Sex chromosome complement interacts with gonadal hormones in determining regional-specific neuroactive steroid levels in plasma, hippocampus, and hypothalamus. A study using the four core genotype mouse model.

An important aspect of the neuromodulatory and neuroprotective actions exerted by neuroactive steroids is that they are sex-specific, as determined by the sexually dimorphic levels of these molecules in plasma and the nervous tissue. Thus, the identification of the factors that generate the sex-dimorphic levels of neuroactive steroids may be crucial from a neuroprotectant perspective. The main driver for sex determination in mammals is the SRY gene and the subsequent presence of a specific gonad: testes for males and ovaries for females, thus producing hormonal compounds, primarily androgens and estrogens, respectively. Nowadays, it is well established that despite the relevance of gonads, other factors control sexual features, and, among them, sex chromosome complement is highly relevant. In this study, neuroactive steroids were evaluated by liquid chromatography-tandem mass spectrometry in the hypothalamus, the hippocampus, and plasma of the four core genotype mouse model, to determine the relative contribution of sex chromosome complement and gonads in determining their sex dimorphic levels. The data obtained reveal that although gonads are the main contributing factor for sex differences in neuroactive steroid levels, the levels of some neuroactive steroids, including testosterone, are also influenced in brain and plasma by tissue-specific actions of sex chromosomes. The data presented here adds a new piece to the puzzle of steroid level regulation, which may be useful in designing sex-specific neuroprotective approaches to pathological conditions affecting the nervous system.

Neonatal inhibition of androgen activity alters the programming of body weight and orexinergic peptides differentially in male and female rats.

The involvement of androgens in the regulation of energy metabolism has been demonstrated. The main objective of the present research was to study the involvement of androgens in both the programming of energy metabolism and the regulatory peptides associated with feeding. For this purpose, androgen receptors and the main metabolic pathways of testosterone were inhibited during the first five days of postnatal life in male and female Wistar rats. Pups received a daily s.c. injection from the day of birth, postnatal day (P) 1, to P5 of Flutamide (a competitive inhibitor of androgen receptors), Letrozole (an aromatase inhibitor), Finasteride (a 5-alpha-reductase inhibitor) or vehicle. Body weight, food intake and fat pads were measured. Moreover, hypothalamic Agouti-related peptide (AgRP), neuropeptide Y (NPY), orexin, and proopiomelanocortin (POMC) were analyzed by quantitative real-time polymerase chain reaction assay. The inhibition of androgenic activity during the first five days of life produced a significant decrease in body weight in females at P90 but did not affect this parameter in males. Moreover, the inhibition of aromatase decreased hypothalamic AgRP mRNA levels in males while the inhibition of 5α-reductase decreased hypothalamic AgRP and orexin mRNA levels in female rats. Finally, food intake and visceral fat, but not subcutaneous fat, were affected in both males and females depending on which testosterone metabolic pathway was inhibited. Our results highlight the differential involvement of androgens in the programming of energy metabolism as well as the AgRP and orexin systems during development in male and female rats.

Interaction of gonadal hormones, dopaminergic system, and epigenetic regulation in the generation of sex differences in substance use disorders: A systematic review.

Substance use disorder (SUD) is a chronic condition characterized by pathological drug-taking and seeking behaviors. Remarkably different between males and females, suggesting that drug addiction is a sexually differentiated disorder. The neurobiological bases of sex differences in SUD include sex-specific reward system activation, influenced by interactions between gonadal hormone level changes, dopaminergic reward circuits, and epigenetic modifications of key reward system genes. This systematic review, adhering to PICOS and PRISMA-P 2015 guidelines, highlights the sex-dependent roles of estrogens, progesterone, and testosterone in SUD. In particular, estradiol elevates and progesterone reduces dopaminergic activity in SUD females, whilst testosterone and progesterone augment SUD behavior in males. Finally, SUD is associated with a sex-specific increase in the rate of opioid and monoaminergic gene methylation. The study reveals the need for detailed research on gonadal hormone levels, dopaminergic or reward system activity, and epigenetic landscapes in both sexes for efficient SUD therapy development.

Kif21B mediates the effect of estradiol on the morphological plasticity of mouse hippocampal neurons.

