Basal Hypothalamus Research Articles

β-Lapachone Exerts Hypnotic Effects via Adenosine A1 Receptor in Mice.

Sleep is one of the most essential physiological phenomena for maintaining health. Sleep disturbances, such as insomnia, are often accompanied by psychiatric or physical conditions such as impaired attention, anxiety, and stress. Medication used to treat insomnia have concerns about potential side effects with long-term use, so interest in the use of alternative medicine is increasing. In this study, we investigated the hypnotic effects of β-lapachone (β-Lap), a natural naphthoquinone compound, using pentobarbital-induced sleep test, immunohistochemistry, real-time PCR, and western blot in mice. Our results indicated that β-Lap exerts a significant hypnotic effect by showing a decrease in sleep onset latency and an increase in total sleep time in pentobarbital-induced sleep model. The results of c-Fos immunostaining showed that β-Lap decreased neuronal activity in the basal forebrain and lateral hypothalamus, which are wakefulness-promoting brain regions, while increasing in the ventrolateral preoptic nucleus, a sleep-promoting region; all these effects were significantly abolished by 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), an adenosine A1 receptor (A1R) antagonist. Western blot analysis showed that β-Lap increased extracellular signalregulated kinase phosphorylation and nuclear factor-kappa B translocation from the cytoplasm to the nucleus; these effects were inhibited by DPCPX. Additionally, β-Lap increased the mRNA levels of A1R. Taken together, these results suggest that β-Lap exerts hypnotic effects, potentially through A1R.

Ventral amygdalofugal pathway as an integrated surgically important network: microsurgical anatomy and segmentation based on fiber dissection.

The ventral amygdalofugal pathway (VAFP) provides afferent and efferent connections to the amygdala and spans along some of the frequently traversed intra-axial surgical corridors as a dominant fiber bundle. This study aimed to reveal the frequently overlooked VAFP fibers by examining their courses and connections to the basal forebrain, septal region, hypothalamus, thalamus, tegmentum, and brainstem. Ten postmortem human brains were used to display the characteristics of the VAFP, and fiber dissection results were compared with those of tractography. From anterior to posterior, the VAFP was separated into 5 different portions: 1) amygdala-substantia innominata; 2) amygdaloseptal (diagonal band of Broca); 3) amygdalo-thalamic; 4) amygdalo-hypothalamic, intermingling with the medial forebrain bundle and extending to the bed nucleus of stria terminalis; and 5) amygdalotegmental. The results of fiber dissections were confirmed with findings obtained from diffusion tensor tractography. This study supports the concept that interconnected forebrain, diencephalic, mesencephalic, and brainstem connections of the VAFP form an integrated surgically important network. The fiber dissection findings also provide the neuroanatomical basis for VAFP segmentation, which may help neurosurgeons better appreciate the complex microsurgical anatomy of the amygdalar connections. Amygdala-substantia innominata and amygdalotegmental connections are demonstrated for the first time and clarified within the structure of the VAFP.

Distribution of gamma-aminobutyric acid immunoreactivity in the brain of the Siberian sturgeon (Acipenser baeri): Comparison with other fishes.

Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the central nervous system (CNS) of vertebrates. Immunohistochemical techniques with specific antibodies against GABA or against its synthesizing enzyme, glutamic acid decarboxylase (GAD) allowed characterizing GABAergic neurons and fibers in the CNS. However, studies on the CNS distribution of GABAergic neurons and fibers of bony fishes are scant and were done in teleost species. With the aim of understanding the early evolution of this system in bony vertebrates, we analyzed the distribution of GABA-immunoreactive (-ir) and GAD-ir neurons and fibers in the CNS of a basal ray-finned fish, the Siberian sturgeon (Chondrostei, Acipenseriformes), using immunohistochemical techniques. Our results revealed the presence and distribution of GABA/GAD-ir cells in different regions of the CNS such as olfactory bulbs, pallium and subpallium, hypothalamus, thalamus, pretectum, optic tectum, tegmentum, cerebellum, central grey, octavolateralis area, vagal lobe, rhombencephalic reticular areas, and the spinal cord. Abundant GABAergic innervation was observed in most brain regions, and GABAergic fibers were very abundant in the hypothalamic floor along the hypothalamo-hypophyseal tract and neurohypophysis. In addition, GABA-ir cerebrospinal fluid-contacting cells were observed in the alar and basal hypothalamus, saccus vasculosus, and spinal cord central canal. The distribution of GABAergic systems in the sturgeon brain shows numerous similarities to that observed in lampreys, but also to those of teleosts and tetrapods.

