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Similar to psychostimulants, the peripheral administration of menthol promotes mouse motor activity, and the neurotransmitter dopamine has been suggested to be involved in this effect. The present study aimed to elucidate the effects of l-menthol on parts of the central nervous system that are involved in motor effects. The subcutaneous administration of l-menthol significantly increased the number of c-Fos-like immunoreactive nuclei in the dorsal striatum of the mice, and motor activity was promoted. It also increased the extracellular dopamine level in the dorsal striatum of the mice. These observations indicated that after subcutaneous administration, l-menthol enhances dopamine-mediated neurotransmission, and activates neuronal activity in the dorsal striatum, thereby promoting motor activity in mice.

BackgroundThe bidirectional selection of high and low anxiety-like behavior is a valuable tool for understanding the neurocircuits that are responsible for anxiety disorders. Our group developed two breeding lines of rats, known as Carioca High- and Low-conditioned Freezing (CHF and CLF), based on defensive freezing in the contextual fear conditioning paradigm. A random selected line was employed as a control (CTL) comparison group for both CHF and CLF lines of animals. The present study performed Fos immunochemistry to investigate changes in neural activity in different brain structures among CHF and CLF rats when they were exposed to contextual cues that were previously associated with footshock.ResultsThe study indicated that CHF rats expressed high Fos expression in the locus coeruleus, periventricular nucleus of the hypothalamus (PVN), and lateral portion of the septal area and low Fos expression in the medial portion of the septal area, dentate gyrus, and prelimbic cortex (PL) compared to CTL animals. CLF rats exhibited a decrease in Fos expression in the PVN, PL, and basolateral nucleus of the amygdala and increase in the cingulate and perirhinal cortices compared to CTL animals.ConclusionsBoth CHF and CLF rats displayed Fos expression changes key regions of the anxiety brain circuitry. The two bidirectional lines exhibit different pattern of neural activation and inhibition with opposing influences on the PVN, the main structure involved in regulating the hypothalamic-pituitary-adrenal neuroendocrine responses observed in anxiety disorders.

Dysfunctions in the GABAergic system lead to various pathological conditions and impaired inhibitory function is one of the causes behind neuropathies characterized by neuronal hyper excitability. TheDlxhomeobox genes are involved in the development of nervous system, neural crest, brachial arches and developing appendages.Dlxgenes also take part in neuronal migration and differentiation during development, more precisely, in the migration and differentiation of GABAergic neurons. Functional analysis ofdlxgenes has mainly been carried out in developing zebrafish embryos and larvae; however information regarding the expression and roles of these genes in the adult zebrafish brain is still lacking. The extensive neurogenesis that takes place in the brain of adult zebrafish makes them a good model for the visualization of mechanisms involvingdlxgenes during adulthood in physiological conditions and during regeneration of the nervous system. We have identified the adult brain regions where transcripts ofdlx1a, dlx2a, dlx5aanddlx6agenes are normally found and have confirmed that within telencephalic domains, there is high overlapping expression of the fourdlxparalogs with a marker for GABAergic neurons. Co-localization analyses carried with the Tg(dlx6a-1.4kbdlx5a/dlx6a:GFP) reporter line have also shown that in some areas of the diencephalon, cells expressing thedlx5a/6abigene may have a neural stem cell identity by co-localizing with a Sox2 antibody. Furthermore, investigations in a response to stab wound lesions, have demonstrated a possible participation of thedlx5a/6abigene, most likely, ofdlx5aduring the regeneration of the adult zebrafish brain. These data suggest a possible participation ofdlx-expressing cells during brain regeneration in adult zebrafish and also provide information on the role ofdlxgenes under normal physiological conditions in adults.

Research of the central actions of prolactin is highly focused on females, but this hormone has also documented roles in male physiology and behaviour. Here, we provide the first description of the pattern of prolactin-derived signalling in the male mouse brain, employing the immunostaining of phosphorylated signal transducer and activator of transcription 5 (pSTAT5) after exogenous prolactin administration. Next, we explore possible sexually dimorphic differences by comparing pSTAT5 immunoreactivity in prolactin-supplemented males and females. We also assess the role of testosterone in the regulation of central prolactin signalling in males by comparing intact with castrated prolactin-supplemented males. Prolactin-supplemented males displayed a widespread pattern of pSTAT5 immunoreactivity, restricted to brain centres showing expression of the prolactin receptor. Immunoreactivity for pSTAT5 was present in several nuclei of the preoptic, anterior and tuberal hypothalamus, as well as in the septofimbrial nucleus or posterodorsal medial amygdala of the telencephalon. Conversely, non-supplemented control males were virtually devoid of pSTAT5-immunoreactivity, suggesting that central prolactin actions in males are limited to situations concurrent with substantial hypophyseal prolactin release (e.g. stress or mating). Furthermore, comparison of prolactin-supplemented males and females revealed a significant, female-biased sexual dimorphism, supporting the view that prolactin has a preeminent role in female physiology and behaviour. Finally, in males, castration significantly reduced pSTAT5 immunoreactivity in some structures, including the paraventricular and ventromedial hypothalamic nuclei and the septofimbrial region, thus indicating a region-specific regulatory role of testosterone over central prolactin signalling.