Introduction: Neurons are polarized cells, and their ability to change their morphology has a functional implication in the development and plasticity of the nervous system in order to establish new connections. Extracellular factors strongly influence neuronal shape and connectivity. For instance, the developmental actions of estradiol on hippocampal neurons are well characterized, and we have demonstrated in previous studies that Ngn3 mediates these actions. On the other hand, Kif21B regulates microtubule dynamics and carries out retrograde transport of the TrkB/brain-derived neurotrophic factor (BDNF) complex, essential for neuronal development. Methods: In the present study, we assessed the involvement of kinesin Kif21B in the estradiol-dependent signaling mechanisms to regulate neuritogenesis through cultured mouse hippocampal neurons. Results: We show that estradiol treatment increases BDNF expression, and estradiol and BDNF modify neuron morphology through TrkB signaling. Treatment with K252a, a TrkB inhibitor, decreases dendrite branching without affecting axonal length, whereas. Combined with estradiol or BDNF, it blocks their effects on axons but not dendrites. Notably, the downregulation of Kif21B abolishes the actions of estradiol and BDNF in both the axon and dendrites. In addition, Kif21B silencing also decreases Ngn3 expression, and downregulation of Ngn3 blocks the effect of BDNF on neuron morphology. Discussion: These results suggest that Kif21B is required for the effects of estradiol and BDNF on neuronal morphology, but phosphorylation-mediated activation of TrkB is essential only for axonal growth. Our results show that the Estradiol/BDNF/TrkB/Kif21B/Ngn3 is a new and essential pathway mediating hippocampal neuron development.

High levels of HDAC expression correlate with microglial aging.

BACKGROUND: Cellular damage gradually accumulates with aging, promoting a time-dependent functional decline of the brain. Microglia play an essential regulatory role in maintaining cognitive activity by phagocytosing cell debris and apoptotic cells during neurogenesis. The activities of different histone deacetylases (HDACs) regulate microglial function during development and neurodegeneration. However, no studies have described the role of HDACs in microglia during physiological aging. RESEARCH DESIGN AND METHODS: HDAC and microglial marker levels were examined in microglial cells after inducing senescence in vitro and in mouse and human hippocampal biopsies in vivo, using quantitative real-time PCR. Publicly available datasets were used to determine HDAC expression in different brain areas during physiological aging. RESULTS: HDAC expression increased upon the induction of senescence with bleomycin or serial passage in microglial cultures. High levels of HDACs were detected in mice and aged human brain samples. Human hippocampal samples showed a positive correlation between the expression of HDAC1, 3, and 7 and microglial and senescence markers. HDAC1 and 3 levels are enriched in the purified aged microglial population. CONCLUSIONS: Several HDACs, particularly HDAC1, are elevated in microglia upon senescence induction in vitro and with aging in vivo, and correlate with microglial and senescence biomarkers.

IGF1 gene therapy in middle-aged female rats delays reproductive senescence through its effects on hypothalamic GnRH and kisspeptin neurons.

The process of aging is the result of progressive loss of homeostasis and functional body impairment, including the central nervous system, where the hypothalamus plays a key role in regulating aging mechanisms. The consequences of aging include a chronic proinflammatory environment in the hypothalamus that leads to decreased secretion of gonadotropin-releasing hormone (GnRH) and impairs kisspeptin neuron functionality. In this work, we investigated the effect of insulin-like growth factor 1 (IGF1) gene therapy on hypothalamic kisspeptin/GnRH neurons and on microglial cells, that mediate the inflammatory process related with the aging process. The results show that IGF1 rats have higher kisspeptin expression in the anteroventral periventricular (AVPV) nucleus and higher immunoreactivity of GnRH in the arcuate nucleus and median eminence. In addition, IGF1-treated animals exhibit increased numbers of Iba1+ microglial cells and MHCII+/Iba1+ in the AVPV and arcuate nuclei. In conclusion, IGF1 gene therapy maintains kisspeptin production in the AVPV nucleus, induces GnRH release in the median eminence, and alters the number and reactivity of microglial cells in middle-aged female rats. We suggest that IGF1 gene therapy may have a protective effect against reproductive decline.

