Female reproductive aging in rats is characterized by reduced gonadotropin releasing hormone (GnRH) neuronal activation under estradiol positive feedback conditions and a delayed and attenuated luteinizing hormone (LH) surge. The newly identified excitatory neuropeptide kisspeptin is proposed to be a critical mediator of the pubertal transition and the ovarian steroid-induced LH surge. We previously showed that estradiol induces less kisspeptin mRNA expression in the anterior hypothalamus [anatomical location of anteroventral periventricular nucleus (AVPV)] in middle-aged than in young rats and intrahypothalamic infusion of kisspeptin restores LH surge amplitude in middle-aged females. Thus, reduced kisspeptin neurotransmission may contribute to age-related LH surge abnormalities. This study tested the hypothesis that middle-aged females will also exhibit reduced numbers of kisspeptin immunopositive neurons in the AVPV under estradiol positive feedback conditions. Using immunohistochemistry, we demonstrate that middle-aged females primed with ovarian steroids have fewer AVPV kisspeptin immunopositive neurons than young females. Age did not affect kisspeptin mRNA expression in the pituitary, numbers of kisspeptin immunopositive neurons in the arcuate nucleus, or estradiol-dependent reductions in kisspeptin mRNA expression in the posterior hypothalamus (containing the arcuate nucleus). These data strongly suggest that age-related LH surge dysfunction results, in part, from a reduced sensitivity of AVPV kisspeptin neurons to estradiol and hence decreased availability of AVPV kisspeptin neurons to activate GnRH neurons under positive feedback conditions.
Aging; Kisspeptin; GPR54; Hypothalamus; AVPV; Arcuate nucleus
The control of reproductive function involves actions of sex steroids upon their nuclear receptors in the hypothalamus and preoptic area (POA). Whether hypothalamic hormone receptors change their expression in aging male mammals has not been extensively pursued, although such changes may underlie functional losses in reproductive physiology occurring with aging. We performed a stereological analysis of immunoreactive androgen receptor (AR) and estrogen receptor alpha (ERα) cells in three POA nuclei of male Sprague-Dawley rats [anteroventral periventricular nucleus (AVPV), median preoptic area (MePO), and medial preoptic nucleus (MPN)], at young (3 mo), middle-aged (12 mo) and old (20 mo) ages. Serum testosterone and estradiol levels were assayed. Testosterone concentrations decreased significantly and progressively with aging. Estradiol concentrations were significantly higher in middle-aged than either young or old rats. Stereologic analyses of the POA demonstrated that AR-immunoreactive cell numbers and density in the AVPV, MePO and MPN were significantly higher in old compared with young or middle-aged rats. No change in the total number or density of ERα immunoreactive cells was detected with age, although when cells were subdivided by intensity of immunolabeling, the most heavily-labeled ERα cells increased in number with aging in the AVPV and MePO, and in density in the AVPV. There are several interpretations to our finding of substantially increased AR cell numbers during aging, including a potential compensatory up-regulation of the AR under diminished testosterone concentrations. These results provide further information about how the neural targets of steroid hormones change with advancing age.
androgen receptor; estrogen receptor; aging; anteroventral periventricular nucleus (AVPV); median preoptic area (MePO); medial preoptic nucleus (MPN)
Males and females exhibit numerous anatomical and physiological differences in the brain which often underlie important sex differences in physiology or behavior, including aspects relating to reproduction. Neural sex differences are both region- and trait-specific and may consist of divergences in synapse morphology, neuron size and number, and specific gene expression levels. In most cases, sex differences are induced by the sex steroid hormonal milieu during early perinatal development. In rodents, the hypothalamic anteroventral periventricular nucleus (AVPV) is sexually differentiated as a result of postnatal sex steroids, and specific neuronal populations in this nucleus are also sexually dimorphic, with females possessing more kisspeptin, dopaminergic, and GABA/glutamate neurons than males. The ability of female rodents, but not males, to display an estrogen-induced luteinizing hormone (LH) surge is consistent with the higher levels of these neuropeptides in the AVPV of females. Of these AVPV populations, the recently-identified kisspeptin system has been most strongly implicated as a critical component of the sexually-dimorphic LH surge mechanism, though GABA and glutamate have also received some attention. New findings have suggested that the sexual differentiation and development of kisspeptin neurons in the AVPV is mediated by developmental estradiol signaling. Although apoptosis is the most common process implicated in neuronal sexual differentiation, it is currently unknown how developmental estradiol acts to differentiate specific neuronal populations in the AVPV, such as kisspeptin or dopaminergic neurons.