The amygdala plays an important functional role in fear and anxiety. Abnormalities in the amygdala are believed to be involved in the neurobiological basis of panic disorder (PD). Previous structural neuroimaging studies have found global volumetric and morphological abnormalities in the amygdala in patients with PD. Very few studies, however, have explored for structural abnormalities in various amygdala sub-regions, which consist of various sub-nuclei, each with different functions. This study aimed to evaluate for volumetric abnormalities in the amygdala sub-nuclei, in order to provide a better understanding neurobiological basis of PD. Thirty-eight patients with PD and 38 matched healthy control (HC) participants underwent structural MRI scanning. The volume of the whole amygdala, as well as its consistent sub-nuclei, were calculated using FreeSurfer software. Relative volumes of these amygdala sub-regions were compared between the two groups. Results showed significantly smaller volumes in the right lateral and basal nuclei in the patients with PD compared with the HC. Lateral and basal nuclei are thought to play crucial role for processing sensory information related with anxiety and fear. Our results suggest that these particular amygdala sub-regions play a role in the development of PD symptoms.

In female mice, the experience of being shipped from the breeder facility or a single injection of the bacterial endotoxin, lipopolysaccharide (LPS), during pubertal development alters the behavioral response to estradiol in adulthood as demonstrated by perturbations of estradiol's effects on sexual behavior, cognitive function, as well as its anxiolytic and anti-depressive properties. Microglia, the primary type of immunocompetent cell within the brain, contribute to brain development and respond to stressors with marked and long-lasting morphological and functional changes. Here, we describe the morphology of microglia and their response to shipping and LPS in peripubertal and adult female mice. Peripubertal mice have more microglia with long, thick processes in the hippocampus, amygdala and hypothalamus as compared with adult mice in the absence of an immune challenge. An immune challenge also increases immunoreactivity (IR) of ionized calcium binding adaptor molecule 1 (Iba1), which is constitutively expressed in microglia. In the hippocampus, the age of animal was without effect on the increase in Iba1- IR following shipping from the breeder facility or LPS exposure. In the amygdala, we observed more Iba1-IR following shipping or LPS treatment in peripubertal mice, compared to adult mice. In the hypothalamus, there was a disassociation of the effects of shipping and LPS treatment as LPS treatment, but not shipping, induced an increase in Iba1-IR. Taken together these data indicate that microglial morphologies differ between pubertal and adult mice; moreover, the microglial response to complex stressors is greater in pubertal mice as compared to adult mice.

Food anticipatory activity (FAA) refers to a daily rhythm of locomotor activity that emerges under conditions of food restriction, whereby animals develop an intense, predictable period of activity in the few hours leading up to a predictable, daily delivery of food. The neural mechanisms by which FAA is regulated are not yet fully understood. Although a number of brain regions appear to be involved in regulating the development and expression of FAA, there is little evidence to date concerning the role of the anterior agranular insular cortex (AICa). The AICa plays a critical role in integrating the perception of visceral states with motivational behaviour such as feeding. We assessed the effect of bilateral electrolytic or ibotenic acid lesions of the AICa on FAA in male Wistar rats receiving food for varying lengths of time (2 h, 3 h, or 5 h) during the middle of the light phase (starting at either ZT4 or ZT6). Contrary to our initial expectations, we found that both electrolytic and ibotenic acid lesions significantly increased, rather than decreased, the amount of FAA expressed in lesioned rats. Despite increased FAA, lesioned rats did not eat significantly more during restricted feeding (RF) periods than control rats. Similar to controls, AlCa-lesioned rats showed negligible anticipatory activity to a restricted treat suggesting that the increased anticipatory activity in lesioned rats is associated with food restriction, rather than the appetitive value of the meal. Monitoring behaviour in an open field indicated that increased FAA in AlCa-lesioned rats was not explained by a general increase in locomotor activity. Together, these findings suggest that the AICa contributes to the network of brain regions involved in FAA.

How the hypothalamus transmits hunger information to other brain regions to govern whole brain function to orchestrate feeding behavior has remained largely unknown. Our present study suggests the importance of a recently found lateral hypothalamic neuropeptide, QRFP, in this signaling. Qrfp-/- mice were hypophagic and lean, and exhibited increased anxiety-like behavior, and were hypoactive in novel circumstances as compared with wild type littermates. They also showed decreased wakefulness time in the early hours of the dark period. Histological studies suggested that QRFP neurons receive rich innervations from neurons in the arcuate nucleus which is a primary region for sensing the body's metabolic state by detecting levels of leptin, ghrelin and glucose. These observations suggest that QRFP is an important mediator that acts as a downstream mediator of the arcuate nucleus and regulates feeding behavior, mood, wakefulness and activity.

