Sex differences in the coexpression of prokineticin receptor 2 and gonadal steroids receptors in mice.

Loss-of-function mutations in prokineticin 2 (PROK2) and the cognate receptor prokineticin receptor 2 (PROKR2) genes have been implicated in reproductive deficits characteristic of Kallmann Syndrome (KS). Knock out of Prokr2 gene produces the KS-like phenotype in mice resulting in impaired migration of gonadotropin releasing hormone (GnRH) neurons, olfactory bulb dysgenesis, and infertility. Beyond a developmental role, pharmacological and genetic studies have implicated PROKR2 in the control of the estrous cycle in mice. However, PROKR2 is expressed in several reproductive control sites but the brain nuclei associated with reproductive control in adult mice have not been defined. We set out to determine if ProkR2 neurons in both male and female mouse brains directly sense changes in the gonadal steroids milieu. We focused on estrogen receptor α (ERα) and androgen receptor (AR) due to their well-described function in reproductive control via actions in the brain. We found that the ProkR2-Cre neurons in the posterior nucleus of the amygdala have the highest colocalization with ERα and AR in a sex-specific manner. Few colocalization was found in the lateral septum and in the bed nucleus of the stria terminalis, and virtually no colocalization was observed in the medial amygdala. Our findings indicate that the posterior nucleus of the amygdala is the main site where PROKR2 neurons may regulate aspects of the reproductive function and social behavior in adult mice.

Modulation of hypothalamic S6K1 and S6K2 alters feeding behavior and systemic glucose metabolism.

The mTOR/S6Ks signaling is one of the intracellular pathways important for metabolic control, acting both peripherally and centrally. In the hypothalamus, mTOR/S6Ks axis mediates the action of leptin and insulin and can modulate the expression of neuropeptides. We analyzed the role of different S6Ks isoforms in the hypothalamic regulation of metabolism. We observed decreased food intake and decreased expression of agouti-related peptide (AgRP) following intracerebroventricular (icv) injections of adenoviral-mediated overexpression of three different S6Ks isoforms. Moreover, mice overexpressing p70-S6K1 in undefined periventricular hypothalamic neurons presented changes in glucose metabolism, as an increase in gluconeogenesis. To further evaluate the hypothalamic role of a less-studied S6K isoform, p54-S6K2, we used a Cre-LoxP approach to specifically overexpress it in AgRP neurons. Our findings demonstrate the potential participation of S6K2 in AgRP neurons regulating feeding behavior.

LPS-Induced Low-Grade Inflammation Increases Hypothalamic JNK Expression and Causes Central Insulin Resistance Irrespective of Body Weight Changes.

Metabolic endotoxemia contributes to low-grade inflammation in obesity, which causes insulin resistance due to the activation of intracellular proinflammatory pathways, such as the c-Jun N-terminal Kinase (JNK) cascade in the hypothalamus and other tissues. However, it remains unclear whether the proinflammatory process precedes insulin resistance or it appears because of the development of obesity. Hypothalamic low-grade inflammation was induced by prolonged lipopolysaccharide (LPS) exposure to investigate if central insulin resistance is induced by an inflammatory stimulus regardless of obesity. Male Wistar rats were treated with single (1 LPS) or repeated injections (6 LPS) of LPS (100 µg/kg, IP) to evaluate the phosphorylation of the insulin receptor substrate-1 (IRS1), Protein kinase B (AKT), and JNK in the hypothalamus. Single LPS increased the expression of pIRS1, pAKT, and pJNK, whereas the repeated LPS treatment failed to recruit pIRS1 and pAKT. The 6 LPS treated rats showed increased total JNK and pJNK. The 6 LPS rats became unresponsive to the hypophagic effect induced by central insulin administration (12 µM/5 µL, ICV). Prolonged exposure to LPS (24 h) impaired the insulin-induced AKT phosphorylation and the translocation of the transcription factor forkhead box protein O1 (FoxO1) from the nucleus to the cytoplasm of the cultured hypothalamic GT1-7 cells. Central administration of the JNK inhibitor (20 µM/5 µL, ICV) restored the ability of insulin to phosphorylate IRS1 and AKT in 6 LPS rats. The present data suggest that an increased JNK activity in the hypothalamus underlies the development of insulin resistance during prolonged exposure to endotoxins. Our study reveals that weight gain is not mandatory for the development of hypothalamic insulin resistance and the blockade of proinflammatory pathways could be useful for restoring the insulin signaling during prolonged low-grade inflammation as seen in obesity.