sex difference; sexually dimorphic; sexual differentiation; hypothalamus; kisspeptin; Kiss1; tyrosine hydroxylase; development; reproduction; AVPV
The loss of reproductive capacity during aging involves changes in the neural regulation of the hypothalamic gonadotropin-releasing hormone (GnRH) neurons controlling reproduction. This neuronal circuitry includes glutamate receptors on GnRH neurons. Previously, we reported an increase in the expression of the NR2b subunit protein of the NMDA receptor on GnRH neurons in middle-aged compared to young female rats. Here, we examined the functional implications of the NR2b subunit on the onset of reproductive aging, using an NR2b-specific antagonist ifenprodil. Young (3–5 mos.) and middle-aged (10–13 mos.) female rats were ovariectomized (OVX), 17β-estradiol (E2) or vehicle (cholesterol) treated, and implanted with a jugular catheter. Serial blood sampling was undertaken every 10 minutes for 4 hours, with ifenprodil (10mg/kg) or vehicle injected (i.p.) after one hour of baseline sampling. The pulsatile release of pituitary LH and levels of GnRH mRNA in hypothalamus were quantified as indices of the reproductive axis. Our results showed effects of ifenprodil on both endpoints. In OVX rats given cholesterol, neither age nor ifenprodil had any effects on LH release. In E2-treated rats, aging was associated with significant decreases in pulsatile LH release. Additionally, ifenprodil stimulated parameters of pulsatile LH release in both young and middle-aged animals. Ifenprodil had few effects on GnRH mRNA; the only significant effect of ifenprodil was found in the middle-aged, cholesterol group. Together, these findings support a role for the NR2b subunit of the NMDAR in GnRH/LH regulation. Because most of these effects were exhibited on pituitary LH release in the absence of a concomitant change in GnRH gene expression, it is likely that NMDA receptors containing the NR2b subunit plays a role in GnRH-induced LH release, independent of de novo GnRH gene expression.
Luteinizing hormone (LH); gonadotropin-releasing hormone (GnRH); glutamate; N-methyl-D-Aspartate receptor (NMDAR); NR2b; reproductive aging; reproductive senescence; estrogen; GnRH mRNA; ifenprodil
Kisspeptin is a product of the Kiss1 gene and is expressed in the forebrain. Neurons that express Kiss1 play a crucial role in the regulation of pituitary luteinizing hormone secretion and reproduction. These neurons are the direct targets for the action of estradiol-17β (E2), which acts through the estrogen receptor alpha isoform (ERα) to regulate Kiss1 expression. In the arcuate nucleus (Arc), where the dynorphin gene (Dyn) is expressed in Kiss1 neurons, E2 inhibits the expression of Kiss1 mRNA. However, E2 induces the expression of Kiss1 in the anteroventral periventricular nucleus (AVPV). The mechanism for differential regulation of Kiss1 in the Arc and AVPV by E2 is unknown. ERα signals through multiple pathways, which can be categorized as either classical, involving the estrogen response element (ERE) or non-classical, involving ERE–independent mechanisms. To elucidate the molecular basis for the action of E2 on Kiss1 and Dyn expression, we studied the effects of E2 on Kiss1 and Dyn mRNAs in the brains of mice bearing targeted alterations in the ERα signaling pathways. We found that stimulation of Kiss1 expression by E2 in the AVPV and inhibition of Dyn in the Arc required an ERE-dependent pathway, whereas the inhibition of Kiss1 expression by E2 in the Arc involved ERE-independent mechanisms. Thus, distinct ERα signaling pathways can differentially regulate the expression of identical genes across different brain regions, and E2 can act within the same neuron through divergent ERα signaling pathways to regulate different neurotransmitter genes.
Kiss1; Dynorphin; ERα; Classical and Non-classical ER signaling; hypothalamus; mouse
Reproductive function requires timely secretion of gonadotropin releasing hormone, which is controlled by a complex excitatory/inhibitory network influenced by sex steroids. Kiss1 neurons are fundamental players in this network, but it is currently unclear whether different conditions of circulating sex steroids directly alters Kiss1 neuronal activity. Here, we show that Kiss1 neurons in the anteroventral periventricular and anterior periventricular nuclei (AVPV/PeN) of males and females exhibit a bimodal resting membrane potential (RMP) influenced by KATP channels, suggesting the presence of two neuronal populations defined as Type I (irregular firing patterns) and Type II (quiescent). Kiss1 neurons in the arcuate nucleus (Arc) are also composed of firing and quiescent cells, but unlike AVPV/PeN neurons, the range of RMPs did not follow a bimodal distribution. Moreover, Kiss1 neuronal activity in the AVPV/PeN, but not in the Arc, is sexually dimorphic. In females, estradiol shifts the firing pattern of AVPV/PeN Kiss1 neurons and alters cell capacitance and spontaneous inhibitory postsynaptic potentials (IPSCs) amplitude of AVPV/PeN and Arc Kiss1 populations in an opposite manner. Notably, mice with selective deletion of estrogen receptor α (ERα) from Kiss1 neurons show cellular activity similar to that observed in ovariectomized females, suggesting that estradiol-induced changes in Kiss1 cellular properties require ERα. We also show that female prepubertal Kiss1 neurons are under higher inhibitory influence while all AVPV/PeN Kiss1 neurons are spontaneously active. Collectively, our findings indicate that changes in cellular activity may underlie Kiss1 action in pubertal initiation and female reproduction.
