AVPV Neurons Research Articles

28 Melanocortin 3 Receptor in Neural Circuits Linking Metabolic State with Reproduction and Growth

Abstract Metabolic state must be communicated to the brain to regulate behavior, growth, nutrient partitioning and development to meet energetic demands. While it is clearly evident that reproduction and energy balance are closely intertwined, the neural circuits integrating these two processes are poorly understood. Recent functional data suggests the melanocortin 3 receptor (MC3R) mediates the exchange of information between reproductive and metabolic state. Additionally, females exhibit uniquely aberrant metabolic and reproductive phenotypes after deletion of MC3R. In this work, we aimed to characterize the role of MC3R neural circuits in linking metabolic state with reproduction and growth in females. We found that MC3R is expressed in brain regions known to have a role in the control of reproductive and metabolic state, including the anteroventral periventricular nucleus (AVPV) and arcuate nucleus (ARH). In the ARH of female mice, significant differences in overall levels of Mc3r mRNA were not observed at postnatal day 16 (P16), P28 or P48, nor did the percentage of ARH Kiss1, Esr1, Sst, Pomc or Agrp neurons doubly-labeled for Mc3r appear to change during postnatal development. However, the percentage of ARH Ghrh neurons labeled for Mc3r significantly increased from P16 to P48. Thus, MC3R in the ARH may play a potential role in conveying metabolic signals critical for growth during development. In the AVPV of female mice, overall abundance of Mc3r mRNA was low at P16, but increased significantly by P28, and was further elevated in mature mice (P48). Additionally, the percentage of Kiss1- and Esr1-expressing neurons in the AVPV labeled for Mc3r increased significantly between P16 and P48, suggesting that Mc3r may have an important role in the timing of sexual maturation. In addition to the direct regulatory action of MC3R expressed in KISS1 neurons in the AVPV, the impact of MC3R signaling may also be transmitted transynaptically via direct neural projections from MC3R neurons to KISS1 neurons in the AVPV, or to GnRH neurons in the rostral preoptic area. Clearly labeled MC3R terminals were observed in close apposition to both KISS1 neurons in the AVPV, and GnRH neurons in the rostral medial preoptic area. Although both the GnRH and KISS1 neuronal populations appeared heavily innervated by MC3R neurons, the density of MC3R terminals onto KISS1 neurons appeared to be substantially greater than that onto GnRH neurons, suggesting that inputs to AVPV neurons may be particularly impactful on reproduction. Thus, MC3R may not only regulate the activity of KISS1 neurons in the AVPV directly, but may also function transynaptically as an important neurobiological substrate for coordination of metabolic state with sexual maturation and reproductive function.

OR09-01 Mini-Puberty Could Be A Key Phase Regulating Reproductive Function In Female Mice

Abstract Disclosure: M. Chester: None. M. Devillers: None. L. Naule: None. F. Souaré: None. R. Corre: None. D. Quintas: None. C. Petrovic: None. J. Cohen-Tannoudji: None. S. Mhaouty-Kodja: None. C.J. Guigon: None. After birth, there is a transient activation of the hypothalamic-pituitary-ovary (HPO) axis, with high secretion of LH and FSH acting on the ovary to promote the secretion of sex steroids and in particular that of estradiol (E2). This period, called mini-puberty, occurs between 10-17 days postnatal (dpn) in mice. During reproductive life, E2 plays an important role on the HPO axis by regulating GnRH pulsatility and, thus, the cyclic fluctuations of gonadotropin hormone levels required for follicular development and ovulation. E2 acts on hypothalamic kisspeptin neurons of the arcuate (ARC) and the anteroventral periventricular nucleus (AVPV), which project to GnRH neurons and regulate their activity by negative and positive feedbacks, respectively. The possibility that E2 contributes, during mini-puberty, to the maturation of hypothalamic structures involved in puberty onset and reproductive function, has never been investigated. The aim of this study was to elucidate the putative roles of mini-puberty on reproduction. We developed a mouse model injected with a GnRH receptor antagonist (GonadoSTOP mice) suppressing LH and FSH surges to cause a 50% decrease in E2 levels during mini-puberty. Our results show that GonadoSTOP mice exhibited later first diestrus 2 (D2) (31.0 dpn versus 29.9 dpn in CTR; n=55-56 mice/group; P=0.0285), suggesting a possible delay in the timing of puberty. During reproductive life, GonadoSTOP mice spent a longer % of time in D2 than control mice, with no alteration in total estrous cycle time (64.4% versus 56% in CTR; n=18-19 mice/group; P=0.0350). Immunohistochemical analysis of kisspeptin neurons in ARC and AVPV of ovariectomized and hormonally-primed mice showed that their numbers were significantly lower in AVPV from GonadoSTOP mice than in control mice (n=5-6 brains/group; P=0.038). In fertility studies, we observed no difference in the time to conception or % of pregnant mice between the two groups at early reproductive life (3, 4 and 5 months). Contrary to these results, at the time of reproductive decline in control mice (11 months), a substantial % of GonadoSTOP mice could still be pregnant (47% versus 18% of CTR mice; P=0.0017). At this age, GonadoSTOP mice had lower LH levels than control mice (P=0.0074), but similar levels to young control mice at 4 months (P>0.05). RT-qPCR analyses of markers reflecting ovarian activity (Cyp19a1, Inhbb, Amh, Gdf9) at 4 and 11 months revealed that ovarian aging occurred similarly in both groups, suggesting that reproductive longevity in GonadoSTOP mice does not have an ovarian origin. Taken together these data suggest that transient and sustained activation of the HPO axis at mini-puberty may regulate key aspects of reproductive function with long-term impact on fertility, possibly by acting on the hypothalamus. Presentation: Friday, June 16, 2023

