GluN3A and excitatory glycine receptors in the adult hippocampus.

The GluN3A subunit of N-methyl-D-aspartate receptors (NMDARs) plays an established role in synapse development, but its contribution to neural circuits in the adult brain is less clear. Recent work has demonstrated that in select cell populations, GluN3A assembles with GluN1 to form GluN1/GluN3A receptors that are insensitive to glutamate and instead serve as functional excitatory glycine receptors (eGlyRs). Our understanding of these eGlyRs, and how they contribute to intrinsic excitability and synaptic communication within relevant networks of the developing and the mature brain, is only beginning to be uncovered. Here, using male and female mice, we demonstrate that GluN3A subunits are enriched in the adult ventral hippocampus (VH), where they localize to synaptic and extrasynaptic sites and can assemble as functional eGlyRs on CA1 pyramidal cells. GluN3A expression was barely detectable in the adult dorsal hippocampus (DH). We also observed a high GluN2B content in the adult VH, characterized by slow NMDAR current decay kinetics and a high sensitivity to the GluN2B-containing NMDAR antagonist ifenprodil. Interestingly, the GluN2B enrichment in the adult VH was dependent on GluN3A as GluN3A deletion accelerated NMDAR decay and reduced ifenprodil sensitivity in the VH, suggesting that GluN3A expression can regulate the balance of conventional NMDAR subunit composition at synaptic sites. Lastly, we found that GluN3A knockout also enhanced both NMDAR-dependent calcium influx and NMDAR-dependent long-term potentiation in the VH. Together, these data reveal a novel role for GluN3A and eGlyRs in the control of ventral hippocampal circuits in the mature brain.Significance statement Glutamate is the brain's most abundant excitatory neurotransmitter. One of its most common receptors is the NMDA receptor (NMDAR), composed of a variety of subunits. Here, we show that an atypical NMDAR subunit, GluN3A, is enriched in the ventral hippocampus, where it forms a receptor that is no longer sensitive to glutamate. Instead, it acts as an excitatory receptor for glycine, normally an inhibitory transmitter. We show that GluN3A plays essential roles in the adult ventral hippocampus, modulating transmitter release probability, conventional NMDAR subunit composition, calcium entry, and synaptic plasticity. These findings are important as they shed light on the role of GluN3A and these newly discovered excitatory glycine receptors in the mature brain.

Prostaglandin E2 activates melanin-concentrating hormone neurons to drive diet-induced obesity.

Hypothalamic inflammation reduces appetite and body weight during inflammatory diseases, while promoting weight gain when induced by high-fat diet (HFD). How hypothalamic inflammation can induce opposite energy balance outcomes remains unclear. We found that prostaglandin E2 (PGE2), a key hypothalamic inflammatory mediator of sickness, also mediates diet-induced obesity (DIO) by activating appetite-promoting melanin-concentrating hormone (MCH) neurons in the hypothalamus in rats and mice. The effect of PGE2 on MCH neurons is excitatory at low concentrations while inhibitory at high concentrations, indicating that these neurons can bidirectionally respond to varying levels of inflammation. During prolonged HFD, endogenous PGE2 depolarizes MCH neurons through an EP2 receptor-mediated inhibition of the electrogenic Na+/K+-ATPase. Disrupting this mechanism by genetic deletion of EP2 receptors on MCH neurons is protective against DIO and liver steatosis in male and female mice. Thus, an inflammatory mediator can directly stimulate appetite-promoting neurons to exacerbate DIO and fatty liver.

High-fat diet-induced elevation of body weight set point in male mice.

