Ghrelin-responsive mediobasal hypothalamic neurons mediate exercise-associated food intake and exercise endurance.

Previous studies have implicated the orexigenic hormone ghrelin as a mediator of exercise endurance and the feeding response postexercise. Specifically, plasma ghrelin levels nearly double in mice when they are subjected to an hour-long bout of high-intensity interval exercise (HIIE) using treadmills. Also, growth hormone secretagogue receptor-null (GHSR-null) mice exhibit decreased food intake following HIIE and diminished running distance (time until exhaustion) during a longer, stepwise exercise endurance protocol. To investigate whether ghrelin-responsive mediobasal hypothalamus (MBH) neurons mediate these effects, we stereotaxically delivered the inhibitory designer receptor exclusively activated by designer drugs virus AAV2-hSyn-DIO-hM4(Gi)-mCherry to the MBH of Ghsr-IRES-Cre mice, which express Cre recombinase directed by the Ghsr promoter. We found that chemogenetic inhibition of GHSR-expressing MBH neurons (upon delivery of clozapine-N-oxide) 1) suppressed food intake following HIIE, 2) reduced maximum running distance and raised blood glucose and blood lactate levels during an exercise endurance protocol, 3) reduced food intake following ghrelin administration, and 4) did not affect glucose tolerance. Further, HIIE increased MBH Ghsr expression. These results indicate that activation of ghrelin-responsive MBH neurons is required for the normal feeding response to HIIE and the usual amount of running exhibited during an exercise endurance protocol.

Evidence of extraganglionic vagal mechanoreceptors in the mouse vagus nerve.

Vagal afferent neuronal somas are in the nodose and jugular ganglia. In this study, we identified extraganglionic neurons in whole-mount preparations of the vagus nerves from Phox2b-Cre-ZsGreen transgenic mice. These neurons are typically arranged in small clusters and monolayers along the cervical vagus nerve. Although infrequent, these neurons were sometimes observed along the thoracic and esophageal vagus. We performed RNAscope in situ hybridization and confirmed that the extraganglionic neurons detected in this transgenic mouse strain expressed vagal afferent markers (i.e., Phox2b and Slc17a6) as well as markers that identify them as potential gastrointestinal mechanoreceptors (i.e., Tmc3 and Glp1r). We also identified extraganglionic neurons in the vagus nerves of wild-type mice that were injected intraperitoneally with Fluoro-Gold, thereby ruling out possible anatomical discrepancies specific for transgenic mice. In wild-type mice, extraganglionic cells were positive for peripherin, confirming their neuronal nature. Taken together, our findings revealed a previously undiscovered population of extraganglionic neurons associated with the vagus nerve. Going forward, it is important to consider the possible existence of extraganglionic mechanoreceptors that transmit signals from the abdominal viscera in future studies related to vagal structure and function.

Delineating a serotonin 1B receptor circuit for appetite suppression in mice.

Triptans are a class of commonly prescribed antimigraine drugs. Here, we report a previously unrecognized role for them to suppress appetite in mice. In particular, frovatriptan treatment reduces food intake and body weight in diet-induced obese mice. Moreover, the anorectic effect depends on the serotonin (5-HT) 1B receptor (Htr1b). By ablating Htr1b in four different brain regions, we demonstrate that Htr1b engages in spatiotemporally segregated neural pathways to regulate postnatal growth and food intake. Moreover, Htr1b in AgRP neurons in the arcuate nucleus of the hypothalamus (ARH) contributes to the hypophagic effects of HTR1B agonists. To further study the anorexigenic Htr1b circuit, we generated Htr1b-Cre mice. We find that ARH Htr1b neurons bidirectionally regulate food intake in vivo. Furthermore, single-nucleus RNA sequencing analyses revealed that Htr1b marks a subset of AgRP neurons. Finally, we used an intersectional approach to specifically target these neurons (Htr1bAgRP neurons). We show that they regulate food intake, in part, through a Htr1bAgRP→PVH circuit.

Suprachiasmatic nucleus-mediated glucose entry into the arcuate nucleus determines the daily rhythm in blood glycemia.

