypothesized that chronic E2 exposure would increase the expression of cytokine and chemokine genes in the AN. To test this, young (Y) and middle aged (MA) female rats were sham implanted or implanted with slow release E2 pellets for 90 days. Ovariectomized Y and MA rats were treated the same way. At the end of treatment, animals were sacrificed and the AN was microdissected from brain sections. RNA extracted from the AN was subjected to a qPCR array. The array consisted of genes related to the inflammatory response like IL‐1β, TNF‐α, IL‐6, CCl2 and iNOS. In Y animals, E2 treatment significantly upregulated the following genes: IL‐6 by 3.73 fold, CCL2 by 4.76 fold, and iNOS by 9.42 fold compared to control (p<0.05). There was no change in the expression of these genes in MA animals after E2 treatment. These results indicate that chronic E2 exposure affects the expression of pro‐inflammatory genes in an age‐associated fashion. Supported by NIH AG027697.

Fasting Reduces KiSS‐1 Expression in the Anteroventral Periventricular Nucleus (AVPV): Effects of Fasting on the Expression of KiSS‐1 and Neuropeptide Y in the AVPV or Arcuate Nucleus of Female Rats

Changes in metabolic state, such as those induced by fasting, have profound effects on reproduction. In rats, the time-course over which fasting inhibits luteinising hormone (LH) release is reduced to 48 h by the presence of oestradiol-17beta (E(2)). Hypothalamic kisspeptin plays a key role in mediating the actions of E(2) on gonadotrophin-releasing hormone (GnRH) neurones, and thereby promotes LH release. KiSS-1-expressing neurones are found in the anteroventral periventricular nucleus (AVPV) and arcuate nucleus (ARC). Extensive evidence implicates the AVPV in GnRH release and the ARC in energy balance. The latter nucleus also contains neurones that express neuropeptide Y (NPY), an orexigenic peptide implicated in GnRH control. To elucidate the involvement of kisspeptin and/or NPY in hypothalamic responses to fasting, their expression was quantified by in situ hybridisation histochemistry in ovariectomised rats, with or without E(2) replacement, before and after 48 h of fasting. In the presence of E(2), but not in its absence, the fasting suppressed plasma LH. In the AVPV, the low level of KiSS-1 expression found in the absence of E(2) was unaffected by fasting. By contrast, the elevated level found in the presence of E(2) was suppressed by fasting. Independent of E(2), fasting had no effect on KiSS-1 expression in the ARC, but increased NPY expression at that site. The present study has identified the AVPV as a site at which KiSS-1 expression can be influenced by fasting. The results suggest that inhibition of KiSS-1 expression in the AVPV may be a significant factor in restraining the gonadotrophic axis in response to negative energy balance in the presence of oestrogen. The extent to which the concurrent rise in NPY expression in the ARC may contribute to the suppression of LH release by influencing AVPV kisspeptin neurones, directly or indirectly, or by actions independent of kisspeptin, remains to be established.

Reproductive Experience Increases Prolactin Responsiveness in the Medial Preoptic Area and Arcuate Nucleus of Female Rats

The experience of pregnancy plus lactation produces long-term enhancements in maternal behavior as well as reduced secretion of prolactin, a key hormone for the initial establishment of maternal care. Given that prolactin acts centrally to induce maternal care as well as regulate its own secretion, we tested whether prolactin receptors in brain regions known to regulate behavioral and neuroendocrine processes were up-regulated and more responsive to prolactin in reproductively experienced females. Diestrous primiparous (8 wk after weaning) and age-matched virgin rats were treated with 250 microg ovine prolactin sc or vehicle and the brains collected 2 h later for measurement of mRNA for genes involved in prolactin signaling. Reproductively experienced rats had lower serum prolactin concentrations, compared with virgin rats, suggesting enhanced prolactin feedback on the arcuate neurons regulating prolactin secretion. In the medial preoptic area and arcuate nucleus (regions involved in regulating maternal behavior and prolactin secretion, respectively), the level of long-form prolactin receptor mRNA was higher in primiparous rats, and prolactin treatment induced a further increase in receptor expression in these animals. In the same regions, suppressors of cytokine signaling-1 and -3 mRNA levels were also markedly increased after prolactin treatment in reproductively experienced but not virgin rats. These results support the idea that reproductive experience increases central prolactin responsiveness. The induction of prolactin receptors and enhanced prolactin responsiveness as a result of pregnancy and lactation may help account for the retention of maternal behavior and shifts in prolactin secretion in reproductively experienced females.