OBJECTIVE: High-fat diets (HFD) are thought to disrupt energy homeostasis to drive overeating and obesity. However, weight loss resistance in individuals with obesity suggests that homeostasis is intact. This study aimed to reconcile this difference by systematically assessing body weight (BW) regulation under HFD. METHODS: Male C57BL/6 N mice were fed diets with varying fat and sugar in different durations and patterns. BW and food intake were monitored. RESULTS: BW gain was transiently accelerated by HFD (≥40%) prior to plateauing. The plateau was consistent regardless of starting age, HFD duration, or fat/sugar content. Reverting to a low-fat diet (LFD) caused transiently accelerated weight loss, which correlated with how heavy mice were before the diet relative to LFD-only controls. Chronic HFD attenuated the efficacy of single or repetitive dieting, revealing a defended BW higher than that of LFD-only controls. CONCLUSIONS: This study suggests that dietary fat modulates the BW set point immediately upon switching from LFD to HFD. Mice defend a new elevated set point by increasing caloric intake and efficiency. This response is consistent and controlled, suggesting that hedonic mechanisms contribute to rather than disrupt energy homeostasis. An elevated floor of the BW set point after chronic HFD could explain weight loss resistance in individuals with obesity.

Short-term Fasting Induces Alternate Activation of Orexin and Melanin-concentrating Hormone Neurons in Rats.

Orexin and melanin-concentrating hormone (MCH) neurons constitute the energy balance circuitry that coordinates the fasting response. Orexin neurons mediate food foraging at the expense of energy storage, while MCH neurons promote energy storage by reducing energy expenditure and increasing food intake. It is unknown if these cell groups undergo plastic changes as hunger and metabolic changes escalate over time during fasting. To address this, we performed in vitro electrophysiological recording on orexin and MCH neurons in the lateral hypothalamus and perifornical area from rats fasted for 12 or 24 h or fed ad-libitum. Orexin neurons showed a transient decrease in presynaptic glutamate release at 12 h. This turned to an increase at 24 h of fasting, while membrane potential depolarized and AMPA receptor conductance increased. In contrast, MCH neurons were transiently depolarized at 12 h fasting along with increased presynaptic glutamate release. These changes reversed at 24 h, while the number of AMPA receptors decreased. Our results indicate that MCH neurons are preferentially activated during the early phase of fasting (12 h), which would protect against weight loss. With a longer fast, orexin neurons become activated, which would promote arousal and exploratory activity required for foraging behaviors. This alternating activation of these cell groups may reflect a dynamic balance of energy conservation and foraging behaviors to optimize energy balance during ongoing fasting.

High-fat diet induces time-dependent synaptic plasticity of the lateral hypothalamus.

OBJECTIVE: Orexin (ORX) and melanin-concentrating hormone (MCH) neurons in the lateral hypothalamus are critical regulators of energy homeostasis and are thought to differentially contribute to diet-induced obesity. However, it is unclear whether the synaptic properties of these cells are altered by obesogenic diets over time. METHODS: Rats and mice were fed a control chow or palatable high-fat diet (HFD) for various durations and then synaptic properties of ORX and MCH neurons were examined using exvivo whole-cell patch clamp recording. Confocal imaging was performed to assess the number of excitatory synaptic contacts to these neurons. RESULTS: ORX neurons exhibited a transient increase in spontaneous excitatory transmission as early as 1 day up to 1 week of HFD, which returned to control levels with prolonged feeding. Conversely, HFD induced a delayed increase in excitatory synaptic transmission to MCH neurons, which progressively increased as HFD became chronic. This increase occurred before the onset of significant weight gain. These synaptic changes appeared to be due to altered postsynaptic sensitivity or the number of active synaptic contacts depending on cell type and feeding duration. However, HFD induced no change in inhibitory transmission in either cell type at any time point. CONCLUSIONS: These results suggest that the effects of HFD on feeding-related neurons are cell type-specific and dynamic. This highlights the importance of considering the feeding duration for research and weight loss interventions. ORX neurons may contribute to early hyperphagia, whereas MCH neurons may play a role in the onset and long-term maintenance of diet-induced obesity.

Prolonged High Fat Diet Worsens the Cellular Response to a Small, Covert-like Ischemic Stroke.