Distribution of the neuronal inputs to the ventral premammillary nucleus of male and female rats.

The ventral premammillary nucleus (PMV) expresses dense collections of sex steroid receptors and receptors for metabolic cues, including leptin, insulin and ghrelin. The PMV responds to opposite sex odor stimulation and projects to areas involved in reproductive control, including direct innervation of gonadotropin releasing hormone neurons. Thus, the PMV is well positioned to integrate metabolic and reproductive cues, and control downstream targets that mediate reproductive function. In fact, lesions of PMV neurons blunt female reproductive function and maternal aggression. However, although the projections of PMV neurons have been well documented, little is known about the neuronal inputs received by PMV neurons. To fill this gap, we performed a systematic evaluation of the brain sites innervating the PMV neurons of male and female rats using the retrograde tracer subunit B of the cholera toxin (CTb). In general, we observed that males and females show a similar pattern of afferents. We also noticed that the PMV is preferentially innervated by neurons located in the forebrain, with very few projections coming from brainstem nuclei. The majority of inputs originated from the medial nucleus of the amygdala, the bed nucleus of the stria terminalis and the medial preoptic nucleus. A moderate to high density of afferents was also observed in the ventral subiculum, the arcuate nucleus and the ventrolateral subdivision of the ventromedial nucleus of the hypothalamus. Our findings strengthen the concept that the PMV is part of the vomeronasal system and integrates the brain circuitry controlling reproductive functions.

The periaqueductal gray as a critical site to mediate reward seeking during predatory hunting.

Previous studies using morphine-treated dams reported a role for the rostral lateral periaqueductal gray (rlPAG) in the behavioral switching between nursing and insect hunting, likely to depend on an enhanced seeking response to the presence of an appetitive rewarding cue (i.e., the roach). To elucidate the neural mechanisms mediating such responses, in the present study, we first observed how the rlPAG influences predatory hunting in male rats. Our behavioral observations indicated that bilateral rlPAG NMDA lesions dramatically interfere with prey hunting, leaving the animal without chasing or attacking the prey, but do not seem to affect the general levels of arousal, locomotor activity and regular feeding. Next, using Phaseolus vulgaris-leucoagglutinin (PHA-L), we have reviewed the rlPAG connection pattern, and pointed out a particularly dense projection to the hypothalamic orexinergic cell group. Double labeled PHA-L and orexin sections showed an extensive overlap between PHA-L labeled fibers and orexin cells, revealing that both the medial/perifornical and lateral hypothalamic orexinergic cell groups receive a substantial innervation from the rlPAG. We have further observed that both the medial/perifornical and lateral hypothalamic orexinergic cell groups up-regulate Fos expression during prey hunting, and that rlPAG lesions blunted this Fos increase only in the lateral hypothalamic, but not in the medial/perifornical, orexinergic group, a finding supposedly associated with the lack of motivational drive to actively pursue the prey. Overall, the present results suggest that the rlPAG should exert a critical influence on reward seeking by activating the lateral hypothalamic orexinergic cell group.

Comparative distribution of cocaine- and amphetamine-regulated transcript (CART) in the hypothalamus of the capuchin monkey (Cebus apella) and the common marmoset (Callithrix jacchus).