The social environment can alter pubertal timing through neuroendocrine mechanisms that are not fully understood; it is thought that stress hormones (e.g., glucocorticoids or corticotropin-releasing hormone) influence the hypothalamic-pituitary-gonadal axis to inhibit puberty. Here, we use the eusocial naked mole-rat, a unique species in which social interactions in a colony (i.e. dominance of a breeding female) suppress puberty in subordinate animals. Removing subordinate naked mole-rats from this social context initiates puberty, allowing for experimental control of pubertal timing. The present study quantified gene expression for reproduction- and stress-relevant genes acting upstream of gonadotropin-releasing hormone in brain regions with reproductive and social functions in pre-pubertal, post-pubertal, and opposite sex-paired animals (which are in various stages of pubertal transition). Results indicate sex differences in patterns of neural gene expression. Known functions of genes in brain suggest stress as a key contributing factor in regulating male pubertal delay. Network analysis implicates neurokinin B (Tac3) in the arcuate nucleus of the hypothalamus as a key node in this pathway. Results also suggest an unappreciated role for the nucleus accumbens in regulating puberty.

The anterior hypothalamic area (AHA) is an important integrative relay structure for a variety of autonomic, endocrine, and behavioral responses including feeding behavior and response to stress. However, changes in the activity of the AHA neurons during stress and feeding in freely moving rats are not clear. The present study investigated the firing rate and burst activity of neurons in the central nucleus of the AHA (cAHA) during sucrose intake in non-stressful conditions and after acute stress in freely behaving rats. Rats were implanted with micro-electrodes into the cAHA, and extracellular multi-unit activity was recorded during 1-h access to 10% sucrose in non-stressful conditions or after acute foot shock stress. Acute stress significantly reduced sucrose intake, total sucrose lick number, and lick frequency in licking clusters, and increased inter-lick intervals. At the cluster start (CS) of sucrose licking, the cAHA neurons increased (CS-excited, 20% of the recorded neurons), decreased (CS-inhibited, 42% of the neurons) or did not change (CS-nonresponsive, 38% of the neurons) their firing rate. Stress resulted in a significant increase in the firing rate of the CS-inhibited neurons by decreasing inter-spike intervals within the burst firing of these neurons. This increase in the stress-induced firing rate of the CS-inhibited neurons was accompanied by a disruption of the correlation between the firing rate of CS-inhibited and CS-nonresponsive neurons that was observed in non-stressful conditions. Stress did not affect the firing rate of the CS-excited and CS-nonresponsive neurons. However, stress changed the pattern of burst firing of the CS-excited and CS-nonresponsive neurons by decreasing and increasing the burst number in the CS-excited and CS-nonresponsive neurons, respectively. These results suggest that the cAHA neurons integrate the signals related to stress and intake of palatable food and play a role in the stress- and eating-related circuitry.

Framingham Heart Study suggests that dysfunction of steroid receptor coactivator-1 may be involved in the development of hypertension. However, there is no functional evidence linking steroid receptor coactivator-1 to the regulation of blood pressure. We used immunohistochemistry to map the expression of steroid receptor coactivator-1 protein in mouse brain, especially in regions implicated in the regulation of blood pressure. Steroid receptor coactivator-1 protein was found in central amygdala, medial amygdala, supraoptic nucleus, arcuate nucleus, ventromedial, dorsomedial, paraventricular hypothalamus, and nucleus of the solitary tract. To determine the effects of steroid receptor coactivator-1 protein on cardiovascular system we measured blood pressures, blood flow velocities, echocardiographic parameters, and aortic input impedance in female steroid receptor coactivator-1 knockout mice and their wild type littermates. Steroid receptor coactivator-1 knockout mice had higher blood pressures and increased aortic stiffness when compared to female wild type littermates. Additionally, the hearts of steroid receptor coactivator-1 knockout mice seem to consume higher energy as evidenced by increased impedance and higher heart rate pressure product when compared to female wild type littermates. Our results demonstrate that steroid receptor coactivator-1 may be functionally involved in the regulation of blood pressure and aortic stiffness through the regulation of sympathetic activation in various neuronal populations.

Task-specific rehabilitative training is commonly used for chronic stroke patients. Axonal remodeling is believed to be one mechanism underlying rehabilitation-induced functional recovery, and significant roles of the corticospinal pathway have previously been demonstrated. Brainstem-spinal pathways, as well as the corticospinal tract, have been suggested to contribute to skilled motor function and functional recovery after brain injury. However, whether axonal remodeling in the brainstem-spinal pathways is a critical component for rehabilitation-induced functional recovery is not known. In this study, rats were subjected to photothrombotic stroke in the caudal forelimb area of the primary motor cortex and received rehabilitative training with a skilled forelimb reaching task for 4 weeks. After completion of the rehabilitative training, the retrograde tracer Fast blue was injected into the contralesional lower cervical spinal cord. Fast blue-positive cells were counted in 32 brain areas located in the cerebral cortex, hypothalamus, midbrain, pons, and medulla oblongata. Rehabilitative training improved motor performance in the skilled forelimb reaching task but not in the cylinder test, ladder walk test, or staircase test, indicating that rehabilitative skilled forelimb training induced task-specific recovery. In the histological analysis, rehabilitative training significantly increased the number of Fast blue-positive neurons in the ipsilesional rostral forelimb area and secondary sensory cortex. However, rehabilitative training did not alter the number of Fast blue-positive neurons in any areas of the brainstem. These results indicate that rehabilitative skilled forelimb training enhances axonal remodeling selectively in the corticospinal pathway, which suggests a critical role of cortical plasticity, rather than brainstem plasticity, in task-specific recovery after subtotal motor cortex destruction.

