Lateral Hypothalamus Orexin Research Articles

Mediation of lateral hypothalamus orexin input to lateral habenula in the inhibitory effects of mechanical stimulation on psychomotor responses induced by cocaine.

The lateral hypothalamus (LH) plays an important physiological role in brain function and also plays an important role in substance abuse. The neuropeptides called orexin (or hypocretins) have been identified as being located exclusively in the cell bodies of the LH. Our previous studies have demonstrated that mechanical stimulation (MS) of the ulnar nerve produces strong inhibitory effects on cocaine addiction-like behaviors through activation of LH projection to the lateral habenula (LHb). Therefore, the present study hypothesized that ulnar MS would suppress the psychomotor responses induced by cocaine through the orexinergic LH-to-LHb pathway. Ulnar MS attenuated cocaine enhancement of locomotor activity and 50-kHz ultrasonic vocalizations, which was prevented by antagonism of orexin-receptor type 2 (OX2R) in the LHb. Injection of orexin-A into the LHb reduced the cocaine-induced psychomotor responses. MS of the ulnar nerve excited LH orexinergic neurons. In addition, the excitation of LHb neurons by MS was blocked by the systemic administration of an OX2R antagonist. These findings suggest that MS applied to the ulnar nerve recruits an orexinergic LH-to-LHb pathway to suppress the psychomotor responses induced by cocaine.

Melanin-concentrating hormone and orexin shape social affective behavior via action in the insular cortex of rat.

In a social context, individuals are able to detect external information from others and coordinate behavioral responses according to the situation, a phenomenon called social decision-making. Social decision-making is multifaceted, influenced by emotional and motivational factors like stress, sickness, and hunger. However, the neurobiological basis for motivational state competition and interaction is not well known. We investigated possible neural mechanisms through which internal states could shape social behavior in a social affective preference (SAP) test. In the SAP test, experimental rats given a choice to interact with naïve or stressed conspecifics exhibit an age-dependent preference to interact with stressed juvenile conspecifics, but avoid stressed adult conspecifics. First, we assessed the effect of food and water deprivation on SAP behavior. Behavior in the SAP test requires the insular cortex, which receives input from the ingestion-related peptides melanin-concentrating hormone (MCH) and orexin neurons of the lateral hypothalamus (LH). This study aimed to evaluate the role of LH and insular MCH and orexin in SAP test. SAP tests were conducted in rats that were sated, food and water deprived or allowed 1 h of access to food and water after 14 h of deprivation (relieved condition). Separate cohorts of sated rats received cannula implants for microinjection of drugs to inhibit the LH or to block or stimulate MCH or orexin receptors in the insula prior to SAP tests or social interaction tests. Food and water deprivation prior to SAP tests with juvenile rats caused a shift in preference away from the stressed rat toward the naïve juveniles. Pharmacological inhibition of LH with muscimol (100 ng/side) abolished the preference for the juvenile-stressed conspecific, as well as the preference for the adult naïve conspecific. The blockade of MCH receptor 1or orexin receptors in the insular cortex with SNAP94847 (50 μM) or TCS1102 (1 μM), respectively, also abolished the preference for the stressed juvenile conspecific, but only the antagonism of orexin receptors was able to abolish the preference for the adult naïve conspecific. Microinjection of increasing doses (50 or 500 nM) of MCH or orexin-A in the insular cortex increased the interaction time in the one-on-one social interaction test with juvenile conspecifics; however, only the microinjection of orexin-A increased the interaction time with adult naïve conspecifics. Taken together, these results suggest that lateral hypothalamus peptides shape the direction of social approach or avoidance via actions MCH and orexin neurotransmission in the insular cortex.

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.

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.

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.

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.

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.

Significant Quantitative Differences in Orexin Neuronal Activation After Pain Assessments in an Animal Model of Sickle Cell Disease.