Organizational Effects of Estrogens and Androgens on Estrogen and Androgen Receptor Expression in Pituitary and Adrenal Glands in Adult Male and Female Rats.

Sex steroid hormones, such as androgens and estrogens, are known to exert organizational action at perinatal periods and activational effects during adulthood on the brain and peripheral tissues. These organizational effects are essential for the establishment of biological axes responsible for regulating behaviors, such as reproduction, stress, and emotional responses. Estradiol (E2), testosterone, and their metabolites exert their biological action through genomic and non-genomic mechanisms, bounding to canonical receptors, such as estrogen receptor (ER)α, ERß, and androgen receptor (AR) or membrane receptors, such as the G protein-coupled estrogen receptor (GPER), respectively. Expression of ERs and AR was found to be different between males and females both in the brain and peripheral tissues, suggesting a sex-dependent regulation of their expression and function. Therefore, studying the ERs and AR distribution and expression levels is key to understand the central and peripheral role of sex steroids in the establishment of sex-specific behaviors in males and females. We investigated the organizational effects of estrogens and androgens in the pituitary and adrenal glands of adult male and female rats. For this, selective blockade of AR with flutamide or 5α-reductase with finasteride or aromatase with letrozole during the first 5 days of life has been performed in male and female pups and then quantification of ERs and AR expression in both glands has been carried out in adulthood. Data show that inhibition of dihydrotestosterone (DHT) and E2 production during the first five postnatal days mainly decreases the ER expression in male to female values and AR expression in female to male levels in the pituitary gland and increases AR expression in female to male levels in the adrenal gland. In contrast, blocking the action of androgens differentially modulates the ERs in males and females and decreases AR in both males and females in both glands. Altogether, the results suggest that neonatal modifications of the androgen and estrogen pathways can potentially lead to permanent modifications of the neuroendocrine functions of the pituitary and adrenal glands in the adulthood of both sexes.

"Shikonin inhibits microglia activation and reduces CFA-induced mechanical hyperalgesia in an animal model of pain".

Shikonin is an ointment produced from Lithospermun erythrorhizon which has been used in traditional medicine both in Europe and Asia for wound healing and is associated with anti-inflammatory properties. The goal of this work is to assess the analgesic properties of Shikonin in the CFA-induced inflammation model of pain. Rats were subjected to inflammation of the hind paw by CFA injection with a preventive injection of Shikonin and compared to either a control group or to a CFA-inflamed group with the vehicle drug solution. Inflammation of the hind paw by CFA was assessed by measurement of the dorsal to plantar diameter. Mechanical thresholds were established by means of the Von Frey filaments which are calibrated filaments that exert a defined force. Finally, the spinal cord of the studied animals was extracted to analyse the microglia population through immunohistochemistry using the specific marker Iba-1. Our results show that Shikonin reduces the paw oedema caused by CFA inflammation. Subsequently, there is a concomitant restoration of the mechanical thresholds reduced by CFA hind paw injection. Additionally, spinal microglia is activated after CFA-induced inflammation. Our results show that microglia is inhibited by Shikonin and has concomitant restoration of the mechanical thresholds. Our findings demonstrate for the first time that Shikonin inhibits microglia morphological changes and thereby ameliorates pain-like behaviour elicited by mechanical stimulation.

A cis-acting structural variation at the ZNF558 locus controls a gene regulatory network in human brain development.

The human forebrain has expanded in size and complexity compared to chimpanzees despite limited changes in protein-coding genes, suggesting that gene expression regulation is an important driver of brain evolution. Here, we identify a KRAB-ZFP transcription factor, ZNF558, that is expressed in human but not chimpanzee forebrain neural progenitor cells. ZNF558 evolved as a suppressor of LINE-1 transposons but has been co-opted to regulate a single target, the mitophagy gene SPATA18. ZNF558 plays a role in mitochondrial homeostasis, and loss-of-function experiments in cerebral organoids suggests that ZNF558 influences developmental timing during early human brain development. Expression of ZNF558 is controlled by the size of a variable number tandem repeat that is longer in chimpanzees compared to humans, and variable in the human population. Thus, this work provides mechanistic insight into how a cis-acting structural variation establishes a regulatory network that affects human brain evolution.