Objectives Assess direct versus indirect action(s) of ghrelin on hypothalamic NPY neurons. Materials and methods Electrophysiology was used to measure ion channel activity in NPY-GFP neurons in slice preparations. Ca2+ imaging was used to monitor ghrelin activation of isolated NPY GFP-labeled neurons. Immunohistochemistry was used to localize Trpm4, SUR1 and Kir6.2 in the hypothalamus. Results Acylated ghrelin depolarized the membrane potential (MP) of NPY-GFP neurons in brain slices. Depolarization resulted from a decreased input resistance (IR) in ~70% of neurons (15/22) or an increased IR in the remainder (7/22), consistent with the opening or closing of ion channels, respectively. Although tetrodotoxin (TTX) blockade of presynaptic action potentials reduced ghrelin-induced changes in MP and IR, ghrelin still significantly depolarized the MP and decreased IR in TTX-treated neurons, suggesting that ghrelin directly opens cation channel(s) in NPY neurons. In isolated NPY-GFP neurons, ghrelin produced a sustained rise of [Ca2+]c, with an EC50 ~110 pM. Pharmacologic studies confirmed that the direct action of ghrelin was through occupation of the growth hormone secretagogue receptor, GHS-R, and demonstrated the importance of the adenylate cyclase/cAMP/protein kinase A (PKA) and phospholipase C/inositol triphosphate (PLC/IP3) pathways as activators of 5' AMP-activated protein kinase (AMPK). Activation of isolated neurons was not affected by CNQX or TTX, but reducing [Na+]o suppressed activation, suggesting a role for Na+-permeable cation channels. SUR1 and two channel partners, Kir6.2 and Trpm4, were identified immunologically in NPY-GFP neurons in situ. The actions of SUR1 and Trpm4 modulators were informative: like ghrelin, diazoxide, a SUR1 agonist, elevated [Ca2+]c and glibenclamide, a SUR1 antagonist, partially suppressed ghrelin action, while 9-phenanthrol and flufenamic acid, selective Trpm4 antagonists, blocked ghrelin actions on isolated neurons. Ghrelin activation was unaffected by nifedipine and ω-conotoxin, inhibitors of L- and N-type Ca2+ channels, respectively, while Ni2+, mibefradil, and TTA-P2 completely or partially inhibited ghrelin action, implicating T-type Ca2+ channels. Activation was also sensitive to a spider toxin, SNX-482, at concentrations selective for R-type Ca2+ channels. Nanomolar concentrations of GABA markedly inhibited ghrelin-activation of isolated NPY-GFP neurons, consistent with chronic suppression of ghrelin action in vivo. Conclusions NPY neurons express all the molecular machinery needed to respond directly to ghrelin. Consistent with recent studies, ghrelin stimulates presynaptic inputs that activate NPY-GFP neurons in situ. Ghrelin can also directly activate a depolarizing conductance. Results with isolated NPY-GFP neurons suggest the ghrelin-activated, depolarizing current is a Na+ conductance with the pharmacologic properties of SUR1/Trpm4 non-selective cation channels. In the isolated neuron model, the opening of SUR1/Trpm4 channels activates T- and SNX482-sensitive R-type voltage dependent Ca2+ channels, which could contribute to NPY neuronal activity in situ.

Background Nutrient deprivation during early development has been associated with the predisposition to metabolic disorders in adulthood. Considering its interaction with metabolism, appetite and behavior, the endocannabinoid (eCB) system represents a promising target of developmental programming. Methods By cross-fostering and variation of litter size, early postnatal nutrition of CB6F1-hybrid mice was controlled during the lactation period (3, 6, or 10 pups/mother). After weaning and redistribution at P21, all pups received standard chow ad libitum. Gene expression analyses (liver, visceral fat, hypothalamus) were performed at P50, eCB concentrations were determined in liver and visceral fat. Locomotor activity and social behavior were analyzed by means of computer-assisted videotracking. Results Body growth was permanently altered, with differences for length, weight, body mass index and fat mass persisting beyond P100 (all 3>6>10,p6>10 (DAGLα p6>10 (FAAH p