Sickle cell disease is a hemoglobinopathy that causes sickling of red blood cells, resulting in vessel blockage, stroke, anemia, inflammation, and extreme pain. The development and treatment of pain, in particular, neuropathic pain in sickle cell disease patients is poorly understood and impedes our progress toward the development of novel therapies to treat pain associated with sickle cell disease. The orexin/hypocretin system offers a novel approach to treat chronic pain and hyperalgesia. These neuropeptides are synthesized in three regions: perifornical area (PFA), lateral hypothalamus (LH), and dorsomedial hypothalamus (DMH). Data suggest that orexin–A neuropeptide has an analgesic effect on inflammatory pain and may affect mechanisms underlying the maintenance of neuropathic pain. The purpose of this study was to determine whether there are neuronal activation differences in the orexin system as a result of neuropathic pain testing in a mouse model of sickle cell disease. Female transgenic sickle mice that express exclusively (99%) human sickle hemoglobin (HbSS-BERK) and age-/gender-matched controls (HbAA-BERK mice; n = 10/group, 20–30 g) expressing normal human hemoglobin A were habituated to each test protocol and environment before collecting baseline measurements and testing. Four measures were used to assess pain-related behaviors: thermal/heat hyperalgesia, cold hyperalgesia, mechanical hyperalgesia, and deep-tissue hyperalgesia. Hypothalamic brain sections from HbAA-BERK and HbSS-BERK mice were processed to visualize orexin and c-Fos immunoreactivity and quantified. The percentage of double labeled neurons in the PFA was significantly higher than the percentage of double labeled neurons in the LH orexin field of HbAA-BERK mice (*p < 0.05). The percentages of double labeled neurons in PFA and DMH orexin fields are significantly higher than those neurons in the LH of HbSS-BERK mice (*p < 0.05). These data suggest that DMH orexin neurons were preferentially recruited during neuropathic pain testing and a more diverse distribution of orexin neurons may be required to produce analgesia in response to pain in the HbSS-BERK mice. Identifying specific orexin neuronal populations that are integral in neuropathic pain processing will allow us to elucidate mechanisms that provide a more selective, targeted approach in treating of neuropathic pain in sickle cell disease.

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.

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.

Cue-induced food seeking after punishment is associated with increased Fos expression in the lateral hypothalamus and basolateral and medial amygdala.

In humans, relapse to unhealthy eating habits following dieting is a significant impediment to obesity treatment. Food-associated cues are one of the main triggers of relapse to unhealthy eating during self-imposed abstinence. Here we report a behavioral method examining cue-induced relapse to food seeking following punishment-induced suppression of food taking. We trained male rats to lever press for food pellets that were delivered after a 10-s conditional stimulus (CS) (appetitive). Following training, 25% of reinforced lever presses resulted in the presentation of a compound stimulus consisting of a novel CS (aversive) and the appetitive CS followed by a pellet and footshock. After punishment-imposed abstinence, we tested the rats in an extinction test where lever pressing resulted in the presentation of either the appetitive or aversive CS. We then compared activity of lateral hypothalamus (LH) and associated extrahypothalamic regions following this test. We also assessed Fos expression in LH orexin and GABA neurons. We found that cue-induced relapse of food seeking on test was higher in rats tested with the appetitive CS compared to the aversive CS. Relapse induced by the appetitive CS was associated with increased Fos expression in LH, caudal basolateral amygdala (BLA), and medial amygdala (MeA). This relapse was also associated with increased Fos expression in LH orexin and VGAT-expressing neurons. These data show that relapse to food seeking can be induced by food-associated cues after punishment-imposed abstinence, and this relapse is associated with increased activity in LH, caudal BLA, and MeA. (PsycINFO Database Record

Lateral hypothalamus orexinergic system modulates the stress effect on pentylenetetrazol induced seizures through corticotropin releasing hormone receptor type 1

Stress is a trigger factor for seizure initiation which activates hypothalamic pituitary adrenal (HPA) axis as well other brain areas. In this respect, corticotropin releasing hormone (CRH) and lateral hypothalamus (LH) orexinergic system are involved in seizure occurrence. In this study, we investigated the role of LH area and orexin expression in (mediation of) stress effect on pentylenetetrazol (PTZ) -induced seizures with hippocampal involvement.Two mild foot shock stresses were applied to intact and adrenalectomized animals; with or without CRHr1 blocking (NBI 27914) in the LH area. Then, changes in orexin production were evaluated by RT-PCR. Intravenous PTZ infusion (25 mg/ml) -induced convulsions were scored upon modified Racine scale. Finally, hippocampal glutamate and GABA were evaluated to study excitability changes.We demonstrated that the duration and severity of convulsions in stress-induced as well as adrenalectomized group were increased. Plasma corticosterone (CRT) level and orexin mRNA expression were built up in the stress and/or seizure groups. Furthermore, glutamate and GABA content was increased and decreased respectively due to stress and seizures. In contrast, rats receiving CRHr1 inhibitor showed reduced severity and duration of seizures, increased GABA, decreased glutamate and corticosterone and also orexin mRNA compared to the inhibitor free rats.Stress and adrenalectomy induced augmenting effect on seizure severity and duration and the subsequent reduction due to CRHr1 blocking with parallel orexin mRNA changes, indicated the likely involvement of CRH1r induced orexin expression of the LH in gating stress effect on convulsions.

Hippocampus ghrelin signaling mediates appetite through lateral hypothalamic orexin pathways.