Hypothalamic Expression of Estrogen Receptor Isoforms Underlies Estradiol Control of Luteinizing Hormone in Female Rats

Luteinizing hormone (LH) secretion during the ovarian cycle is governed by fluctuations in circulating estradiol (E2) that oppositely regulate kisspeptin neurons in the anteroventral periventricular nucleus (AVPV) and arcuate nucleus (ARC) of the hypothalamus. However, how these effects are orchestrated to achieve fertility is unknown. Here, we have tested the hypothesis that AVPV and ARC neurons have different sensitivities to E2 to coordinate changes in LH secretion. Cycling and ovariectomized rats with low and high E2 levels were used. As an index of E2 responsiveness, progesterone receptor (PR) was expressed only in the AVPV of rats with high E2, showing the preovulatory LH surge. On the other hand, kisspeptin neurons in the ARC responded to low E2 levels sufficient to suppress LH release. Notably, the Esr1/Esr2 ratio of gene expression was higher in the ARC than AVPV, regardless of E2 levels. Accordingly, the selective pharmacological activation of estrogen receptor α (ERα) required lower doses to induce PR in the ARC. The activation of ERβ, in turn, amplified E2-induced PR expression in the AVPV and the LH surge. Thus, ARC and AVPV neurons are differently responsive to E2. Lower E2 levels activate ERα in the ARC, whereas ERβ potentiates the E2 positive feedback in the AVPV, which appears related to the differential Esr1/Esr2 ratio in these 2 brain areas. Our findings provide evidence that the distinct expression of ER isoforms in the AVPV and ARC plays a key role in the control of periodic secretion of LH required for fertility in females.

Progesterone receptor-Src kinase signaling pathway mediates neuroprogesterone induction of the luteinizing hormone surge in female rats.

Neural circuits in female rats are exposed to sequential estradiol and progesterone to regulate the release of luteinizing hormone (LH) and ultimately ovulation. Estradiol induces progesterone receptors (PGRs) in anteroventral periventricular nucleus (AVPV) kisspeptin neurons, and as estradiol reaches peak concentrations, neuroprogesterone (neuroP) synthesis is induced in hypothalamic astrocytes. This local neuroP signals to PGRs expressed in kisspeptin neurons to trigger the LH surge. We tested the hypothesis that neuroP-PGR signaling through Src family kinase (Src) underlies the LH surge. As observed in vitro, PGR and Src are co-expressed in AVPV neurons. Estradiol treatment increased the number of PGR immunopositive cells and PGR and Src colocalization. Furthermore, estradiol treatment increased the number of AVPV cells that had extranuclear PGR and Src in close proximity (<40nm). Infusion of the Src inhibitor (PP2) into the AVPV region of ovariectomized/adrenalectomized (ovx/adx) rats attenuated the LH surge in trunk blood collected 53h post-estradiol (50µg) injection that induced neuroP synthesis. Although PP2 reduced the LH surge in estradiol benzoate treated ovx/adx rats, activation of either AVPV PGR or Src in 2µg estradiol-primed animals significantly elevated LH concentrations compared to dimethyl sulfoxide infused rats. Finally, antagonism of either AVPV PGR or Src blocked the ability of PGR or Src activation to induce an LH surge in estradiol-primed ovx/adx rats. These results indicate that neuroP, which triggers the LH surge, signals through an extranuclear PGR-Src signaling pathway.

