Lateral Hypothalamic Orexin Neurons Research Articles

Lateral hypothalamic orexin neurons mediate electroacupuncture-induced anxiolytic effects in a rat model of post-traumatic stress disorder

The lateral hypothalamus' orexinergic system has been associated with anxiety-related behaviors, and electroacupuncture (EA) modifies orexin neurons to control the anti-anxiety process. However, in a rat model of post-traumatic stress disorder (PTSD), the important role of LH orexin neurons (OXNs) in the anxiolytic effects induced by EA has not been explored. In this study, rats underwent modified single prolonged stress (MSPS) for seven days before developing EA. The rats were then subjected to elevated plus maze (EPM) and open field (OFT) tests, and western blot and c-Fos/orexin double labeling investigations were carried out to determine the functional activation of LH orexinergic neurons. Compared to MSPS model rats, it has been demonstrated that EA stimulation enhanced the amount of time spent in the central zone (TSCZ) in OFT and the amount of time spent in the open arm (TSOA) in EPM in MSPS model rats (P < 0.01). After behavioral testing, MSPS model rats had decreased activated c-Fos positive OXNs. Still, EA in SPS rats increased that number and elevated orexin type 1 receptors (OXR1) protein expression in the LH. Furthermore, after administering SB334867 (an OXR1 antagonist) to MSPS model rats, the effects of EA therapy on anxiety-like behaviors (ALBs) were significantly diminished. Additionally, when low-dose orexin-A (LORXA) was administered intracerebroventricularly together with EA stimulation in MSPS rats, the anxiolytic effects of the stimulation were substantially enhanced (P < 0.05). The results of this study reveal the mechanisms by which acupuncture may reduce PTSD and advance our understanding of the function of LH orexin signaling in EA's anxiolytic effects.

Effects of glucose modulation in lateral hypothalamus on motivated behavior to obtain sucrose in an operant task

Orexin neurons in the Lateral Hypothalamus (LH) play an important role in food seeking behavior. Approximately 60 percent of LH orexin neurons are inhibited by elevated extracellular glucose. It has been shown that elevated LH glucose decreases conditioned place preference for a food associated chamber. However, it has never been shown how modulation of LH extracellular glucose effects a rat's motivation to work for food. In this experiment we used reverse microdialysis to modulate extracellular glucose levels in LH during an operant task. Results from a progressive ratio task demonstrated that 4 mM glucose perfusion significantly decreased the animal's motivation to work for sucrose pellets while not effecting the hedonic value of the pellets. In a second experiment we demonstrated that 4 mM but not 2.5 mM glucose perfusion was sufficient to significantly decrease the number of sucrose pellets earned. Finally, we showed that modulating LH extracellular glucose mid-session from 0.7 mM to 4 mM did not affect behavior. This indicates that once feeding behavior has begun the animal becomes unresponsive to changes in extracellular glucose levels in LH. Taken together these experiments indicate that LH glucose sensing neurons play an important role in motivation to initiate feeding. However, once consumption has begun it is likely that feeding is controlled by brain regions downstream of LH.

Orexin receptors regulate hippocampal sharp wave-ripple complexes in ex vivo slices

Orexin is a neuromodulatory peptide produced by lateral hypothalamic orexin neurons and binds to G-protein-coupled orexin-1 receptor and orexin-2 receptors. Whether orexin modulates learning and memory is not fully understood. Orexin has biphasic effects on learning and memory: promoting learning and memory at homeostatic levels and inhibiting at supra- and sub-homeostatic levels. Hippocampal sharp wave-ripples encode memory information and are essential for memory consolidation and retrieval. The role of orexin on sharp wave-ripples in hippocampal CA1 remains unknown. Here, we used multi-electrode array recordings in acute ex vivo hippocampal slices to determine the effects of orexin receptor antagonists on sharp wave-ripples. Bath-application of either the orexin-1 receptor antagonist N-(2-Methyl-6-benzoxazolyl)-N'-1,5-naphthyridin-4-yl urea (SB-334867) or the orexin-2 receptor antagonist N-Ethyl-2-[(6-methoxy-3-pyridinyl)[(2-methylphenyl)sulfonyl]amino]-N-(3-pyridinylmethyl)-acetamide (EMPA) reduced sharp wave and ripple incidence, sharp wave amplitude, and sharp wave duration. SB-334867 and EMPA effects on sharp wave amplitude and duration were equivalent, whereas EMPA exhibited a greater reduction of sharp wave and ripple incidence. EMPA also increased ripple duration, whereas SB-334867 had no effect. Inhibition of both orexin receptors with a dual orexin receptor antagonist N-[1,1'-Biphenyl]-2-yl-1-[2-[(1-methyl-1H-benzimidazol-2-yl)thio]acetyl-2-pyrrolidinedicarboxamide (TCS-1102) had effects similar to EMPA, however, sharp wave amplitude and duration were unaffected. Region-specific expression of orexin receptors suggests orexin may regulate sharp wave generation in CA3, dentate gyrus-mediated sharp wave modification, sharp wave propagation to CA1, and local ripple emergence in CA1. Our study indicates an orexin contribution to hippocampal sharp wave-ripple complexes and suggests a mechanism by which sub-homeostatic concentrations of orexin may inhibit learning and memory function.