HPG axis; puberty; hypothalamus; neuroendocrine regulation; estrogen feedback
The differential expression and secretion of the neuropeptide kisspeptin from neurons in the arcuate (Arc) and anteroventral periventricular (AVPV) nuclei of the hypothalamus coordinate the temporal release of pituitary gonadotropins that control the female reproductive cycle. However, the molecular basis for this differential regulation is incompletely understood. Here, we report that liver receptor homolog-1 (LRH-1), a member of the nuclear receptor superfamily, is expressed in kisspeptin neurons in the Arc but not in the AVPV in female mice. LRH-1 binds directly to the kisspeptin (Kiss1) promoter and stimulates Kiss1 transcription. Deletion of LRH-1 from kisspeptin neurons in mice decreased Kiss1 expression in the Arc, leading to reduced plasma FSH levels, dysregulated follicle maturation, and prolongation of the estrous cycle. Conversely, overexpression of LRH-1 in kisspeptin neurons increased Arc Kiss1 expression and plasma FSH concentrations. These studies provide a molecular basis for the differential regulation of basal kisspeptin expression in Arc and AVPV neurons and reveal a prominent role for LRH-1 in hypothalamus in regulating the female reproductive axis.
Fibroblast growth factor (FGF) signaling is essential for the development of the gonadotropin-releasing hormone (GnRH) system. Mice harboring deficiencies in Fgf8 or Fgf receptor 1 (Fgfr1) suffer a significant loss of GnRH neurons, but their reproductive phenotypes have not been examined. This study examined if female mice hypomorphic for Fgf8, Fgfr1, or both (compound hypomorphs) exhibited altered parameters of pubertal onset, estrous cyclicity, and fertility. Further, we examined the number of kisspeptin (KP)-immunoreactive (ir) neurons in the anteroventral periventricular/periventricular nuclei (AVPV/PeV) of these mice to assess if changes in the KP system, which stimulates the GnRH system, could contribute to the reproductive phenotypes. Single hypomorphs (Fgfr1+/− or Fgf8+/−) had normal timing for vaginal opening (VO) but delayed first estrus. However, after achieving the first estrus, they underwent normal expression of estrous cycles. In contrast, the compound hypomorphs underwent early VO and normal first estrus, but had disorganized estrous cycles that subsequently reduced their fertility. KP immunohistochemistry on Postnatal Day 15, 30, and 60 transgenic female mice revealed that female compound hypomorphs had significantly more KP-ir neurons in the AVPV/PeV compared to their wild-type littermates, suggesting increased KP-ir neurons may drive early VO but could not maintain the cyclic changes in GnRH neuronal activity required for female fertility. Overall, these data suggest that Fgf signaling deficiencies differentially alter the parameters of female pubertal onset and cyclicity. Further, these deficiencies led to changes in the AVPV/PeV KP-ir neurons that may have contributed to the accelerated VO in the compound hypomorphs.
Single or combined deficiencies in Fgfr1 and Fgf8 differentially alter reproduction and the kisspeptin system in female mice.
female infertility; gonadal function; gonadotropin-releasing hormone (GnRH/GnRH receptor); kisspeptin
Humans and mice with loss-of-function mutations of the genes encoding kisspeptins (Kiss1) or kisspeptin receptors (Kiss1r) are infertile due to hypogonadotropic hypogonadism. Within the hypothalamus, Kiss1 mRNA is expressed in the anteroventral periventricular nucleus (AVPV) and the arcuate nucleus (Arc). In order to better study the different populations of kisspeptin cells we generated Kiss1-Cre transgenic mice. We obtained one line with Cre activity specifically within Kiss1 neurons (line J2-4), as assessed by generating mice with Cre-dependent expression of green fluorescent protein or β-galactosidase. Also, we demonstrated Kiss1 expression in the cerebral cortex and confirmed previous data showing Kiss1 mRNA in the medial nucleus of amygdala and anterodorsal preoptic nucleus. Kiss1 neurons were more concentrated towards the caudal levels of the Arc and higher leptin-responsivity was observed in the most caudal population of Arc Kiss1 neurons. No evidence for direct action of leptin in AVPV Kiss1 neurons was observed. Melanocortin fibers innervated subsets of Kiss1 neurons of the preoptic area and Arc, and both populations expressed MC4R. Specifically in the preoptic area, 18–28% of Kiss1 neurons expressed MC4R. In the Arc, 90% of Kiss1 neurons were glutamatergic, 50% of which also were GABAergic. In the AVPV, 20% of Kiss1 neurons were glutamatergic whereas 75% were GABAergic. The differences observed between the Kiss1 neurons in the preoptic area and the Arc likely represent neuronal evidence for their differential roles in metabolism and reproduction.