Gpr149 is involved in energy homeostasis in the male mouse.

GPR149 is an orphan receptor about which little is known. Accordingly, in the present study, we mapped the tissue expression of Gpr149 in mice using three complementary approaches: quantitative PCR, in situ hybridization, and a newly generated Gpr149-Cre reporter mouse model. The strongest expressions of Gpr149 were observed in neurons of the islands of Calleja, the ventromedial hypothalamus, and the rostral interpeduncular nucleus. Moderate-to-low expression was also observed in the basal forebrain, striatum, hypothalamus, brainstem, and spinal cord. Some Gpr149 expression was also detected in the primary afferent neurons, enteric neurons, and pituitary endocrine cells. This expression pattern is consistent with the involvement of GPR149 signaling in the regulation of energy balance. To explore the physiological function of GPR149 in vivo, we used CRISPR-Cas9 to generate a global knockout allele with mice lacking Gpr149 exon 1. Preliminary metabolic findings indicated that Gpr149-/- mice partially resist weight gain when fed with a high-fat diet and have greater sensitivity to insulin than control mice. In summary, our data may serve as a resource for future in vivo studies on GPR149 in the context of diet-induced obesity.

Anton de Haen (1704-1776) and his extraordinary "portentosum infundibulum" case: the futile skull cauterization of a blind patient with a craniopharyngioma.

Anton de Haen (1704-1776) became one of the most influential physicians in the Habsburg Empire as a reformer of clinical instruction at Vienna Citizen's Hospital (Bürgerspital), where he introduced the bedside teaching method he had learned from Herman Boerhaave in Leyden, Holland. He also promoted the meticulous recording of clinical observations and the use of postmortem studies to identify the cause of death in hospitalized patients. Among the numerous clinicopathological reports compiled in his monumental 18-volume work Rationis Medendi in Nosocomio Practico, published in 1761, was the first documented patient with amenorrhea caused by a pituitary tumor, appearing in the 6th volume. This 20-year-old amaurotic woman, who had suffered from chronic excruciating headache, died after the unsuccessful application of a cauterizing iron to her temporal bone. At the autopsy, a large solid-cystic and calcified tumor with gross characteristics typical of adamantinomatous craniopharyngioma was found encroaching on the infundibulum and third ventricle. This is the first known account of an infundibulo-tuberal lesion associated with the impairment of sexual functions, predating by 140 years the pathological evidence for a sexual brain center sited at the basal hypothalamus. In this paper, the authors analyze the historical importance and impact of de Haen's foundational report on the fields of neuroendocrinology and neurosurgery.

Sustained Pituitary T3 Production Explains the T4-mediated TSH Feedback Mechanism.

The regulation of thyroid activity and thyroid hormone (TH) secretion is based on feedback mechanisms that involve the anterior pituitary TSH and medial basal hypothalamus TSH-releasing hormone. Plasma T3 levels can be "sensed" directly by the anterior pituitary and medial basal hypothalamus; plasma T4 levels require local conversion of T4 to T3, which is mediated by the type 2 deiodinase (D2). To study D2-mediated T4 to T3 conversion and T3 production in the anterior pituitary gland, we used mouse pituitary explants incubated with 125I-T4 for 48 hours to measure T3 production at different concentrations of free T4. The results were compared with cultures of D1- or D2-expressing cells, as well as freshly isolated mouse tissue. These studies revealed a unique regulation of the D2 pathway in the anterior pituitary gland, distinct from that observed in nonpituitary tissues. In the anterior pituitary, increasing T4 levels reduced D2 activity slightly but caused a direct increase in T3 production. However, the same changes in T4 levels decreased T3 production in human HSkM cells and murine C2C12 cells (both skeletal muscle) and mouse bone marrow tissue, which reached zero at 50 pM free T4. In contrast, the increase in T4 levels caused the pig kidney LLC-PK1 cells and kidney fragments to proportionally increase T3 production. These findings have important implications for both physiology and clinical practice because they clarify the mechanism by which fluctuations in plasma T4 levels are transduced in the anterior pituitary gland to mediate the TSH feedback mechanism.