Previous reports have shown that the early postnatal environment has the ability to modify the obesity phenotype of Otsuka Long-Evans Tokushima Fatty (OLETF) rats. To determine whether this early postnatal environment affects hypothalamic signaling systems involved in energy balance, OLETF pups and lean Long-Evans Tokushima Otsuka (LETO) pups were cross-fostered to same or opposite strain Dams (designated as LdLp: LETO pups with LETO dams; LdOp: OLETF pups with LETO dams; OdLp: LETO pups with OLETF dams; and OdOp: OLETF pups with OLETF dams). Hypothalamic gene expression was examined at postnatal day 23 (PND 23) and PND 90 as OdOp rats started to gain more body weight at PND 23 and developed obesity at PND 90 relative to lean control LdLp rats. On PND 23, neuropeptide Y (Npy) gene expression was significantly increased in the dorsomedial hypothalamus (DMH) in both LdOp and OdOp pups compared to LdLp pups. Maternal environment did not affect DMH Npy expression in LETO weanlings. On PND 90, maternal environment during the cross-fostering period had a major effect on DMH Npy expression. Levels were significantly increased in both OdOp and OdLp rats relative to those in LdOp rats and LdLp controls. Reduced expression of Npy in the DMH of LdOp rats was consistent with their reduction of body weight compared to OdOp rats. In contrast to DMH Npy, gene expression for Npy and proopiomelanocortin in the arcuate nucleus appeared to appropriately respond to alterations in body weight and plasma leptin levels. Levels of oxytocin gene expression in the paraventricular nucleus were lower in offspring raised by LETO dams apparently responding to the higher DMH NPY levels. Together, our results demonstrate effects of both genotype and early postnatal environment on obesity of OLETF rats and further suggest an important role of DMH NPY in the development of obesity of OLETF rats.

Despite rhythmic expression of clock genes being found throughout the central nervous system, very little is known about their function outside of the suprachiasmatic nucleus. Determining the pattern of clock gene expression across neuronal subpopulations is a key step in understanding their regulation and how they may influence the functions of various brain structures. Using immunofluorescence and confocal microscopy, we quantified the co-expression of the clock proteins BMAL1 and PER2 with two neuropeptides, Substance P (SubP) and Enkephalin (Enk), expressed in distinct neuronal populations throughout the forebrain. Regions examined included the limbic forebrain (dorsal striatum, nucleus accumbens, amygdala, stria terminalis), thalamus medial habenula of the thalamus, paraventricular nucleus and arcuate nucleus of the hypothalamus and the olfactory bulb. In most regions examined, BMAL1 was homogeneously expressed in nearly all neurons (~90%), and PER2 was expressed in a slightly lower proportion of cells. There was no specific correlation to SubP- or Enk- expressing subpopulations. The olfactory bulb was unique in that PER2 and BMAL1 were expressed in a much smaller percentage of cells, and Enk was rarely found in the same cells that expressed the clock proteins (SubP was undetectable). These results indicate that clock genes are not unique to specific cell types, and further studies will be required to determine the factors that contribute to the regulation of clock gene expression throughout the brain.

Background: The neuropeptide S receptor (NPSR1) and its ligand neuropeptide S (NPS) have received increased attention in the last few years, as both establish a previously unknown system of neuromodulation. Animal research studies have suggested that NPS may be involved in arousal/wakefulness and may also have a crucial role in sleep regulation. The single nucleotide polymorphism (SNP) rs324981 in NPSR1 has begun to shed light on a function of the NPS-system in human sleep regulation. Due to an amino acid exchange, the T-allele leads to an increased sensitivity of the NPSR1. In the only genomewide association study to date on circadian sleep parameters in humans, an association was found between rs324981 and regular bedtime. However, the sleep parameters in this study were only measured by self-rating. Therefore, our study aimed to replicate these findings using an objective measure of sleep. Methods: The study included n = 393 white subjects (62–79 years) who participated in an actigraphic assessment for determining sleep duration, rest duration, sleep onset, rest onset and sleep onset latency. Genotyping of the SNP rs324981 was performed using the TaqMan OpenArray System. Results: The genotype at rs324981 was not significantly associated with rest onset (bedtime) or sleep onset (p = .146 and p = .199, respectively). However, the SNP showed a significant effect on sleep- and rest duration (p = .007 and p = .003, respectively). Subjects that were homozygous for the minor T-allele had a significantly decreased sleep- and rest duration compared to A-allele carriers. Conclusion: The results of this study indicate that the sleep pattern in humans is influenced by the NPS-system. However, the previously reported association between bedtime and rs324981 could not be confirmed. The current finding of decreased sleep duration in T/T allele carriers is in accordance with studies in rodents reporting similar results after NPS application.:Background; Methods; Results; Conclusions