Prolactin-induced activation of STAT5 within the hypothalamic arcuate nucleus

Prolactin signalling within hypothalamic areas associated with the control of fertility was examined in male and lactating female rats. Following exogenous prolactin treatment, phosphorylation of STAT5 (signal transducer and activator of transcription) within the arcuate nucleus was measured using a highly sensitive immunoblotting strategy. A significant increase in phosphorylated STAT5 was detected in the arcuate nucleus of female rats compared with same-sex controls. No such effect was apparent in the males. Phosphorylation of STAT5 was not observed in the liver of either males or females. These results show that prolactin-induced intracellular signalling within the hypothalamus involves activation of the Janus tyrosine kinase/signal transducer and activator of transcription pathway, and that this signalling mechanism can be readily triggered in lactating females where prolactin receptors are known to be upregulated and fertility impaired.

The estrogen receptor (ER) α, but not ER β, gene is expressed in hypothalamic growth hormone-releasing hormone neurons of the adult female rat

Growth hormone (GH) synthesis and release from pituitary somatotropes is controlled by the opposing actions of the hypothalamic neuropeptides, GH-releasing hormone (GHRH) in the arcuate nucleus (ARC), and somatostatin in the periventricular nucleus (PeV) and ARC. There is a striking sex difference in the pattern of GH secretion in rats. We have previously demonstrated in male rats that 70% of GHRH neurons in the ARC contain the estrogen receptor α (ER α) gene, whereas less than 5% of somatostatin neurons in the ARC and PeV expressed the ER α or ER β gene. In addition, it has been reported that the PeV somatostatin neurons of neither sex possess ER immunoreactivity. However, there is no available data about colocalization of ERs and GHRH and/or somatostatin in the ARC of female rats. In this study, we used in situ hybridization in the adult female rat brain to determine whether GHRH neurons and/or somatostatin neurons in the ARC coexpress the ER α or ER β gene. In the ARC, ER α mRNA was seen in the ventrolateral region where GHRH mRNA signals were also observed, and in the dorsomedial region where somatostatin mRNA signals were also observed. From studies using adjacent sections through these areas, the distribution of these cells appeared to overlap in part with that of cells containing ER α mRNA. On the other hand, few positive cells for ER β mRNA were observed in the ARC. The double-label in situ hybridization studies showed that in the ARC, 73.4% of GHRH neurons contain ER α mRNA, whereas less than 5% of somatostatin neurons express the ER α gene. These results indicated that the majority of the GHRH neurons in ARC have ER α, but not ER β, and few somatostatin neurons in ARC have ER α or ER β in either adult female or male rats, suggesting that colocalization with ERs in GHRH and/or somatostatin neurons is not an important determinant of the gender specific pattern of GH secretion.

Estrogen-induced mu-opioid receptor internalization in the medial preoptic nucleus is mediated via neuropeptide Y-Y1 receptor activation in the arcuate nucleus of female rats.

The endogenous peptides beta-endorphin (beta-END) and neuropeptide Y (NPY) have been implicated in regulating sexual receptivity. Both beta-END and NPY systems are activated by estrogen and inhibit female sexual receptivity. The initial estrogen-induced sexual nonreceptivity is correlated with the activation and internalization of mu-opioid receptors (MORs), in the medial preoptic nucleus (MPN). Progesterone reverses the estrogen-induced activation/internalization of MOR and induces the sexual receptive behavior lordosis. To determine whether NPY and endogenous opioids interact, we tested the hypothesis that estrogen-induced MOR activation is mediated through NPY-Y1 receptor (Y1R) activation. Retrograde tract tracing demonstrated Y1Ron beta-END neurons that projected to the MPN. Sex steroid modulation of MOR in the MPN acts through NPY and the Y1R. Estradiol administration or intracerebroventricular injection of NPY activated/internalized Y1R in the arcuate nucleus and MOR in the MPN of ovariectomized (OVX) rats. Moreover, the selective Y1R agonist [Leu31, Pro34]-Neuropeptide Y (LPNY) internalized MOR in the MPN of OVX rats. The Y1R antagonist (Cys31, Nva34)-Neuropeptide Y (27-36)2 prevented estrogen-induced Y1R and MOR activation/internalization. NPY reversed the progesterone blockade of estradiol-induced Y1R and MOR internalization in the arcuate nucleus and MPN, respectively. Behaviorally, LPNY inhibited estrogen plus progesterone-induced lordosis, and the MOR-selective antagonist D-Phe-Cys-Tyr-d-Trp-Orn-Thr-Pen-Thr amide reversed LPNY-induced inhibition of lordosis. These results suggest that a sequential sex steroid activation of NPY and MOR circuits regulates sexual receptivity.

