The Pituitary-Adrenal Response to Paradoxical Sleep Deprivation Is Similar to a Psychological Stressor, Whereas the Hypothalamic Response Is Unique.

Stressors of different natures induce activation of the hypothalamic-pituitary-adrenal (HPA) axis at different magnitudes. Moreover, the HPA axis response to repeated exposure is usually distinct from that elicited by a single session. Paradoxical sleep deprivation (PSD) augments ACTH and corticosterone (CORT) levels, but the nature of this stimulus is not yet defined. The purpose of the present study was to qualitatively compare the stress response of animals submitted to PSD to that of rats exposed once or four times to cold, as a physiological stress, movement restraint (RST) as a mixed stressor and predator odour (PRED) as the psychological stressor, whilst animals were submitted for 1 or 4 days to PSD and respective control groups. None of the stressors altered corticotropin releasing factor immunoreactivity in the paraventricular nucleus of the hypothalamus (PVN), median eminence (ME) or central amygdala, compared to control groups, whereas vasopressin immunoreactivity in PSD animals was decreased in the PVN and increased in the ME, indicating augmented activity of this system. ACTH levels were higher after repeated stress or prolonged PSD than after single- or 1 day-exposure and control groups, whereas the CORT response was habituated by repeated stress, but not by 4-days PSD. This dissociation resulted in changes in the CORT : ACTH ratio, with repeated cold and RST decreasing the ratio compared to single exposure, but no change was seen in PRED and PSD groups. Comparing the magnitude and pattern of pituitary-adrenal response to the different stressors, PSD-induced responses were closer to that shown by PRED-exposed rats. In contrast, the hypothalamic response of PSD-exposed rats was unique, inasmuch as this was the only stressor which increased the activity of the vasopressin system. In conclusion, we propose that the pituitary-adrenal response to PSD is similar to that induced by a psychological stressor.

Anatomical Markers of Activity in Hypothalamic Neurons.

The scientific community has searched for years for ways of examining neuronal tissue to track neural activity with reliable anatomical markers for stimulated neuronal activity. Existing studies that focused on hypothalamic systems offer a few options but do not always compare approaches or validate them for dependence on cell firing, leaving the reader uncertain of the benefits and limitations of each method. Thus, in this article, potential markers will be presented and, where possible, placed into perspective in terms of when and how these methods pertain to hypothalamic function. An example of each approach is included. In reviewing the approaches, one is guided through how neurons work, the consequences of their stimulation, and then the potential markers that could be applied to hypothalamic systems are discussed. Approaches will use features of neuronal glucose utilization, water/oxygen movement, changes in neuron-glial interactions, receptor translocation, cytoskeletal changes, stimulus-synthesis coupling that includes expression of the heteronuclear or mature mRNA for transmitters or the enzymes that make them, and changes in transcription factors (immediate early gene products, precursor buildup, use of promoter-driven surrogate proteins, and induced expression of added transmitters. This article includes discussion of methodological limitations and the power of combining approaches to understand neuronal function. © 2020 American Physiological Society. Compr Physiol 10:549-575, 2020.

Impairments in the reproductive axis of female mice lacking estrogen receptor ß in GnRH neurons.

The effect of estrogen on the differentiation and maintenance of reproductive tissues is mediated by two nuclear estrogen receptors (ERs), ERα, and ERß. Lack of functional ERα and ERß genes in vivo significantly affects reproductive function; however, the target tissues and signaling pathways in the hypothalamus are not clearly defined. Here, we describe the generation and reproductive characterization of a complete-ERß KO (CERßKO) and a GnRH neuron-specific ERßKO (GERßKO) mouse models. Both ERßKO mouse models displayed a delay in vaginal opening and first estrus. Hypothalamic gonadotropin-releasing hormone (GnRH) mRNA expression levels in both ERßKO mice were similar to control mice; however female CERßKO and GERßKO mice had lower basal and surge serum gonadotropin levels. Although a GnRH stimulation test in both female ERßKO models showed preserved gonadotropic function in the same animals, a kisspeptin stimulation test revealed an attenuated response by GnRH neurons, suggesting a role for ERß in normal GnRH neuron function. No alteration in estrogen-negative feedback was observed in either ERßKO mouse models after ovariectomy and estrogen replacement. Further, abnormal development of ovarian follicles with low serum estradiol levels and impairment of fertility were observed in both ERßKO mouse models. In male ERßKO mice, no differences in the timing of pubertal onset or serum luteinizing hormone and follicle-stimulating hormone levels were observed as compared with controls. Taken together, these data provide in vivo evidence for a role of ERß in GnRH neurons in modulating puberty and reproduction, specifically through kisspeptin responsiveness in the female hypothalamic-pituitary-gonadal axis.

Jak2 is necessary for neuroendocrine control of female reproduction.

Gonadotropin-releasing hormone (GnRH) neurons represent the final common output of signals from the brain that regulates reproductive function. A wide range of environmental factors impact GnRH neuron activity including disease, stress, nutrition, and seasonal cues, as well as gonadal steroid hormones. The CNS response is thought to be mediated, at least in part, through intermediate signaling molecules that affect GnRH neuronal activity. In vitro, GnRH neuronal cell lines respond to a variety of ligands that activate the Jak (Janus-activated kinase)/STAT (signal transducers and activators of transcription) intracellular signaling pathway. To determine its biological function in reproduction, we used Cre (cAMP response element)/LoxP technology to generate GnRH neuron-specific Jak2 conditional knock-out (Jak2 G(-/-)) mice. GnRH mRNA levels were reduced in Jak2 G(-/-) mice when compared with controls, while the number of GnRH neurons was equivalent, indicating a reduction in GnRH gene expression. Secretion of GnRH is also reduced as basal serum luteinizing hormone (LH) levels were significantly lower in female Jak2 G(-/-) mice while the pituitary responded normally to exogenous GnRH. Preovulatory LH surge levels were blunted in Jak2 G(-/-) mice, which was correlated with reduced GnRH neuronal activation as assessed by c-Fos. However, the activation of GnRH neurons following release from estrogen-negative feedback is retained. Female Jak2 G(-/-) mice exhibited significantly delayed puberty and first estrus, abnormal estrous cyclicity, and impaired fertility. These results demonstrate an essential role for Jak2 signaling in GnRH neurons for normal reproductive development and fertility in female mice.