Obesity is associated with worse neurological outcomes following overt ischemic strokes. The majority of strokes however, are covert, small strokes that often evade detection. How obesity impacts the cellular response to covert strokes is unclear. Here, we used a diet-induced obesity model by feeding mice a high fat diet (HFD) and examining its impact on the behavioral and cellular responses to either an Endothelin-1-induced focal ischemic stroke or a saline injection (control). Specifically, we examined cells in regions with different levels of blood perfusion: the non-perfused core, the hypo-perfused surround and the perfused region around the infarct. We show that HFD selectively exacerbated the response to stroke but not to saline injections. Stroke affected the composition of microglia/macrophages, astrocytes and neurons within each region of perfusion. In the non-perfused core, the majority of cells were Iba-1+ microglia and macrophages. HFD resulted in a greater infiltration of CD68+ macrophages into the infarct core while CD68+ /TMEM119+ microglia were reduced. Furthermore, there was a trend towards an increased spread of the astrogliosis scar from the infarct border in the HFD condition. Within the hypo-perfused region, significantly fewer neurons survived in HFD-fed mice than Chow-fed mice, suggesting that neurons in the HFD condition have an increased vulnerability. In summary, diet-induced obesity exacerbates covert-like stroke injuries by worsening the cellular responses in the varying levels of perfusion across the infarct.

Dopamine modulates excitatory transmission to orexin neurons in a receptor subtype-specific manner.

Dopamine (DA) can promote or inhibit consummatory and reward-related behaviors by activating different receptor subtypes in the lateral hypothalamus and perifornical area (LH/PF). Because orexin neurons are involved in reward and localized in the LH/PF, DA may modulate these neurons to influence reward-related behaviors. To determine the cellular mechanism underlying dopaminergic modulation of orexin neurons, the effect of DA on excitatory transmission to these neurons was investigated using in vitro electrophysiology on rat brain slices. We found that low concentrations (0.1-1 µM) of DA increased evoked excitatory postsynaptic current amplitude while decreasing paired-pulse ratio. In contrast, high concentrations (10-100 µM) of DA did the opposite. The excitatory effect of low DA was blocked by the D1 receptor antagonist SCH-23390, whereas the inhibitory effect of high DA was blocked by the D2 receptor antagonist sulpiride. These results indicate distinct roles of D1 and D2 receptors in bidirectional presynaptic modulation of excitatory transmission. DA had stronger effects on isolated synaptic activity than repetitive ones, suggesting that sensitivity to dopaminergic modulation depends on the level of network activity. In orexin neurons from high-fat diet-fed rats, a high concentration of DA was less effective in suppressing repetitive synaptic activity compared with chow controls. Therefore, in diet-induced obesity, intense synaptic inputs may preferentially reach orexin neurons while intermittent signals are inhibited by high DA levels. In summary, our study provides a cellular mechanism by which DA may exert opposite behavioral effects in the LH/PF through bidirectional modulation of orexin neurons via different DA receptors.

Sleep deprivation-induced pre- and postsynaptic modulation of orexin neurons.

Sleep/wake states are controlled by sleep- and wake-promoting systems, and transitions between states are thought to be regulated by their reciprocal inhibition and homeostatic sleep need. Orexin neurons are known to promote wake maintenance and stabilize the sleep/wake switch. Thus, we asked whether orexin neurons are modulated by homeostatic sleep need. Rats were sleep deprived or left undisturbed to rest for 6 h, then acute brain slices were generated for patch clamp recordings. We found that sleep deprivation increased firing and reduced spike frequency adaptation in response to excitatory drive in orexin neurons. These changes were specific to D-type orexin neurons which, unlike H-type orexin neurons, lack A-type current. In D-type orexin neurons, sleep deprivation decreased afterhyperpolarizing potential, which was associated with increased gain, measured as the slope of the input-output relationship. These effects were mimicked by inhibition of SK channels. Furthermore, sleep deprivation resulted in presynaptic inhibition of excitatory inputs to both D-type and H-type orexin neurons, which preferentially affected sparse synaptic inputs while sparing high frequency synaptic activities. Taken together, our results indicate that sleep deprivation modulates the gain control and synaptic gating in orexin neurons. These pre-and postsynaptic changes would tune orexin neurons to strong wake-promoting excitatory signals, while dampening weak synaptic inputs to allow transition to sleep in the absence of such strong signals. These mechanisms are consistent with a role of orexin neurons not only as a key state stabilizer, but also as a homeostatic wake integrator in the sleep/wake switch. This article is part of the Special Issue entitled 'Hypothalamic Control of Homeostasis'.