SAT434 Estradiol Controls GnRH-II Gene Expression In The Hypothalamus And Pituitary Gland Of Female Rhesus Macaques (Macaca Mulatta)

Abstract Disclosure: A. Lomniczi: None. M. Appleman: None. H.F. Urbanski: None. It has been known for many years that rhesus macaques express two forms of gonadotropin-releasing hormone (GnRH-I and GnRH-II) in the medial basal hypothalamus (MBH), and that both forms are capable of stimulating the release of luteinizing hormone (LH) in vivo. However, while much has been published about the role of GnRH-I in reproduction, very little is known about the function of GnRH-II. To shed light on this issue, we studied the expression pattern of these two genes in different parts of the monkey hypothalamus, median eminence and pituitary gland. Quantitative PCR (qPCR) analysis revealed that while GnRH-I is preferentially expressed in the Arcuate Nucleus (ARC), Median Eminence (ME), Supraoptic Nucleus (SON) and Suprachiasmatic Nucleus (SCN); GnRH-II was highly expressed in the SON, ARC-ME and Paraventricular Nucleus (PVN), confirming our previously published in situ hybridization studies. Strikingly, GnRH-II mRNA was also detected in the pituitary gland, a site where GnRH-I was not found. We have previously demonstrated that maximum hypothalamic GnRH-II expression occurs around the time of the preovulatory LH surge, when estrogen (E2) levels are maximal, but no significant changes in GnRH-I mRNA levels occur. Here using an ovariectomized (OVX) macaque model subjected to E2 replacement paradigm, designed to mimic the plasma E2 levels observed during the preovulatory estradiol peak, we performed RNAseq and qPCR from the PVN and pituitary gland to identify genes differentially regulated by E2. While GnRH-I mRNA was not affected by E2 treatment in any of the tissues studied, pituitary GnRH-II expression highly correlated with estrogen levels (r>0.8; p<0.01), with over a hundred-fold increase in mRNA expression. Taken together, these data suggest that pituitary GnRH-II (but not GnRH-I) gene expression is induced by a positive feedback action of estradiol. This novel finding raises the possibility that GnRH-II plays a major role in triggering the preovulatory LH surge in primates, not only at the ARC-ME but also at the pituitary level. Presentation Date: Saturday, June 17, 2023

Anatomical Connectivity of the Intercalated Cells of the Amygdala.

The intercalated cells of the amygdala (ITCs) are a fundamental processing structure in the amygdala that remain relatively understudied. They are phylogenetically conserved from insectivores through primates, inhibitory, and project to several of the main processing and output stations of the amygdala and basal forebrain. Through these connections, the ITCs are best known for their role in conditioned fear, where they are required for fear extinction learning and recall. Prior work on ITC connectivity is limited, and thus holistic characterization of their afferent and efferent connectivity in a genetically defined manner is incomplete. The ITCs express the FoxP2 transcription factor, affording genetic access to these neurons for viral input-output mapping. To fully characterize the anatomic connectivity of the ITCs, we used cre-dependent viral strategies in FoxP2-cre mice to reveal the projections of the main (mITC), caudal (cITC), and lateral (lITC) clusters along with their presynaptic sources of innervation. Broadly, the results confirm many known pathways, reveal previously unknown ones, and demonstrate important novel insights about each nucleus's unique connectivity profile and relative distributions. We show that the ITCs receive information from a wide range of cortical, subcortical, basal, amygdalar, hippocampal, and thalamic structures, and project broadly to areas of the basal forebrain, hypothalamus, and entire extent of the amygdala. The results provide a comprehensive map of their connectivity and suggest that the ITCs could potentially influence a broad range of behaviors by integrating information from a wide array of sources throughout the brain.