Feeding behavior rarely occurs in direct response to metabolic deficit, yet the overwhelming majority of research on the biology of food intake control has focused on basic metabolic and homeostatic neurobiological substrates. Most animals, including humans, have habitual feeding patterns in which meals are consumed based on learned and/or environmental factors. Here we illuminate a novel neural system regulating higher-order aspects of feeding through which the gut-derived hormone ghrelin communicates with ventral hippocampus (vHP) neurons to stimulate meal-entrained conditioned appetite. Additional results show that the lateral hypothalamus (LHA) is a critical downstream substrate for vHP ghrelin-mediated hyperphagia and that vHP ghrelin activated neurons communicate directly with neurons in the LHA that express the neuropeptide, orexin. Furthermore, activation of downstream orexin-1 receptors is required for vHP ghrelin-mediated hyperphagia. These findings reveal novel neurobiological circuitry regulating appetite through which ghrelin signaling in hippocampal neurons engages LHA orexin signaling.

Orexin receptor type-1 antagonist SB-334867 decreases morphine-induced antinociceptive effect in formalin test

Orexin-A and orexin-B are two neuropeptides selectively synthesized in the lateral hypothalamus (LH), a region involved in morphine induced analgesia and pain modulation. Furthermore, orexin-A has been reported to produce an analgesic effect in pain models, which was blocked by orexin-1 receptor antagonist SB-334867, but not naloxone. We studied the effects of intracerebroventricular (ICV) injection of SB-334867, a selective orexin receptor type-1 antagonist, on morphine-induced antinociceptive effect in formalin test in rats. Morphine injection at a dose of 1.5mg/kg caused a significant decrease in the formalin-induced nociceptive behaviors in phase 1, interphase, and phase 2A, whereas at doses of 3, 6, and 10mg/kg, a significant reduction in the formalin-induced nociceptive behaviors was observed in all phases. The ICV injection of SB-334867 alone had no effect on the formalin-induced nociceptive behaviors. Pre-treatment with SB-334867 at a dose of 0.5nmol significantly attenuated the analgesia induced by morphine (at dose 1.5mg/kg of morphine; interphase and phase 2B and at dose 3mg/kg of morphine just phase 2B of formalin test). Also, pre-treatment with SB-334867 at a dose of 5nmol considerably attenuated the morphine-induced analgesia (at dose 1.5mg/kg of morphine; phase 1, interphase, and phase 2, at dose 3 and 6mg/kg of morphine just phase 2 of formalin test). Pre-treatment with SB-334867 at a dose of 50nmol remarkably attenuated the morphine-induced analgesia (at dose 1.5 and 3mg/kg of morphine; in phase 1, interphase, and phase 2 and also at dose 6mg/kg of morphine; phase 1 and phase 2B of formalin test). These data suggest that the antinociceptive effects of morphine in formalin test might be associated with orexin receptor type-1. Our findings reveal a new role for the lateral hypothalamus orexin neurons in the morphine-induced analgesia.

Regulation of the ventral tegmental area by the bed nucleus of the stria terminalis is required for expression of cocaine preference

Lateral hypothalamus (LH) orexin neurons are essential for the expression of a cocaine place preference. However, the afferents that regulate the activity of these orexin neurons during reward behaviors are not completely understood. Using tract tracing combined with Fos staining, we examined LH afferents for Fos induction during cocaine preference in rats. We found that the ventral bed nucleus of the stria terminalis (vBNST) was a major input to the LH orexin cell field that was significantly Fos-activated during cocaine conditioned place preference (CPP). Inactivation of the vBNST with baclofen plus muscimol blocked expression of cocaine CPP. Surprisingly, such inactivation of the vBNST also increased Fos induction in LH orexin neurons; as activity in these cells is normally associated with increased preference, this result indicates that a vBNST-orexin connection is unlikely to be responsible for CPP that is dependent on vBNST activity. Because previous studies have revealed that vBNST regulates dopamine cells in the ventral tegmental area (VTA), which is known to be involved in CPP and other reward functions, we tested whether vBNST afferents to the VTA are necessary for cocaine CPP. We found that disconnection of the vBNST and VTA (using local microinjections of baclofen plus muscimol unilaterally into the vBNST and contralateral VTA) significantly attenuated expression of cocaine preference. However, blocking ionotropic glutamatergic afferents to the VTA from the vBNST did not significantly reduce cocaine preference. These results indicate that a non-glutamatergic vBNST-VTA projection is involved in expression of cocaine preference.

A septal-hypothalamic pathway drives orexin neurons, which is necessary for conditioned cocaine preference.