Macrophage migration inhibitory factor mediates metabolic dysfunction induced by atypical antipsychotic therapy.

Atypical antipsychotics are highly effective antischizophrenic medications but their clinical utility is limited by adverse metabolic sequelae. We investigated whether upregulation of macrophage migration inhibitory factor (MIF) underlies the insulin resistance that develops during treatment with the most commonly prescribed atypical antipsychotic, olanzapine. Olanzapine monotherapy increased BMI and circulating insulin, triglyceride, and MIF concentrations in drug-naive schizophrenic patients with normal MIF expression, but not in genotypic low MIF expressers. Olanzapine administration to mice increased their food intake and hypothalamic MIF expression, which led to activation of the appetite-related AMP-activated protein kinase and Agouti-related protein pathway. Olanzapine also upregulated MIF expression in adipose tissue, which reduced lipolysis and increased lipogenic pathways. Increased plasma lipid concentrations were associated with abnormal fat deposition in liver and skeletal muscle, which are important determinants of insulin resistance. Global MIF-gene deletion protected mice from olanzapine-induced insulin resistance, as did intracerebroventricular injection of neutralizing anti-MIF antibody, supporting the role of increased hypothalamic MIF expression in metabolic dysfunction. These findings uphold the potential pharmacogenomic value of MIF genotype determination and suggest that MIF may be a tractable target for reducing the metabolic side effects of atypical antipsychotic therapy.

Electrophysiological Properties of Melanin-Concentrating Hormone and Orexin Neurons in Adolescent Rats.

Orexin and melanin-concentrating hormone (MCH) neurons have complementary roles in various physiological functions including energy balance and the sleep/wake cycle. in vitro electrophysiological studies investigating these cells typically use post-weaning rodents, corresponding to adolescence. However, it is unclear whether these neurons are functionally mature at this period and whether these studies can be generalized to adult cells. Therefore, we examined the electrophysiological properties of orexin and MCH neurons in brain slices from post-weaning rats and found that MCH neurons undergo an age-dependent reduction in excitability, but not orexin neurons. Specifically, MCH neurons displayed an age-dependent hyperpolarization of the resting membrane potential (RMP), depolarizing shift of the threshold, and decrease in excitatory transmission, which reach the adult level by 7 weeks of age. In contrast, basic properties of orexin neurons were stable from 4 weeks to 14 weeks of age. Furthermore, a robust short-term facilitation of excitatory synapses was found in MCH neurons, which showed age-dependent changes during the post-weaning period. On the other hand, a strong short-term depression was observed in orexin neurons, which was similar throughout the same period. These differences in synaptic responses and age dependence likely differentially affect the network activity within the lateral hypothalamus where these cells co-exist. In summary, our study suggests that orexin neurons are electrophysiologically mature before adolescence whereas MCH neurons continue to develop until late adolescence. These changes in MCH neurons may contribute to growth spurts or consolidation of adult sleep patterns associated with adolescence. Furthermore, these results highlight the importance of considering the age of animals in studies involving MCH neurons.

Sleep Deprivation Distinctly Alters Glutamate Transporter 1 Apposition and Excitatory Transmission to Orexin and MCH Neurons.

