Optogenetic Stimulation of Arcuate Nucleus Kiss1 Neurons Reveals a Steroid-Dependent Glutamatergic Input to POMC and AgRP Neurons in Male Mice.

Kisspeptin (Kiss1) neurons are essential for reproduction, but their role in the control of energy balance and other homeostatic functions remains unclear. Proopiomelanocortin (POMC) and agouti-related peptide (AgRP) neurons, located in the arcuate nucleus (ARC) of the hypothalamus, integrate numerous excitatory and inhibitory inputs to ultimately regulate energy homeostasis. Given that POMC and AgRP neurons are contacted by Kiss1 neurons in the ARC (Kiss1(ARC)) and they express androgen receptors, Kiss1(ARC) neurons may mediate the orexigenic action of testosterone via POMC and/or AgRP neurons. Quantitative PCR analysis of pooled Kiss1(ARC) neurons revealed that mRNA levels for Kiss1 and vesicular glutamate transporter 2 were higher in castrated male mice compared with gonad-intact males. Single-cell RT-PCR analysis of yellow fluorescent protein (YFP) ARC neurons harvested from males injected with AAV1-EF1α-DIO-ChR2:YFP revealed that 100% and 88% expressed mRNAs for Kiss1 and vesicular glutamate transporter 2, respectively. Whole-cell, voltage-clamp recordings from nonfluorescent postsynaptic ARC neurons showed that low frequency photo-stimulation (0.5 Hz) of Kiss1-ChR2:YFP neurons elicited a fast glutamatergic inward current in POMC and AgRP neurons. Paired-pulse, photo-stimulation revealed paired-pulse depression, which is indicative of greater glutamate release, in the castrated male mice compared with gonad-intact male mice. Group I and group II metabotropic glutamate receptor agonists depolarized and hyperpolarized POMC and AgRP neurons, respectively, which was mimicked by high frequency photo-stimulation (20 Hz) of Kiss1(ARC) neurons. Therefore, POMC and AgRP neurons receive direct steroid- and frequency-dependent glutamatergic synaptic input from Kiss1(ARC) neurons in male mice, which may be a critical pathway for Kiss1 neurons to help coordinate energy homeostasis and reproduction.

Short photoperiod-induced decrease of histamine H3 receptors facilitates activation of hypothalamic neurons in the Siberian hamster.

Nonhibernating seasonal mammals have adapted to temporal changes in food availability through behavioral and physiological mechanisms to store food and energy during times of predictable plenty and conserve energy during predicted shortage. Little is known, however, of the hypothalamic neuronal events that lead to a change in behavior or physiology. Here we show for the first time that a shift from long summer-like to short winter-like photoperiod, which induces physiological adaptation to winter in the Siberian hamster, including a body weight decrease of up to 30%, increases neuronal activity in the dorsomedial region of the arcuate nucleus (dmpARC) assessed by electrophysiological patch-clamping recording. Increased neuronal activity in short days is dependent on a photoperiod-driven down-regulation of H3 receptor expression and can be mimicked in long-day dmpARC neurons by the application of the H3 receptor antagonist, clobenproprit. Short-day activation of dmpARC neurons results in increased c-Fos expression. Tract tracing with the trans-synaptic retrograde tracer, pseudorabies virus, delivered into adipose tissue reveals a multisynaptic neuronal sympathetic outflow from dmpARC to white adipose tissue. These data strongly suggest that increased activity of dmpARC neurons, as a consequence of down-regulation of the histamine H3 receptor, contributes to the physiological adaptation of body weight regulation in seasonal photoperiod.

Photoperiodic changes in hypothalamic insulin receptor gene expression are regulated by gonadal testosterone.