leptin; melanocortin; Cre-recombinase; reporter mouse; GAD-67; vGluT2
Throughout the hypothalamus there are several regions known to contain sex differences in specific cellular, neurochemical, or cell grouping characteristics. The current study examined the potential origin of sex differences in calbindin expression in the preoptic area and hypothalamus as related to sources of nitric oxide. Specific cell populations were defined by immunoreactive (ir) calbindin and neuronal nitric oxide synthase (nNOS) in the preoptic area/anterior hypothalamus (POA/AH), anteroventral periventricular nucleus (AVPv), and ventromedial nucleus of the hypothalamus (VMN). The POA/AH of adult mice was characterized by a striking sex difference in the distribution of cells with ir-calbindin. Examination of the POA/AH of androgen receptor deficient Tfm mice suggests that this pattern was in part androgen receptor dependent, since Tfm males had reduced ir-calbindin compared with wild-type males and more similar to wild-type females. At P0 ir-calbindin was more prevalent than in adulthood, with males having significantly more ir-calbindin and nNOS than have females. Cells that contained either ir-calbindin or ir-nNOS in the POA/AH were in adjacent cell groups, suggesting that NO derived from the enzymatic activity of nNOS may influence the development of ir-calbindin cells. In the region of AVPv, at P0, there was a sex difference with males having more ir-nNOS fibers than have females while ir-calbindin was not detected. In the VMN, at P0, ir-nNOS was greater in females than in males, with no significant difference in ir-calbindin. We suggest that NO as an effector molecule and calbindin as a molecular biomarker illuminate key aspects of sexual differentiation in the developing mouse brain.
sex differences; hypothalamus; preoptic area; nitric oxide; calbindin
Disruption of reproductive function is a hallmark of abuse of anabolic androgenic steroids (AAS) in female subjects. To understand the central actions of AAS, patch clamp recordings were made in estrous, diestrous and AAS-treated mice from gonadotropin releasing hormone (GnRH) neurons, neurons in the medial preoptic area (mPOA) and neurons in the anteroventroperiventricular nucleus (AVPV); regions known to provide GABAergic and kisspeptin inputs to the GnRH cells. Action potential (AP) frequency was significantly higher in GnRH neurons of estrous mice than in AAS-treated or diestrous animals. No significant differences in AAS-treated, estrous or diestrous mice were evident in the amplitude or kinetics of spontaneous postsynaptic currents (sPCSs), miniature PSCs or tonic currents mediated by GABAA receptors or in GABAA receptor subunit expression in GnRH neurons. In contrast, the frequency of GABAA receptor-mediated sPSCs in GnRH neurons showed an inverse correlation with AP frequency across the three hormonal states. Surprisingly, AP activity in the medial preoptic area (mPOA), a likely source of GABAergic afferents to GnRH cells, did not vary in concert with the sPSCs in the GnRH neurons. Furthermore, pharmacological blockade of GABAA receptors did not alter the pattern in which there was lower AP frequency in GnRH neurons of AAS-treated and diestrous versus estrous mice. These data suggest that AAS do not impose their effects either directly on GnRH neurons or on putative GABAergic afferents in the mPOA. AP activity recorded from neurons in kisspeptin-rich regions of the anteroventroperiventricular nucleus (AVPV) and the expression of kisspeptin mRNA and peptide did vary coordinately with AP activity in GnRH neurons. Our data demonstrate that AAS treatment imposes a “diestrous-like” pattern of activity in GnRH neurons and suggest that this effect may arise from suppression of presynaptic kisspeptin-mediated excitatory drive arising from the AVPV. The actions of AAS on neuroendocrine regulatory circuits may contribute the disruption of reproductive function observed in steroid abuse.
Anabolic steroid; GnRH; GABAA receptor; drug abuse; kisspeptin; reproduction
NMDA (N-methyl-D-aspartic acid) is a widely known agonist for a class of glutamate receptors, the NMDA type. Synthetic NMDA elicits very strong activity for the induction of hypothalamic factors and hypophyseal hormones in mammals. Moreover, endogenous NMDA has been found in rat, where it has a role in the induction of GnRH (Gonadotropin Releasing Hormone) in the hypothalamus, and of LH (Luteinizing Hormone) and PRL (Prolactin) in the pituitary gland.
In this study we show evidence for the occurrence of endogenous NMDA in the amphioxus Branchiostoma lanceolatum. A relatively high concentration of NMDA occurs in the nervous system of this species (3.08 ± 0.37 nmol/g tissue in the nerve cord and 10.52 ± 1.41 nmol/g tissue in the cephalic vesicle). As in rat, in amphioxus NMDA is also biosynthesized from D-aspartic acid (D-Asp) by a NMDA synthase (also called D-aspartate methyl transferase).
Given the simplicity of the amphioxus nervous and endocrine systems compared to mammalian, the discovery of NMDA in this protochordate is important to gain insights into the role of endogenous NMDA in the nervous and endocrine systems of metazoans and particularly in the chordate lineage.