Cocaine- and amphetamine-regulated transcript (CART) is widely distributed in the brain of many species. In the hypothalamus, CART neurotransmission has been implicated in diverse functions including energy balance, stress response, and temperature and endocrine regulation. Although some studies have been performed in primates, very little is known about the distribution of CART neurons in New World monkeys. New World monkeys are good models for systems neuroscience, as some species have evolved several behavioral and anatomical characteristics shared with humans, including diurnal and social habits, intense maternal care, complex manipulative abilities and well-developed frontal cortices. In the present study, we assessed the distribution of CART mRNA and peptide in the hypothalamus of the capuchin monkey (Cebus apella) and the common marmoset (Callithrix jacchus). We found that the distribution of hypothalamic CART neurons in these monkeys is similar to what has been described for rodents and humans, but some relevant differences were noticed. Only in capuchin monkeys CART neurons were observed in the suprachiasmatic and the intercalatus nuclei, whereas only in marmoset CART neurons were observed in the dorsal anterior nucleus. We also found that the only in marmoset displayed CART neurons in the periventricular preoptic nucleus and in an area seemingly comprising the premammillary nucleus. These hypothalamic sites are both well defined in rodents but poorly defined in humans. Our findings indicate that CART expression in hypothalamic neurons is conserved across species but the identified differences suggest that CART is also involved in the control of species-specific related functions.

The ventral premammillary nucleus links metabolic cues and reproduction.

The amount of body fat and the energy balance are important factors that influence the timing of puberty and the normal reproductive function. Leptin is a key hormone that conveys to the central nervous system information about the individual energy reserve and modulates the hypothalamus-pituitary-gonad (HPG) axis. Recent findings suggest that the ventral premammillary nucleus (PMV) mediates the effects of leptin as a permissive factor for the onset of puberty and the coordinated secretion of luteinizing hormone during conditions of negative energy balance. In this review, we will summarize the existing literature about the potential role played by PMV neurons in the regulation of the HPG axis.

The PI3K signaling pathway mediates the biological effects of leptin.

The activation of the leptin receptor recruits several intracellular signaling pathways, including the phosphatidylinositol 3-kinase (PI3K) pathway. While some of the leptin-induced signaling pathways, such as the JAK2/STAT3 pathway, induce cellular responses primarily through changes in gene expression, the PI3K pathway affects cellular properties more rapidly, through post-translational changes such as protein phosphorylation. Accordingly, several studies have shown that the PI3K pathway is required for the acute effects of leptin, such as a leptin-induced decrease in food intake. Leptin signaling through PI3K also affects the electrophysiological properties of neurons, including changes in their membrane potential and firing rates. In this review, we summarize the recent advances in our understanding of the role played by the PI3K signaling pathway in controlling food intake and energy balance. In particular, we focus on the importance of the PI3K signaling pathway as a mediator of the effects of leptin on hypothalamic neurons.

The PI3K signaling pathway mediates the biological effects of leptin / A via de sinalização intracelular da PI3K medeia os efeitos biológicos da leptina

The activation of the leptin receptor recruits several intracellular signaling pathways, including the phosphatidylinositol 3-kinase (PI3K) pathway. While some of the leptin-induced signaling pathways, such as the JAK2/STAT3 pathway, induce cellular responses primarily through changes in gene expression, the PI3K pathway affects cellular properties more rapidly, through post-translational changes such as protein phosphorylation. Accordingly, several studies have shown that the PI3K pathway is required for the acute effects of leptin, such as a leptin-induced decrease in food intake. Leptin signaling through PI3K also affects the electrophysiological properties of neurons, including changes in their membrane potential and firing rates. In this review, we summarize the recent advances in our understanding of the role played by the PI3K signaling pathway in controlling food intake and energy balance. In particular, we focus on the importance of the PI3K signaling pathway as a mediator of the effects of leptin on hypothalamic neurons.

A ativação do receptor de leptina recruta diversas vias de sinalização intracelular, entre elas a via da fosfatidilinositol 3-quinase (PI3K). Enquanto algumas dessas vias, como a sinalização pelo JAK2/STAT3, induzem respostas celulares por meio de mudanças na transcrição gênica, a via da PI3K altera propriedades celulares de forma rápida, via fosforilação de proteínas. Em concordância, estudos mostraram que a via da PI3K é necessária para que a leptina induza seus efeitos agudos, como redução da ingestão alimentar, após administração de leptina. A ativação da PI3K pela leptina também afeta as propriedades fisiológicas de neurônios, incluindo mudanças no potencial de membrana e no potencial de ação. Nesta revisão, resumimos os recentes avanços na compreen-são do papel desempenhado pela via de sinalização da PI3K no controle da ingestão alimentar e do balanço energético. Discutimos, principalmente, como a via da PI3K é importante para mediar os efeitos da leptina sobre os neurônios hipotalâmicos.

