Maternal and offspring pools of osteocalcin influence brain development and functions.

The powerful regulation of bone mass exerted by the brain suggests the existence of bone-derived signals modulating this regulation or other functions of the brain. We show here that the osteoblast-derived hormone osteocalcin crosses the blood-brain barrier, binds to neurons of the brainstem, midbrain, and hippocampus, enhances the synthesis of monoamine neurotransmitters, inhibits GABA synthesis, prevents anxiety and depression, and favors learning and memory independently of its metabolic functions. In addition to these postnatal functions, maternal osteocalcin crosses the placenta during pregnancy and prevents neuronal apoptosis before embryos synthesize this hormone. As a result, the severity of the neuroanatomical defects and learning and memory deficits of Osteocalcin(-/-) mice is determined by the maternal genotype, and delivering osteocalcin to pregnant Osteocalcin(-/-) mothers rescues these abnormalities in their Osteocalcin(-/-) progeny. This study reveals that the skeleton via osteocalcin influences cognition and contributes to the maternal influence on fetal brain development.

FoxO1 target Gpr17 activates AgRP neurons to regulate food intake.

Hypothalamic neurons expressing Agouti-related peptide (AgRP) are critical for initiating food intake, but druggable biochemical pathways that control this response remain elusive. Thus, genetic ablation of insulin or leptin signaling in AgRP neurons is predicted to reduce satiety but fails to do so. FoxO1 is a shared mediator of both pathways, and its inhibition is required to induce satiety. Accordingly, FoxO1 ablation in AgRP neurons of mice results in reduced food intake, leanness, improved glucose homeostasis, and increased sensitivity to insulin and leptin. Expression profiling of flow-sorted FoxO1-deficient AgRP neurons identifies G-protein-coupled receptor Gpr17 as a FoxO1 target whose expression is regulated by nutritional status. Intracerebroventricular injection of Gpr17 agonists induces food intake, whereas Gpr17 antagonist cangrelor curtails it. These effects are absent in Agrp-Foxo1 knockouts, suggesting that pharmacological modulation of this pathway has therapeutic potential to treat obesity.

Peripheral oxytocin activates vagal afferent neurons to suppress feeding in normal and leptin-resistant mice: a route for ameliorating hyperphagia and obesity.

Oxytocin (Oxt), a neuropeptide produced in the hypothalamus, is implicated in regulation of feeding. Recent studies have shown that peripheral administration of Oxt suppresses feeding and, when infused subchronically, ameliorates hyperphagic obesity. However, the route through which peripheral Oxt informs the brain is obscure. This study aimed to explore whether vagal afferents mediate the sensing and anorexigenic effect of peripherally injected Oxt in mice. Intraperitoneal Oxt injection suppressed food intake and increased c-Fos expression in nucleus tractus solitarius to which vagal afferents project. The Oxt-induced feeding suppression and c-Fos expression in nucleus tractus solitarius were blunted in mice whose vagal afferent nerves were blocked by subdiaphragmatic vagotomy or capsaicin treatment. Oxt induced membrane depolarization and increases in cytosolic Ca(2+) concentration ([Ca(2+)]i) in single vagal afferent neurons. The Oxt-induced [Ca(2+)]i increases were markedly suppressed by Oxt receptor antagonist. These Oxt-responsive neurons also responded to cholecystokinin-8 and contained cocaine- and amphetamine-regulated transcript. In obese diabetic db/db mice, leptin failed to increase, but Oxt increased [Ca(2+)]i in vagal afferent neurons, and single or subchronic infusion of Oxt decreased food intake and body weight gain. These results demonstrate that peripheral Oxt injection suppresses food intake by activating vagal afferent neurons and thereby ameliorates obesity in leptin-resistant db/db mice. The peripheral Oxt-regulated vagal afferent neuron provides a novel target for treating hyperphagia and obesity.

Endogenous GLP-1 acts on paraventricular nucleus to suppress feeding: projection from nucleus tractus solitarius and activation of corticotropin-releasing hormone, nesfatin-1 and oxytocin neurons.