Oxytocin and orexin systems bidirectionally regulate the ability of opioid cues to bias reward seeking

As opioid-related fatalities continue to rise, the need for novel opioid use disorder (OUD) treatments could not be more urgent. Two separate hypothalamic neuropeptide systems have shown promise in preclinical OUD models. The oxytocin system, originating in the paraventricular nucleus (PVN), may protect against OUD severity. By contrast, the orexin system, originating in the lateral hypothalamus (LH), may exacerbate OUD severity. Thus, activating the oxytocin system or inhibiting the orexin system are potential therapeutic strategies. The specific role of these systems with regard to specific OUD outcomes, however, is not fully understood. Here, we probed the therapeutic efficacy of pharmacological interventions targeting the orexin or oxytocin system on two distinct metrics of OUD severity in rats—heroin choice (versus choice for natural reward, i.e., food) and cued reward seeking. Using a preclinical model that generates approximately equal choice between heroin and food reward, we examined the impact of exogenously administered oxytocin, an oxytocin receptor antagonist (L-368,899), and a dual orexin receptor antagonist (DORA-12) on opioid choice. Whereas these agents did not alter heroin choice when rewards (heroin and food) were available, oxytocin and DORA-12 each significantly reduced heroin seeking in the presence of competing reward cues when no rewards were available. In addition, the number of LH orexin neurons and PVN oxytocin neurons correlated with specific behavioral economic variables indicative of heroin versus food motivation. These data identify a novel bidirectional role of the oxytocin and orexin systems in the ability of opioid-related cues to bias reward seeking.

Dual orexin receptor antagonist drug suvorexant can help in amelioration of predictable chronic mild stress-induced hyperalgesia

AimsThis study aimed to evaluate the involvement of the orexin system in predictable chronic mild stress (PCMS) and the effects of suvorexant, a dual orexin receptor antagonist, on nociceptive behavior in PCMS. Materials and methodsMale C57BL/6 J mice were separated into various PCMS groups: a control group with sawdust on the floor of the rearing cage (C), a group with mesh wire on the floor (M), and a group with water just below the mesh wire (W). Activation of lateral hypothalamic orexin neurons was assessed using immunofluorescence. In another experiment, half of the mice in each group were administered an intraperitoneal injection of suvorexant (10 mg/kg), and the remaining mice were injected with the same amount of vehicle (normal saline). Thermal hyperalgesia was examined using tail immersion and hot plate tests, while mechanical hyperalgesia was investigated using the tail pinch test after 21 days of PCMS. Key findingsAnimals subjected to PCMS showed an increased percentage of activated orexin neurons in the lateral hypothalamic region after 21 days. Mice raised in the PCMS environment showed increased pain sensitivity in several pain tests; however, the symptoms were significantly reduced by suvorexant administration. SignificanceThe findings revealed that PCMS activates hypothalamic orexin neuronal activity, and the use of suvorexant can help attenuate PCMS-induced thermal and mechanical hyperalgesia.

Lateral Hypothalamic Orexin Neurons Mediate the Reward Effects of Pain Relief Induced by Electroacupuncture.