Glutamate transporter 1 (GLT1) is the main astrocytic transporter that shapes glutamatergic transmission in the brain. However, whether this transporter modulates sleep-wake regulatory neurons is unknown. Using quantitative immunohistochemical analysis, we assessed perisomatic GLT1 apposition with sleep-wake neurons in the male rat following 6 h sleep deprivation (SD) or following 6 h undisturbed conditions when animals were mostly asleep (Rest). We found that SD decreased perisomatic GLT1 apposition with wake-promoting orexin neurons in the lateral hypothalamus compared with Rest. Reduced GLT1 apposition was associated with tonic presynaptic inhibition of excitatory transmission to these neurons due to the activation of Group III metabotropic glutamate receptors, an effect mimicked by a GLT1 inhibitor in the Rest condition. In contrast, SD resulted in increased GLT1 apposition with sleep-promoting melanin-concentrating hormone (MCH) neurons in the lateral hypothalamus. Functionally, this decreased the postsynaptic response of MCH neurons to high-frequency synaptic activation without changing presynaptic glutamate release. The changes in GLT1 apposition with orexin and MCH neurons were reversed after 3 h of sleep opportunity following 6 h SD. These SD effects were specific to orexin and MCH neurons, as no change in GLT1 apposition was seen in basal forebrain cholinergic or parvalbumin-positive GABA neurons. Thus, within a single hypothalamic area, GLT1 differentially regulates excitatory transmission to wake- and sleep-promoting neurons depending on sleep history. These processes may constitute novel astrocyte-mediated homeostatic mechanisms controlling sleep-wake behavior.SIGNIFICANCE STATEMENT Sleep-wake cycles are regulated by the alternate activation of sleep- and wake-promoting neurons. Whether and how astrocytes can regulate this reciprocal neuronal activity are unclear. Here we report that, within the lateral hypothalamus, where functionally opposite wake-promoting orexin neurons and sleep-promoting melanin-concentrating hormone neurons codistribute, the glutamate transporter GLT1, mainly present on astrocytes, distinctly modulates excitatory transmission in a cell-type-specific manner and according to sleep history. Specifically, GLT1 is reduced around the somata of orexin neurons while increased around melanin-concentrating hormone neurons following sleep deprivation, resulting in different forms of synaptic plasticity. Thus, astrocytes can fine-tune the excitability of functionally discrete neurons via glutamate transport, which may represent novel regulatory mechanisms for sleep.

Short-term high-fat diet primes excitatory synapses for long-term depression in orexin neurons.

KEY POINTS: High-fat diet consumption is a major cause of obesity. Orexin neurons are known to be activated by a high-fat diet and in turn promote further consumption of a high-fat diet. Our study shows that excitatory synapses to orexin neurons become amenable to long-term depression (LTD) after 1 week of high-fat diet feeding. However, this effect reverses after 4 weeks of a high-fat diet. This LTD may be a homeostatic response to a high-fat diet to curb the activity of orexin neurons and hence caloric consumption. Adaptation seen after prolonged high-fat diet intake may contribute to the development of obesity. ABSTRACT: Overconsumption of high-fat diets is one of the strongest contributing factors to the rise of obesity rates. Orexin neurons are known to be activated by a palatable high-fat diet and mediate the activation of the mesolimbic reward pathway, resulting in further food intake. While short-term exposure to a high-fat diet is known to induce synaptic plasticity within the mesolimbic pathway, it is unknown if such changes occur in orexin neurons. To investigate this, 3-week-old male rats were fed a palatable high-fat western diet (WD) or control chow for 1 week and then in vitro patch clamp recording was performed. In the WD condition, an activity-dependent long-term depression (LTD) of excitatory synapses was observed in orexin neurons, but not in chow controls. This LTD was presynaptic and depended on postsynaptic metabotropic glutamate receptor 5 (mGluR5) and retrograde endocannabinoid signalling. WD also increased extracellular glutamate levels, suggesting that glutamate spillover and subsequent activation of perisynaptic mGluR5 may occur more readily in the WD condition. In support of this, pharmacological inhibition of glutamate uptake was sufficient to prime chow control synapses to undergo a presynaptic LTD. Interestingly, these WD effects are transient, as extracellular glutamate levels were similar to controls and LTD was no longer observed in orexin neurons after 4 weeks of WD. In summary, excitatory synapses to orexin neurons become amenable to LTD under a palatable high-fat diet, which may represent a homeostatic mechanism to prevent overactivation of these neurons and to curtail high-fat diet consumption.