Expression of SATB1 and SATB2 in the brain of bony fishes: what fish reveal about evolution

Satb1 and Satb2 belong to a family of homeodomain proteins with highly conserved functional and regulatory mechanisms and posttranslational modifications in evolution. However, although their distribution in the mouse brain has been analyzed, few data exist in other non-mammalian vertebrates. In the present study, we have analyzed in detail the sequence of SATB1 and SATB2 proteins and the immunolocalization of both, in combination with additional neuronal markers of highly conserved populations, in the brain of adult specimens of different bony fish models at key evolutionary points of vertebrate diversification, in particular including representative species of sarcopterygian and actinopterygian fishes. We observed a striking absence of both proteins in the pallial region of actinopterygians, only detected in lungfish, the only sarcopterygian fish. In the subpallium, including the amygdaloid complex, or comparable structures, we identified that the detected expressions of SATB1 and SATB2 have similar topologies in the studied models. In the caudal telencephalon, all models showed significant expression of SATB1 and SATB2 in the preoptic area, including the acroterminal domain of this region, where the cells were also dopaminergic. In the alar hypothalamus, all models showed SATB2 but not SATB1 in the subparaventricular area, whereas in the basal hypothalamus the cladistian species and the lungfish presented a SATB1 immunoreactive population in the tuberal hypothalamus, also labeled with SATB2 in the latter and colocalizing with the gen Orthopedia. In the diencephalon, all models, except the teleost fish, showed SATB1 in the prethalamus, thalamus and pretectum, whereas only lungfish showed also SATB2 in prethalamus and thalamus. At the midbrain level of actinopterygian fish, the optic tectum, the torus semicircularis and the tegmentum harbored populations of SATB1 cells, whereas lungfish housed SATB2 only in the torus and tegmentum. Similarly, the SATB1 expression in the rhombencephalic central gray and reticular formation was a common feature. The presence of SATB1 in the solitary tract nucleus is a peculiar feature only observed in non-teleost actinopterygian fishes. At these levels, none of the detected populations were catecholaminergic or serotonergic. In conclusion, the protein sequence analysis revealed a high degree of conservation of both proteins, especially in the functional domains, whereas the neuroanatomical pattern of SATB1 and SATB2 revealed significant differences between sarcopterygians and actinopterygians, and these divergences may be related to the different functional involvement of both in the acquisition of various neural phenotypes.

The Gut Microbiome Influences the Gene Expression Profile of Neuroendocrine and Enteroendocrine Systems

Objective/Hypothesis: The gut microbiome influences metabolism, but the underlying mechanisms are not completely understood. We hypothesized that the gut microbiome modulates metabolism by regulating the expression of key neuroendocrine and enteroendocrine genes such as neuropeptide Y (Npy) which is a potent brain orexigenic peptide and G-protein-coupled receptor-17 (Gpr17) which decreases incretin secretion in the gut.
 Approach: The gene expression profiles in mouse hypothalamus and gastrointestinal tract segments (duodenum, jejunum, ileum, and colon) were compared from germ-free (GF) mice (n=9 females, n=9 males) and conventional mice (n=9 females, n=9 males). Gene transcription was determined through total RNA yield extracted from the various tissues using qRT-qPCR. The data was analyzed using two-way ANOVA and unpaired t-test.
 Results: We found that RNA yield in brain and gut tissues differed in a sex and microbiome dependent manner. The medial basal hypothalamus of the female germ-free mice had lower RNA yield compared with conventional female mice (p=0.0082). In the intestine, the RNA concentrations of the germ-free mice showed a decrease from the proximal to distal gut, however, an opposite trend was observed in the conventional mice with an overall increase of concentration from the proximal to distal gut. This trend may reflect the high concentration of the microbiota in the distal gut. Regarding gene expression of the neuroendocrine and enteroendocrine systems, Npy had lower expression in the medial basal hypothalamus (MBH) of conventional males (p=0.004), Gpr17 showed moderate duodenal expression in conventional mice (p=0.029), and Gpr17 showed increased duodenal expression in germ-free females (p=0.044).
 Summary/Conclusions: With enriched microbiota in the ileum and colon, our results indicate that gut microbiota increase host gene transcription in the distal gut segments. Future studies will aim at investigating the mechanistic link between the microbiome and regulation of gene expression in neuroendocrine and enteroendocrine tissues.