Glucagon-like peptide-1 (GLP-1) receptor agonists have been used to treat type 2 diabetic patients and shown to reduce food intake and body weight. The anorexigenic effects of GLP-1 and GLP-1 receptor agonists are thought to be mediated primarily via the hypothalamic paraventricular nucleus (PVN). GLP-1, an intestinal hormone, is also localized in the nucleus tractus solitarius (NTS) of the brain stem. However, the role of endogenous GLP-1, particularly that in the NTS neurons, in feeding regulation remains to be established. The present study examined whether the NTS GLP-1 neurons project to PVN and whether the endogenous GLP-1 acts on PVN to restrict feeding. Intra-PVN injection of GLP-1 receptor antagonist exendin (9-39) increased food intake. Injection of retrograde tracer into PVN combined with immunohistochemistry for GLP-1 in NTS revealed direct projection of NTS GLP-1 neurons to PVN. Moreover, GLP-1 evoked Ca(2+) signaling in single neurons isolated from PVN. The majority of GLP-1-responsive neurons were immunoreactive predominantly to corticotropin-releasing hormone (CRH) and nesfatin-1, and less frequently to oxytocin. These results indicate that endogenous GLP-1 targets PVN to restrict feeding behavior, in which the projection from NTS GLP-1 neurons and activation of CRH and nesfatin-1 neurons might be implicated. This study reveals a neuronal basis for the anorexigenic effect of endogenous GLP-1 in the brain.

Prolyl endopeptidase-deficient mice have reduced synaptic spine density in the CA1 region of the hippocampus, impaired LTP, and spatial learning and memory.

Prolyl endopeptidase (PREP) is a phylogenetically conserved serine protease and, in humans and rodents, is highly expressed in the brain. Several neuropeptides associated with learning and memory and neurodegenerative disorders have been proposed to be the substrates for PREP, suggesting a possible role for PREP in these processes. However, its physiological function remains elusive. Combining genetic, anatomical, electrophysiological, and behavioral approaches, we show that PREP genetrap mice have decreased synaptic spine density in the CA1 region of the hippocampus, reduced hippocampal long-term potentiation, impaired hippocampal-mediated learning and memory, and reduced growth-associated protein-43 levels when compared with wild-type controls. These observations reveal a role for PREP in mediating hippocampal plasticity and spatial memory formation, with implications for its pharmacological manipulation in diseases related to cognitive impairment.

Repeated in vivo exposure of cocaine induces long-lasting synaptic plasticity in hypocretin/orexin-producing neurons in the lateral hypothalamus in mice.

Hypocretin (orexin), a neuropeptide synthesized exclusively in the perifornical/lateral hypothalamus, is critical for drug seeking and relapse, but it is not clear how the circuitry centred on hypocretin-producing neurons (hypocretin neurons) is modified by drugs of abuse and how changes in this circuit might alter behaviours related to drug addiction. In this study, we show that repeated, but not single, in vivo cocaine administration leads to a long-lasting, experience-dependent potentiation of glutamatergic synapses on hypocretin neurons in mice following a cocaine-conditioned place preference (CPP) protocol. The synaptic potentiation occurs postsynaptically and probably involves up-regulation of AMPA-type glutamate receptors on hypocretin neurons. Phosphorylation of cAMP response element-binding protein (CREB) is also significantly increased in hypocretin neurons in cocaine-treated animals, suggesting that CREB-mediated pathways may contribute to synaptic potentiation in these cells. Furthermore, the potentiation of synaptic efficacy in hypocretin neurons persists during cocaine withdrawal, but reverses to baseline levels after prolonged abstinence. Finally, the induction of long-term potentiation (LTP) triggered by a high-frequency stimulation is facilitated in hypocretin neurons in cocaine-treated mice, suggesting that long-lasting changes in synapses onto hypocretin neurons would probably be further potentiated by other stimuli (such as concurrent environmental cues) paired with the drug. In summary, we show here that hypocretin neurons undergo experience-dependent synaptic potentiation that is distinct from that reported in other reward systems, such as the ventral tegmental area, following exposure to cocaine. These findings support the idea that the hypocretin system is important for behavioural changes associated with cocaine administration in animals and humans.

Fasting inhibits excitatory synaptic input on paraventricular oxytocin neurons via neuropeptide Y and Y1 receptor, inducing rebound hyperphagia, and weight gain.