Glycemia is maintained within very narrow boundaries with less than 5% variation at a given time of the day. However, over the circadian cycle, glycemia changes with almost 50% difference. How the suprachiasmatic nucleus, the biological clock, maintains these day-night variations with such tiny disparities remains obscure. We show that via vasopressin release at the beginning of the sleep phase, the suprachiasmatic nucleus increases the glucose transporter GLUT1 in tanycytes. Hereby GLUT1 promotes glucose entrance into the arcuate nucleus, thereby lowering peripheral glycemia. Conversely, blocking vasopressin activity or the GLUT1 transporter at the daily trough of glycemia increases circulating glucose levels usually seen at the peak of the rhythm. Thus, biological clock-controlled mechanisms promoting glucose entry into the arcuate nucleus explain why peripheral blood glucose is low before sleep onset.

Identification of Leptin Receptor-Expressing Cells in the Nodose Ganglion of Male Mice.

Leptin has been proposed to modulate viscerosensory information directly at the level of vagal afferents. In support of this view, broad expression for the leptin receptor (Lepr) has previously been reported in vagal afferents. However, the exact identity and distribution of leptin-sensitive vagal afferents has not been elucidated. Using quantitative PCR, we found that the whole mouse nodose ganglion was predominantly enriched in the short form of Lepr, rather than its long form. Consistent with this observation, the acute administration of leptin did not stimulate JAK-STAT signaling in the nodose ganglion. Using chromogenic in situ hybridization in wild-type mice and several reporter mouse models, we demonstrated that Lepr mRNA was restricted to nonneuronal cells in the epineurium and parenchyma of the nodose ganglion and a subset of vagal afferents, which accounted for only 3% of all neuronal profiles. Double labeling studies further established that Lepr-expressing vagal afferents were Nav1.8-negative fibers that did not supply the peritoneal cavity. Finally, double chromogenic in situ hybridization revealed that many Lepr-expressing neurons coexpressed the angiotensin 1a receptor (At1ar), which is a gene expressed in baroreceptors. Taken together, our data challenge the commonly held view that Lepr is broadly expressed in vagal afferents. Instead, our data suggest that leptin may exert a previously unrecognized role, mainly via its short form, as a direct modulator of a very small group of At1ar-positive vagal fibers.

Suprachiasmatic Nucleus Interaction with the Arcuate Nucleus; Essential for Organizing Physiological Rhythms.

The suprachiasmatic nucleus (SCN) is generally considered the master clock, independently driving all circadian rhythms. We recently demonstrated the SCN receives metabolic and cardiovascular feedback adeptly altering its neuronal activity. In the present study, we show that microcuts effectively removing SCN-arcuate nucleus (ARC) interconnectivity in Wistar rats result in a loss of rhythmicity in locomotor activity, corticosterone levels, and body temperature in constant dark (DD) conditions. Elimination of these reciprocal connections did not affect SCN clock gene rhythmicity but did cause the ARC to desynchronize. Moreover, unilateral SCN lesions with contralateral retrochiasmatic microcuts resulted in identical arrhythmicity, proving that for the expression of physiological rhythms this reciprocal SCN-ARC interaction is essential. The unaltered SCN c-Fos expression following glucose administration in disconnected animals as compared to a significant decrease in controls demonstrates the importance of the ARC as metabolic modulator of SCN neuronal activity. Together, these results indicate that the SCN is more than an autonomous clock, and forms an essential component of a larger network controlling homeostasis. The present novel findings illustrate how an imbalance between SCN and ARC communication through circadian disruption could be involved in the etiology of metabolic disorders.

The Arcuate Nucleus: A Site of Fast Negative Feedback for Corticosterone Secretion in Male Rats.