Chemical identity and connections of medial preoptic area neurons expressing melanin-concentrating hormone during lactation.

Lactation is an energy-demanding process characterized by massive food and water consumption, cessation of the reproductive cycle and induction of maternal behavior. During lactation, melanin-concentrating hormone (MCH) mRNA and peptide expression are increased in the medial preoptic area (MPO) and in the anterior paraventricular nucleus of the hypothalamus. Here we show that MCH neurons in the MPO coexpress the GABA synthesizing enzyme GAD-67 mRNA. We also show that MCH neurons in the MPO of female rats are innervated by neuropeptides that control energy homeostasis including agouti-related protein (AgRP), alpha-melanocyte stimulating hormone (alphaMSH) and cocaine- and amphetamine-regulated transcript (CART). Most of these inputs originate from the arcuate nucleus neurons. Additionally, using injections of retrograde tracers we found that CART neurons in the ventral premammillary nucleus also innervate the MPO. We then assessed the projections of the female MPO using injections of anterograde tracers. The MPO densely innervates hypothalamic nuclei related to reproductive control including the anteroventral periventricular nucleus, the ventrolateral subdivision of the ventromedial nucleus (VMHvl) and the ventral premammillary nucleus (PMV). We found that the density of MCH-ir fibers is increased in the VMHvl and PMV during lactation. Our findings suggest that the expression of MCH in the MPO may be induced by changing levels of neuropeptides involved in metabolic control. These MCH/GABA neurons may, in turn, participate in the suppression of cyclic reproductive function and/or sexual behavior during lactation through projections to reproductive control sites.

Male and female odors induce Fos expression in chemically defined neuronal population.

Olfactory information modulates innate and social behaviors in rodents and other species. Studies have shown that the medial nucleus of the amygdala (MEA) and the ventral premammillary nucleus (PMV) are recruited by conspecific odor stimulation. However, the chemical identity of these neurons is not determined. We exposed sexually inexperienced male rats to female or male odors and assessed Fos immunoreactivity (Fos-ir) in neurons expressing NADPH diaphorase activity (NADPHd, a nitric oxide synthase), neuropeptide urocortin 3, or glutamic acid decarboxylase mRNA (GAD-67, a GABA-synthesizing enzyme) in the MEA and PMV. Male and female odors elicited Fos-ir in the MEA and PMV neurons, but the number of Fos-immunoreactive neurons was higher following female odor exposure, in both nuclei. We found no difference in odor induced Fos-ir in the MEA and PMV comparing fed and fasted animals. In the MEA, NADPHd neurons colocalized Fos-ir only in response to female odors. In addition, urocortin 3 neurons comprise a distinct population and they do not express Fos-ir after conspecific odor stimulation. We found that 80% of neurons activated by male odors coexpressed GAD-67 mRNA. Following female odor, 50% of Fos neurons coexpressed GAD-67 mRNA. The PMV expresses very little GAD-67, and virtually no colocalization with Fos was observed. We found intense NADPHd activity in PMV neurons, some of which coexpressed Fos-ir after exposure to both odors. The majority of the PMV neurons expressing NADPHd colocalized cocaine- and amphetamine-regulated transcript (CART). Our findings suggest that female and male odors engage distinct neuronal populations in the MEA, thereby inducing contextualized behavioral responses according to olfactory cues. In the PMV, NADPHd/CART neurons respond to male and female odors, suggesting a role in neuroendocrine regulation in response to olfactory cues.

Increased expression of GluR2-flip in the hippocampus of the Wistar audiogenic rat strain after acute and kindled seizures.