Kisspeptin (KP)- and neurokinin B (NKB)- synthesizing neurons of the hypothalamic arcuate nucleus play a pivotal role in the regulation of pulsatile gonadotropin-releasing hormone (GnRH) secretion. Unlike in rodents and sheep, the homologous KP and NKB neurons in the human infundibular region rarely express dynorphin- but often exhibit Substance P (SP) immunoreactivity, indicating remarkable species differences in the neurochemical phenotype of these neurons. In search for additional neuropeptides in human KP and NKB neurons, we carried out immunofluorescent studies on hypothalamic sections obtained from five postmenopausal women. Colocalization experiments provided evidence for the presence of cocaine- and amphetamine-regulated transcript (CART) in 47.9 ± 6.6% of KP-immunoreactive (IR) and 30.0 ± 4.9% of NKB-IR perikarya and in 17.0 ± 2.3% of KP-IR and 6.2 ± 2.0% of NKB-IR axon varicosities. All three neuropeptides were present in 33.3 ± 4.9% of KP-IR and 28.2 ± 4.6% of NKB-IR somata, respectively, whereas triple-labeling showed lower incidences in KP-IR (14.3 ± 1.8%) and NKB-IR (5.9 ± 2.0%) axon varicosities. CART-IR KP and NKB neurons established contacts with other peptidergic cells, including GnRH-IR neurons and also sent projections to the infundibular stalk. KP and NKB fibers with CART often contained SP as well, while being distinct from CART fibers co-containing the orexigenic peptide agouti-related protein. Presence of CART in human, but not rodent, KP and NKB neurons represents a new example of species differences in the neuropeptide repertoire of mediobasal hypothalamic KP and NKB neurons. Target cells, receptor sites and physiological significance of CART in the efferent communication of KP and NKB neurons in primates require clarification.

ObjectiveLeptin resistance is a common hallmark of obesity. Rats on a free-choice high-fat high-sugar (fcHFHS) diet are resistant to peripherally administered leptin. The aim of this study was to investigate feeding responses to central leptin as well as the associated changes in mRNA levels in hypothalamic and mesolimbic brain areas.Design and methodsRats on a CHOW or fcHFHS diet for 8 days received leptin or vehicle intracerebro(lateral)ventricularly (ICV) and food intake was measured 5 h and 24 h later. Four days later, rats were sacrificed after ICV leptin or vehicle and mRNA levels were quantified for hypothalamic pro-opiomelanocortin (POMC) and neuropeptide Y (NPY) and for preproenkephalin (ppENK) in nucleus accumbens and tyrosine hydroxylase (TH) in ventral tegmental area (VTA).ResultsICV leptin decreased caloric intake both in CHOW and fcHFHS rats. In fcHFHS, leptin preferentially decreased chow and fat intake. Leptin increased POMC and decreased NPY mRNA in CHOW, but not in fcHFHS rats. In CHOW rats, leptin had no effect on ppENK mRNA and decreased TH mRNA. In fcHFHS, leptin decreased ppENK mRNA and increased TH mRNA.ConclusionDespite peripheral and arcuate leptin resistance, central leptin suppresses feeding in fcHFHS rats. As the VTA and nucleus accumbens are still responsive to leptin, these brain areas may therefore, at least partly, account for the leptin-induced feeding suppression in rats on a fcHFHS diet.

Ghrelin, the endogenous growth hormone secretagogue, has an important role in metabolic homeostasis. It exists in two major molecular forms: acylated (AG) and unacylated (UAG). Many studies suggest different roles for these two forms of ghrelin in energy balance regulation. In the present study, we compared the effects of acute intracerebroventricular administration of AG, UAG and their combination (AG+UAG) to young adult Wistar rats on food intake and central melanocortin system modulation. Although UAG did not affect food intake it significantly increased the number of c-Fos positive neurons in the arcuate (ARC), paraventricular (PVN) and solitary tract (NTS) nuclei. In contrast, UAG suppressed AG-induced neuronal activity in PVN and NTS. Central UAG also modulated hypothalamic expression of Mc4r and Bmp8b, which were increased and Mc3r, Pomc, Agrp and Ucp2, which were decreased. Finally, UAG, AG and combination treatments caused activation of c-Fos in POMC expressing neurons in the arcuate, substantiating a physiologic effect of these peptides on the central melanocortin system. Together, these results demonstrate that UAG can act directly to increase neuronal activity in the hypothalamus and is able to counteract AG-induced neuronal activity in the PVN and NTS. UAG also modulates expression of members of the melanocortin signaling system in the hypothalamus. In the absence of an effect on energy intake, these findings indicate that UAG could affect energy homeostasis by modulation of the central melanocortin system.

Female sexual behavior is controlled by central estrogenic action in the ventromedial nucleus of the hypothalamus (VMN). This region plays a pivotal role in facilitating sex-related behavior in response to estrogen stimulation via neural activation by several neurotransmitters, including histamine, which participates in this mechanism through its strong neural potentiating action. However, the mechanism through which estrogen signaling is linked to the histamine system in the VMN is unclear. This study was undertaken to investigate the relationship between estrogen and histamine receptor subtype H1 (H1R), which is a potent subtype among histamine receptors in the brain. We show localization of H1R exclusively in the ventrolateral subregion of the female VMN (vl VMN), and not in the dorsomedial subregion. In the vl VMN, abundantly expressed H1R were mostly colocalized with estrogen receptor α. Intriguingly, H1R mRNA levels in the vl VMN were significantly elevated in ovariectomized female rats treated with estrogen benzoate. These data suggest that estrogen can amplify histamine signaling by enhancing H1R expression in the vl VMN. This enhancement of histamine signaling might be functionally important for allowing neural excitation in response to estrogen stimulation of the neural circuit and may serve as an accelerator of female sexual arousal.