Evidence that TGF May Directly Modulate POMC mRNA Expression in the Female Rat Arcuate Nucleus

Evidence that TGF May Directly Modulate POMC mRNA Expression in the Female Rat Arcuate Nucleus

Evidence that TGF beta may directly modulate POMC mRNA expression in the female rat arcuate nucleus.

The purpose of the present study was to determine whether TGF beta, a cytokine secreted by hypothalamic astrocytes, was able to regulate POMC neurons in the arcuate nucleus. In a first set of experiments, mediobasal hypothalamic fragments were exposed to TGF beta(1), and the relative POMC mRNA expression was assessed by in situ hybridization using a radiolabeled POMC riboprobe. The results showed that 4 x 10(-10) M TGF beta(1) was efficient in decreasing significantly the amounts of POMC mRNA (P < 0.01). Interestingly, the decrease of relative POMC mRNA levels was higher in the rostral than in the caudal parts of the arcuate nucleus. In a second set of experiments, we examined the occurrence of TGF beta receptors expression in arcuate POMC neurons. Dual labeling in situ hybridization and in situ hybridization, coupled to immunohistochemical labeling, were performed to examine mRNA expression of the type I serine-threonine kinase receptor for TGF beta and the presence of type II receptor for TGF beta, respectively, in POMC neurons. The results indicated that TGF beta receptor I mRNA and TGF beta receptor II protein were expressed in numerous POMC neurons. Regional analysis revealed that the highest proportion of POMC neurons expressing TGF beta receptors was located in the rostral part of the arcuate nucleus. Using dual labeling immunohistochemistry, we also found that Smad2/3 immunoreactivity, a TGF beta(1) downstream signaling molecule, was present in the cytoplasm and nucleus of some POMC (beta-endorphin) neurons. We next examined whether the number of POMC neurons expressing TGF beta-RI mRNA was affected by sex steroids. Quantification of the number of POMC neurons expressing TGF beta receptor I mRNA in ovariectomized, ovariectomized E2-treated, and ovariectomized E2 plus progesterone-treated animals revealed that estrogen treatment decreased the expression of TGF beta receptor I mRNA in POMC neurons located in the rostral half of the arcuate nucleus, an effect reversed by progesterone in a subset of the most rostral cells. Taken together, these data reveal that TGF beta(1) may directly modulate the activity of POMC neurons through the activation of TGF beta receptors. Therefore, the present study provides additional evidence for the involvement of TGF beta(1) in the regulation of neuroendocrine functions and supports the existence of a glial-to-neurons communication within the arcuate nucleus.

The highly sialylated isoform of the neural cell adhesion molecule is required for estradiol-induced morphological synaptic plasticity in the adult arcuate nucleus.

The large quantities of polysialic acid (PSA) characterizing highly sialylated isoform of the neural cell adhesion molecule (PSA-NCAM), greatly reduce cell adhesion and render this particular cell surface adhesion molecule a likely candidate to intervene in dynamic neuronal phenomena, such as synaptic plasticity. The hypothalamic arcuate nucleus expresses high levels of PSA-NCAM and maintains a high capacity for neuroplastic changes in the adult. Thus, in the arcuate nucleus of female rats, varying circulating levels of estrogen give rise to a reversible reduction in the number of axo-somatic GABA synapses, together with a changing ensheathing of neuronal somata by astrocytes. To examine the role of PSA in such changes, we perturbed its expression, either by blockade with antibodies raised against this carbohydrate moiety (delivered intracerebroventricularly), or by its enzymatic cleavage after microinjection of endoneuraminidase N over the arcuate nucleus. Either procedure was performed in ovariectomized adult rats that received concurrent treatment with 17 beta-estradiol. Morphological synaptic plasticity was analysed using the unbiased disector method to assess synaptic densities in ultrathin sections of the arcuate nucleus immunogold-labelled for GABA. As expected, 17 beta-estradiol induced a significant reduction in the number of GABAergic axo-somatic synapses, a reduction which did not occur after infusion of anti-PSA antibodies or in vivo enzymatic removal of PSA from NCAM. Taken together, our results provide strong evidence that the presence of large quantities of the PSA moiety on NCAM is a necessary prerequisite for estrogen-induced phasic remodelling of synapses in the adult female arcuate nucleus.