RF18 | PSAT143 Brain Determinants of Dynamic Bone Remodeling in Females

Abstract Central estrogen signaling in the medial basal hypothalamus (MBH) coordinates energy expenditure, food intake, and reproduction, as well as skeletal homeostasis. Prior work from our lab found that estrogen signaling via Estrogen Receptor alpha (ERa) in the arcuate (ARC) nucleus normally restrains bone formation in female mice only. We asked if the high bone mass in the Esr1Nkx2-1Cre mouse model, which ablates ERa in the MBH, is maintained or deteriorates with chronic overnutrition, as this is known to increase bone frailty in humans. To carry out this study, Esr1Nkx2-1Cre mutant female mice were fed HFD (Research Diets, D12492, 60% fat) or chow (SCD, 5058; LabDiet, 4% fat) for 17 weeks beginning at 10 weeks of age and housed at 22C. Although neither cohort gained significant weight on HFD nor exhibited marked changes in other metabolic parameters (%fat, GTT, ITT, etc.), this dietary challenge reversed and normalized the high bone mass in mutant females. Specifically, the ratio of BV/TV in the distal femur decreased dramatically from 36% to 6%, as measured by 3D-microcomputed tomography (micro-CT); cortical bone also decreased similarly. For comparison, control females fed HFD showed a non-significant decrease in bone mass (4.9% to 3.9% BV/TV). Unexpectedly, the large drop in bone mass in Ers1Nkx2-1Cre females fed HFD was uncoupled from an increase in bone marrow adipose tissue (BMAT). In contrast, control cohorts showed a marked 9-fold increase in BMAT following HFD. Thus, the high bone mass in mutant Ers1Nkx2-1Cre females, which persists in aged and estrogen-depleted females, quickly disintegrates with a dietary challenge. Recent RNA-Seq data from microdissected ARC tissue reveals several secreted factors that are dramatically elevated in mutant females but drop 10-20-fold after HFD. Current studies are underway to ask if these candidate factors from the ARC impact bone mass or the osteogenic capacity of skeletal stem cells. Our findings are in stark contrast to the notion that dietary excess reduces bone health by increasing bone fat at the expense of osteogenesis. Instead, based on our model, we posit that dietary excess directly determines dynamic bone remodeling by acting on the brain. Presentation: Saturday, June 11, 2022 1:00 p.m. - 3:00 p.m., Sunday, June 12, 2022 12:30 p.m. - 12:35 p.m.

Glutamatergic pedunculopontine tegmental neurons control wakefulness and locomotion via distinct axonal projections.

The pedunculopontine tegmental (PPT) nucleus is implicated in many brain functions, ranging from sleep/wake control and locomotion, to reward mechanisms and learning. The PPT contains cholinergic, GABAergic, and glutamatergic neurons with extensive ascending and descending axonal projections. Glutamatergic PPT (PPTvGlut2) neurons are thought to promote wakefulness, but the mechanisms through which this occurs are unknown. In addition, some researchers propose that PPTvGlut2 neurons promote locomotion, yet even though the PPT is a target for deep brain stimulation in Parkinson's disease, the role of the PPT in locomotion is debated. We hypothesized that PPTvGluT2 neurons drive arousal and specific waking behaviors via certain projections and modulate locomotion via others. We mapped the axonal projections of PPTvGlut2 neurons using conditional anterograde tracing and then photostimulated PPTvGlut2 soma or their axon terminal fields across sleep/wake states and analyzed sleep/wake behavior, muscle activity, and locomotion in transgenic mice. We found that stimulation of PPTvGlut2 soma and their axon terminals rapidly triggered arousals from non-rapid eye movement sleep, especially with activation of terminals in the basal forebrain (BF) and lateral hypothalamus (LH). With photoactivation of PPTvGlut2 terminals in the BF and LH, this wakefulness was accompanied by locomotion and other active behaviors, but stimulation of PPTvGlut2 soma and terminals in the substantia nigra triggered only quiet wakefulness without locomotion. These findings demonstrate the importance of the PPTvGluT2 neurons in driving various aspects of arousal and show that heterogeneous brain nuclei, such as the PPT, can promote a variety of behaviors via distinct axonal projections.

A versatile transcription factor: Multiple roles of orthopedia a (otpa) beyond its restricted localization in dopaminergic systems of developing and adult zebrafish (Danio rerio) brains.