High-fat diet-induced downregulation of anorexic leukemia inhibitory factor in the brain stem.

OBJECTIVE: High-fat diet (HFD) is known to induce low-grade hypothalamic inflammation. Whether inflammation occurs in other brain areas remains unknown. This study tested the effect of short-term HFD on cytokine gene expression and identified leukemia inhibitory factor (LIF) as a responsive cytokine in the brain stem. Thus, functional and cellular effects of LIF in the brain stem were investigated. METHODS: Male rats were fed chow or HFD for 3 days, and then gene expression was analyzed in different brain regions for IL-1ß, IL-6, TNF-α, and LIF. The effect of intracerebroventricular injection of LIF on chow intake and body weight was also tested. Patch clamp recording was performed in the nucleus tractus solitarius (NTS). RESULTS: HFD increased pontine TNF-α mRNA while downregulating LIF in all major parts of the brain stem, but not in the hypothalamus or hippocampus. LIF injection into the cerebral aqueduct suppressed food intake without conditioned taste aversion, suggesting that LIF can induce anorexia via lower brain regions without causing malaise. In the NTS, a key brain stem nucleus for food intake regulation, LIF induced acute changes in neuronal excitability. CONCLUSIONS: HFD-induced downregulation of anorexic LIF in the brain stem may provide a permissive condition for HFD overconsumption. This may be at least partially mediated by the NTS.

Concentration-dependent activation of dopamine receptors differentially modulates GABA release onto orexin neurons.

Dopamine (DA) and orexin neurons play important roles in reward and food intake. There are anatomical and functional connections between these two cell groups: orexin peptides stimulate DA neurons in the ventral tegmental area and DA inhibits orexin neurons in the hypothalamus. However, the cellular mechanisms underlying the action of DA on orexin neurons remain incompletely understood. Therefore, the effect of DA on inhibitory transmission to orexin neurons was investigated in rat brain slices using the whole-cell patch-clamp technique. We found that DA modulated the frequency of spontaneous and miniature IPSCs (mIPSCs) in a concentration-dependent bidirectional manner. Low (1 µM) and high (100 µM) concentrations of DA decreased and increased IPSC frequency, respectively. These effects did not accompany a change in mIPSC amplitude and persisted in the presence of G-protein signaling inhibitor GDPßS in the pipette, suggesting that DA acts presynaptically. The decrease in mIPSC frequency was mediated by D2 receptors whereas the increase required co-activation of D1 and D2 receptors and subsequent activation of phospholipase C. In summary, our results suggest that DA has complex effects on GABAergic transmission to orexin neurons, involving cooperation of multiple receptor subtypes. The direction of dopaminergic influence on orexin neurons is dependent on the level of DA in the hypothalamus. At low levels DA disinhibits orexin neurons whereas at high levels it facilitates GABA release, which may act as negative feedback to curb the excitatory orexinergic output to DA neurons. These mechanisms may have implications for consummatory and motivated behaviours.

Single rapamycin administration induces prolonged downward shift in defended body weight in rats.

Manipulation of body weight set point may be an effective weight loss and maintenance strategy as the homeostatic mechanism governing energy balance remains intact even in obese conditions and counters the effort to lose weight. However, how the set point is determined is not well understood. We show that a single injection of rapamycin (RAP), an mTOR inhibitor, is sufficient to shift the set point in rats. Intraperitoneal RAP decreased food intake and daily weight gain for several days, but surprisingly, there was also a long-term reduction in body weight which lasted at least 10 weeks without additional RAP injection. These effects were not due to malaise or glucose intolerance. Two RAP administrations with a two-week interval had additive effects on body weight without desensitization and significantly reduced the white adipose tissue weight. When challenged with food deprivation, vehicle and RAP-treated rats responded with rebound hyperphagia, suggesting that RAP was not inhibiting compensatory responses to weight loss. Instead, RAP animals defended a lower body weight achieved after RAP treatment. Decreased food intake and body weight were also seen with intracerebroventricular injection of RAP, indicating that the RAP effect is at least partially mediated by the brain. In summary, we found a novel effect of RAP that maintains lower body weight by shifting the set point long-term. Thus, RAP and related compounds may be unique tools to investigate the mechanisms by which the defended level of body weight is determined; such compounds may also be used to complement weight loss strategy.