Distinct subcellular autophagy impairments in induced neurons from patients with Huntington's disease.

Huntington's disease is a neurodegenerative disorder caused by CAG expansions in the huntingtin (HTT) gene. Modelling Huntington's disease is challenging, as rodent and cellular models poorly recapitulate the disease as seen in ageing humans. To address this, we generated induced neurons through direct reprogramming of human skin fibroblasts, which retain age-dependent epigenetic characteristics. Huntington's disease induced neurons (HD-iNs) displayed profound deficits in autophagy, characterized by reduced transport of late autophagic structures from the neurites to the soma. These neurite-specific alterations in autophagy resulted in shorter, thinner and fewer neurites specifically in HD-iNs. CRISPRi-mediated silencing of HTT did not rescue this phenotype but rather resulted in additional autophagy alterations in control induced neurons, highlighting the importance of wild-type HTT in normal neuronal autophagy. In summary, our work identifies a distinct subcellular autophagy impairment in adult patient derived Huntington's disease neurons and provides a new rationale for future development of autophagy activation therapies.

G Protein-Coupled Estrogen Receptor Immunoreactivity in the Rat Hypothalamus Is Widely Distributed in Neurons, Astrocytes, and Oligodendrocytes, Fluctuates during the Estrous Cycle, and Is Sexually Dimorphic.

INTRODUCTION: The membrane-associated G protein-coupled estrogen receptor 1 (GPER) mediates the regulation by estradiol of arginine-vasopressin immunoreactivity in the supraoptic and paraventricular hypothalamic nuclei of female rats and is involved in the estrogenic control of hypothalamic regulated functions, such as food intake, sexual receptivity, and lordosis behavior. OBJECTIVE: To assess GPER distribution in the rat hypothalamus. METHODS: GPER immunoreactivity was assessed in different anatomical subdivisions of five selected hypothalamic regions of young adult male and cycling female rats: the arcuate nucleus, the lateral hypothalamus, the paraventricular nucleus, the supraoptic nucleus, and the ventromedial hypothalamic nucleus. GPER immunoreactivity was colocalized with NeuN as a marker of mature neurons, GFAP as a marker of astrocytes, and CC1 as a marker of mature oligodendrocytes. RESULTS: GPER immunoreactivity was detected in hypothalamic neurons, astrocytes, and oligodendrocytes. Sex and regional differences and changes during the estrous cycle were detected in the total number of GPER-immunoreactive cells and in the proportion of neurons, astrocytes, and oligodendrocytes that were GPER-immunoreactive. CONCLUSIONS: These findings suggest that estrogenic regulation of hypothalamic function through GPER may be different in males and females and may fluctuate during the estrous cycle in females.

G Protein-Coupled Estrogen Receptor Immunoreactivity Fluctuates During the Estrous Cycle and Show Sex Differences in the Amygdala and Dorsal Hippocampus.

G protein-coupled estrogen receptor (GPER) in the amygdala and the dorsal hippocampus mediates actions of estradiol on anxiety, social recognition and spatial memory. In addition, GPER participates in the estrogenic regulation of synaptic function in the amygdala and in the process of adult neurogenesis in the dentate gyrus. While the distribution of the canonical estrogen receptors α and ß in the amygdala and dorsal hippocampus are well characterized, little is known about the regional distribution of GPER in these brain regions and whether this distribution is affected by sex or the stages of the estrous cycle. In this study we performed a morphometric analysis of GPER immunoreactivity in the posterodorsal medial, anteroventral medial, basolateral, basomedial and central subdivisions of the amygdala and in all the histological layers of CA1 and the dentate gyrus of the dorsal hippocampal formation. The number of GPER immunoreactive cells was estimated in these different structures. GPER immunoreactivity was detected in all the assessed subdivisions of the amygdaloid nucleus and dorsal hippocampal formation. The number of GPER immunoreactive cells was higher in males than in estrus females in the central (P = 0.001) and the posterodorsal medial amygdala (P < 0.05); higher in males than in diestrus females in the strata orients (P < 0.01) and radiatum-lacunosum-moleculare (P < 0.05) of CA1-CA3 and in the molecular layer of the dentate gyrus (P < 0.01); higher in diestrus females than in males in the basolateral amygdala (P < 0.05); higher in diestrus females than in estrus females in the central (P < 0.01), posterodorsal medial (P < 0.01) and basolateral amygdala (P < 0.01) and higher in estrus females than in diestrus females in the strata oriens (P < 0.05) and radiatum-lacunosum-moleculare (P < 0.05) of CA1-CA3 and in the molecular layer (P < 0.05) and the hilus of the dentate gyrus (P < 0.05). The findings suggest that estrogenic regulation of the amygdala and hippocampus through GPER may be different in males and in females and may fluctuate during the estrous cycle.