SUN-243 Deletion of KNDy Neuron-Specific KISS1 Disrupts Estrous Cyclicity and LH Pulsatility in Female Mice

Kisspeptin (encoded by Kiss1), a neuropeptide critically involved in neuroendocrine regulation of reproduction, is primarily synthesized in two discrete hypothalamic nuclei: the anteroventral periventricular area (AVPV) and arcuate nucleus (ARC). AVPV Kiss1 is important for the pre-ovulatory luteinizing hormone (LH) surge unique to females as well as estrogen-induced positive feedback control of GnRH and LH. In contrast, ARC Kiss1 neurons, which largely co-express the neuropeptides NKB and dynorphin (collectively known as KNDy neurons), are major regulators of pulsatile release of GnRH and LH, and mediate estrogen-induced negative feedback control of both GnRH and LH. Previous studies have not fully separated the specific roles for Kiss1 in the AVPV versus KNDy-ARC neurons in the downstream control of GnRH and LH release. Therefore, we generated a Pdyn-Cre/Kiss1fl/fl (KO) mouse model to target Kiss1 in the KNDy neurons to differentiate KNDy neuron-specific function from AVPV Kiss1 function in the maturation and maintenance of the reproductive axis. qRT-PCR data documented a significant reduction of Kiss1 expression in the mediobasal hypothalamus (containing ARC) compared to controls, whereas Kiss1 in the preoptic area (containing AVPV) was similar in both KO and controls. Immunofluorescent IHC confirmed a loss of kisspeptin immunoreactivity in the ARC of KO animals while expression in the AVPV remained intact. Markers of pubertal onset (day of vaginal opening and first estrus in females; day of preputial separation in males) were normal in KO mice, suggesting that AVPV Kiss1 and/or other neural signals may be sufficient for pubertal onset. In addition, body weight throughout pubertal growth was comparable between KO and control animals of both sexes. Interestingly, KO female mice had disrupted estrous cycles presenting with persistent diestrus and a small vaginal opening. In order to test our hypothesis that conditional deletion of Kiss1 in KNDy neurons disrupts or ablates episodic GnRH/LH pulsatile release, we collected serial tail blood samples from mice at diestrus and measured LH. KO female mice exhibited significantly fewer LH pulses in a 3-hour timespan compared to controls, suggesting that KNDy neurons were functionally compromised. These observations indicate the central role of KNDy neurons in the regulation of GnRH/LH pulsatility and estrous cyclicity. The functional effects of disrupted estrous cyclicity and slower LH pulses observed in KO females are currently under study to assess potential abnormalities in ovarian folliculogenesis and fertility. Future experiments will determine whether ARC Kiss1 deletion disrupts the KNDy-driven negative feedback response of LH to gonadectomy, as well as address potential sex differences in ARC Kiss1-mediated negative feedback control of LH release.

Neurochemical Characterization of Neurons Expressing Estrogen Receptor β in the Hypothalamic Nuclei of Rats Using in Situ Hybridization and Immunofluorescence.

Estrogens play an essential role in multiple physiological functions in the brain, including reproductive neuroendocrine, learning and memory, and anxiety-related behaviors. To determine these estrogen functions, many studies have tried to characterize neurons expressing estrogen receptors known as ERα and ERβ. However, the characteristics of ERβ-expressing neurons in the rat brain still remain poorly understood compared to that of ERα-expressing neurons. The main aim of this study is to determine the neurochemical characteristics of ERβ-expressing neurons in the rat hypothalamus using RNAscope in situ hybridization (ISH) combined with immunofluorescence. Strong Esr2 signals were observed especially in the anteroventral periventricular nucleus (AVPV), bed nucleus of stria terminalis, hypothalamic paraventricular nucleus (PVN), supraoptic nucleus, and medial amygdala, as previously reported. RNAscope ISH with immunofluorescence revealed that more than half of kisspeptin neurons in female AVPV expressed Esr2, whereas few kisspeptin neurons were found to co-express Esr2 in the arcuate nucleus. In the PVN, we observed a high ratio of Esr2 co-expression in arginine-vasopressin neurons and a low ratio in oxytocin and corticotropin-releasing factor neurons. The detailed neurochemical characteristics of ERβ-expressing neurons identified in the current study can be very essential to understand the estrogen signaling via ERβ.

Activity of neurons in the preoptic area and their participation in reproductive senescence: Preliminary findings

Alterations in the hypothalamic–pituitary–gonadal axis in females determine the transition from regular to irregular reproductive cycles, with loss of fertility. Stimulation of noradrenergic neurons of the anteroventral periventricular neurons (AVPV) is essential for regular reproductive cycles. Therefore, we examined the activity of neurons of the AVPV and measure the noradrenaline (NE) of acyclic rats, in constant estrus, and compared it with that of cyclic rats in estrus. Female cyclic (4–5months) and acyclic (17–18months) rats were euthanized at 10, 14, and 18h in estrus. Brains were processed for immunoreactivity to antigens related to Fos (FRA) in AVPV, and the NE was determined by HPLC-ED. Plasma concentrations of LH, FSH, E2 and P4 were determined. In the acyclic animals, plasma LH was higher but the FSH was lower. There was decreasing P4 at different times, while the E2 was constant and lower in acyclic rats. FRA-ir expression in AVPV neurons of acyclic rats as well as turnover of NE was higher when compared with cyclic group. The preliminary findings showed increased activity in AVPV neurons in aging contribute to changes in the temporal pattern of neuroendocrine signaling, compromising the accuracy of inhibitory and stimulatory effects, causing irregularity in the estrous cycle and determining reproductive senescence.