AMPA receptors colocalize with neuropeptide-Y- and galanin-containing, but not with dopamine, neurons of the female rat arcuate nucleus: a semiquantitative immunohistochemical colocalization study.

It is well established that excitatory amino acid (EAA) neurotransmission is an essential component in the regulation of the gonadotropin-releasing hormone (GnRH) delivery system. However, the morphological interconnection of these systems is not fully understood. The objective of the present study was to determine whether or not alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors--as indicators of aspartate/glutamatergic innervation--are present in the major neuronal populations, such as the neuropeptide-Y-(NPY), galanin- (GAL) and tyrosine-hydroxylase- (TH) containing neurons of the arcuate nucleus (AN) of the female rat. Colocalization experiments using the "mirror" technique demonstrated that: (1) AN neurons containing GluR1 are also immunoreactive (IR) for GluR2/3; (2) 38.32% of AMPA-IR cells contain NPY and 31.72% of AMPA-containing neurons are also IR for GAL; in turn, 79.41% of NPY- and 56.19% of GAL-containing neurons are IR for AMPA receptors; none of the neurons are IR for both AMPA receptors and TH. These data suggest that an excitatory aspartate/glutamatergic input is implicated in the regulation of the examined neuropeptide-containing AN neurons but not in that of TH-IR cells of the same area.

Synaptic density in the arcuate nucleus of female rats approaching middle age

Synaptic number and synapses/neuronal cell membrane were evaluated from ultrastructural micrographs of the arcuate nucleus from 90-, 120-, 180-, and 240-day female rats grouped into ovary-intact or ovariectomized animals treated with peanut oil vehicle or estradiol benzoate (10 μg/100 g body weight) for 3 days. In ovary-intact rats, synaptic density was significantly less in middle aged 240 day animals than in 90-, 120-, or 180-day animals with greatest decrease occurring between 180- and 240-day animals. Ovary-intact and ovariectomized animals treated with estradiol benzoate had significantly higher sera estradiol levels, but the estradiol was ineffective in increasing synaptic density in the middle aged animals. Logistical regression confirmed a correlation between a decrease in synapses and increasing age but not estradiol treatment.

Enlarging Effects of Estradiol on the Nuclear Volume of Neurons in the Hypothalamus during Aging

Neuronal nuclear volumes (NNVs) were measured in the medial preoptic nucleus (MPN), anterior hypothalamic area (AHA) and arcuate nucleus (ARN) of young adult, middle-aged, and old rats of both sexes. The NNVs in the darkly stained sexual-dimorphic nucleus of the preoptic area (SDN-POA) and the lighter staining surrounding area (non-SDN-POA) within the MPN were measured separately. Intact young and middle-aged female rats had larger NNVs than those of the males in SDN-POA, non-SDN-POA and AHA but not in ARN. During aging, only intact old female rats manifested significant NNV shrinkage in all the measured areas. Long-term treatment with estradiol benzoate (EB) caused a significant enlargement of the NNVs in non-SDN-POA and ARN of middle-aged and old male rats as well as the NNVs in SDN-POA, non-SDN-POA and ARN of old female rats. The enlarging effect of EB on NNVs in both SDN-POA and non-SDN-POA of female rats could be prevented by ovariectomy. Furthermore, NNVs in SDN-POA and non-SDN-POA of ovariectomized female rats were even smaller than those of the age-matched intact female rats. These results indicate that: (1) the NNVs of MPN and ARN in male and female rats were enlarged after long-term exposure of physiological dose of estradiol; (2) the enlarging effects of EB on NNV in MPN can explain why the NNV of intact female rats is larger than that of males, and (3) during aging, the sex-specific shrinkage of NNVs in MPN, AHA and ARN of female rats may be due to an intrinsic aging process rather than long-term effects of EB.