Kisspeptin and its G protein-coupled receptor (GPR) 54 are essential for activation of the hypothalamo-pituitary-gonadal axis. In the rat, the kisspeptin neurons critical for gonadotropin secretion are located in the hypothalamic arcuate (ARC) and anteroventral periventricular (AVPV) nuclei. As the ARC is known to be the site of the gonadotropin-releasing hormone (GnRH) pulse generator we explored whether kisspeptin-GPR54 signalling in the ARC regulates GnRH pulses.
We examined the effects of kisspeptin-10 or a selective kisspeptin antagonist administration intra-ARC or intra-medial preoptic area (mPOA), (which includes the AVPV), on pulsatile luteinizing hormone (LH) secretion in the rat. Ovariectomized rats with subcutaneous 17β-estradiol capsules were chronically implanted with bilateral intra-ARC or intra-mPOA cannulae, or intra-cerebroventricular (icv) cannulae and intravenous catheters. Blood samples were collected every 5 min for 5–8 h for LH measurement. After 2 h of control blood sampling, kisspeptin-10 or kisspeptin antagonist was administered via pre-implanted cannulae. Intranuclear administration of kisspeptin-10 resulted in a dose-dependent increase in circulating levels of LH lasting approximately 1 h, before recovering to a normal pulsatile pattern of circulating LH. Both icv and intra-ARC administration of kisspeptin antagonist suppressed LH pulse frequency profoundly. However, intra-mPOA administration of kisspeptin antagonist did not affect pulsatile LH secretion.
These data are the first to identify the arcuate nucleus as a key site for kisspeptin modulation of LH pulse frequency, supporting the notion that kisspeptin-GPR54 signalling in this region of the mediobasal hypothalamus is a critical neural component of the hypothalamic GnRH pulse generator.
Kisspeptin, a neuropeptide product of the KiSS-1 gene, has recently been implicated in the regulation of seasonal breeding in a number of species, including Siberian hamsters. In this species, kisspeptin expression is reduced in the anteroventral periventricular nucleus (AVPV) following exposure to inhibitory day lengths, and exogenous kisspeptin activates the reproductive neuroendocrine axis of reproductively quiescent animals. Because sex steroids can impact kisspeptin expression, it is unclear whether changes in kisspeptin occur in direct response to photoperiodic cues or secondarily in response to changes in sex steroid concentrations resulting from the transition to reproductive quiescence. The present study aimed to assess the relative contributions of photoperiod and testosterone in regulating kisspeptin expression in Siberian hamsters. Animals housed in long or short day lengths for 8 weeks were either castrated or received sham surgeries. Half of the hamsters in each photoperiod were given testosterone to mimic long-day sex steroid concentrations. The results obtained indicate that kisspeptin neurones in the AVPV and arcuate nuclei were influenced by both photoperiod and testosterone. In the AVPV, removal of testosterone or exposure to inhibitory day lengths led to a marked reduction in kisspeptin-immunoreactive cells, and testosterone treatment increased cell numbers across conditions. Importantly, long-day castrates exhibited significantly more kisspeptin cells than short-day castrates or intact short-day animals with empty capsules, suggesting the influences of photoperiod, independent of gonadal steroids. In general, the opposite pattern emerged for the arcuate nuclei. Collectively, these data suggest a role for both gonadal-dependent and independent (i.e. photoperiodic) mechanisms regulating seasonal changes in kisspeptin expression in Siberian hamsters.
KiSS-1; reproduction; RF amide; seasonal breeding; gonadal steroids
Gonadotrophin-releasing hormone (GnRH) neurones located within the brain are the final neuroendocrine output regulating the reproductive hormone axis. Their small number and scattered distribution in the hypothalamus make them particularly difficult to study in vivo. The Cre/loxP system is a valuable tool to delete genes in specific cells and tissues. We report the production of two mouse lines that express the CRE bacteriophage recombinase in a GnRH-specific manner. The first line, the GnRH-CRE mouse, contains a transgene in which CRE is under the control of the murine GnRH promoter and targets CRE expression specifically to GnRH neurones in the hypothalamus. The second line, the GnRH-CRETeR mouse, uses the same murine GnRH promoter to target CRE expression to GnRH neurones, but is modified to be constitutively repressed by a tetracycline repressor (TetR) expressed from a downstream tetracycline repressor gene engineered within the transgene. GnRH neurone-specific CRE expression can therefore be induced by treatment with doxycycline which relieves repression by TetR. These GnRH-CRE and GnRH-CRETeR mice can be used to study the function of genes expressed specifically in GnRH neurones. The GnRH-CRETeR mouse can be used to study genes that may have distinct roles in reproductive physiology during the various developmental stages.