Profiling of lincRNAs in human pluripotent stem cell derived forebrain neural progenitor cells.

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) can be differentiated into many different cell types of the central nervous system. One challenge when using pluripotent stem cells is to develop robust and efficient differentiation protocols that result in homogenous cultures of the desired cell type. Here, we have utilized the SMAD-inhibitors SB431542 and Noggin in a fully defined monolayer culture model to differentiate human pluripotent cells into homogenous forebrain neural progenitors. Temporal fate analysis revealed that this protocol results in forebrain-patterned neural progenitor cells that start to express early neuronal markers after two weeks of differentiation, allowing for the analysis of gene expression changes during neurogenesis. Using this system, we were able to identify many previously uncharacterized long intergenic non-coding RNAs that display dynamic expression during human forebrain neurogenesis.

Blocking of Estradiol Receptors ERα, ERß and GPER During Development, Differentially Alters Energy Metabolism in Male and Female Rats.

Estradiol not only participates in the regulation of energy metabolism in adulthood, but also during the first stages of life as it modulates the alterations induced by under- and over-nutrition. The objectives of the present study were to determine: 1) If estradiol is involved in the normal programming of energy metabolism in rats; 2) If there is a specific window of time for this programming and 3) If males and females are differentially vulnerable to the action of this hormone. Estrogen receptors (ER) α, ERß and GPER were blocked by their specific antagonists MPP, PHTPP and G15, respectively, from postnatal day (P) 1 (the day of birth) to P5 or from P5 to P13. Physiological parameters such as body weight, fat depots and caloric intake were then analysed at P90. Hypothalamic AgRP, POMC, MC4R, ERα, ERß and GPER mRNA levels and plasma levels of estradiol, were also studied. We found that blocking ER receptors from P5 to P13 significantly decreases long-term body weight in males and hypothalamic POMC mRNA levels in females. The blocking of ERs from P1 to P5 only affected plasma estradiol levels in females. The present results indicate programming actions of estradiol from P5 to P13 on body weight in male and POMC expression in female rats and emphasize the importance of including both sexes in metabolic studies. It is necessary to unravel the mechanisms that underlie the actions of estradiol on food intake, both during development and in adulthood, and to determine how this programming differentially takes place in males and females.

Estrogen receptor beta and G protein-coupled estrogen receptor 1 are involved in the acute estrogenic regulation of arginine-vasopressin immunoreactive levels in the supraoptic and paraventricular hypothalamic nuclei of female rats.

The ovarian hormone 17ß-estradiol is known to regulate the release, expression and immunoreactivity of arginine-vasopressin (AVP) in the supraoptic and paraventricular hypothalamic nuclei of rodents. Previous studies have shown that estrogen receptor α is involved in the effects of chronic estradiol administration on arginine-vasopressin immunoreactivity in the female rat hypothalamus. In this study we have examined the effect of an acute administration of estradiol or specific agonists for estrogen receptors α, ß and G protein-coupled estrogen receptor 1 on the immunoreactivity of arginine-vasopressin in the hypothalamus of adult ovariectomized female rats. Acute estradiol administration resulted in a significant decrease in the number of arginine-vasopressin immunoreactive neurons in the supraoptic and paraventricular nuclei after 24 h. The effects of the specific estrogen receptors agonists suggest that the action of estradiol on arginine-vasopressin immunoreactivity is mediated in the supraoptic nucleus by G protein-coupled estrogen receptor 1 and in the paraventricular nucleus by both estrogen receptor ß and G protein-coupled estrogen receptor 1. Thus, in contrast to previous studies on the effect of chronic estrogenic treatments, the present findings suggest that estrogen receptor ß and G protein-coupled estrogen receptor 1 mediate the acute effects of estradiol on arginine-vasopressin immunoreactivity in the hypothalamus of ovariectomized rats.