The Wistar Audiogenic Rat (WAR) is an epileptic-prone strain developed by genetic selection from a Wistar progenitor based on the pattern of behavioral response to sound stimulation. Chronic acoustic stimulation protocols of WARs (audiogenic kindling) generate limbic epileptogenesis, confirmed by ictal semiology, amygdale, and hippocampal EEG, accompanied by hippocampal and amygdala cell loss, as well as neurogenesis in the dentate gyrus (DG). In an effort to identify genes involved in molecular mechanisms underlying epileptic process, we used suppression-subtractive hybridization to construct normalized cDNA library enriched for transcripts expressed in the hippocampus of WARs. The most represented gene among the 133 clones sequenced was the ionotropic glutamate receptor subunit II (GluR2), a member of the alpha-amino-3-hydroxy-5-methyl-4-isoxazoleopropionic acid (AMPA) receptor. Although semiquantitative RT-PCR analysis shows that the hippocampal levels of the GluR2 subunits do not differ between naïve WARs and their Wistar counterparts, we observed that the expression of the transcript encoding the splice-variant GluR2-flip is increased in the hippocampus of WARs submitted to both acute and kindled audiogenic seizures. Moreover, using in situ hybridization, we verified upregulation of GluR2-flip mainly in the CA1 region, among the hippocampal subfields of audiogenic kindled WARs. Our findings on differential upregulation of GluR2-flip isoform in the hippocampus of WARs displaying audiogenic seizures is original and agree with and extend previous immunohistochemical for GluR2 data obtained in the Chinese P77PMC audiogenic rat strain, reinforcing the association of limbic AMPA alterations with epileptic seizures.

Melanin-concentrating hormone is expressed in the laterodorsal tegmental nucleus only in female rats.

Melanin-concentrating hormone (MCH) is a neuropeptide originating from prepro-MCH. In male rats, neurons expressing MCH are found in the lateral hypothalamic area and medial zona incerta, as well as, sparsely, in the olfactory tubercle and pontine reticular formation. The wide distribution of MCH fibers suggests the involvement of this neuropeptide in a variety of functions, including arousal, neuroendocrine control and energy homeostasis. In lactating females, MCH is expressed in the preoptic area, indicating sexual dimorphism in MCH gene activation according to the female reproductive state. We hypothesized that MCH is also expressed differentially in the brainstem of female rats. Adult male rats and female rats (in the afternoon of diestrus and proestrus days; ovariectomized; or on lactation days 5, 12 and 19) were perfused between 2 and 4 p.m., and the brainstems were processed for in situ hybridization using a 35S-labeled prepro-MCH riboprobe. As described in males, prepro-MCH was expressed in the pontine reticular formation of females. We also observed consistent prepro-MCH expression in the caudal laterodorsal tegmental nucleus (LDT) of females but no differential expression comparing the various female reproductive states. Using dual-label immunohistochemistry or dual-label in situ hybridization, we found that brainstem MCH neurons coexpress glutamic acid decarboxylase mRNA, the gamma aminobutyric acid (GABA) processing enzyme, but do not colocalize choline acetyl transferase (acetylcholine processing enzyme). Since changes in LDT GABAergic cell activity are associated with rapid eye movement (REM) sleep, our findings suggest that MCH interacts with LDT GABAergic neurons and plays a role in REM sleep regulation.

Distribution of urocortin 3 neurons innervating the ventral premammillary nucleus in the rat brain.

Urocortin 3 (Ucn 3) is a recently described peptide of the corticotropin-releasing factor family. Neurons expressing Ucn 3 mRNA and peptide are distributed in specific brain areas, including the median preoptic nucleus, the perifornical area (PFx), and the medial nucleus of the amygdala (MEA). Fibers immunoreactive to Ucn 3 are confined to certain brain nuclei, being particularly dense in the ventral premammillary nucleus (PMV). In studies involving electrolytic lesions and analysis of Fos distribution according to behavioral paradigms, the PMV has been potentially implicated in conspecific aggression and sexual behavior. However, the role that Ucn 3 plays in this pathway has not been explored. Therefore, we investigated the origins of the urocortinergic innervation of the PMV of Wistar rat in an attempt to map the brain circuitry and identify likely related functions. We injected the retrograde tracer cholera toxin b subunit into the PMV and found that 88% of the Ucn 3-immunoreactive fibers in the PMV originate in the dorsal MEA, and that few originate in the PFx. As a control, we injected the anterograde tracer biotin dextran amine into both regions. We observed that the PMV is densely innervated by the MEA, and scarcely innervated by the PFx. The MEA is a secondary relay of the vomeronasal system and projects amply to hypothalamic nuclei related to hormonal and behavioral adjustments, including the PMV. Although physiological studies should also be performed, we hypothesize that Ucn 3 participates in such pathways, conveying sensory information to the PMV, which in turn modulates behavioral and neuroendocrine responses.