GnRH; LHRH; CRE recombinase; transgenic mouse
The hypothalamic-pituitary-gonadal (HPG) axis undergoes a number of changes throughout the reproductive life cycle that are responsible for the development, puberty, adulthood, and senescence of reproductive systems. This natural progression is dictated by the neural network controlling the hypothalamus including the cells that synthesize and release gonadotropin-releasing hormone (GnRH) and their regulatory neurotransmitters. Glutamate and GABA are the primary excitatory and inhibitory neurotransmitters in the central nervous system, and as such contribute a great deal to modulating this axis throughout the lifetime via their actions on receptors in the hypothalamus, both directly on GnRH neurons as well as indirectly though other hypothalamic neural networks. Interactions among GnRH neurons, glutamate, and GABA, including the regulation of GnRH gene and protein expression, hormone release, and modulation by estrogen, are critical to age-appropriate changes in reproductive function. Here, we present evidence for the modulation of GnRH neurosecretory cells by the balance of glutamate and GABA in the hypothalamus, and the functional consequences of these interactions on reproductive physiology across the life cycle.
GnRH; NMDA receptor; glutamate; GABA; hypothalamus; reproductive aging; puberty
Polychlorinated biphenyls (PCBs) can disrupt the reproductive axis, particularly when the exposure occurs during the vulnerable developmental periods. Some effects of environmental endocrine disruptors such as PCBs may be exerted through binding to estrogen receptors (ERs). In this study we examined the endocrine-disrupting effects of Aroclor 1221 (a commercial PCB mixture), focusing on its actions on the ER-ss, which has been implicated in mediating effects of endocrine-disrupting chemicals. A low, ecologically relevant dose of Aroclor 1221 or vehicle (ethanol) was administered three times each to rat dams, on gestational day 16 and on postpartum days 1 and 4, a developmental period during which steroid hormones have permanent effects on adult brain structure and function. Effects on ER-ss cell number in the anteroventral periventricular nucleus (AVPV) were quantified; this sexually dimorphic nucleus of the brain is essential to female reproductive function. For comparison, we quantified ER-ss cell number in another hypothalamic region, the supraoptic nucleus (SON). Using a stereologic approach, we found that Aroclor 1221 caused a highly significant down-regulation of the number of ER-ss-expressing cells in the AVPV, but had no effect in the SON. Thus, PCB exposure has consequences for neural ER expression, and these findings have implications for wildlife and humans that have been exposed to environmental estrogens, particularly during the susceptible periods of early development.
N-methyl--aspartate (NMDA) receptors are glutamate-gated cation channels that mediate excitatory neurotransmission in the central nervous system. In addition to glutamate, NMDA receptors are also activated by coagonist binding of the gliotransmitter, -serine. Neuronal NMDA receptors mediate activity-dependent blood flow regulation in the brain. Our objective was to determine whether NMDA receptors expressed by brain endothelial cells can induce vasodilation of isolated brain arteries. Adult mouse middle cerebral arteries (MCAs) were isolated, pressurized, and preconstricted with norepinephrine. N-methyl--aspartate receptor agonists, glutamate and NMDA, significantly dilated MCAs in a concentration-dependent manner in the presence of -serine but not alone. Dilation was significantly inhibited by NMDA receptor antagonists, -2-amino-5-phosphonopentanoate and 5,7-dichlorokynurenic acid, indicating a response dependent on NMDA receptor glutamate and -serine binding sites, respectively. Vasodilation was inhibited by denuding the endothelium and by selective inhibition or genetic knockout of endothelial nitric oxide synthase (eNOS). We also found evidence for expression of the pan-NMDA receptor subunit, NR1, in mouse primary brain endothelial cells, and for the NMDA receptor subunit NR2C in cortical arteries in situ. Overall, we conclude that NMDA receptor coactivation by glutamate and -serine increases lumen diameter in pressurized MCA in an endothelial and eNOS-dependent mechanism.
-serine; eNOS; glutamate; middle cerebral artery; NMDA receptor; NR2C
N-methyl-D-aspartate (NMDA) receptors are associated with many forms of synaptic plasticity. Their expression level and subunit composition undergo developmental changes in several brain regions. In the mouse cerebellum, beside a developmental switch between NR2B and NR2A/C subunits in granule cells, functional postsynaptic NMDA receptors are seen in Purkinje cells of neonate and adult but not juvenile rat and mice. A presynaptic effect of NMDA on GABA release by cerebellar interneurons was identified recently. Nevertheless whereas NMDA receptor subunits are detected on parallel fiber terminals, a presynaptic effect of NMDA on spontaneous release of glutamate has not been demonstrated. Using mouse cerebellar cultures and patch-clamp recordings we show that NMDA facilitates glutamate release onto Purkinje cells in young cultures via a presynaptic mechanism, whereas NMDA activates extrasynaptic receptors in Purkinje cells recorded in old cultures. The presynaptic effect of NMDA on glutamate release is also observed in Purkinje cells recorded in acute slices prepared from juvenile but not from adult mice and requires a specific protocol of NMDA application.