The reward of pain relief caused by acupuncture has been found to be clinically significant. However, the molecular mechanisms underlying acupuncture-induced reward of pain relief in chronic pain remain unclear and have not been analyzed in suitable preclinical models. Here, we investigated whether acupuncture could potentially induce the reward of pain relief and orexin neuronal signaling in the lateral hypothalamus (LH) and exhibit a possible role in electroacupuncture (EA)-induced reward in spared nerve injury (SNI) rats. Therefore, by using conditioned place preference (CPP) paradigm, we noticed that EA induced the preference for cues associated with EA-induced pain relief in the early, but not late, phase of chronic pain. These observations were different from the immediate antihyperalgesic effects of EA. c-Fos/orexin double labeling revealed that EA stimulation on 14 days but not on 28 days after SNI modeling activated greater numbers of c-Fos positive orexin neurons in the LH after the CPP test. Moreover, the administration of an orexin-A antagonist in the LH significantly blocked the reward effects of pain relief induced by EA. Furthermore, by using cholera toxin b subunit combined with c-Fos detection, we found that the orexin circuit from the LH to the nucleus accumbens (NAc) shell was significantly activated after EA induced CPP. Microinjection of the orexin antagonist into the NAc shell substantially attenuated the CPP induced by EA. Intravenous injection of low-dose orexin-A together with EA resulted in significantly greater antihyperalgesia effects and CPP scores. Together, these findings clearly demonstrated that LH orexin signaling could potentially play a critical role in the reward effects of pain relief induced by acupuncture. The observations of the present study extended our understanding of orexin signaling in the LH and its role in EA-induced reward, providing new insights into the mechanisms of acupuncture analgesia.

Orexin, serotonin, and energy balance.

The lateral hypothalamus is critical for the control of ingestive behavior and spontaneous physical activity (SPA), as lesion or stimulation of this region alters these behaviors. Evidence points to lateral hypothalamic orexin neurons as modulators of feeding and SPA. These neurons affect a broad range of systems, and project to multiple brain regions such as the dorsal raphe nucleus, which contains serotoninergic neurons (DRN) important to energy homeostasis. Physical activity is comprised of intentional exercise and SPA. These are opposite ends of a continuum of physical activity intensity and structure. Non‐goal‐oriented behaviors, such as fidgeting, standing, and ambulating, constitute SPA in humans, and reflect a propensity for activity separate from intentional activity, such as high‐intensity voluntary exercise. In animals, SPA is activity not influenced by rewards such as food or a running wheel. Spontaneous physical activity in humans and animals burns calories and could theoretically be manipulated pharmacologically to expend calories and protect against obesity. The DRN neurons receive orexin inputs, and project heavily onto cortical and subcortical areas involved in movement, feeding and energy expenditure (EE). This review discusses the function of hypothalamic orexin in energy‐homeostasis, the interaction with DRN serotonin neurons, and the role of this orexin‐serotonin axis in regulating food intake, SPA, and EE. In addition, we discuss possible brain areas involved in orexin–serotonin cross‐talk; the role of serotonin receptors, transporters and uptake‐inhibitors in the pathogenesis and treatment of obesity; animal models of obesity with impaired serotonin‐function; single‐nucleotide polymorphisms in the serotonin system and obesity; and future directions in the orexin–serotonin field.This article is categorized under:Metabolic Diseases > Molecular and Cellular Physiology

Topographical Differences in Orexin Activation after Pain Assessments in Hb AA and HbSS Sickle Mice

The orexins are neuropeptides that are synthesized in the perifornical area (PFA), lateral hypothalamus (LH) and dorsomedial hypothalamus (DMH) and mediate arousal, sleep, energy balance, food and drug reward. The LH region is associated with reward, while the PFA and DMH are more associated with stress and arousal. Data suggest that orexin-A has an analgesic effect on inflammatory pain and it may affect mechanisms underlying the maintenance of neuropathic pain. To date, there have been no published studies to investigate this system as a potential target to treat neuropathic pain in a mouse model of sickle cell disease (SCD). Therefore, we seek to determine activational and topographical differences in neuronal activation of the orexin system in a mouse model of SCD by using an array of behavioral measures to characterize pain in transgenic mice expressing human hemoglobin.Female transgenic sickle mice that express exclusively (99%) human sickle hemoglobin (HbSS) and age-/gender-matched controls (HbAA mice; n = 5/group, 20-30g) expressing normal human hemoglobin A are used in the study. All behavioral tests are performed in a quiet room maintained at a constant temperature of 23- 25°C. All mice are habituated to each test protocol and environment for at least three consecutive days before collecting baseline measurements and testing. Three measures are used to assess pain-related behaviors: thermal/heat hyperalgesia (test for heat sensitivity), cold hyperalgesia (for cold sensitivity), and mechanical hyperalgesia (sensitivity to mechanical stimulus). Ninety minutes after the pain assessment testing, brains from HbAA and HbSS mice are quick frozen and hypothalamic brain sections from the mice are processed to visualize orexin-A and c-Fos using double label immunohistochemistry.Preliminary analyses show that significant topographical differences are observed in LH versus PFA for HbAA and HbSS mice (p<0.05) and LH versus DMH for HbAA and HbSS mice (p<0.05). This indicates that the number of double labeled neurons in the LH is significantly lower than the number of double labeled neurons in the PFA and DMH of HbAA and HbSS mice. Additionally, a significant topographical difference is observed in LH versus DMH for only HbSS mice (p<0.05). These data demonstrate that the number of activated orexin neurons is greater after pain testing in the DMH (a region more associated with stress and arousal) versus LH in HbSS mice. There is a trend towards a significant increase in activated LH orexin neurons in HbAA versus HbSS mice. Additional studies are underway to determine whether fewer reward-associated, orexin neurons in the LH are activated in HbSS versus HbAA mice after pain testing, and whether this decrease in orexin activation may provide a possible explanation for the increased hyperalgesia in HbSS sickle mice, which contributes to neuropathic pain observed in SCD. DisclosuresGupta:Tautona Group: Consultancy, Honoraria; Fera Pharmaceuticals LLC: Consultancy, Honoraria.