Brain Circuit Compels Maternal Behavior Through Oxytocin

A neural circuit in the brain that is different in female and male mice controls maternal behaviors such as retrieving lost pups—control that is exerted via the secretion of oxytocin. This new finding showcases a direct link between a sexually dimorphic brain region and sex-specific behavior [1]. It has long been assumed that sex differences in behavior arise from neuronal differences in the brain, but only a few studies have shown such a link. For instance, researchers have identified transcription factors that have sexually dimorphic patterns of expression in the hypothalamus and amygdala, resulting in regionally restricted expression in response to the sex hormones that activate these factors. Knocking out each of these factors results in distinct effects on behavior, such as aggression in males or maternal behavior in females. In the new study, Niv Scott et al. took a close look at neurons in a sexually dimorphic region of the hypothalamus, the AVPV (anteroventral periventricular nucleus). In this region, females (compared with males) have a larger number of neurons that contain tyrosine hydroxylase (TH), generally an indicator of a dopaminergic cell. The researchers showed that over 90 percent of these neurons expressed an enzyme responsible for the production of dopamine, which is known to enhance interactions between mother and pups. Consistent with previous studies, males had low numbers of these neurons and virgin females had more (about 493 neurons). Postpartum females—mothers—had even more TH-expressing neurons in this region (about 725 neurons). The researchers then dissected the function of these neurons using three approaches: they ablated these neurons, drove the expression of dopamine in the neurons, and performed optogenetic experiments to activate these neurons with light. The researchers found that this region controlled maternal care in females, such as the tendency of both virgin and post-partum females to retrieve pups wandering from the nest. Ablating neurons in this region quelled such behaviors, and activating the neurons—or driving dopamine expression—promoted the behaviors. Male mice will usually ignore stray pups or attack them, and experiments altering the state of these neurons did not change this behavior. Instead, in males, it seems that this neural circuit controls male-male aggression. Finally, the researchers linked this region to the secretion of oxytocin, which is known to promote maternal behaviors. They found that activating the AVPV neurons in females increased circulating oxytocin, and they showed that the AVPV neurons provided synaptic input to oxytocin-expressing neurons in another region of the periventricular nucleus. The researchers conclude that when a female sees a wandering pup, her AVPV neurons are activated—causing an uptick in circulating oxytocin and compelling her to retrieve the pup.

KNDy Neurons Modulate the Magnitude of the Steroid-Induced Luteinizing Hormone Surges in Ovariectomized Rats.

Kisspeptin is the most potent stimulator of LH release. There are two kisspeptin neuronal populations in the rodent brain: in the anteroventral periventricular nucleus (AVPV) and in the arcuate nucleus. The arcuate neurons coexpress kisspeptin, neurokinin B, and dynorphin and are called KNDy neurons. Because estradiol increases kisspeptin expression in the AVPV whereas it inhibits KNDy neurons, AVPV and KNDy neurons have been postulated to mediate the positive and negative feedback effects of estradiol on LH secretion, respectively. Yet the role of KNDy neurons during the positive feedback is not clear. In this study, ovariectomized rats were microinjected bilaterally into the arcuate nucleus with a saporin-conjugated neurokinin B receptor agonist for targeted ablation of approximately 70% of KNDy neurons. In oil-treated animals, ablation of KNDy neurons impaired the rise in LH after ovariectomy and kisspeptin content in both populations. In estradiol-treated animals, KNDy ablation did not influence the negative feedback of steroids during the morning. Surprisingly, KNDy ablation increased the steroid-induced LH surges, accompanied by an increase of kisspeptin content in the AVPV. This increase seems to be due to lack of dynorphin input from KNDy neurons to the AVPV as the following: 1) microinjections of a dynorphin antagonist into the AVPV significantly increased the LH surge in estradiol-treated rats, similar to KNDy ablation, and 2) intra-AVPV microinjections of dynorphin in KNDy-ablated rats restored LH surge levels. Our results suggest that KNDy neurons provide inhibition to AVPV kisspeptin neurons through dynorphin and thus regulate the amplitude of the steroid-induced LH surges.

Collapsin response mediator protein 4 affects the number of tyrosine hydroxylase‐immunoreactive neurons in the sexually dimorphic nucleus in female mice