N-methyl-D-aspartate (NMDA) receptors are ligand-gated ion channels activated by the neurotransmitter glutamate. These channels are highly expressed by brain neurons and are critically involved in excitatory synaptic transmission. Results from previous studies show that both native and recombinant NMDA receptors are inhibited by ethanol at concentrations associated with signs of behavioral impairment and intoxication. Given the important role that NMDA receptors play in synaptic transmission and brain function, it is important to understand the factors that regulate the ethanol inhibition of these receptors. One dynamic mechanism for regulating ethanol action may be via phosphorylation of NMDA subunits by serine-threonine and tyrosine kinases. Both NR1 and NR2 subunits contain multiple sites of phosphorylation and in the NR1 subunit, most of these are contained within the C1 domain, a carboxy-terminal cassette that is subject to alternative splicing. While results from our previous studies suggest that single phosphorylation sites do not greatly affect ethanol sensitivity of NMDA receptors, it is likely that in vivo, these subunits are phosphorylated at multiple sites by different kinases. In the present study, we constructed a series of NMDA receptor mutants at serine (S) or threonine (T) residues proposed to be sites of phosphorylation by PKA and various isoforms of PKC. Ethanol (100 mM) inhibited currents from wild-type NR1/2A and NR1/2B receptors expressed in HEK293 cells by approximately 25% and 30% respectively. This inhibition was not different in single site mutants expressing alanine (A) or aspartate/glutamate (D/E) at positions T879, S896 or T900. The mutant NR1(S890D) showed greater ethanol inhibition than NR1(890A) containing receptors although this was only observed when it was combined with the NR2A subunit. Ethanol inhibition was not altered by aspartate substitution at four serines (positions 889, 890, 896, 897) or when T879D was added to the four serine-substituted mutant. Ethanol inhibition was increased when T900E was added to the five serine/threonine substituted mutant but again this was selective for NR2A containing receptors. Together with previously published data, these findings suggest that modification of putative phosphorylation sites could contribute to the overall acute ethanol sensitivity of recombinant NMDA receptors. Supported by R37 AA009986.
PKA; PKC; phosphorylation; electrophysiology; alcohol
The nervous system (both central and peripheral) is anatomically and physiologically differentiated between the sexes, ranging from gender-based differences in the cerebral cortex to motoneuron number in the spinal cord. Although genetic factors may play a role in the development of some sexually differentiated traits, most identified sex differences in the brain and behavior are produced under the influence of perinatal sex steroid signaling. In many species, the ability to display an estrogen-induced luteinizing hormone (LH) surge is sexually differentiated, yet the specific neural population(s) that allows females but not males to display such estrogen-mediated “positive feedback” has remained elusive. Recently, the Kiss1/kisspeptin system has been implicated in generating the sexually-dimorphic circuitry underlying the LH surge. Specifically, Kiss1 gene expression and kisspeptin protein levels in the anteroventral periventricular (AVPV) nucleus of the hypothalamus are sexually differentiated, with females displaying higher levels than males, even under identical hormonal conditions as adults. These findings, in conjunction with accumulating evidence implicating kisspeptins as potent secretagogues of gonadotropin-releasing hormone (GnRH), suggest that the sex-specific display of the LH surge (positive feedback) reflects sexual differentiation of AVPV Kiss1 neurons. In addition, developmental kisspeptin signaling via its receptor GPR54 appears to be critical in males for the proper sexual differentiation of a variety of sexually dimorphic traits, ranging from complex social behavior to specific forebrain and spinal cord neuronal populations. This review discusses the recent data, and their implications, regarding the bidirectional relationship between the Kiss1 system and the process of sexual differentiation.
metastin; kisspeptin; Kiss1; GPR54; sexual differentiation; sex differences; AVPV; tyrosine hydroxylase; development
The effects of ethanol on brain function are thought to be due in part to alterations in the activity of ion channels that regulate synaptic activity. Results from previous studies from this lab and others have shown that ethanol inhibits the function of the N-methyl-D-aspartate (NMDA) receptors, a calcium-permeable ion channel activated by the neurotransmitter glutamate. Factors that alter the acute sensitivity of NMDA receptors to ethanol may be critical in determining how neurons and neuronal networks respond to the presence of ethanol. In this study, we have examined the effect of physiologically relevant concentrations of magnesium on the ethanol sensitivity of recombinant NMDA receptors and how ethanol inhibition under these conditions is influenced by the NR3A subunit.
Recombinant cDNAs encoding NMDA receptor subunits were expressed in human embryonic kidney (HEK) 293 cells. Whole-cell patch-clamp electrophysiology was used to measure currents induced by rapid application of glutamate in the absence and presence of ethanol.
In magnesium-free recording solution, ethanol inhibited glutamate-mediated currents in cells transfected with NMDA receptor subunits. The magnitude of ethanol inhibition was significantly enhanced when recordings were carried out in media containing 1 mM magnesium. This effect was reversible and required magnesium-sensitive receptors. Magnesium did not enhance ethanol inhibition of glycine-activated NR1/NR3A/NR3B receptors. However, NR3A co-expression prevented the enhancement of ethanol's inhibitory effect on receptors composed of NR2A but not NR2B subunits.