Fasting with varying intensities is used to treat obesity-related diseases. Re-feeding after fasting exhibits hyperphagia and often rebound weight gain. However, the mechanisms underlying the hyperphagia and rebound remain elusive. Here we show that 24 h food restriction (24 h FR) and milder 50% FR, both depress synaptic transmission in the hypothalamic paraventricular nucleus (PVN) and induce acute hyperphagia in rats. 24 h FR is followed by weight rebound but 50% FR is not. Orexigenic neuropeptide Y (NPY) via the Y1 receptor (Y1R) inhibited the miniature excitatory postsynaptic current (mEPSC) on anorexigenic oxytocin neurons in the PVN. 24 h FR and 50% FR activated this neuronal pathway to induce acute hyperphagia on Days 1-3 and Days 1-2 after FR, respectively. 24 h FR induced large mEPSC depression, recurrent hyperphagia on Days 9-12 and rebound weight gain on Days 12-17, whereas 50% FR induced moderate mEPSC depression and sustained weight reduction. Transverse data analysis on Day 1 after 24 h FR and 50% FR demonstrated saturation kinetics for the mEPSC depression-hyperphagiacurve, implying hysteresis. The results reveal FR-driven synaptic plasticity in the NPY-Y1R-oxytocin neurocircuit that drives acute hyperphagia. FR with the intensity that regulates the synapse-feeding relay without hysteresis is the key for successful dieting.

Intracellular energy status regulates activity in hypocretin/orexin neurones: a link between energy and behavioural states.

The hypocretin/orexin (Hcrt)-containing neurones within the lateral hypothalamus integrate nutritional, energetic and behavioural cues to generate the final output in order to exert their functions. It is still not clear how Hcrt neurones monitor changes in energy status in animals. In brain slices from transgenic mice expressing green fluorescent protein (GFP) exclusively in Hcrt neurones, we examined the roles of intracellular levels of ATP ([ATP](i)) in regulating activities in these cells with conventional and perforated whole-cell recording. By using 'ATP clamp' we demonstrated that membrane potential (V(m)) correlated with the [ATP](i) in Hcrt neurones. Perforated recording revealed a V(m) of -46.1 ± 1.6 mV (n = 18), close to the level measured with an [ATP](i) equal to 5-6 mm (-48.7 ± 1.4 mV, n = 16, 5 mm ATP), suggesting that a unique demand for energy is required to maintain normal functionality in Hcrt cells. A direct disruption of ATP production or reduction in ambient glucose levels resulted in an inhibition of activity in Hcrt neurones. The V(m) was significantly depolarized in Hcrt neurones in sleep-deprived mice as compared with controls (P < 0.01, t test), which was eliminated by experimental manipulations causing the same level of [ATP](i) and K(ATP) channel opening in both groups, suggesting a decrease during sleep and an increase during sustained wakefulness in [ATP](i) in Hcrt cells. In summary, these data demonstrate that a delicate control of activity by monitoring the availability of intracellular energy stores in Hcrt cells may serve as a novel mechanism regulating energy expenditure and behavioural state dependent upon the energy state in animals.

[Roles and functional interplay of the gut, brain stem, hypothalamus and limbic system in regulation of feeding].

The brain controls feeding via two components; homeostatic and accessory regulation. Homeostatic regulation is executed by the hypothalamic centers. Accessory regulation occurs in response to environmental conditions and stimuli such as memory, stress, emotion, reward and hedonic feeling, which are operated by the limbic system, particularly the hippocampus and amygdala. Nutrients and visceral hormones, representing peripheral metabolic states, regulate activity of these brain areas, which is performed by direct action after entering through blood-brain area and by sending information via the vagus nerve and brain stem. We here review how the hippocampus and amygdala as well as gut, brain stem and hypothalamus function and interact with each other to achieve integrative regulation of feeding.

Leptin transiently antagonizes ghrelin and long-lastingly orexin in regulation of Ca2+ signaling in neuropeptide Y neurons of the arcuate nucleus.