In the sexually dimorphic anteroventral periventricular nucleus (AVPV) of the hypothalamus, females have a greater number of tyrosine hydroxylase-immunoreactive (TH-ir) and kisspeptin-immunoreactive (kisspeptin-ir) neurons than males. In this study, we used proteomics analysis and gene-deficient mice to identify proteins that regulate the number of TH-ir and kisspeptin-ir neurons in the AVPV. Analysis of protein expressions in the rat AVPV on postnatal day 1 (PD1; the early phase of sex differentiation) using two-dimensional fluorescence difference gel electrophoresis followed by MALDI-TOF-MS identified collapsin response mediator protein 4 (CRMP4) as a protein exhibiting sexually dimorphic expression. Interestingly, this sexually differential expressions of CRMP4 protein and mRNA in the AVPV was not detected on PD6. Prenatal testosterone exposure canceled the sexual difference in the expression of Crmp4 mRNA in the rat AVPV. Next, we used CRMP4-knockout (CRMP4-KO) mice to determine the in vivo function of CRMP4 in the AVPV. Crmp4 knockout did not change the number of kisspeptin-ir neurons in the adult AVPV in either sex. However, the number of TH-ir neurons was increased in the AVPV of adult female CRMP4-KO mice as compared with the adult female wild-type mice. During development, no significant difference in the number of TH-ir neurons was detected between sexes or genotypes on embryonic day 15, but a female-specific increase in TH-ir neurons was observed in CRMP4-KO mice on PD1, when the sex difference was not yet apparent in wild-type mice. These results indicate that CRMP4 regulates the number of TH-ir cell number in the female AVPV.

Dopamine Regulation of Gonadotropin-Releasing Hormone Neuron Excitability in Male and Female Mice

Numerous in vivo studies have shown that dopamine is involved in the regulation of LH secretion in mammals. However, the mechanisms through which this occurs are not known. In this study, we used green fluorescent protein-tagged GnRH neurons to examine whether and how dopamine may modulate the activity of adult GnRH neurons in the mouse. Bath-applied dopamine (10-80 μm) potently inhibited the firing of approximately 50% of GnRH neurons. This resulted from direct postsynaptic inhibitory actions through D1-like, D2-like, or both receptors. Further, one third of GnRH neurons exhibited an increase in their basal firing rate after administration of SCH23390 (D1-like antagonist) and/or raclopride (D2-like antagonist) indicating tonic inhibition by endogenous dopamine in the brain slice. The role of dopamine in presynaptic modulation of the anteroventral periventricular nucleus (AVPV) γ-aminobutyric acid/glutamate input to GnRH neurons was examined. Exogenous dopamine was found to presynaptically inhibit AVPV-evoked γ-aminobutyric acid /glutamate postsynaptic currents in about 50% of GnRH neurons. These effects were, again, mediated by both D1- and D2-like receptors. Neither postsynaptic nor presynaptic actions of dopamine were found to be different between diestrous, proestrous, and estrous females, or males. Approximately 20% of GnRH neurons were shown to receive a dopaminergic input from AVPV neurons in male and female mice. Together, these observations show that dopamine is one of the most potent inhibitors of GnRH neuron excitability and that this is achieved through complex pre- and postsynaptic actions that each involve D1- and D2-like receptor activation.

Kisspeptin Neurons in the Anteroventral Periventricular Nucleus as Temporal Gate-Keepers to the Preovulatory Gonadotropin-Releasing Hormone Surge.

In rodents, the suprachiasmatic nucleus (SCN) plays a critical role in timing the preovulatory surge of gonadotropin-releasing hormone (GnRH), restricting its occurrence to a narrow temporal window; however, neither the pathway nor the mechanism by which temporal control occurs has been elucidated. Vasopressin (AVP) projections from the SCN to the anteroventral periventricular nucleus (AVPV) may provide the temporal gating mechanism that governs the GnRH surge. Kisspeptin (Kiss1) neurons in AVPV are thought to directly innervate GnRH neurons and trigger the GnRH surge. To explore the role of Kiss1 neurons in timing the preovulatory surge, we used single-cell RT-PCR and whole-cell recordings to investigate endogenous conductances underlying rhythmic burst firing and the effects of AVP in eGFP-labeled Kiss1 neurons of ovariectomized, estradiol-treated mice. AVP signals primarily via the Gq-coupled receptor V1a in the brain, and the expression of V1a was confirmed in analysis of individual Kiss1 neurons (6 animals, 5-19 cells/animal), suggesting that Kiss1 neurons are direct targets for AVP. To assess the functional significance of V1a receptor in Kiss1 neurons, we performed whole cell recordings of the AVPV Kiss1 neurons. Kiss1 neurons had a resting membrane potential of -52.9 ± 2.6 mV (n=27), and 25% of these neurons displayed burst firing. Bath application of AVP (1uM) to hypothalamic slice preparations resulted in rapid depolarization and increased firing activity in a subset of AVPV Kiss1 neurons. Moreover, Kiss1 neurons were also found to express the molecular constituents of a cell-autonomous circadian clock using single cell RT-PCR. Bmal1, a critical molecular component of the transcriptional feedback loop, was detected in 67% (7 animals, 5-19 cells/animal) of Kiss1 cells in the AVPV. Another molecular regulator of the circadian clock, Per2, was also identified in 63% of Kiss1 neurons in this region (6 animals, 5-18cells/animal). Per2 was confirmed in 52% of Bmal1+ cells, and few cells (17%) expressed neither Bmal1 nor Per2 (6 animals, 5-18 cells/animal). We conclude that AVPV Kiss1 neurons are direct targets for activation by AVP - likely arising from afferent projections from the SCN. Taken together, based on the observed expression of key components of molecular clock by these Kiss1 neurons, we infer that exogenous and autonomous temporal regulation gating mechanisms within Kiss1 neurons guide the activation of Kiss1 neurons to generate the preovulatory GnRH surge. (poster)