Alternative pathways for catecholamine action in oral motor control.

Orofacial movement is a complex function performed by facial and jaw muscles. Jaw movement is enacted through the triggering of motoneurons located primarily in the trigeminal motor nucleus (Mo5). The Mo5 is located in the pontine reticular formation, which is encircled by premotor neurons. Previous studies using retrograde tracers have demonstrated that premotor neurons innervating the Mo5 are distributed in brainstem areas, and electrophysiological studies have suggested the existence of a subcortical relay in the corticofugal-Mo5 pathway. Various neurotransmitters have been implicated in oral movement. Dopamine is of special interest since its imbalance may produce changes in basal ganglia activity, which generates abnormal movements, including jaw motor dysfunction, as in oral dyskinesia and possibly in bruxism. However, the anatomical pathways connecting the dopaminergic systems with Mo5 motoneurons have not been studied systematically. After injecting retrograde tracer fluorogold into the Mo5, we observed retrograde-labeled neurons in brainstem areas and in a few forebrain nuclei, such as the central nucleus of the amygdala, and the parasubthalamic nucleus. By using dual-labeled immunohistochemistry, we found tyrosine hydroxylase (a catecholamine-processing enzyme) immunoreactive fibers in close apposition to retrograde-labeled neurons in brainstem nuclei, in the central nucleus of the amygdala and the parasubthalamic nucleus, suggesting the occurrence of synaptic contacts. Therefore, we suggested that catecholamines may regulate oralfacial movements through the premotor brainstem nuclei, which are related to masticatory control, and forebrain areas related to autonomic and stress responses.

Projeçöes diencefálicas para o córtex motor e o núcleo pedunculopontino do tegmento, imunorreativas ao hormônio concentrador de melanina (MCH) e/ou ao neuropeptídeo EI (NEI): um estudo com múltiplos traçadores neuronais / Diencephalic origns of melanin-concentrating hormone-and neuropeptide EI - immunoreactive projections to pedunculopontine nucleous and motor cortex: a study with multiple neuronal tracers

O hormônio concentrador de melanina (MCH) é um heptadecapeptídeo cíclico descrito pela primeira vez em hipófise de salmäo e posteriormente em alguns mamíferos. Os corpos celulares McH-ir encontram-se localizados em regiöes específicas do sistema nervoso central (SNC) do rato, como a Zona Incerta (EI) e a Área Hipotalâmica Lateral (AHL) enquanto que as fibras apresentam-se espalhadas por todo o neuro-eixo. Indica que a inervaçäo cortical é densa e homogênea por todo o neocórtex, assim como, a inervaçäo de várias regiöes da formaçäo reticular, como o núcleo pedunculopontino do tegmento (PPTg). Faz um estudo com múltiplos traçadores neuronais em ratos albinos machos (Wistar) usando 6 procedimentos distintos. Utiliza injeçöes de Diamidino Yellow (DY) no córtex motor (CxM) e de True Blue (TB) no PPTg. Demonstra a existência de neurônios MCH-ir marcados com ambos os traçadores distintamente, tanto na ZI quanto na AHL. Realiza controle com leucoaglutimina do Phaseolus vulgaris (PHA-L) injetado nestes territórios, demonstrando a inervaçäo do CxM e do PPTg originários das regiöes onde as células dupla e triplamente marcada foram encontradas. Conclui que estas células säo também NEI-ir; os resultados envidenciam a presença de colaterais de AHL e ZI para 2 regiöes de SNC envolvidas com o comportamento locomotor. Discute a pesquisa em termos de envolvimento do MCH com o comportamento alimentar, que justamente com o locomotor indica uma atuaçäo na busca do alojamento (a maior quantidade de células marcadas säo originárias de fibras localizadas na regiäo motora das vibrissas), e no sistema reticular ativador ascendente