Orexin in Dorsal Raphe Nucleus enhances Physical Activity and Energy Expenditure

Background Lateral hypothalamic (LH) orexin neurons modulate spontaneous physical activity (SPA), and energy expenditure (EE). Orexin deficiency results in weight gain, whereas heightened LH orexin sensitivity results in obesity resistance. Orexins act on multiple brain sites, including the serotonergic dorsal raphe nucleus (DRN), which receives excitatory projections from the orexin neurons. Our earlier studies show presence of both orexin-1 (OX1R) and orexin-2 receptors (OX2R) in DRN, and higher expression of OX1R and OX2R in DRN of obesity resistant (OR) rats. We hypothesized that orexin in DRN enhances SPA and EE. Secondarily, we hypothesized that DRN GABA (gamma-aminobutyric acid) signaling mediates the effect of orexin on SPA and EE. Methods We manipulated orexin tone in DRN either by direct injections of orexin A into DRN or by chemogenetic activation of LH orexin neurons in aged (~15 mo) orexin-cre mice. Mice were implanted with a guide cannula targeting DRN, and simultaneously prepared with the stimulatory (hM3Dq floxed) designer receptors exclusively activated by designer drugs (DREADD) virus in LH (N = 16). All mice recovered for 3 weeks to allow transfection of the DREADD virus, and randomly assigned to saline or clozapine N-oxide (CNO, DREADD activator) treatment. Mice were transferred to Promethion indirect calorimetry cages (Sable Systems International) to continuously monitor SPA and EE. Body weight and food intake were measured daily. After acclimation, mice received either IP vehicle or CNO (1 mg/kg) in a repeated measure design. Fifteen minutes prior to IP CNO or saline, animals were infused with either the GABA agonist muscimol (32.5 pmol/0.2 μl), GABA antagonist bicuculline (32.5 pmol/0.2 μl) or vehicle into DRN. In a separate experiment, either orexin A (250 pmol/0.2 μl) or saline was injected into DRN without IP CNO injection, to study the direct effect of DRN orexin on SPA and EE for 24h post-injection. Results We found that DREADD activation of LH orexin neurons increases SPA and EE. Manipulation of GABA receptors (by muscimol or bicuculline) in DRN did not affect orexin neuron activation induced enhancement of SPA. On the other hand, intra-DRN OXA enhanced SPA and EE in these mice up to 4h post-injection, without affecting food intake. Conclusions These results support the idea that OXA in DRN facilitates negative energy balance by increasing physical activity-induced energy expenditure. In addition, these data suggest that the DRN is a prominent site modulating OXA-stimulated SPA and EE, and that modulation of DRN OXA is a potential strategy to mitigate age-induced reductions in SPA and EE.