Multivesicular release underlies short term synaptic potentiation independent of release probability change in the supraoptic nucleus.

Magnocellular neurons of the supraoptic nucleus receive glutamatergic excitatory inputs that regulate the firing activity and hormone release from these neurons. A strong, brief activation of these excitatory inputs induces a lingering barrage of tetrodotoxin-resistant miniature EPSCs (mEPSCs) that lasts for tens of minutes. This is known to accompany an immediate increase in large amplitude mEPSCs. However, it remains unknown how long this amplitude increase can last and whether it is simply a byproduct of greater release probability. Using in vitro patch clamp recording on acute rat brain slices, we found that a brief, high frequency stimulation (HFS) of afferents induced a potentiation of mEPSC amplitude lasting up to 20 min. This amplitude potentiation did not correlate with changes in mEPSC frequency, suggesting that it does not reflect changes in presynaptic release probability. Nonetheless, neither postsynaptic calcium chelator nor the NMDA receptor antagonist blocked the potentiation. Together with the known calcium dependency of HFS-induced potentiation of mEPSCs, our results imply that mEPSC amplitude increase requires presynaptic calcium. Further analysis showed multimodal distribution of mEPSC amplitude, suggesting that large mEPSCs were due to multivesicular glutamate release, even at late post-HFS when the frequency is no longer elevated. In conclusion, high frequency activation of excitatory synapses induces lasting multivesicular release in the SON, which is independent of changes in release probability. This represents a novel form of synaptic plasticity that may contribute to prolonged excitatory tone necessary for generation of burst firing of magnocellular neurons.

Nociceptin induces hypophagia in the perifornical and lateral hypothalamic area.

Nociceptin/orphanin FQ (N/OFQ) is known to induce food intake when administered into the lateral ventricle or certain brain areas. This is somewhat contradictory to its reward-suppressing role, as food is a strong rewarding stimulus. This discrepancy may be due to the functional diversity of N/OFQ's target brain areas. N/OFQ has been shown to inhibit orexin and melanin-concentrating hormone (MCH) neurons, both of which are appetite-inducing cells. As the expression of these neurons is largely confined to the lateral hypothalamus/perifornical area (LH/PFA), we hypothesized that N/OFQ inhibits food intake by acting in this area. To test this hypothesis, we examined the effect of local N/OFQ infusion within the LH/PFA on food intake in the rat and found that N/OFQ decreased sugar pellet as well as chow intake. This effect was not seen when the injection site was outside of the LH/PFA, suggesting a site-specific effect. Next, to determine a possible cellular mechanism of N/OFQ action on food intake, whole cell patch clamp recordings were performed on rat orexin neurons. As previously reported in mice, N/OFQ induced a strong and long lasting hyperpolarization. Pharmacological study indicated that N/OFQ directly inhibited orexin neurons by activating ATP-sensitive potassium (KATP) channels. This effect was partially but significantly attenuated by the inhibitors of PI3K, PKC and PKA, suggesting that the N/OFQ signaling is mediated by these protein kinases. In summary, our results demonstrate a KATP channel-dependent N/OFQ signaling and that N/OFQ is a site-specific anorexic peptide.

ATP-sensitive potassium channels mediate the thermosensory response of orexin neurons.