Frequency-Dependent Recruitment of Fast Amino Acid and Slow Neuropeptide Neurotransmitter Release Controls Gonadotropin-Releasing Hormone Neuron Excitability

The anteroventral periventricular nucleus (AVPV) is thought to play a key role in regulating the excitability of gonadotropin-releasing hormone (GnRH) neurons that control fertility. Using an angled, parahorizontal brain slice preparation we have undertaken a series of electrophysiological experiments to examine how the AVPV controls GnRH neurons in adult male and female mice. More than half (59%) of GnRH neurons located in the rostral preoptic area were found to receive monosynaptic inputs from the AVPV in a sex-dependent manner. AVPV stimulation frequencies <1 Hz generated short-latency action potentials in GnRH neurons with GABA and glutamate mediating >90% of the evoked fast synaptic currents. The AVPV GABA input was dominant and found to excite or inhibit GnRH neurons in a cell-dependent manner. Increasing the AVPV stimulation frequency to 5-10 Hz resulted in the appearance of additional poststimulus inhibitory as well as delayed excitatory responses in GnRH neurons that were independent of ionotropic amino acid receptors. The inhibition observed immediately following the end of the stimulation period was mediated partly by GABA(B) receptors, while the delayed activation was mediated by the neuropeptide kisspeptin. The latter response was essentially absent in Gpr54 knock-out mice and abolished by a Gpr54 antagonist. Together, these studies show that AVPV neurons provide direct amino acid and neuropeptidergic inputs to GnRH neurons. Low-frequency activation generates predominant GABA/glutamate release with higher frequency activation recruiting release of kisspeptin. This frequency-dependent release of amino acid and neuropeptide neurotransmitters greatly expands the range of AVPV control of GnRH neuron excitability.

AVPV neurons containing estrogen receptor-beta in adult male rats are influenced by soy isoflavones

BackgroundIsoflavones, the most abundant phytoestrogens in soy foods, are structurally similar to 17beta-estradiol. It is known that 17beta-estradiol induces apoptosis in anteroventral periventricular nucleus (AVPV) in rat brain. Also, there is evidence that consumption of soy isoflavones reduces the volume of AVPV in male rats. Therefore, in this study, we examined the influence of dietary soy isoflavones on apoptosis in AVPV of 150 day-old male rats fed either a soy isoflavone-free diet (Phyto-free) or a soy isoflavone-rich diet (Phyto-600).ResultsThe occurrence of apoptosis in AVPV was examined by TUNEL staining. The incidence of apoptosis was about 10 times higher in the Phyto-600 group (33.1 ± 1.7%) than in the Phyto-free group (3.6 ± 1.0%). Furthermore, these apoptotic cells were identified as neurons by dual immunofluorescent staining of GFAP and NeuN as markers of astrocytes and neurons, respectively. Then the dopaminergic neurons in AVPV were detected by immunohistochemistry staining of tyrosine hydroxylase (TH). No significant difference in the number of TH neurons was observed between the diet treatment groups. When estrogen receptor (ER) alpha and beta were examined by immunohistochemistry, we observed a 22% reduction of ERbeta-positive cell numbers in AVPV with consumption of soy isoflavones, whereas no significant change in ERalpha-positive cell numbers was detected. Furthermore, almost all the apoptotic cells were ERbeta-immunoreactive (ir), but not ERalpha-ir. Last, subcutaneous injections of equol (a major isoflavone metabolite) that accounts for approximately 70–90% of the total circulating plasma isoflavone levels did not alter the volume of AVPV in adult male rats.ConclusionIn summary, these findings provide direct evidence that consumption of soy isoflavones, but not the exposure to equol, influences the loss of ERbeta-containing neurons in male AVPV.

Neuropeptide Glutamic Acid-Isoleucine May Induce Luteinizing Hormone Secretion via Multiple Pathways