Synchronous neuronal interactions in rat hypothalamic culture: a novel model for the study of network dynamics in metabolic disorders

Synchronous neural activity is a feature of normal brain function, and altered synchronization is observed in several neurological diseases. Dysfunction in hypothalamic pathways leads to obesity, suggesting that hypothalamic neural synchrony is critical for energy homeostasis. The lateral hypothalamic orexin neurons are extensively interconnected with other brain structures and are important for energy balance. Earlier studies show that rats with higher orexin sensitivity are obesity resistant. Similarly, topiramate, an anti-epileptic drug, has been shown to reduce weight in humans. Since orexin enhances neuronal excitation, we hypothesized that obesity-resistant rats with higher orexin sensitivity may exhibit enhanced hypothalamic synchronization. We further hypothesized that anti-obesity agents such as orexin and topiramate will enhance hypothalamic synchronization. To test this, we examined neural synchronicity in primary embryonic hypothalamic cell cultures, obtained from embryonic day 18 (E18) obesity-susceptible Sprague-Dawley (SD) and obesity-resistant rats. Hypothalamic tissue was cultured in multielectrode array (MEA), and recordings were performed twice weekly, from 4th to 32nd day in vitro (DIV). Next, we tested the effects of orexin and topiramate application on neural synchronicity of hypothalamic cultures obtained from SD rat embryos. Signals were analyzed for synchronization using cross correlation. Our results showed that (1) obesity-resistant hypothalamus exhibits significantly higher synchronization compared to obesity-sensitive hypothalamus; and (2) orexin and topiramate enhance hypothalamic synchronization. These results support that enhanced orexin sensitivity is associated with greater neural synchronization, and that anti-obesity treatments enhance network synchronization, thus constrain variability in hypothalamic output signals, to extrahypothalamic structures involved in energy homeostasis.

Stress induces reinstatement of extinguished cocaine conditioned place preference by a sequential signaling via neuropeptide S, orexin, and endocannabinoid.

Neurons containing neuropeptide S (NPS) and orexins are activated during stress. Previously, we reported that orexins released during stress, via orexin OX1 receptors (OX1 Rs), contribute to the reinstatement of cocaine seeking through endocannabinoid/CB1 receptor (CB1 R)-mediated dopaminergic disinhibition in the ventral tegmental area (VTA). Here, we further demonstrated that NPS released during stress is an up-stream activator of this orexin-endocannabinoid cascade in the VTA, leading to the reinstatement of cocaine seeking. Mice were trained to acquire cocaine conditioned place preference (CPP) by context-pairing cocaine injections followed by the extinction training with context-pairing saline injections. Interestingly, the extinguished cocaine CPP in mice was significantly reinstated by intracerebroventricular injection (i.c.v.) of NPS (1 nmol) in a manner prevented by intraperitoneal injection (i.p.) of SHA68 (50 mg/kg), an NPS receptor antagonist. This NPS-induced cocaine reinstatement was prevented by either i.p. or intra-VTA microinjection (i.vta.) of SB-334867 (15 mg/kg, i.p. or 15 nmol, i.vta.) and AM 251 (1.1 mg/kg, i.p. or 30 nmol, i.vta.), antagonists of OX1 Rs and CB1 Rs, respectively. Besides, NPS (1 nmol, i.c.v.) increased the number of c-Fos-containing orexin neurons in the lateral hypothalamus (LH) and increased orexin-A level in the VTA. The latter effect was blocked by SHA68. Furthermore, a 30-min restraint stress in mice reinstated extinguished cocaine CPP and was prevented by SHA68. These results suggest that NPS is released upon stress and subsequently activates LH orexin neurons to release orexins in the VTA. The released orexins then reinstate extinguished cocaine CPP via an OX1 R- and endocannabinoid-CB1 R-mediated signaling in the VTA.

Orexin neurons and inhibitory Agrp→orexin circuits guide spatial exploration in mice.

Photoinhibition of endogenous activity of lateral hypothalamic orexin neurons causes place preference and reduces innate avoidance Endogenous activity of orexin neurons correlates with place preference Mediobasal hypothalamic Agrp neurons inhibit orexin neurons via GABA, and chemogenetic suppression of Agrp neurons increases avoidance in an orexin receptor-dependent manner. Hypothalamic orexin/hypocretin neurons integrate multiple sensory cues and project brain-wide to orchestrate diverse innate behaviours. Their loss impairs many context-appropriate actions, but the motivational characteristics of orexin cell activity remain unclear. We and others previously approached this question by artificial orexin stimulation, which could induce either rewarding (positive valence) or aversive (negative valence) brain activity. It is unknown to what extent such approaches replicate natural/endogenous orexin signals, which rapidly fluctuate during wakefulness. Here we took an alternative approach, focusing on observing and silencing natural orexin cell signals associated with a fundamental innate behaviour, self-paced spatial exploration. We found that mice are more likely to stay in places paired with orexin cell optosilencing. The orexin cell optosilencing also reduced avoidance of places that mice find innately aversive. Correspondingly, calcium recordings revealed that orexin cell activity rapidly reduced upon exiting the innately aversive places. Furthermore, we provide optogenetic evidence for an inhibitory GABAergic Agrp→orexin hypothalamic neurocircuit, and find that Agrp cell suppression increases innate avoidance behaviour, consistent with orexin disinhibition. These results imply that exploration may be motivated and oriented by a need to reduce aversive orexin cell activity, and suggest a hypothalamic circuit for fine-tuning orexin signals to changing ethological priorities.