Estradiol upregulates Bcl-2 expression in adult brain neurons.

Bcl-2, a protein which negatively modulates apoptosis, is up-regulated by estrogen in several tissues. To determine the effect of estradiol on Bcl-2 in the adult brain, its immunoreactive distribution was examined in the hypothalamic arcuate nucleus of female rats under different endocrine conditions. The number of Bcl-2-immunoreactive neurons was significantly increased (p < 0.001) on the day of estrus compared with proestrus, diestrus and metestrus, was decreased by ovariectomy and showed a dose-response increase after estradiol administration to ovariectomized rats. Progesterone, when injected simultaneously with estradiol, reduced the effect of estradiol. These findings indicate that ovarian hormones regulate Bcl-2 in hypothalamic neurons and suggest that this protein may be involved in the neuroprotective effects of estrogen.

Lack of gonadotropin-positive feedback in the male rat is associated with lack of estrogen-induced synaptic plasticity in the arcuate nucleus.

We have demonstrated that estrogen induces synaptic reorganization in the hypothalamic arcuate nucleus of female rats during the ovarian cycle and proposed that estrogen-induced synaptic retraction plays a role in the disinhibition of gonadotropin secretion that occurs during the afternoon of proestrus. This so-called positive feedback of gonadotropins is developmentally determined. It is present in female rats and absent in males. To confirm the role of the estrogen-induced synaptic retraction in positive feedback, we tested whether administration of estrogen to male rats also fails to induce synaptic remodeling of the arcuate nucleus. Male and female rats were gonadectomized and studied as follows. One month following gonadectomy, animals received either a single injection of estradiol (100 micrograms/animal in sesame oil; 12 males and 12 females) or vehicle (6 males and 6 females). Twenty-four hours following injections, all animals in the vehicle-injected group and 6 animals of each sex in the treatment groups were sacrificed, while the rest of the animals were killed 48 h following the hormone injections (6 per group). As expected, quantitative electron microscopic analysis of the female arcuate nuclei revealed that compared to oil-injected controls, estradiol induced drastic decreases in the overall synapse counts by 24 h (121 +/- 10 vs. 74 +/- 5 synapses/1,000 microns membrane; p < 0.05). Synaptic counts had recovered to control levels by 48 h. On the contrary, in males, estradiol treatment did not cause changes in the total synapse counts at either time. As a further control, the lack of an estrogen-induced gonadotropin surge in long-term castrate males was also confirmed. Our study confirmed that in males estradiol does not alter the net synaptology of the arcuate nucleus or cause gonadotropin positive feedback. This is in clear contradistinction to females which show both synaptic plasticity and gonadotropin-positive feedback upon receiving exogenous or endogenous estrogen. The lack of estrogen-induced synaptic plasticity may be an underlying mechanism in the abolishment of positive gonadotropin, feedback in developing males and the development of constant estrus in aging female rats.

Neuropeptide Y gene expression in the arcuate nucleus is increased during preovulatory luteinizing hormone surges

Recent studies have suggested that neuropeptide Y (NPY) plays an important role in the induction of the preovulatory LH surge. The present study was performed in order to determine if a change in NPY gene expression within arcuate nucleus NPY neurons is associated with the generation of the preovulatory LH surge. In Exp 1, in situ hybridization was used to measure NPY messenger RNA (mRNA) levels in the arcuate nucleus of female rats at 0900 h and every 2 h from 1400-2200 h on the day of proestrus (PRO). Comparisons between groups showed a clear, stepwise increase in NPY gene expression throughout the day of PRO. At 1600 h, when LH values were significantly greater than 0900 h values, NPY mRNA labeling intensities in the arcuate nucleus were significantly greater than 0900 h levels (P < 0.01). By 1800 h, the time at which the LH surge peaked, NPY mRNA levels also peaked and were nearly 3-fold greater than levels observed at 0900 h (P < 0.01). NPY mRNA levels at 2000 h and 2200 h remained elevated above 0900 h levels (P < 0.01) but by 2000 h had decreased significantly from 1800 h levels (P < 0.05). In Exp 2, NPY mRNA levels were measured once again at 0900 h and 1800 h on PRO, and then at 0900 h and 1800 h on metestrus (MET), in order to determine if the change in gene expression seen in Exp 1 was unique to the day of PRO, or if it simply reflected a daily rhythm of gene expression in the nucleus. Analysis of mRNA levels showed no difference in NPY mRNA levels between 0900-1800 h on MET. Also, NPY mRNA levels at 0900 h and 1800 h on MET were significantly less than levels at 1800 h on PRO (P < 0.01). These results are consistent with the hypothesis that NPY neurons participate in the generation of LH surges through increased production of NPY and subsequent potentiation of the release and/or actions of LHRH.