AIM: To explore the mechanism for interactions of leptin with ghrelin and orexin in the arcuate nucleus (ARC) activating neuropeptide Y (NPY) neurons during physiological regulation of feeding. METHODS: Single neurons from ARC of adult rats with matured feeding function were isolated. [Ca2+]i was measured to monitor their activities. The time course of leptin effects on ghrelin-induced versus orexin-induced [Ca2+]i increases in NPY neurons was studied. RESULTS: Administration of ghrelin or orexin-A at 10(-10) mol/L increased cytosolic Ca2+ concentration ([Ca2+]i) in NPY neurons isolated from the ARC of adult rats. Upon administration of leptin at 10(-14)-10(-12) mol/L, ghrelin-induced [Ca2+]i increases were initially (<10 min) inhibited but later restored, exhibiting a transient pattern of inhibition. In contrast, orexin-induced [Ca2+]i increases were inhibited by leptin in a long-lasting manner. Furthermore, a prior administration of leptin inhibited orexin action but not ghrelin action to increase [Ca2+]i. CONCLUSION: Leptin counteracted ghrelin effects transiently and orexin effects long-lastingly in NPY neurons. The transient property with which leptin counteracts ghrelin action in NPY neurons may allow the fasting-associated increase in ghrelin levels to activate NPY neurons in the presence of physiological leptin and to stimulate feeding.

New insight into GABAergic neurons in the hypothalamic feeding regulation.

Several lines of study have suggested that GABA in the hypothalamic feeding center plays a role in promoting food intake. Recent studies revealed that not only NPY/AgRP neurons in the hypothalamic arcuate nucleus (ARC) that co-express GABA but also other GABAergic neurons act as an orexigenic. Here, we review the progress of studies on hypothalamic GABAergic neurons distributed in ARC, dorsomedial hypothalamus (DMH), and lateral hypothalamus (LH). Three advanced technologies have been applied and greatly contributed to the recent progress. Optogenetic (and chemogenetic) approaches map input and output pathways of particular subpopulations of GABAergic neurons. In vivo Ca2+ imaging using GRIN lens and GCaMP can correlate the activity of GABAergic neuron subpopulations with feeding behavior. Single-cell RNA-seq approach clarifies precise transcriptional profiles of GABAergic neuron subpopulations. These approaches have shown diversity of GABAergic neurons and the subpopulation-dependent role in feeding regulation.

Synaptic vesicle dynamics in the mossy fiber-CA3 presynaptic terminals of mouse hippocampus.

The mossy fiber (MF)-CA3 synapse in the hippocampus is unique in the CNS because of its wide dynamic range of transmitter release during short- and long-term plasticity. The presynaptic mechanisms underlying the fidelity of transmission were investigated for the MF-CA3 synapses. The relative size of readily releasable pool (RRP) of vesicles was estimated by counting the number of docked vesicles at an active zone (AZ) on the transmission electron microscopy (TEM) image. The size of the releasable pool and the exo-endocytosis kinetics were directly measured from individual large MF boutons in hippocampal slices of transgenic mice that selectively express synaptopHluorin (SpH), a pH-sensitive GFP fused to the lumenal aspect of one of the vesicular membrane proteins, VAMP-2, in these boutons. Here we found (1) there are distinct two vesicle pools, the resting pool which is resistant to exocytosis, and the releasable pool, (2) the initially docked vesicles are easily depleted and the RRP is maintained by refilling from the reserve subpopulation of releasable pool ("reserve" releasable pool), and (3) the contribution of rapid reuse of recycled vesicles is relatively small. Therefore, the fidelity of transmission is suggested to be ensured by the rapid refilling rate of RRP.

Adiponectin at physiological level glucose-independently enhances inhibitory postsynaptic current onto NPY neurons in the hypothalamic arcuate nucleus.

Adiponectin regulates glucose and lipid metabolism, acting against atherosclerosis and metabolic syndrome. Accumulating evidences suggest that adiponectin acts on the brain including the arcuate nucleus of hypothalamus (ARC). The ARC contains orexigenic neuropeptide Y (NPY)/agouti related peptide (AgRP) neurons and anorexigenic proopiomelanocortin (POMC) neurons, the first order neurons for feeding regulation. We recently reported that intracerebroventricular injection of adiponectin at low glucose level suppressed food intake, while at elevated glucose level it promoted food intake, exhibiting glucose-dependent reciprocal effects. As an underlying neuronal mechanism, physiological level of adiponectin at low glucose activated ARC POMC neurons and at high glucose inactivated them. Now, whether physiological level of adiponectin also affects NPY/AgRP neurons is essential for fully understanding the adiponectin action, but it remains to be clarified. We here report that a physiological dose of adiponectin, in both high and low glucose conditions, attenuated action potential firing without altering resting membrane potential in ARC NPY neurons. This adiponectin effect was abolished by GABAA receptor blockade. Adiponectin enhanced amplitude but not frequency of inhibitory postsynaptic current (IPSC) onto NPY neurons. These results demonstrate that adiponectin enhances IPSC onto NPY neurons to attenuate action potential firing in NPY neurons in a glucose-independent manner, being contrasted to its glucose-dependent effect on POMC neurons.