Neuropeptide glutamic acid-isoleucine (NEI) is a 14-amino acid peptide processed from prepro-melanin-concentrating hormone (ppMCH). In males, the localization of NEI is almost identical to that of MCH, the cell bodies of both being located primarily in the lateral hypothalamic area and zona incerta, projecting fibers throughout the brain. Although MCH has been widely studied, the role that NEI plays in brain circuitry has been poorly investigated. Recently, we showed that intracerebroventricular injection of NEI increases serum luteinizing hormone (LH) levels. In order to identify the anatomical substrate underlying this effect, we used combined immunohistochemistry methods to analyze the forebrains of females on the diestrus and proestrus days, as well as those of ovariectomized females treated with estradiol benzoate, with estradiol benzoate plus progesterone or with sesame oil (control animals). We found that ovariectomized females with no steroid treatment showed an increased number of NEI-immunoreactive neurons in the medial zona incerta. In addition, we observed dense to moderate NEI innervation of areas related to reproduction, including the organum vasculosum of the lamina terminalis, the anteroventral periventricular nucleus (AVPV) and the median eminence. The NEI fibers were in close apposition with the AVPV and gonadotropin-releasing hormone (GnRH) neurons expressing Fos in the afternoon of the proestrus day or following administration of estradiol benzoate plus progesterone. In the median eminence, NEI varicosities and terminal-like structures were in close proximity to blood vessels and GnRH fibers. Our results suggest that NEI might induce LH secretion in one of the following ways: by direct release into the median eminence, by modulation of GnRH neurons located in the preoptic area, by modulation of the GnRH terminals located in the median eminence or by an additive effect involving other neurotransmitters or neurohormones. Release of NEI might also induce LH secretion indirectly by modulating AVPV neurons.

Increased Fos Immunoreactivity in Suprachiasmatic Nucleus before Luteinizing Hormone Surge in Estrogen-Treated Ovariectomized Female Rats

Background/Aims: The suprachiasmatic nucleus (SCN) is thought to control the timing of luteinizing hormone (LH) surges. The present study was designed to examine temporal patterns of Fos expression in the dorsomedial and ventrolateral parts of the SCN (SCNdm and SCNvl) of female rats during an LH surge. It also included examination of temporal changes in plasma LH levels and temporal changes in Fos levels in the anteroventral periventricular nucleus (AVPV) and gonadotropin-releasing hormone (GnRH) neurons. Methods: Ovariectomized rats injected with 20 µg estradiol benzoate (EB) or vehicle were sacrificed at various times from Zeitgeber time (ZT) 8:00 to 16:30 h (ZT8–16.5; ZT0 = lights on; ZT12 = lights off) on the 2nd day after the injection. Immunohistochemical analyses for Fos and GnRH and enzyme-linked immunosorbent assays for LH were then performed. Results: In both the SCNdm and SCNvl of EB rats, the number of Fos-immunoreactive cells significantly increased between ZT9.5–10.5 and ZT11–12. On the other hand, in EB rats there were significant peaks of LH levels and Fos levels in GnRH neurons and the AVPV between ZT11–12 and ZT13–14. There was no significant difference in the number of Fos-immunoreactive cells between EB and control rats in either the SCNdm or SCNvl at ZT9.5–10.5, or in the SCNdm at ZT11–12, whereas the SCNvl of EB rats contained more Fos-immunoreactive cells than that of control rats at ZT11–12. Conclusion: These results suggest that in female rats during an LH surge, a peak in the Fos level in the SCN precedes peaks in Fos levels in the AVPV and GnRH neurons.

Both Estrogen Receptor-α and -β Are Required for Sexual Differentiation of the Anteroventral Periventricular Area in Mice

Sexual dimorphisms in the hypothalamus are mediated in several cases by local aromatization of androgens to estrogens during the perinatal period. In this series of experiments, the contributions of the two estrogen receptors (ERs), ERalpha and ERbeta, to the differentiation of the sexually dimorphic subpopulation of dopaminergic neurons in the anteroventral periventricular area (AVPV) was examined. In the first experiment, numbers of tyrosine hydroxylase (TH) immunoreactive (-ir) AVPV neurons in ERbeta knockout and wild-type (WT) mice of both sexes were measured. In the second experiment, the average number of TH-ir neurons in the medial portion of the AVPV in ERalpha knockout, ERbeta knockout, double-ER knockout, and WT mice of both sexes was calculated. In both experiments TH-ir cell numbers were sexually dimorphic as expected, with female individuals of all genotypes exhibiting more TH-ir neurons than WT males. Interestingly the average number of TH-ir neurons in all knockout males was significantly higher than in WT male littermates. In fact, TH-ir cell numbers in all knockout males were equivalent to females. In a final experiment, C57BL/6J female mice were treated during the first 3 postnatal days with either estradiol, or a specific agonist for one of the two ERs. Additional male and female pups received vehicle injections. Treatments with estradiol or either ER-specific agonist significantly reduced the number of TH-ir AVPV neurons in female brains. Our data demonstrate that both ERalpha and ERbeta are involved in the sexual differentiation of the AVPV in mice.