In order to adapt to seasonal changes, animals exhibit robust changes in their reproductive status, body weight, and molt. However, the molecular mechanisms regulating such seasonal changes in physiology and behavior are not fully understood. Here, we report the photoperiodic regulation of the insulin receptor (IR) gene in the infundibular nucleus (anatomically homologous to the mammalian arcuate nucleus) of the Japanese quail. When the birds were transferred from short-day to long-day conditions, a significant increase in the level of IR mRNA was observed on the 10th long day, whereas that in testicular length was observed on the 5th long day. Castration abolished IR mRNA expression induced by long-day conditions, whereas the testosterone administration mimicked induction of IR mRNA expression induced by long-day conditions. These results suggested that the photoperiodic regulation of the IR mRNA in the infundibular nucleus is mediated by testosterone from the testes. It has been known that the central administration of insulin increases luteinizing hormone (LH) secretion, and neuron-specific disruption of IR gene causes impaired gonadal function due to the dysregulation of LH and increased food intake and body weight. Together with these results, the photoperiodic regulation of the IR mRNA in the hypothalamus may enhance the effect of long days in the seasonal response of reproduction and body weight changes.

Up-regulation of the expression of cocaine and amphetamine-regulated transcript peptide by electroacupuncture in the arcuate nucleus of diet-induced obese rats.

It was reported that acupuncture or electro-acupuncture (EA) is effective in reducing the body weight for obese patients, although the mechanisms remain obscure. In a previous study, we have found that rats fed with high-fat (HIF) diet developed diet-induced obesity (DIO) with a concomitant decrease in the hypothalamic content of the cocaine and amphetamine-regulated transcript (CART) peptide, a peptide with anorexiogenic effect. To assess the central effect of EA on DIO rat, we revealed that EA up-regulated the expression of CART peptide in the arcuate nucleus (ARC) of the DIO rats. After feeding with HIF diet for 14 weeks, the DIO rats received EA stimulation three times per week for 4 weeks. The expression of CART peptide in ARC was measured using immunohistochemistry. The plasma ACTH was measured with ELISA. EA caused a reduction of both body weight and energy intake in DIO rats and increased the expression of CART peptide in ARC. The plasma ACTH was increased in response to restraint stress, but EA produced no further increase in ACTH levels. The results suggest that EA can up-regulate the expression of CART peptide to approach normal level, resulting in an inhibition of food intake and a reduction of body weight in DIO rats.

Neuropeptide Y gene expression in male sheep: influence of photoperiod and testosterone.

The frequency of pulsatile release of gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) is high in the breeding season and low in the nonbreeding season. These alterations in the patterns of GnRH and LH release are due to an interaction of daylength and gonadal steroid negative feedback. A vast amount of data indicates that steroid-responsive neural systems may play a role in regulating seasonal changes in GnRH release. One candidate system is neuropeptide Y (NPY). To determine the independent and interactive influences of photoperiod and steroid exposure on NPY mRNA levels, we used hypothalamic tissue from four groups (n = 4 per group) of castrated male sheep that were simultaneously housed in photochambers and exposed to: (1) a 16L:8D photoperiod (LD); (2) LD and implanted with testosterone (LD + T); (3) a 10L:14D photoperiod (SD), and (4) SD + T. Circulating levels of T averaged 2.8 +/- 0.2 ng/ml in implanted animals, but were undetectable in nonimplanted males. Mean LH levels were significantly reduced (p < 0.01) in the LD + T group as compared with the other groups which did not differ from each other. The silver grain area per NPY neuron in the arcuate nucleus, as assessed by in situ hybridization, was inversely related to mean LH values, with the grain area per cell being significantly greater (p < 0.05) for LD + T males than for all other groups which did not differ from each other. NPY cell numbers were not significantly different (p > 0.10) among the treatment groups. These results show that NPY mRNA expression is increased in male sheep during a LD photoperiod in a T-dependent manner. Our data are consistent with the idea that NPY is involved in the seasonal regulation of GnRH and LH release in the male sheep.

Control of rat hypothalamic pro-opiomelanocortin neurons by a circadian clock that is entrained by the daily light-off signal.