Nesfatin-1 is a recently discovered metabolic peptide hormone that decreases food intake after lateral, third, or fourth brain ventricle; cisterna magna; or paraventricular nucleus (PVN) injection in ad libitum fed rats. Additional micro-injection studies will improve the understanding of how nesfatin-1 acts on the brain and define specific nuclei responsive to nesfatin-1, which will provide insight on its effects on food intake. We evaluated how nesfatin-1 injection into the dorsal vagal complex (DVC) modulates food intake response in rats during the dark phase. Consistent with previous observations, nesfatin-1-injected rats significantly reduced cumulative food intake over a 5-h period in rats. Chronic administration of nesfatin-1 into the DVC reduced body weight gain over a 10-day period. Because glucosensing neurons in the DVC are involved in glucoprivic feeding and homeostatic control of blood glucose, we examined the effect of nesfatin-1 on the excitability of DVC glucosensing neurons. Nesfatin-1 inhibited most of the glucose-inhibitory (GI) neurons and excited most of the glucose-excitatory (GE) neurons in the DVC. Current-clamp electrophysiology recordings from DVC glucosensing neurons in slice preparation showed that bath applied nesfatin-1(10 nM) increased the firing frequency of GE neurons and inhibited the firing rate of GI-neurons. Nesfatin-1 inhibited 88.9% (16/18) of gastric distension inhibitory (GD-INH) neurons and excited 76.2% (32/42) of gastric distension excitatory (GD-EXC) neurons. Thus, nesfatin-1 may control food intake by modulating the excitability of glucosensing neurons in the DVC.

Leptin acts via neuronal leptin receptors to control energy balance. Hypothalamic pro-opiomelanocortin (POMC) and agouti-related peptide (AgRP)/Neuropeptide Y (NPY)/GABA neurons produce anorexigenic and orexigenic neuropeptides and neurotransmitters, and express the long signaling form of the leptin receptor (LepRb). Despite progress in the understanding of LepRb signaling and function, the sub-cellular localization of LepRb in target neurons has not been determined, primarily due to lack of sensitive anti-LepRb antibodies. Here we applied light microscopy (LM), confocal-laser scanning microscopy (CLSM), and electron microscopy (EM) to investigate LepRb localization and signaling in mice expressing a HA-tagged LepRb selectively in POMC or AgRP/NPY/GABA neurons. We report that LepRb receptors exhibit a somato-dendritic expression pattern. We further show that LepRb activates STAT3 phosphorylation in neuronal fibers within several hypothalamic and hindbrain nuclei of wild-type mice and rats, and specifically in dendrites of arcuate POMC and AgRP/NPY/GABA neurons of Leprb (+/+) mice and in Leprb (db/db) mice expressing HA-LepRb in a neuron specific manner. We did not find evidence of LepRb localization or STAT3-signaling in axon-fibers or nerve-terminals of POMC and AgRP/NPY/GABA neurons. Three-dimensional serial EM-reconstruction of dendritic segments from POMC and AgRP/NPY/GABA neurons indicates a high density of shaft synapses. In addition, we found that the leptin activates STAT3 signaling in proximity to synapses on POMC and AgRP/NPY/GABA dendritic shafts. Taken together, these data suggest that the signaling-form of the leptin receptor exhibits a somato-dendritic expression pattern in POMC and AgRP/NPY/GABA neurons. Dendritic LepRb signaling may therefore play an important role in leptin's central effects on energy balance, possibly through modulation of synaptic activity via post-synaptic mechanisms.

Neurons synthesizing neurokinin B (NKB) and kisspeptin (KP) in the hypothalamic arcuate nucleus represent important upstream regulators of pulsatile gonadotropin-releasing hormone (GnRH) neurosecretion. In search of neuropeptides co-expressed in analogous neurons of the human infundibular nucleus (Inf), we have carried out immunohistochemical studies of the tachykinin peptide Substance P (SP) in autopsy samples from men (21-78 years) and postmenopausal (53-83 years) women. Significantly higher numbers of SP-immunoreactive (IR) neurons and darker labeling were observed in the Inf of postmenopausal women than in age-matched men. Triple-immunofluorescent studies localized SP immunoreactivity to considerable subsets of KP-IR and NKB-IR axons and perikarya in the infundibular region. In postmenopausal women, 25.1% of NKB-IR and 30.6% of KP-IR perikarya contained SP and 16.5% of all immunolabeled cell bodies were triple-labeled. Triple-, double- and single-labeled SP-IR axons innervated densely the portal capillaries of the infundibular stalk. In quadruple-labeled sections, these axons formed occasional contacts with GnRH-IR axons. Presence of SP in NKB and KP neurons increases the functional complexity of the putative pulse generator network. First, it is possible that SP modulates the effects of KP and NKB in axo-somatic and axo-dendritic afferents to GnRH neurons. Intrinsic SP may also affect the activity and/or neuropeptide release of NKB and KP neurons via autocrine/paracrine actions. In the infundibular stalk, SP may influence the KP and NKB secretory output via additional autocrine/paracrine mechanisms or regulate GnRH neurosecretion directly. Finally, possible co-release of SP with KP and NKB into the portal circulation could underlie further actions on adenohypophysial gonadotrophs.