Neuropeptide Y gene expression in the arcuate nucleus is increased during preovulatory luteinizing hormone surges.

Recent studies have suggested that neuropeptide Y (NPY) plays an important role in the induction of the preovulatory LH surge. The present study was performed in order to determine if a change in NPY gene expression within arcuate nucleus NPY neurons is associated with the generation of the preovulatory LH surge. In Exp 1, in situ hybridization was used to measure NPY messenger RNA (mRNA) levels in the arcuate nucleus of female rats at 0900 h and every 2 h from 1400-2200 h on the day of proestrus (PRO). Comparisons between groups showed a clear, stepwise increase in NPY gene expression throughout the day of PRO. At 1600 h, when LH values were significantly greater than 0900 h values, NPY mRNA labeling intensities in the arcuate nucleus were significantly greater than 0900 h levels (P < 0.01). By 1800 h, the time at which the LH surge peaked, NPY mRNA levels also peaked and were nearly 3-fold greater than levels observed at 0900 h (P < 0.01). NPY mRNA levels at 2000 h and 2200 h remained elevated above 0900 h levels (P < 0.01) but by 2000 h had decreased significantly from 1800 h levels (P < 0.05). In Exp 2, NPY mRNA levels were measured once again at 0900 h and 1800 h on PRO, and then at 0900 h and 1800 h on metestrus (MET), in order to determine if the change in gene expression seen in Exp 1 was unique to the day of PRO, or if it simply reflected a daily rhythm of gene expression in the nucleus. Analysis of mRNA levels showed no difference in NPY mRNA levels between 0900-1800 h on MET. Also, NPY mRNA levels at 0900 h and 1800 h on MET were significantly less than levels at 1800 h on PRO (P < 0.01). These results are consistent with the hypothesis that NPY neurons participate in the generation of LH surges through increased production of NPY and subsequent potentiation of the release and/or actions of LHRH.

Role of dopamine in the regulation of proopiomelanocortin (POMC) mRNA levels in the arcuate nucleus and pituitary gland of the female rat as studied by in situ hybridization

The effects of the dopaminergic antagonist haloperidol (HAL) as well as the D 2 dopamine receptor agonist bromocriptine (BRO) on proopiomelanocortin (POMC) mRNA levels in the female rat arcuate nucleus and pituitary were investigated by quantitative in situ hybridization. Since we had already shown that sex steroids could induce a decrease in POMC mRNA levels in the arcuate nucleus of castrated rats, the involvement of the dopaminergic system in the inhibitory effect of estradiol (E 2) was also investigated. In situ hybridization was performed on paraformaldehyde-fixed cryostat sections through the arcuate nucleus and whole pituitary gland using a 35S-labelled cDNA probe encoding for POMC. In the arcuate nucleus of intact animals, a 14-day treatment with BRO increased by 54% the number of silver grains/unit of surface of labelled neurons while HAL decreased by 30% the value of this parameter. Hypophysectomy which induced a 20% decrease in the hybridization signal could not prevent the effects of BRO or HAL. Ovariectomy performed 14 days earlier increased by 20% the number of silver grains while a 14-day treatment of ovariectomized animals with E 2 decreased the hybridization signal by 32%. On the other hand, the concomitant administration of HAL and E 2 did not induce significant variations in POMC mRNA levels compared to those obtained following HAL administration, but slightly decreased the hybridization signal by 20% compared to that induced by E 2 alone. In the intermediate lobe of the pituitary, BRO markedly depressed (30% of control values) and HAL increased by 50% the levels of POMC mRNA. The present data clearly demonstrate that POMC mRNA levels are differently regulated by dopamine in the intermediate lobe of the pituitary and the arcuate nucleus and that the effects of BRO and HAL on arcuate POMC mRNA are not mediated by the pituitary gland. They do not allow to draw any definite conclusion about the possible involvement of the dopaminergic system in the inhibitory role of E 2 on POMC gene regulation.