Plasticity of calcium-permeable AMPA glutamate receptors in Pro-opiomelanocortin neurons.

POMC neurons integrate metabolic signals from the periphery. Here, we show in mice that food deprivation induces a linear current-voltage relationship of AMPAR-mediated excitatory postsynaptic currents (EPSCs) in POMC neurons. Inhibition of EPSCs by IEM-1460, an antagonist of calcium-permeable (Cp) AMPARs, diminished EPSC amplitude in the fed but not in the fasted state, suggesting entry of GluR2 subunits into the AMPA receptor complex during food deprivation. Accordingly, removal of extracellular calcium from ACSF decreased the amplitude of mEPSCs in the fed but not the fasted state. Ten days of high-fat diet exposure, which was accompanied by elevated leptin levels and increased POMC neuronal activity, resulted in increased expression of Cp-AMPARs on POMC neurons. Altogether, our results show that entry of calcium via Cp-AMPARs is inherent to activation of POMC neurons, which may underlie a vulnerability of these neurons to calcium overload while activated in a sustained manner during over-nutrition.

Endothelial HIF-1α Enables Hypothalamic Glucose Uptake to Drive POMC Neurons.

Glucose is the primary driver of hypothalamic proopiomelanocortin (POMC) neurons. We show that endothelial hypoxia-inducible factor 1α (HIF-1α) controls glucose uptake in the hypothalamus and that it is upregulated in conditions of undernourishment, during which POMC neuronal activity is decreased. Endothelium-specific knockdown of HIF-1α impairs the ability of POMC neurons to adapt to the changing metabolic environment in vivo, resulting in overeating after food deprivation in mice. The impaired functioning of POMC neurons was reversed ex vivo or by parenchymal glucose administration. These observations indicate an active role for endothelial cells in the central control of metabolism and suggest that central vascular impairments may cause metabolic disorders.

Optogenetic activation of leptin- and glucose-regulated GABAergic neurons in dorsomedial hypothalamus promotes food intake via inhibitory synaptic transmission to paraventricular nucleus of hypothalamus.

OBJECTIVE: The dorsomedial hypothalamus (DMH) has been considered an orexigenic nucleus, since the DMH lesion reduced food intake and body weight and induced resistance to diet-induced obesity. The DMH expresses feeding regulatory neuropeptides and receptors including neuropeptide Y (NPY), cocaine- and amphetamine-regulated transcript (CART), cholecystokinin (CCK), leptin receptor, and melanocortin 3/4 receptors. However, the principal neurons generating the orexigenic function in the DMH remain to be defined. This study aimed to clarify the role of the DMH GABAergic neurons in feeding regulation by using optogenetics and electrophysiological techniques. METHODS: We generated the mice expressing ChRFR-C167A, a bistable chimeric channelrhodopsin, selectively in GABAergic neurons of DMH via locally injected adeno-associated virus 2. Food intake after optogenetic activation of DMH GABAergic neurons was measured. Electrophysiological properties of DMH GABAergic neurons were measured using slice patch clamp. RESULTS: Optogenetic activation of DMH GABAergic neurons promoted food intake. Leptin hyperpolarized and lowering glucose depolarized half of DMH GABAergic neurons, suggesting their orexigenic property. Optical activation of axonal terminals of DMH GABAergic neurons at the paraventricular nucleus of hypothalamus (PVN), where anorexigenic neurons are localized, increased inhibitory postsynaptic currents on PVN neurons and promoted food intake. CONCLUSION: DMH GABAergic neurons are regulated by metabolic signals leptin and glucose and, once activated, promote food intake via inhibitory synaptic transmission to PVN.

Glucose level determines excitatory or inhibitory effects of adiponectin on arcuate POMC neuron activity and feeding.