These results suggest that under physiological conditions, NR3A may be an important regulator of the acute ethanol sensitivity of brain NMDA receptors
electrophysiology; addiction; glutamate; HEK cells
The supraoptic nucleus (SON) of the hypothalamus contains magnocellular neurosecretory neurons (MNC) which synthesize and release the peptide hormones vasopressin and oxytocin. Glutamate is a prominent excitatory neurotransmitter in the SON and regulates MNC excitability. NMDA receptors (NMDAR), a type of ionotropic glutamate receptor, mediate synaptic plasticity of MNCs and are necessary for characteristic burst firing patterns which serve to maximize hormone release. NMDARs are di- or tri-heteromeric complexes of NR1 and NR2 subunits. Receptor properties depend on NR2 subunit composition and variable splicing of NR1. We investigated the expression profile of NR1 and NR2 subunits in the SON at the mRNA and protein levels, plus protein expression of NR1 splice variants in control and salt-loaded adult rats. There was robust mRNA expression of all subunits, with NR2D levels being the highest. At the protein level, NR1, NR2B and NR2D were robustly expressed, while NR2A was weakly expressed. NR2C protein was not detected with either of two antibodies. All four NR1 splice variant cassettes (N1, C1, C2, C2’) were detected in the SON, though NR1 N1 expression was too low for accurate analysis. Three days of salt-loading did not alter mRNA, protein or splice variant expression of NMDAR subunits in the SON. Robust NR2D protein expression has not been previously shown in MNCs, and is uncommon in the adult brain. Though the functional significance of this unusual expression profile is unknown, it may contribute to important physiological characteristics of SON neurons, such as burst firing and resistance to excitotoxicity.
hypothalamo-neurohypophyseal system; posterior pituitary; NR2B; NR2D; vasopressin; oxytocin
Recent work identified novel progestin signaling molecules, including progesterone receptor membrane component 1 (Pgrmc1), Pgrmc2, serpine mRNA binding protein 1 (Serbp1), progestin and adiponectin receptors 7 (Paqr7) and Paqr8. These molecules mediate rapid progesterone (P4) effects in non-neural tissue and we recently mapped their expression in the brain. Many rapid effects of P4 require 17β-estradiol (E2) and P4 priming; therefore, we examined the effects of ovarian hormones on the expression of these non-classical progestin signaling molecules. We focused specifically on the anteroventral periventricular nucleus (AVPV), the sexually dimorphic nucleus of the preoptic area (SDN-POA) and the ventrolateral portion of the ventromedial nucleus (VMNvl). These brain nuclei are important for female reproduction. Ovariectomized adult female rats were implanted with capsules containing sesame oil or E2, and injected 48 hours later with sesame oil or P4. Brains were collected eight hours later and RNA was isolated from the AVPV, SDN-POA and VMNvl. We assessed the effects of ovarian hormones on mRNA levels using quantitative polymerase chain reaction (QPCR). In the AVPV, Serbp1 mRNA levels were increased by P4 in the presence of E2, and Paqr8 was downregulated by P4 alone. In the SDN-POA, combined E2 and P4 increased Pgrmc1 and Serbp1 mRNA levels, and E2 alone increased Paqr8 mRNA levels. Finally, in the VMNvl, P4 increased mRNA levels encoding Pgrmc1, Pgrmc2 and Serbp1, and the combination of E2 and P4 increased Pgrmc1 and Serbp1 mRNA levels. Paqr7 was not regulated by E2 or P4 in any brain region examined. In summary, we showed that ovarian hormones regulate novel progestin signaling molecules in brain regions important for the neuroendocrine control of reproduction.
Estradiol; progesterone; neuroendocrine; non-classical signaling; receptor
Caloric restriction enhances N-methyl-D-aspartate (NMDA) receptor binding and upregulates messenger RNA expression of the GluN1 subunit during aging. Old growth hormone receptor knockout mice resemble old calorically restricted rodents in enhanced life span and brain function, as compared with aged controls. This study examined whether aged growth hormone receptor knockout mice also show enhanced expression of NMDA receptors. Six or 23- to 24-month-old male normal-sized control or dwarf growth hormone receptor knockout mice were assayed for NMDA-displaceable [3H]glutamate binding (autoradiography) and GluN1 subunit messenger RNA (in situ hybridization). There was slight sparing of NMDA receptor binding densities within aged medial prefrontal and motor cortices, similar to caloric restriction, but there were greater age-related declines in GluN1 messenger RNA in growth hormone receptor knockout versus control mice. These results suggest that some of the functional improvements in aged mice with altered growth hormone signaling may be due to enhancement of NMDA receptors, but not through the upregulation of messenger RNA for the GluN1 subunit.
NMDA; GluN1; Laron mice; Prefrontal cortex; Hippocampus