Estrogen and progesterone do not activate Fos in AVPV or LHRH neurons in male rats

In rodents, females but not males, in response to escalating levels of estrogen, express a luteinizing hormone (LH) surge that is prompted by a surge in luteinizing hormone-releasing hormone (LHRH). It cannot take place if estrogen-sensitive afferents located in the anteroventral periventricular nucleus (AVPV) are either absent or disabled. Males appear to lack the ability to exhibit an LH surge, but it is unclear what level of the CNS contributes to this dimorphic response. This study was conducted to determine whether estrogen followed by progesterone treatment (E + P) of gonadectomized males evokes Fos activation in LHRH and AVPV neurons as it does in females. The results indicated that, consistent with the males' inability to express an LH surge in response to E + P treatment, LHRH and AVPV neurons in males failed to show increased Fos activation. Examination of neuron nuclear antigen (NeuN, a neuron-specific marker), estrogen receptor (ERα) and progesterone receptor (PR) neurons in AVPV neurons indicated that, while essentially all the neurons of the caudal AVPV in males and females are steroid responsive, the male possessed half the number of steroid responsive neurons within the caudal AVPV (where activation of Fos is maximal in females) compared to the female. Together, these data indicate that the male lacks a substantial population of steroid receptive AVPV neurons and is unable to respond to the presence of E and P and activate either AVPV or LHRH neurons.

Overexpression of Bcl-2 Reduces Sex Differences in Neuron Number in the Brain and Spinal Cord

Several sex differences in the nervous system depend on differential cell death during development in males and females. The anti-apoptotic protein, Bcl-2, promotes the survival of many types of neurons during development and in response to injury. To determine whether Bcl-2 might similarly control cell death in sexually dimorphic regions, we compared neuron number in wild-type mice and transgenic mice overexpressing Bcl-2 under the control of a neuron-specific promoter. Three neural areas were examined: the spinal nucleus of the bulbocavernosus (SNB), in which neuron number is greater in males; the retrodorsolateral nucleus (RDLN) of the spinal cord, which exhibits no sex difference in neuron number; and the anteroventral periventricular nucleus (AVPV) of the hypothalamus, in which both overall cell density and the number of tyrosine hydroxylase immunoreactive (TH-ir) neurons are greater in females. Bcl-2 overexpression significantly increased SNB cell number in females, overall cell density of AVPV in males, and RDLN cell number in both sexes. Bcl-2 overexpression did not alter the number of TH-ir neurons in AVPV of males or females. These findings indicate that Bcl-2 can regulate sexually dimorphic cell number in the brain and spinal cord and suggest that Bcl-2 may mediate effects of testosterone on cell survival during neural development. In contrast to the regulation of overall cell density in AVPV, the sex difference in TH cell number apparently is not caused by a Bcl-2-dependent mechanism.

Ebb Tide: Brain center linked to decrease in egg-producing waves

As mammals age, one of the first things to go is reproductive capacity. Ovulation becomes irregular, because waves of luteinizing hormone (LH) don't crest as high as before. New results indicate that a reduction in nerve impulses from a particular part of the brain is partly responsible for lowering the levels of the hormone that triggers LH production. Release of luteinizing hormone-releasing hormone (LH-RH) from neurons in the hypothalamus, a part of the brain that governs a host of hormone-controlled bodily functions, spurs the pituitary gland to produce waves of LH. These so-called LH surges stimulate egg release from the ovaries, but they diminish during aging because production of LH-RH by hypothalamic neurons decreases. Exactly what causes this decline is unknown, but some suspect that neurons from elsewhere in the hypothalamus might be to blame. Previous work implicated a region known as the AVPv: AVPv nerve cells connect to LH-RH-producing neurons, and damaging the AVPv blocks LH surges. But it hasn't been clear if a decrease in AVPv activity with advancing years prevents LH-RH synthesis. In search of a missing link, Le and colleagues looked for activity in AVPv and LH-RH neurons in the brains of young and middle-aged rats sacrificed in the midst of an LH surge. The team used antibodies to detect the presence of the Fos protein, an indicator of neuron activity. A smaller percentage of LH-RH neurons in middle-aged rat brains contained Fos as compared with young rat brains. In addition, a similarly reduced fraction of neurons in the AVPv region of middle-aged brains lit up with Fos. The researchers then damaged AVPv in young rats and monitored LH-RH neuron activity. LH-RH neurons on the same side of the brain as the lesion took a hit, expressing no Fos; LH-RH neurons on the uninjured side of the brain carried on normally. The results add to evidence that nerves in the AVPv stimulate LH-RH production. They also suggest that reduced AVPv firing accounts in part for the waning LH-RH production--and, in turn, the declining LH and loss of ovulation--that comes with age. Why AVPv activity falls over time is unclear, and the answer is not likely to be simple. The AVPv takes in signals from multiple hormones and neurons, so the change probably involves more than one trigger. And other brain centers could play roles as well. Further elucidating this pathway will help researchers understand how once-swelling hormonal seas flatten. --R. John Davenport W.-W. Le, P. M. Wise, A. Z. Murphy, L. M. Coolen, G. E. Hoffman, Parallel declines in Fos activation of the medial anteroventral periventricular nucleus and LHRH neurons in middle-aged rats. Endocrinology 142 , 4976-4982 (2001). [Abstract] [Full Text]