Orexins (also called hypocretins) have been shown to be importantly involved in reward and addiction, but little is known about the circuitry that regulates orexin neuronal activity during drug-seeking behaviors. Here, we examined inputs to the lateral hypothalamus (LH) orexin cell field from the lateral septum (LS) using tract-tracing and Fos immunohistochemistry after cocaine (10 mg/kg) conditioned place preference (CPP) in Sprague Dawley rats. We found that neurons in rostral LS (LSr) that project to LH are Fos-activated in proportion to cocaine CPP, and that inhibition of LSr neurons with local baclofen and muscimol microinjection (0.3/0.03 nmol) blocks expression of Fos in LH orexin cells and cocaine preference. In addition, using local inactivation in LS and orexin antisense morpholinos in LH, we found that LSr influences on LH orexin neurons are critical for the expression of cocaine preference. These results indicate that LSr activates LH orexin neurons during cocaine place preference, and that this circuit is essential for expression of cocaine place preference.

Role of orexin/hypocretin in reward-seeking and addiction: Implications for obesity

Orexins (also named hypocretins) are recently discovered neuropeptides made exclusively in the hypothalamus. Recent studies have shown that orexin cells located specifically in lateral hypothalamus (LH) are involved in motivated behavior for drugs of abuse as well as natural rewards. Administration of orexin has been shown to stimulate food consumption, and orexin signaling in VTA has been implicated in intake of high-fat food. In self-administration studies, the orexin 1 receptor antagonist SB-334867 (SB) attenuated operant responding for high-fat pellets, sucrose pellets and ethanol, but not cocaine, demonstrating that signaling at orexin receptors is necessary for reinforcement of specific rewards. The orexin system is also implicated in associations between rewards and relevant stimuli. For example, Fos expression in LH orexin neurons varied in proportion to conditioned place preference (CPP) for food, morphine, or cocaine. This Fos expression was altered accordingly for CPP administered during protracted abstinence from morphine or cocaine, when preference for natural rewards was decreased and drug preference was increased. Additionally, orexin has been shown to be involved in reward–stimulus associations in the self-administration paradigm, where SB attenuated cue-induced reinstatement of extinguished sucrose- or cocaine-seeking. Although the specific circuitry mediating the effects of orexin on food reward remains unknown, VTA seems likely to be a critical target for at least some of these orexin actions. Thus, recent studies have established a role for orexin in reward-based feeding, and further investigation is warranted for determining whether function/dysfunction of the orexin system may contribute to the overeating associated with obesity. The paper represents an invited review by a symposium, award winner or keynote speaker at the Society for the Study of Ingestive Behavior [SSIB] Annual Meeting in Portland, July 2009.

Lateral hypothalamic orexin/hypocretin neurons: A role in reward-seeking and addiction

Orexins (synonymous with hypocretins) are recently discovered neuropeptides made exclusively in hypothalamus. Behavioral, anatomical, and neurophysiological studies show that a subset of these cells, specifically those in lateral hypothalamus (LH), are involved in reward processing and addictive behaviors. Fos expression in LH orexin neurons varied in proportion to conditioned place preference (CPP) for morphine, cocaine, or food. This relationship occurred both in drug-naïve rats and in animals during protracted morphine withdrawal, when drug preference was elevated but food preference was decreased. Inputs to the LH orexin cell field from lateral septum and bed nucleus of the stria terminalis were Fos-activated during cocaine CPP in proportion to the preference expressed in each animal. This implies that these inputs may be involved in driving the conditioned responses in LH orexin neurons. Related studies showed that LH orexin neurons that project to ventral tegmental area (VTA) had greater Fos induction in association with elevated morphine preference during protracted withdrawal than non-VTA-projecting orexin neurons, indicating that the VTA is an important site of action for orexin's role in reward processing. In addition, stimulation of LH orexin neurons, or microinjection of orexin into VTA, reinstated an extinguished morphine preference. In self-administration studies, the orexin 1 receptor antagonist SB-334867 (SB) blocked cocaine-seeking induced by discrete or contextual cues previously associated with cocaine, but not by a priming injection of cocaine. There was no effect of SB on cocaine self-administration itself, indicating that it did not interfere with the drug's reinforcing properties. Neurophysiological studies revealed that locally applied orexin often augmented responses of VTA dopamine (DA) neurons to activation of the medial prefrontal cortex (mPFC), consistent with the view that orexin facilitates activation of VTA DA neurons by stimulus–reward associations. This LH-to-VTA orexin pathway was found to be necessary for learning a morphine place preference. These findings are consistent with results showing that orexin facilitates glutamate-mediated responses, and is necessary for glutamate-dependent long-term potentiation in VTA DA neurons. We surmise from these studies that LH orexin neurons play an important role in reward processing and addiction and that LH orexin cells are an important input to VTA for behavioral effects associated with reward-paired stimuli.