Kisspeptin regulates prolactin release through hypothalamic dopaminergic neurons.

Prolactin (PRL) is tonically inhibited by dopamine (DA) released from neurons in the arcuate and periventricular nuclei. Kisspeptin plays a pivotal role in LH regulation. In rodents, kisspeptin neurons are found mostly in the anteroventral periventricular and arcuate nuclei, but the physiology of arcuate kisspeptin neurons is not completely understood. We investigated the role of kisspeptin in the control of hypothalamic DA and pituitary PRL secretion in adult rats. Intracerebroventricular kisspeptin-10 (Kp-10) elicited PRL release in a dose-dependent manner in estradiol (E2)-treated ovariectomized rats (OVX+E2), whereas no effect was found in oil-treated ovariectomized rats (OVX). Kp-10 increased PRL release in males and proestrous but not diestrous females. Associated with the increase in PRL release, intracerebroventricular Kp-10 reduced Fos-related antigen expression in tyrosine hydroxylase-immunoreactive (ir) neurons of arcuate and periventricular nuclei in OVX+E2 rats, with no effect in OVX rats. Kp-10 also decreased 3,4-dihydroxyphenylacetic acid concentration and 3,4-dihydroxyphenylacetic acid-DA ratio in the median eminence but not striatum in OVX+E2 rats. Double-label immunofluorescence combined with confocal microscopy revealed kisspeptin-ir fibers in close apposition to and in contact with tyrosine hydroxylase-ir perikarya in the arcuate. In addition, Kp-10 was not found to alter PRL release from anterior pituitary cell cultures regardless of E2 treatment. We provide herein evidence that kisspeptin regulates PRL release through inhibition of hypothalamic dopaminergic neurons, and that this mechanism is E2 dependent in females. These findings suggest a new role for central kisspeptin with possible implications for reproductive physiology.

Sleep deprivation of rats: the hyperphagic response is real.

STUDY OBJECTIVES: Chronic sleep deprivation of rats causes hyperphagia without body weight gain. Sleep deprivation hyperphagia is prompted by changes in pathways governing food intake; hyperphagia may be adaptive to sleep deprivation hypermetabolism. A recent paper suggested that sleep deprivation might inhibit ability of rats to increase food intake and that hyperphagia may be an artifact of uncorrected chow spillage. To resolve this, a palatable liquid diet (Ensure) was used where spillage is insignificant. DESIGN: Sleep deprivation of male Sprague Dawley rats was enforced for 10 days by the flowerpot/platform paradigm. Daily food intake and body weight were measured. On day 10, rats were transcardially perfused for analysis of hypothalamic mRNA expression of the orexigen, neuropeptide Y (NPY). SETTING: Morgan State University, sleep deprivation and transcardial perfusion; University of Maryland, NPY in situ hybridization and analysis. MEASUREMENTS AND RESULTS: Using a liquid diet for accurate daily measurements, there was no change in food intake in the first 5 days of sleep deprivation. Importantly, from days 6-10 it increased significantly, peaking at 29% above baseline. Control rats steadily gained weight but sleep-deprived rats did not. Hypothalamic NPY mRNA levels were positively correlated to stimulation of food intake and negatively correlated with changes in body weight. CONCLUSION: Sleep deprivation hyperphagia may not be apparent over the short term (i.e., < or = 5 days), but when extended beyond 6 days, it is readily observed. The timing of changes in body weight and food intake suggests that the negative energy balance induced by sleep deprivation prompts the neural changes that evoke hyperphagia.

Changes in hypothalamic corticotropin-releasing hormone, neuropeptide Y, and proopiomelanocortin gene expression during chronic rapid eye movement sleep deprivation of rats.

Chronic rapid eye movement (paradoxical) sleep deprivation (REM-SD) of rats leads to two conspicuous pathologies: hyperphagia coincident with body weight loss, prompted by elevated metabolism. Our goals were to test the hypotheses that 1) as a stressor, REM-SD would increase CRH gene expression in the hypothalamus and that 2) to account for hyperphagia, hypothalamic gene expression of the orexigen neuropeptide Y (NPY) would increase, but expression of the anorexigen proopiomelanocortin (POMC) would decrease. Enforcement of REM-SD of adult male rats for 20 d with the platform (flowerpot) method led to progressive hyperphagia, increasing to approximately 300% of baseline; body weight steadily declined by approximately 25%. Consistent with changes in food intake patterns, NPY expression rapidly increased in the hypothalamic arcuate nucleus by d 5 of REM-SD, peaking at d 20; by contrast, POMC expression decreased progressively during REM-SD. CRH expression was increased by d 5, both in mRNA and ability to detect neuronal perikaryal staining in paraventricular nucleus with immunocytochemistry, and it remained elevated thereafter with modest declines. Taken together, these data indicate that changes in hypothalamic neuropeptides regulating food intake are altered in a manner consistent with the hyperphagia seen with REM-SD. Changes in CRH, although indicative of REM-SD as a stressor, suggest that the anorexigenic actions of CRH are ineffective (or disabled). Furthermore, changes in NPY and POMC agree with current models of food intake behavior, but they are opposite to their acute effects on peripheral energy metabolism and thermogenesis.