Variations in circulating corticosterone (Cort) are driven by the paraventricular nucleus of the hypothalamus (PVN), mainly via the sympathetic autonomic nervous system (ANS) directly stimulating Cort release from the adrenal gland and via corticotropin-releasing hormone targeting the adenohypophysis to release adrenocorticotropic hormone (ACTH). Cort feeds back through glucocorticoid receptors (GRs). Here we show in male Wistar rats that PVN neurons projecting to the adrenal gland do not express GRs, leaving the question of how the ANS in the PVN gets information about circulating Cort levels to control the adrenal. Since the arcuate nucleus (ARC) shows a less restrictive blood-brain barrier, expresses GRs, and projects to the PVN, we investigated whether the ARC can detect and produce fast adjustments of circulating Cort. In low Cort conditions (morning), local microdialysis in the ARC with type I GR antagonist produced a fast and sustained increase of Cort. This was not observed with a type II antagonist. At the circadian peak levels of Cort (afternoon), a type II GR antagonist, but not a type I antagonist, increased Cort levels but not ACTH levels. Antagonist infusions in the PVN did not modify circulating Cort levels, demonstrating the specificity of the ARC to give Cort negative feedback. Furthermore, type I and II GR agonists in the ARC prevented the increase of Cort after stress, demonstrating the role of the ARC as sensor to modulate Cort release. Our findings show that the ARC may be essential to sense blood levels of Cort and adapt Cort secretion depending on such conditions as stress or time of day.

The Circadian System: A Regulatory Feedback Network of Periphery and Brain.

Circadian rhythms are generated by the autonomous circadian clock, the suprachiasmatic nucleus (SCN), and clock genes that are present in all tissues. The SCN times these peripheral clocks, as well as behavioral and physiological processes. Recent studies show that frequent violations of conditions set by our biological clock, such as shift work, jet lag, sleep deprivation, or simply eating at the wrong time of the day, may have deleterious effects on health. This infringement, also known as circadian desynchronization, is associated with chronic diseases like diabetes, hypertension, cancer, and psychiatric disorders. In this review, we will evaluate evidence that these diseases stem from the need of the SCN for peripheral feedback to fine-tune its output and adjust physiological processes to the requirements of the moment. This feedback can vary from neuronal or hormonal signals from the liver to changes in blood pressure. Desynchronization renders the circadian network dysfunctional, resulting in a breakdown of many functions driven by the SCN, disrupting core clock rhythms in the periphery and disorganizing cellular processes that are normally driven by the synchrony between behavior and peripheral signals with neuronal and humoral output of the hypothalamus. Consequently, we propose that the loss of synchrony between the different elements of this circadian network as may occur during shiftwork and jet lag is the reason for the occurrence of health problems.

Role of the Suprachiasmatic and Arcuate Nuclei in Diurnal Temperature Regulation in the Rat.

In mammals, daily changes in body temperature (Tb) depend on the integrity of the suprachiasmatic nucleus (SCN). Fasting influences the Tb in the resting period and the presence of the SCN is essential for this process. However, the origin of this circadian/metabolic influence is unknown. We hypothesized that, not only the SCN but also the arcuate nucleus (ARC), are involved in the Tb setting through afferents to the thermoregulatory median preoptic nucleus (MnPO). Therefore, we investigated by neuronal tracing and microdialysis experiments the possible targeting of the MnPO by the SCN and the ARC in male Wistar rats. We observed that vasopressin release from the SCN decreases the temperature just before light onset, whereas α-melanocyte stimulating hormone release, especially at the end of the dark period, maintains high temperature. Both peptides have opposite effects on the brown adipose tissue activity through thermoregulatory nuclei such as the dorsomedial nucleus of the hypothalamus and the dorsal raphe nucleus. The present study indicates that the coordination between circadian and metabolic signaling within the hypothalamus is essential for an adequate temperature control. SIGNIFICANCE STATEMENT: When circadian and metabolic systems are not well synchronized, individuals may develop metabolic diseases. The underlying mechanisms are unknown. Here, we demonstrate that the balance between the releases of neuropeptides derived from the biological clock and from a metabolic sensory organ as the arcuate nucleus, are essential for an adequate temperature control. These observations show that brain areas involved in circadian and metabolic functions of the body need to interact to produce a coherent arrangement of physiological processes associated with temperature control.