The dorsomedial nucleus of the hypothalamus (DMH) contributes to the regulation of overall energy homeostasis by modulating energy intake as well as energy expenditure. Despite the importance of the DMH in the control of energy balance, DMH-specific genetic markers or neuronal subtypes are poorly defined. Here we demonstrate the presence of cholinergic neurons in the DMH using genetically modified mice that express enhanced green florescent protein (eGFP) selectively in choline acetyltransferase (Chat)-neurons. Overnight food deprivation increases the activity of DMH cholinergic neurons, as shown by induction of fos protein and a significant shift in the baseline resting membrane potential. DMH cholinergic neurons receive both glutamatergic and GABAergic synaptic input, but the activation of these neurons by an overnight fast is due entirely to decreased inhibitory tone. The decreased inhibition is associated with decreased frequency and amplitude of GABAergic synaptic currents in the cholinergic DMH neurons, while glutamatergic synaptic transmission is not altered. As neither the frequency nor amplitude of miniature GABAergic or glutamatergic postsynaptic currents is affected by overnight food deprivation, the fasting-induced decrease in inhibitory tone to cholinergic neurons is dependent on superthreshold activity of GABAergic inputs. This study reveals that cholinergic neurons in the DMH readily sense the availability of nutrients and respond to overnight fasting via decreased GABAergic inhibitory tone. As such, altered synaptic as well as neuronal activity of DMH cholinergic neurons may play a critical role in the regulation of overall energy homeostasis.

The CacyBP/SIP target S100A6 is widely present in the nervous system, and its up-regulation is associated with certain neurodegenerative diseases. Here, we examined the involvement of S100A6 protein in stress responses in mice. Using Western blotting, we observed a marked change in brainstem structures, whereby stressed mice showed approximately one-third the protein level produced in the control group. A decreased level of S100A6 protein in stressed animals was also detected in the olfactory bulb and the cerebellum and stress-related structures such as the hippocampus and the hypothalamus. Additionally, using immunohistochemistry, high levels of S100A6 expression were observed in astrocytes localized in the border zones of all brain ventricles, tanycytes of the ventro-lateral walls of the hypothalamus, including the arcuate nucleus (ARH) and low levels of this protein were in neurons of the olfactory bulb, the hippocampus, the thalamus, the cerebral cortex, the brainstem and the cerebellum. Although S100A6-expressing cells in all these brain structures did not change their phenotype in response to stress, the intensity of immunofluorescent labeling in all studied structures was lower in stressed mice than in control animals. For example, in the ARH, where extremely strong immunostaining was observed, the number of immunolabeled fibers was decreased by approximately half in the stressed group compared with the controls. Although these results are descriptive and do not give clue about functional role of S100A6 in stress, they indicate that the level of S100A6 decreases in several brain structures in response to chronic mild stress, suggesting that this protein may modify stress responses.

Exposure of females to a male pheromone accelerates pulsatile gonadotropin-releasing hormone (GnRH) secretion in goats. Recent evidence has suggested that neurons in the arcuate nucleus (ARC) containing kisspeptin and neurokinin B (NKB) play a pivotal role in the control of GnRH secretion. Therefore, we hypothesized that these neurons may be the central target of the male pheromone. To test this hypothesis, we examined whether NKB signaling is involved in the pheromone action, and whether ARC kisspeptin/NKB neurons receive input from the medial nucleus of the amygdala (MeA)--the nucleus suggested to relay pheromone signals. Ovariectomized goats were implanted with a recording electrode aimed at a population of ARC kisspeptin/NKB neurons, and GnRH pulse generator activity, represented by characteristic increases in multiple-unit activity (MUA) volleys, was measured. Pheromone exposure induced an MUA volley and luteinizing hormone (LH) pulse in control animals, whereas the MUA and LH responses to the pheromone were completely suppressed by the treatment with an NKB receptor antagonist. These results indicate that NKB signaling is a prerequisite for pheromone action. In ovariectomized goats, an anterograde tracer was injected into the MeA, and possible connections between the MeA and ARC kisspeptin/NKB neurons were examined. Histochemical observations demonstrated that a subset of ARC kisspeptin/NKB neurons receive efferent projections from the MeA. These results suggest that the male pheromone signal is conveyed via the MeA to ARC kisspeptin neurons, wherein the signal stimulates GnRH pulse generator activity through an NKB signaling-mediated mechanism in goats.