TRIM28 and the control of transposable elements in the brain.

TRIM28 is an epigenetic co-repressor protein that mediates transcriptional silencing. TRIM28 participates, together with the large family of Kruppel-associated box domain zinc finger proteins (KRAB-ZFP) transcription factors, in the repression of transposable elements (TE). Recent advances indicate that TRIM28-based repression of TEs occurs in the mammalian brain and may provide beneficial effects through the regulation of transcriptional networks. Here, we provide an overview of TRIM28-related functions, highlighting the role of controlling TEs in neural progenitor cells and discuss how this mechanism may have contributed to the evolution of the complex human brain. Finally, we outline future considerations for the field.

REST suppression mediates neural conversion of adult human fibroblasts via microRNA-dependent and -independent pathways.

Direct conversion of human fibroblasts into mature and functional neurons, termed induced neurons (iNs), was achieved for the first time 6 years ago. This technology offers a promising shortcut for obtaining patient- and disease-specific neurons for disease modeling, drug screening, and other biomedical applications. However, fibroblasts from adult donors do not reprogram as easily as fetal donors, and no current reprogramming approach is sufficiently efficient to allow the use of this technology using patient-derived material for large-scale applications. Here, we investigate the difference in reprogramming requirements between fetal and adult human fibroblasts and identify REST as a major reprogramming barrier in adult fibroblasts. Via functional experiments where we overexpress and knockdown the REST-controlled neuron-specific microRNAs miR-9 and miR-124, we show that the effect of REST inhibition is only partially mediated via microRNA up-regulation. Transcriptional analysis confirmed that REST knockdown activates an overlapping subset of neuronal genes as microRNA overexpression and also a distinct set of neuronal genes that are not activated via microRNA overexpression. Based on this, we developed an optimized one-step method to efficiently reprogram dermal fibroblasts from elderly individuals using a single-vector system and demonstrate that it is possible to obtain iNs of high yield and purity from aged individuals with a range of familial and sporadic neurodegenerative disorders including Parkinson's, Huntington's, as well as Alzheimer's disease.

Neuronal Activity, TGFß-Signaling and Unpredictable Chronic Stress Modulate Transcription of Gadd45 Family Members and DNA Methylation in the Hippocampus.

Neuronal activity is altered in several neurological and psychiatric diseases. Upon depolarization not only neurotransmitters are released but also cytokines and other activators of signaling cascades. Unraveling their complex implication in transcriptional control in receiving cells will contribute to understand specific central nervous system (CNS) pathologies and will be of therapeutically interest. In this study we depolarized mature hippocampal neurons in vitro using KCl and revealed increased release not only of brain-derived neurotrophic factor (BDNF) but also of transforming growth factor beta (TGFB). Neuronal activity together with BDNF and TGFB controls transcription of DNA modifying enzymes specifically members of the DNA-damage-inducible (Gadd) family, Gadd45a, Gadd45b, and Gadd45g. MeDIP followed by massive parallel sequencing and transcriptome analyses revealed less DNA methylation upon KCl treatment. Psychiatric disorder-related genes, namely Tshz1, Foxn3, Jarid2, Per1, Map3k5, and Arc are transcriptionally activated and demethylated upon neuronal activation. To analyze whether misexpression of Gadd45 family members are associated with psychiatric diseases, we applied unpredictable chronic mild stress (UCMS) as established model for depression to mice. UCMS led to reduced expression of Gadd45 family members. Taken together, our data demonstrate that Gadd45 family members are new putative targets for UCMS treatments.

NADPH-Diaphorase Colocalizes with GPER and Is Modulated by the GPER Agonist G1 in the Supraoptic and Paraventricular Nuclei of Ovariectomized Female Rats.