Adiponectin regulates glucose and lipid metabolism, acting against metabolic syndrome and atherosclerosis. Accumulating evidence suggest that adiponectin acts on the brain including hypothalamic arcuate nucleus (ARC), where proopiomelanocortin (POMC) neurons play key roles in feeding regulation. Several studies have examined intracerebroventricular (ICV) injection of adiponectin and reported opposite effects, increase or decrease of food intake. These reports used different nutritional states. The present study aimed to clarify whether adiponectin exerts distinct effects on food intake and ARC POMC neurons depending on the glucose concentration. Adiponectin was ICV injected with or without glucose for feeding experiments and administered to ARC slices with high or low glucose for patch clamp experiments. We found that adiponectin at high glucose inhibited POMC neurons and increased food intake while at low glucose it exerted opposite effects. The results demonstrate that glucose level determines excitatory or inhibitory effects of adiponectin on arcuate POMC neuron activity and feeding.

Fasted/fed states regulate postsynaptic hub protein DYNLL2 and glutamatergic transmission in oxytocin neurons in the hypothalamic paraventricular nucleus.

The neurons in the hypothalamus regulate food intake and energy metabolism on reception of systemic energy states. Accumulating evidences have indicated that synaptic transmission on the hypothalamic neurons is modulated by the metabolic condition related to fasted/fed states, and that this modulation of synaptic plasticity plays a role in regulation of feeding. It has been shown that oxytocin (Oxt) neurons in the paraventricular nucleus (PVN) of the hypothalamus sense and integrate various peripheral and central signals and thereby induce satiety. However, whether metabolic conditions regulate the synaptic transmission on Oxt neurons in PVN remains unclear. The present study examined whether the fasted/fed states regulate synaptic transmission on Oxt neurons in PVN. The miniature excitatory postsynaptic currents (mEPSCs) onto Oxt neurons in PVN were increased under ad lib fed condition compared to 24h fasted condition. Furthermore, the NMDA receptor-mediated EPSC on Oxt neurons was increased under fed, compared to fasted, condition. In Oxt neurons, dynein light chain 2 (DYNLL2), a protein suggested to be implicated in the NMDA receptor trafficking to the postsynaptic site, was increased under fed, compared to fasted, condition. The present results suggest that feeding increases excitatory synaptic input on PVN Oxt neurons via mechanisms involving DYNLL2 upregulation and NMDA receptor-mediated synaptic reorganization.

Leptin signaling in astrocytes regulates hypothalamic neuronal circuits and feeding.

We found that leptin receptors were expressed in hypothalamic astrocytes and that their conditional deletion led to altered glial morphology and synaptic inputs onto hypothalamic neurons involved in feeding control. Leptin-regulated feeding was diminished, whereas feeding after fasting or ghrelin administration was elevated in mice with astrocyte-specific leptin receptor deficiency. These data reveal an active role of glial cells in hypothalamic synaptic remodeling and control of feeding by leptin.

Automated test of behavioral flexibility in mice using a behavioral sequencing task in IntelliCage.

There has been a long-standing need to develop efficient and standardized behavioral test methods for evaluating higher-order brain functions in mice. Here, we developed and validated a behavioral flexibility test in mice using IntelliCage, a fully automated behavioral analysis system for mice in a group-housed environment. We first developed a "behavioral sequencing task" in the IntelliCage that enables us to assess the learning ability of place discrimination and behavioral sequence for reward acquisition. In the serial reversal learning using the task, the discriminated spatial patterns of the rewarded and never-rewarded places were serially reversed, and thus, mice were accordingly expected to realign the previously acquired behavioral sequence. In general, the tested mice showed rapid acquisition of the behavioral sequencing task and behavioral flexibility in the subsequent serial reversal stages both in intra- and inter-session analyses. It was found that essentially the same results were obtained among three different laboratories, which confirm the high stability of the present test protocol in different strains of mice (C57BL/6, DBA/2, and ICR). In particular, the most trained cohort of C57BL/6 mice achieved a markedly rapid adaptation to the reversal task in the final phase of the long-term serial reversal test, which possibly indicated that the mice adapted to the "reversal rule" itself. In conclusion, the newly developed behavioral test was shown to be a valid assay of behavioral flexibility in mice, and is expected to be utilized in tests of mouse models of cognitive deficits.