Previous studies have clearly demonstrated that the immediate-early gene, c-fos can regulate, through its protein product Fos, the expression of the pro-opiomelanocortin gene. In the present study, immunohistochemistry for Fos and beta-endorphin was used to assess the basal activity of hypothalamic pro-opiomelanocortin-producing neurons throughout a 12 h light/12 h dark cycle. Here, we showed that Fos is undetectable in most beta-endorphin neurons from late morning until 30 min after light offset in the evening, whereas Fos is spontaneously expressed in these neurons after 1 h following dark onset. The number of beta-endorphin neurons expressing Fos increases continuously during the first half of the dark phase, is maximal at the middle of this phase and decreases through late night and early morning, reaching a nadir 2-3 h after light onset. Acute shifts of lighting parameters allowed us to demonstrate that the light-off signal per se is neither sufficient nor necessary for Fos expression in beta-endorphin neurons. However, when recurrent, this signal is able to entrain Fos expression after a period of adaptation to the new light/dark schedule. Moreover, an expression of Fos in beta-endorphin neurons persists during subjective night in rat exposed to constant light or constant dark for two to three days. Thus, the occurrence of the daily rhythmic increase in the expression of Fos protein in hypothalamic pro-opiomelanocortin neurons exclusively at (subjective) night suggests that these neurons are, most likely, controlled by a (circadian) nocturnal oscillator. Our data also reveal an interesting property of this oscillator: its entrainment by the daily light-to-dark transition signal.

Ultrastructural changes of hypothalamic endocrine zones after irradiation with high energy carbon ions, neutrons, and gamma-rays*.

Ultrastructural lesions in the cells of hypothalamicendocrine zones of the rat brain were investigated 2 and 5 weeks after the exposure to accelerated carbon ions (E = 300 MeV/nucleon) and gamma-rays at a dose of 1.0 Gy and 11 weeks following irradiation with fast neutrons (1.5 MeV) at the doses of 0.25 and 1.0 Gy. The development of degenerative changes in cells of the rat's median eminence and arcuate nucleus after relatively low doses of carbon ions and neutrons was demonstrated. These changes in the hypothalamic endocrine structures could be the reason of the hormonal homeostasis disturbance.

[Ultrastructural changes in neurons of the arcuate nucleus-median eminence complex in rats irradiated with carbon ions and gamma rays]. / Ul'trastrukturnye izmeneniia neironov kompleksa arkuatnoe iadrosredinnoe vozvyshenie u krys, obluchennykh ionami ugleroda i gamma-izlucheniem.

Ultrastructural changes in the arcuate nucleus and medial eminence area of rats were examined 5 weeks after their exposure to accelerated carbon ions with an energy of 300 MeV/nuclon and 60Co gamma-rays at a dose of 2.0 Gy. In carbon-irradiated animals the ultrastructural changes of arcuate nucleus neurons, their processes and synapses were more significant and diversified than in gamma-irradiated rats.

[Karyometric changes in the neurons of the hypothalamic and ependymal nuclei of the 3d cerebral ventricle in sheep after administration of Gn-RH and subsequent irradiation]. / Karyometrické zmeny neurónov jadier hypotalamu a ependýmu tretej mozgovej komory oviec po podaní Gn-RH a následnom oziarení.

Caryometric analysis was used for the study of changes in the cell nucleus volume of the neurons of nucleus paraventricularis, nucleus arcuatus, and ependyma of the third cerebral ventricle of sheep after the administration of Gn-RH, followed by exposure to X-rays. The test animals were 12 ewes in physiological anoestrus and two rams. The trials were conducted in spring. The first group of four sheep and two rams were left as controls; in the ewes of the second group the hypothalamo-hypophysial region was irradiated by exposure to 516.5 mC/kg (200 R); in the four ewes of the third group, ovaries were directly irradiated at laparotomy by exposure to 64.4 mC/kg (250 R). The ewes of the second and third group were treated with an i. m. administration of 400 micrograms Gn-RH per head before irradiation. The excisions were collected and processed the tenth day from irradiation. The studied material was fixed by injection of 10% formalin and finished by another dose of 10% formalin after the excision of the brains. The paraffin slices were stained with haematoxylin-eosine. The caryometric analysis was performed at 3000-fold magnification, 200 cells being measured in each sample. Changes in neurosecretory cells were described in the regions of nucleus paraventricularis, nucleus arcuatus and in the ependyma of the third cerebral ventricle. The results of the caryometric analysis of nucleus paraventricularis and nucleus arcuatus suggest that the administration of Gn-RH and irradiation of the hypothalamo-hypophysial region, and direct irradiation of ovaries, stimulate the studied cerebral structures. The changes observed in the ependyma of the third cerebral ventricle after the administration of Gn-RH and subsequent irradiation of the hypothalamo-hypophysial region are insignificant; it is only after the direct irradiation of ovaries that these cells are inhibited by an indirect effect through the feedback mechanism.