Neuropeptide S-initiated sequential cascade mediated by OX1, NK1, mGlu5 and CB1 receptors: a pivotal role in stress-induced analgesia

BackgroundStress-induced analgesia (SIA) is an evolutionarily conserved phenomenon during stress. Neuropeptide S (NPS), orexins, substance P, glutamate and endocannabinoids are known to be involved in stress and/or SIA, however their causal links remain unclear. Here, we reveal an unprecedented sequential cascade involving these mediators in the lateral hypothalamus (LH) and ventrolateral periaqueductal gray (vlPAG) using a restraint stress-induced SIA model.MethodsMale C57BL/6 mice of 8–12 week-old were subjected to intra-cerebroventricular (i.c.v.) and/or intra-vlPAG (i.pag.) microinjection of NPS, orexin-A or substance P alone or in combination with selective antagonists of NPS receptors (NPSRs), OX1 receptors (OX1Rs), NK1 receptors (NK1Rs), mGlu5 receptors (mGlu5Rs) and CB1 receptors (CB1Rs), respectively. Antinociceptive effects of these mediators were evaluated via the hot-plate test. SIA in mice was induced by a 30-min restraint stress. NPS levels in the LH and substance P levels in vlPAG homogenates were compared in restrained and unrestrained mice.ResultsNPS (i.c.v., but not i.pag.) induced antinociception. This effect was prevented by i.c.v. blockade of NPSRs. Substance P (i.pag.) and orexin-A (i.pag.) also induced antinociception. Substance P (i.pag.)-induced antinociception was prevented by i.pag. Blockade of NK1Rs, mGlu5Rs or CB1Rs. Orexin-A (i.pag.)-induced antinociception has been shown previously to be prevented by i.pag. blockade of OX1Rs or CB1Rs, and here was prevented by NK1R or mGlu5R antagonist (i.pag.). NPS (i.c.v.)-induced antinociception was prevented by i.pag. blockade of OX1Rs, NK1Rs, mGlu5Rs or CB1Rs. SIA has been previously shown to be prevented by i.pag. blockade of OX1Rs or CB1Rs. Here, we found that SIA was also prevented by i.c.v. blockade of NPSRs or i.pag. blockade of NK1Rs or mGlu5Rs. Restrained mice had higher levels of NPS in the LH and substance P in the vlPAG than unrestrained mice.ConclusionsThese results suggest that, during stress, NPS is released and activates LH orexin neurons via NPSRs, releasing orexins in the vlPAG. Orexins then activate OX1Rs on substance P-containing neurons in the vlPAG to release substance P that subsequently. Activates NK1Rs on glutamatergic neurons to release glutamate. Glutamate then activates perisynaptic mGlu5Rs to initiate the endocannabinoid retrograde inhibition of GABAergic transmission in the vlPAG, leading to analgesia.

The number of lateral hypothalamus orexin/hypocretin neurons contributes to individual differences in cocaine demand.

Lateral hypothalamus (LH) orexin neuron signaling has been implicated in the motivation to seek and take drugs of abuse. The number of LH orexin neurons has beenshown to be upregulated with exposure to drugs of abuse. We sought to determine if the number of LH orexin neurons related to individual differences in motivation (demand) for cocaine in our behavioral economics (BE) paradigm, and whether knockdown of these cells predicted changes in economic demand. We quantified LH orexin cell numbers in animals immediately following our BE paradigm, as well as after a 2-week period of abstinence, to relate the number of LH orexin cells to economic demand for cocaine. We also knocked down LH orexin expression with an orexin morpholino antisense to determine how reduced orexin numbers impacted cocaine demand. We found that animals with greater baseline motivation for cocaine (lower demand elasticity) had more LH orexin neurons. Following a 2-week abstinence from cocaine, the number of LH orexin neurons predicted economic demand for cocaine prior to abstinence, indicating that orexin expression is a persistent marker for demand. Reducing LH orexin cell numbers with antisense decreased motivation for cocaine (increased demand elasticity) without affecting baseline consumption. In addition, the number of spared LH orexin neurons after antisense treatment correlated with individual motivation for cocaine. These studies point to a role for the endogenous number of LH orexin neurons in individual differences in motivation for cocaine.