Many transcription factors boost neural development and differentiation in specific directions and serve for identifying similar or homologous structures across species. The expression of Orthopedia (Otp) is critical for the development of certain cell groups along the vertebrate neuraxis, for example, the medial amygdala or hypothalamic neurosecretory neurons. Therefore, the primary focus of the present study is the distribution of Orthopedia a (Otpa) in the larval and adult zebrafish (Danio rerio) brain. Since Otpa is also critical for the development of zebrafish basal diencephalic dopaminergic cells, colocalization of Otpa with the catecholamine synthesizing enzyme tyrosine hydroxylase (TH) is studied. Cellular colocalization of Otpa and dopamine is only seen in magnocellular neurons of the periventricular posterior tubercular nucleus and in the posterior tuberal nucleus. Otpa-positive cells occur in many additional structures along the zebrafish neuraxis, from the secondary prosencephalon down to the hindbrain. Furthermore, Otpa expression is studied in shh-GFP and islet1-GFP transgenic zebrafish. Otpa-positive cells only express shh in dopaminergic magnocellular periventricular posterior tubercular cells, and only colocalize with islet1-GFP in the ventral zone and prerecesscaudal periventricular hypothalamic zone and the perilemniscal nucleus. The scarcity of cellular colocalization of Otpa in islet1-GFP cells indicates that the Shh-islet1 neurogenetic pathway is not active in most Otpa-expressing domains. Our analysis reveals detailed correspondences between mouse and zebrafish forebrain territories including the zebrafish intermediate nucleus of the ventral telencephalon and the mouse medial amygdala. The zebrafish preoptic Otpa-positive domain represents the neuropeptidergic supraopto-paraventricular region of all tetrapods. Otpa domains in the zebrafish basal plate hypothalamus suggest that the ventral periventricular hypothalamic zone corresponds to the otp-expressing basal hypothalamic tuberal field in the mouse. Furthermore, the mouse otp domain in the mammillary hypothalamus compares partly to our Otpa-positive domain in the prerecess caudal periventricular hypothalamic zone (Hc-a).

SURG-07. The impact of early targeted therapy on the neurosurgical approach to pediatric low-grade glioma

We report two cases of pediatric low-grade glioma (pLGG) treated with vemurafenib, an oral BRAF-inhibitor: a 12-year-old girl with involvement of basal ganglia, hypothalamus, and diencephalic junction; a 3-year-old boy, with an optic pathway/hypothalamic glioma extending along the left optic tract and basal ganglia. Both received a biopsy and molecular analysis was performed in the girl’s tumor, showing BRAF V600E mutation. Therefore, instead of surgical removal planned by neurosurgeons, first-line treatment with vemurafenib was started: after one month 45% reduction of the mass according to RANO criteria was found, as well as better balance control and strong reduction of the right arm paresis; five months later, a 70% shrinkage was detected, stabilized to 76% after a year. The young boy first started chemotherapy with vincristine and carboplatin, but at the end of the induction phase the tumor had increased and ascites, hydrocephalus, and visual impairment occurred. Molecular testing showing BRAF V600E mutation on the initial tumor biopsy was obtained; therefore, the surgical option was postponed and therapy with vemurafenib started. After only three days, visual acuity and muscle tone improved; brain MRI showed a 34% reduction of the mass after one week, increased up to 65% after six months. Therefore, no ulterior surgery was necessary. In pLGG, the neurosurgical biopsy is essential to let an early and rapid molecular diagnosis of BRAF mutations and guide subsequent targeted therapies. Our cases demonstrate how a prompt radiological response to vemurafenib and the related clinical improvement can influence both therapeutic and surgical decisions, hopefully reducing the occurrence of second neurosurgery with associated risks of neurological sequelae. To our knowledge, this is the first report assessing such a quick shrinkage in pLGG treated with vemurafenib, highlighting the importance of an early investigation of BRAF status in all cases of LGG in children.

Revisiting TSHβ's Role in Avian Seasonal Reproduction, Insights, and Challenges from Mammalian Models.

The core neuroendocrine pathways regulating seasonal reproduction in vertebrates were characterized over a decade ago. This has led to the development of a "consensus" model of seasonal reproduction that appears to be largely conserved across mammals, birds, amphibians, reptile and fish. This model centers around the photoinduced increase in TSHβ expression in the pars tuberalis of the anterior pituitary gland as the key transducer of photic information from sensory cells to the critical switch in hypothalamic deiodinase enzyme expression that drives changes in localized thyroid hormone signaling. These changes in localized thyroid hormone signaling in the medial basal hypothalamus ultimately activate the reproductive axis. This model has in turn, been consistently supported by studies in a variety of taxa. As such, it has become the standard against which subsequent work is compared, particularly in the non-mammalian literature. However, as new studies move away from the handful of canonical model systems and begin to explore the effects of naturalistic rather than artificial photoperiod manipulations, a more nuanced picture has begun to emerge. Yet, progress in elucidating the detailed events of reproductive photostimulation has been uneven across the research community. In this perspective, I draw on emerging data from studies in free living animals that challenges some of the established assumptions of the avian consensus model of reproduction. Specifically, the role of TSHβ and its dissociation from deiodinase signaling. I then discuss how these apparently surprising findings can be contextualized within the context of the mammalian seasonal literature. In turn, this ability to contextualize from the mammalian literature highlights the breadth of the current gap versus our understanding of the molecular neuroendocrine mechanisms of seasonality in mammals versus birds and other non-mammalian seasonal breeders.