High body temperatures are generally associated with somnolence, lethargy, hypophagia and anhedonia. Orexin neurons have been suggested to play a role in such sickness behaviours due to their known functions in appetite, behavioural and autonomic activation. Furthermore, the activity of orexin neurons is inhibited by lipopolysaccharide that induces fever. However, the cellular mechanism(s) underlying this suppression of orexin neurons was unknown. We used patch-clamp recordings in acute rat brain slices to demonstrate that orexin neurons, including those projecting to the wake-promoting locus coeruleus, are inhibited by increasing the ambient temperature by a 2-4°C increment between 26 and 40°C. This effect was not mediated by conventional thermosensing mechanisms but instead involved the activation of ATP-sensitive potassium (KATP) channels. Since KATP channels can also sense energy substrate levels and cellular metabolism, our results suggest that orexin neurons can integrate the state of energy balance and body temperature, and adjust their output accordingly. Thus, the thermosensitivity of orexin neurons may be an important part of maintaining energy homeostasis during hyperthermia and fever.

Dopamine acts as a partial agonist for α2A adrenoceptor in melanin-concentrating hormone neurons.

Melanin-concentrating hormone (MCH) is a hypothalamic neuropeptide that promotes positive energy balance and anxiety. Since dopamine (DA) is also closely implicated in these functions, the present study investigated the effect of DA on MCH neurons. Using whole-cell patch-clamp recordings in rat brain slices, we found that DA hyperpolarizes MCH neurons by activating G-protein-activated inwardly rectifying K(+) (GIRK) channels. Pharmacological study indicated that the effect was mediated by α2A adrenoceptors, not DA receptors. DA-induced outward current was also observed in the presence of tetrodotoxin or the dopamine ß-hydroxylase inhibitor fusaric acid, suggesting that DA directly binds to α2A receptors on MCH neurons, rather than acting presynaptically or being transformed into norepinephrine (NE) in the slice preparation. The effects of NE and DA were concentration-dependent with EC(50) of 5.9 and 23.7 µm, respectively, and a maximal effect of 106.6 and 57.2 pA, respectively, suggesting that DA functions as a partial agonist. Prolonged (5 min) activation of α2A receptors by either DA or NE attenuated the subsequent response to DA or NE, while 5 s applications were not sufficient to induce desensitization. Therefore, a history of α2A receptor activation by DA or NE can have a lasting inhibitory effect on the catecholaminergic transmission to MCH neurons. Our study suggests that α2A receptors expressed by MCH neurons may be one of the pathways by which DA and NE can interact and modulate mood and energy homeostasis, and this cross talk may have functional implications in mood disorders and obesity.

Local network regulation of orexin neurons in the lateral hypothalamus.

Obesity and inadequate sleep are among the most common causes of health problems in modern society. Thus, the discovery that orexin (hypocretin) neurons play a pivotal role in sleep/wake regulation, energy balance, and consummatory behaviors has sparked immense interest in understanding the regulatory mechanisms of these neurons. The local network consisting of neurons and astrocytes within the lateral hypothalamus and perifornical area (LH/PFA), where orexin neurons reside, shapes the output of orexin neurons and the LH/PFA. Orexin neurons not only send projections to remote brain areas but also contribute to the local network where they release multiple neurotransmitters to modulate its activity. These neurotransmitters have opposing actions, whose balance is determined by the amount released and postsynaptic receptor desensitization. Modulation and negative feedback regulation of excitatory glutamatergic inputs as well as release of astrocyte-derived factors, such as lactate and ATP, can also affect the excitability of orexin neurons. Furthermore, distinct populations of LH/PFA neurons express neurotransmitters with known electrophysiological actions on orexin neurons, such as melanin-concentrating hormone, corticotropin-releasing factor, thyrotropin-releasing hormone, neurotensin, and GABA. These LH/PFA-specific mechanisms may be important for fine tuning the firing activity of orexin neurons to maintain optimal levels of prolonged output to sustain wakefulness and stimulate consummatory behaviors. Building on these exciting findings should shed further light onto the cellular mechanisms of energy balance and sleep-wake regulation.