Activation of medial hypothalamic orexin neurons during a Go/No-Go task

Orexin neurons (Orx; also referred to as hypocretin) are found exclusively in the hypothalamus, and release the neuropeptides orexin A and orexin B (also referred to as hypocretin 1 and 2) throughout the CNS. With its widespread targets, the orexin system is involved in a number of functions including, but not limited to stress, reward, wakefulness, and food seeking. Our laboratory has previously proposed that the dorsomedial hypothalamus (DMH) and perifornical (PFA) orexin neurons function in stress and arousal whereas those in lateral hypothalamus (LH) participate in reward processes (Harris and Aston-Jones, 2006). In the current study, we compared Fos activation in orexin neurons located in medial hypothalamus (DMH and PFA) to those in LH during a Go/No-Go task for a highly palatable food reward, a task that would likely activate regions for arousal/attention as well as reward. The Go/No-Go paradigm is a useful behavioral tool to measure behavioral inhibition, impulsivity, learning, and reaction time. Our results revealed increased activation of medial hypothalamic orexin neurons correlated with greater accuracy on the Go/No-Go task. No correlation was found between Go/No-Go accuracy and activation of lateral hypothalamic orexin neurons. This study supports a functional dichotomy of medial vs lateral orexin neurons, and indicates a role for medial orexin neurons in behavioral performance that requires response inhibition.

Increased Number and Activity of a Lateral Subpopulation of Hypothalamic Orexin/Hypocretin Neurons Underlies the Expression of an Addicted State in Rats

BackgroundThe orexin (hypocretin) system is important for reward-driven motivation but has not been implicated in the expression of a multiphenotype addicted state. MethodsRats were assessed for economic demand for cocaine before and after 14 days of short access, long access, or intermittent access (IntA) to cocaine. Rats were also assessed for a number of other DSM-5–relevant addiction criteria following differential access conditions. Orexin system function was assessed by quantification of numbers and activity of orexin cells, pharmacological blockade of the orexin-1 receptor, and subregion-specific knockdown of orexin cell populations. ResultsIntA produced a cluster of addiction-like behaviors that closely recapitulate key diagnostic criteria for addiction to a greater extent than long access or short access. IntA was accompanied by an increase in number and activity of orexin-expressing neurons within the lateral hypothalamic subregion. This increase in orexin cell number and activity persisted during protracted withdrawal from cocaine for at least 150 days and was accompanied by enhanced incubation of craving in the same rats. Selective knockdown of lateral hypothalamic orexin neurons reduced motivation for cocaine, and orexin-1 receptor signaling played a larger role in drug seeking after IntA. ConclusionsWe provide the first evidence that lateral hypothalamic orexin system function extends beyond general reward seeking to play a critical role in expression of a multiphenotype addiction-like state. Thus, the orexin system is a potential novel target for pharmacotherapies designed to treat cocaine addiction. In addition, these data point to the IntA model as a preferred approach to modeling addiction-like behavior in rats.

Activation of mesolimbic reward system via laterodorsal tegmental nucleus and hypothalamus in exercise-induced hypoalgesia

Ventral tegmental area (VTA) dopamine (DA) neurons are the primary source of dopamine in target structures that constitute the mesolimbic reward system. Previous studies demonstrated that voluntary wheel running (VWR) by neuropathic pain (NPP) model mice produces exercise-induced hypoalgesia (EIH), and that activation of mesolimbic reward system may lead to EIH. However, the neuronal mechanism by which the mesolimbic reward system is activated by VWR is unknown. Here, we found that VWR produces EIH effects and reverses the marked reduction in activated lateral VTA (lVTA)-DA neurons induced by NPP. The proportions of activated laterodorsal tegmental nucleus (LDT)-cholinergic and lateral hypothalamus-orexin neurons were significantly enhanced by VWR. Retrograde tracing and dual immunostaining revealed that VWR activates lVTA-projecting LDT-cholinergic/non-cholinergic and lateral hypothalamic area (LHA)-orexin/non-orexin neurons. Therefore, EIH effects may be produced, at least in part, by activation of the mesolimbic reward system via activation of LDT and LHA neurons.