Long-term priming of hypothalamic microglia is associated with energy balance disturbances under diet-induced obesity.

Exposure of microglia to an inflammatory environment may lead to their priming and exacerbated response to future inflammatory stimuli. Here we aimed to explore hypothalamic microglia priming and its consequences on energy balance regulation. A model of intracerebroventricular administration of neuraminidase (NA, which is present in various pathogens such as influenza virus) was used to induce acute neuroinflammation. Evidences of primed microglia were observed 3 months after NA injection, namely (1) a heightened response of microglia located in the hypothalamic arcuate nucleus after an in vivo inflammatory challenge (high fat diet [HFD] feeding for 10 days), and (2) an enhanced response of microglia isolated from NA‐treated mice and challenged in vitro to LPS. On the other hand, the consequences of a previous NA‐induced neuroinflammation were further evaluated in an alternative inflammatory and hypercaloric scenario, such as the obesity generated by continued HDF feeding. Compared with sham‐injected mice, NA‐treated mice showed increased food intake and, surprisingly, reduced body weight. Besides, NA‐treated mice had enhanced microgliosis (evidenced by increased number and reactive morphology of microglia) and a reduced population of POMC neurons in the basal hypothalamus. Thus, a single acute neuroinflammatory event may elicit a sustained state of priming in microglial cells, and in particular those located in the hypothalamus, with consequences in hypothalamic cytoarchitecture and its regulatory function upon nutritional challenges.

The effects of feed restriction, time of day, and time since feeding on behavioral and physiological indicators of hunger in broiler breeder hens

Broiler breeder chickens are commercially feed restricted to slow their growth and improve their health and production, however, there is research demonstrating that this leads to chronic hunger resulting in poor welfare. A challenge in these studies is to account for possible daily rhythms or the effects of time since last meal on measures relating hunger. To address this, we used 3 feed treatments: AL (ad libitum fed), Ram (restricted, fed in the morning), and Rpm (restricted, fed in the afternoon) to control for diurnal effects. We then conducted foraging motivation tests and collected home pen behavior and physiological samples at 4 times relative to feeding throughout a 24-h period. The feed treatment had the largest influence on the data, with AL birds weighing more, having lower concentrations of plasma NEFA, and mRNA expression of AGRP and NPY alongside higher expression of POMC in the basal hypothalamus than Ram or Rpm birds (P < 0.001). R birds were more successful at and had a shorter latency to complete the motivation test, and did more walking and less feeding than AL birds in the home pen (P < 0.01). There was little effect of time since last meal on many measures (P > 0.05) but AGRP expression was highest in the basal hypothalamus shortly after a meal (P < 0.05), blood plasma NEFA was higher in R birds just before feeding (P < 0.001) and glucose was higher in Ram birds just after feeding (P < 0.001), and the latency to complete the motivation test was shortest before the next meal (P < 0.05). Time of day effects were mainly found in the difference in activity levels in the home pen when during lights on and lights off periods. In conclusion, many behavioral and physiological hunger measures were not significantly influenced by time of day or time since the last meal. For the measures that do change, future studies should be designed so that sampling is balanced in such a way as to minimize bias due to these effects.