Nitric oxide is produced in the brain by the neuronal nitric oxide synthase (nNOS) and carries out a wide range of functions by acting as a neurotransmitter-like molecule. Gonadal hormones are involved in the regulation of the brain nitrergic system. We have previously demonstrated that estradiol, via classical estrogen receptors (ERs), regulates NOS activity in the supraoptic (SON) and paraventricular (PVN) nuclei of the hypothalamus, acting through both ERα and ERß. Magnocellular and parvocellular neurons in the SON and PVN also express the G protein-coupled ER (GPER). In this study, we have assessed whether GPER is also involved in the regulation of nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase in the SON and PVN. Adult female ovariectomized rats were treated with G1, a selective GPER agonist, or with G1 in combination with G15, a selective GPER antagonist. G1 treatment decreased NADPH-diaphorase expression in the SON and in all PVN subnuclei. The treatment with G1 + G15 effectively rescued the G1-dependent decrease in NADPH-diaphorase expression in both brain regions. In addition, the activation of extracellular signal-regulated kinase (ERK) 1/2, one of the kinases involved in the GPER-dependent intracellular signaling pathway and in NOS phosphorylation, was assessed in the same brain nuclei. Treatment with G1 significantly decreased the number of p-ERK 1/2-positive cells in the SON and PVN, while the treatment with G1 + G15 significantly recovered its number to control values. These findings suggest that the activation of GPER in the SON and PVN inhibits the phosphorylation of ERK 1/2, which induces a decrease in NADPH-diaphorase expression.

Genetic inactivation of glutamate neurons in the rat sublaterodorsal tegmental nucleus recapitulates REM sleep behaviour disorder.

SEE SCHENCK AND MAHOWALD DOI101093/AWW329 FOR A SCIENTIFIC COMMENTARY ON THIS ARTICLE: Idiopathic REM sleep behaviour disorder is characterized by the enactment of violent dreams during paradoxical (REM) sleep in the absence of normal muscle atonia. Accumulating clinical and experimental data suggest that REM sleep behaviour disorder might be due to the neurodegeneration of glutamate neurons involved in paradoxical sleep and located within the pontine sublaterodorsal tegmental nucleus. The purpose of the present work was thus to functionally determine first, the role of glutamate sublaterodorsal tegmental nucleus neurons in paradoxical sleep and second, whether their genetic inactivation is sufficient for recapitulating REM sleep behaviour disorder in rats. For this goal, we first injected two retrograde tracers in the intralaminar thalamus and ventral medulla to disentangle neuronal circuits in which sublaterodorsal tegmental nucleus is involved; second we infused bilaterally in sublaterodorsal tegmental nucleus adeno-associated viruses carrying short hairpin RNAs targeting Slc17a6 mRNA [which encodes vesicular glutamate transporter 2 (vGluT2)] to chronically impair glutamate synaptic transmission in sublaterodorsal tegmental nucleus neurons. At the neuroanatomical level, sublaterodorsal tegmental nucleus neurons specifically activated during paradoxical sleep hypersomnia send descending efferents to glycine/GABA neurons within the ventral medulla, but not ascending projections to the intralaminar thalamus. These data suggest a crucial role of sublaterodorsal tegmental nucleus neurons rather in muscle atonia than in paradoxical sleep generation. In line with this hypothesis, 30 days after adeno-associated virus injections into sublaterodorsal tegmental nucleus rats display a decrease of 30% of paradoxical sleep daily quantities, and a significant increase of muscle tone during paradoxical sleep concomitant to a tremendous increase of abnormal motor dream-enacting behaviours. These animals display symptoms and behaviours during paradoxical sleep that closely mimic human REM sleep behaviour disorder. Altogether, our data demonstrate that glutamate sublaterodorsal tegmental nucleus neurons generate muscle atonia during paradoxical sleep likely through descending projections to glycine/GABA premotor neurons in the ventral medulla. Although playing a role in paradoxical sleep regulation, they are, however, not necessary for inducing the state itself. The present work further validates a potent new preclinical REM sleep behaviour disorder model that opens avenues for studying and treating this disabling sleep disorder, and advances potential regions implicated in prodromal stages of synucleinopathies such as Parkinson's disease.