Nucleus accumbens controls wakefulness by a subpopulation of neurons expressing dopamine D1 receptors

Nucleus accumbens (NAc) is involved in behaviors that depend on heightened wakefulness, but its impact on arousal remains unclear. Here, we demonstrate that NAc dopamine D1 receptor (D1R)-expressing neurons are essential for behavioral arousal. Using in vivo fiber photometry in mice, we find arousal-dependent increases in population activity of NAc D1R neurons. Optogenetic activation of NAc D1R neurons induces immediate transitions from non-rapid eye movement sleep to wakefulness, and chemogenetic stimulation prolongs arousal, with decreased food intake. Patch-clamp, tracing, immunohistochemistry, and electron microscopy reveal that NAc D1R neurons project to the midbrain and lateral hypothalamus, and might disinhibit midbrain dopamine neurons and lateral hypothalamus orexin neurons. Photoactivation of terminals in the midbrain and lateral hypothalamus is sufficient to induce wakefulness. Silencing of NAc D1R neurons suppresses arousal, with increased nest-building behaviors. Collectively, our data indicate that NAc D1R neuron circuits are essential for the induction and maintenance of wakefulness.

Fast and Slow Oscillations Recruit Molecularly-Distinct Subnetworks of Lateral Hypothalamic Neurons In Situ.

Electrical signals generated by molecularly-distinct classes of lateral hypothalamus (LH) neurons have distinct physiological consequences. For example, LH orexin neurons promote net body energy expenditure, while LH non-orexin neurons [VGAT, melanin-concentrating hormone (MCH)] drive net energy conservation. Appropriate switching between such physiologically-opposing LH outputs is traditionally thought to require cell-type-specific chemical modulation of LH firing. However, it was recently found that, in vivo, the LH neurons are also physiologically exposed to electrical oscillations of different frequency bands. The role of the different physiological oscillation frequencies in firing of orexin vs non-orexin LH neurons remains unknown. Here, we used brain-slice whole-cell patch-clamp technology to target precisely-defined oscillation waveforms to individual molecularly-defined classes LH cells (orexin, VGAT, MCH, GAD65), while measuring the action potential output of the cells. By modulating the frequency of sinusoidal oscillatory input, we found that high-frequency oscillations (γ, ≈30–200 Hz) preferentially silenced the action potential output orexinLH cells. In contrast, low frequencies (δ-θ, ≈0.5–7 Hz) similarly permitted outputs from different LH cell types. This differential control of orexin and non-orexin cells by oscillation frequency was mediated by cell-specific, impedance-unrelated resonance mechanisms. These results substantiate electrical oscillations as a novel input modality for cell-type-specific control of LH firing, which offers an unforeseen way to control specific cell ensembles within this highly heterogeneous neuronal cluster.

Anatomical and electrophysiological development of the hypothalamic orexin neurons from embryos to neonates.

The amount, quality, and diurnal pattern of sleep change greatly during development. Developmental changes of sleep/wake architecture are in a close relationship to brain development. The fragmentation of wake episodes is one of the salient features in the neonatal period, which is also observed in mature animals and human individuals lacking neuropeptide orexin/hypocretin signaling. This raises the possibility that developmental changes of lateral hypothalamic orexin neurons are relevant to the development of sleep/wake architecture. However, little information is available on morphological and physiological features of developing orexin neurons. To address the cellular basis for maturation of the sleep/wake regulatory system, we investigated the functional development of orexin neurons in the lateral hypothalamus. The anatomical development as well as the changes in the electrophysiological characteristics of orexin neurons was examined from embryonic to postnatal stages in orexin-EGFP mice. Prepro-orexin promoter activity was detectable at embryonic day (E) 12.0, followed by expression of orexin A after E14.0. The number of orexin neurons and their membrane capacitance reached similar levels to adults by postnatal day (P) 7, while their membrane potentials, firing rates, and action potential waveforms were developed by P21. The hyperpolarizing effect of serotonin, which is a major inhibitory signal for adult orexin neurons, was detected after E18.0 and matured at P1. These results suggest that the expression of orexin peptides precedes the maturation of electrophysiological activity of orexin neurons. The function of orexin neurons gradually matures by 3 weeks after birth, coinciding with maturation of sleep/wake architecture.