Critical changes in hypothalamic gene networks in response to pancreatic cancer as found by single-cell RNA sequencing

ObjectiveCancer cachexia is a devastating chronic condition characterized by involuntary weight loss, muscle wasting, abnormal fat metabolism, anorexia, and fatigue. However, the molecular mechanisms underlying this syndrome remain poorly understood. In particular, the hypothalamus may play a central role in cachexia, given that it has direct access to peripheral signals because of its anatomical location and attenuated blood–brain barrier. Furthermore, this region has a critical role in regulating appetite and metabolism. MethodsTo provide a detailed analysis of the hypothalamic response to cachexia, we performed single-cell RNA-seq combined with RNA-seq of the medial basal hypothalamus (MBH) in a mouse model for pancreatic cancer. ResultsWe found many cell type-specific changes, such as inflamed endothelial cells, stressed oligodendrocyes and both inflammatory and moderating microglia. Lcn2, a newly discovered hunger suppressing hormone, was the highest induced gene. Interestingly, cerebral treatment with LCN2 not only induced many of the observed molecular changes in cachexia but also affected gene expression in food-intake decreasing POMC neurons. In addition, we found that many of the cachexia-induced molecular changes found in the hypothalamus mimic those at the primary tumor site. ConclusionOur data reveal that multiple cell types in the MBH are affected by tumor-derived factors or host factors that are induced by tumor growth, leading to a marked change in the microenvironment of neurons critical for behavioral, metabolic, and neuroendocrine outputs dysregulated during cachexia. The mechanistic insights provided in this study explain many of the clinical features of cachexia and will be useful for future therapeutic development.

Analysis of Islet-1, Nkx2.1, Pax6, and Orthopedia in the forebrain of the sturgeon Acipenser ruthenus identifies conserved prosomeric characteristics.

The distribution patterns of a set of conserved brain developmental regulatory transcription factors were analyzed in the forebrain of the basal actinopterygian fish Acipenser ruthenus, consistent with the prosomeric model. In the telencephalon, the pallium was characterized by ventricular expression of Pax6. In the subpallium, the combined expression of Nkx2.1/Islet-1 (Isl1) allowed to propose ventral and dorsal areas, as the septo-pallidal (Nkx2.1/Isl1+) and striatal derivatives (Isl1+), respectively, and a dorsal portion of the striatal derivatives, ventricularly rich in Pax6 and devoid of Isl1 expression. Dispersed Orthopedia (Otp) cells were found in the supracommissural and posterior nuclei of the ventral telencephalon, related to the medial portion of the amygdaloid complex. The preoptic area was identified by the Nkx2.1/Isl1 expression. In the alar hypothalamus, an Otp-expressing territory, lacking Nkx2.1/Isl1, was identified as the paraventricular domain. The adjacent subparaventricular domain (Spa) was subdivided in a rostral territory expressing Nkx2.1 and an Isl1+ caudal one. In the basal hypothalamus, the tuberal region was defined by the Nkx2.1/Isl1 expression and a rostral Otp-expressing domain was identified. Moreover, the Otp/Nkx2.1 combination showed an additional zone lacking Isl1, tentatively identified as the mamillary area. In the diencephalon, both Pax6 and Isl1 defined the prethalamic domain, and within the basal prosomere 3, scattered Pax6- and Isl1-expressing cells were observed in the posterior tubercle. Finally, a small group of Pax6 cells was observed in the pretectal area. These results improve the understanding of the forebrain evolution and demonstrate that its basic bauplan is present very early in the vertebrate lineage.

MKRN3 inhibits the reproductive axis through actions in kisspeptin-expressing neurons

The identification of loss-of-function mutations in MKRN3 in patients with central precocious puberty in association with the decrease in MKRN3 expression in the medial basal hypothalamus of mice before the initiation of reproductive maturation suggests that MKRN3 is acting as a brake on gonadotropin-releasing hormone (GnRH) secretion during childhood. In the current study, we investigated the mechanism by which MKRN3 prevents premature manifestation of the pubertal process. We showed that, as in mice, MKRN3 expression is high in the hypothalamus of rats and nonhuman primates early in life, decreases as puberty approaches, and is independent of sex steroid hormones. We demonstrated that Mkrn3 is expressed in Kiss1 neurons of the mouse hypothalamic arcuate nucleus and that MKRN3 repressed promoter activity of human KISS1 and TAC3, 2 key stimulators of GnRH secretion. We further showed that MKRN3 has ubiquitinase activity, that this activity is reduced by MKRN3 mutations affecting the RING finger domain, and that these mutations compromised the ability of MKRN3 to repress KISS1 and TAC3 promoter activity. These results indicate that MKRN3 acts to prevent puberty initiation, at least in part, by repressing KISS1 and TAC3 transcription and that this action may involve an MKRN3-directed ubiquitination-mediated mechanism.