Targeting the orexin/hypocretin system for the treatment of neuropsychiatric and neurodegenerative diseases: From animal to clinical studies.

Orexins (also known as hypocretins) are neuropeptides located exclusively in hypothalamic neurons that have extensive projections throughout the central nervous system and bind two different G protein-coupled receptors (OX1R and OX2R). Since its discovery in 1998, the orexin system has gained the interest of the scientific community as a potential therapeutic target for the treatment of different pathological conditions. Considering previous basic science research, a dual orexin receptor antagonist, suvorexant, was the first orexin agent to be approved by the US Food and Drug Administration to treat insomnia. In this review, we discuss and update the main preclinical and human studies involving the orexin system with several psychiatric and neurodegenerative diseases. This system constitutes a nice example of how basic scientific research driven by curiosity can be the best route to the generation of new and powerful pharmacological treatments.

Respiratory safety of lemborexant in adult and elderly subjects with moderate-to-severe chronic obstructive pulmonary disease.

Because some hypnotics worsen respiratory conditions, it was important to determine the respiratory safety of lemborexant, a competitive dual orexin-receptor antagonist approved to treat adults with insomnia, in subjects with moderate-to-severe chronic obstructive pulmonary disease. E2006-A001-113 (Study 113; NCT04647383) was a multicentre, multiple-dose, randomised, double-blind, placebo-controlled, two-period crossover study in adult subjects with moderate or severe chronic obstructive pulmonary disease (per spirometry-based Global Initiative for Chronic Obstructive Lung Disease [GOLD] criteria). Subjects (N = 30) were randomised to two treatment sequences comprising 8-night treatment periods (washout ≥ 14 days) with lemborexant 10 mg or placebo. Peripheral oxygen saturation (SpO2; primary endpoint), apnea-hypopnea index, objective sleep parameters and sleep architecture measures were assessed after single (Day 1) and multiple (Day 8) doses. There was no significant difference in least-squares mean SpO2 after a single dose of lemborexant (91.1%) versus placebo (91.5%). Although a statistically significant difference in SpO2 was observed after multiple doses (least-squares mean: lemborexant, 91.3%; placebo, 90.8%) favouring lemborexant, this was not considered clinically meaningful. Apnea-hypopnea index was not significantly different between treatments after single or multiple doses. Total sleep time and total rapid eye movement sleep were significantly greater on Days 1 and 8 with lemborexant versus placebo. Treatment-emergent adverse events were reported in five (16.7%) subjects when taking lemborexant and four (13.3%) subjects when taking placebo; treatment-emergent adverse events were mostly mild. Lemborexant was well tolerated and did not adversely impact SpO2 or apnea-hypopnea index after single and multiple doses relative to placebo in subjects with moderate-to-severe chronic obstructive pulmonary disease.

Current models of insomnia disorder: a theoretical review on the potential role of the orexinergic pathway with implications for insomnia treatment.

Insomnia disorder is considered as a stress-related disorder associated with hyperarousal, stress and emotion dysregulation and the instability of the 'flip-flop' switch system. The orexinergic system is well known for its key role in sleep and arousal processes but also in the allostatic system regulating stress and emotions and may thus be of major interest for insomnia and its treatment. Accordingly, we discuss the potential role of orexins on sleep processes, brain systems modulating stress and emotions with potential implications for insomnia pathophysiology. We reviewed available data on the effect of dual orexin receptor antagonists (DORAs) on sleep and brain systems modulating stress/emotions with implications for insomnia treatment. We present our findings as a narrative review. Few data in animals and humans have reported that disrupted sleep and insomnia may be related to the overactivation of orexinergic system, while some more consistent data in humans and animals reported the overactivation of orexins in response to acute stress and in stress-related disorders. Taken together these findings may let us hypothesise that an orexins overactivation may be associated with stress-related hyperarousal and the hyperactivation of arousal-promoting systems in insomnia. On the other hand, it is possible that by rebalancing orexins with DORAs we may regulate both sleep and allostatic systems, in turn, contributing to a 'switch off' of hyperarousal in insomnia. Nevertheless, more studies are needed to clarify the role of the orexin system in insomnia and to evaluate the effects of DORAs on sleep, stress and emotions regulating systems.

Inhibition of the retinal orexin receptors affects the hypothalamic-pituitary-gonadal axis through retinal pituitary adenylate cyclase activating polypeptide (PACAP) in male Wistar rats.

Orexins A and B (OXA and OXB) and their receptors are expressed in the retina of both human and rodents and play a vital role in regulating signal transmission circuits in the retina. There is an anatomical-physiological relationship between the retinal ganglion cells and suprachiasmatic nucleus (SCN) through glutamate as a neurotransmitter and retinal pituitary adenylate cyclase-activating polypeptide (PACAP) as a co-transmitter. SCN is the main brain center for regulating the circadian rhythm, which governs the reproductive axis. The impact of retinal orexin receptors on the hypothalamic-pituitary-gonadal axis has not been investigated. Retinal OX1R or/and OX2R in adult male rats by 3 µl of SB-334867 (1 µg) or/and 3 µl of JNJ-10397049 (2 µg) were antagonized via intravitreal injection (IVI). Four time-periods were considered (3, 6, 12, and 24 h) for the controls without any treatment, SB-334867, JNJ-10397049, and SB-334867 + JNJ-10397049 groups. Antagonizing retinal OX1R or/and OX2R resulted in a significant elevation of retinal PACAP expression compared to control animals. In addition, expression of GnRH increased non-significantly in the hypothalamus over the 6 h of the study, and the serum concentration of LH decreased significantly in the SB-334867 group after 3 h of injection. Furthermore, testosterone serum levels declined significantly, especially within 3 h of injection; serum levels of progesterone were also exposed to a significant rise at least within 3 h of injection. However, the retinal PACAP expression changes were mediated by OX1R more effectively than by OX2R. In this study, we report the retinal orexins and their receptors as light-independent factors by which the retina affects the hypothalamic-pituitary-gonadal axis.

Does EGFR Signaling Mediate Orexin System Activity in Sleep Initiation?

Sleep-wake cycle disorders are an important symptom of many neurological diseases, including Parkinson's disease, Alzheimer's disease, and multiple sclerosis. Circadian rhythms and sleep-wake cycles play a key role in maintaining the health of organisms. To date, these processes are still poorly understood and, therefore, need more detailed elucidation. The sleep process has been extensively studied in vertebrates, such as mammals and, to a lesser extent, in invertebrates. A complex, multi-step interaction of homeostatic processes and neurotransmitters provides the sleep-wake cycle. Many other regulatory molecules are also involved in the cycle regulation, but their functions remain largely unclear. One of these signaling systems is epidermal growth factor receptor (EGFR), which regulates the activity of neurons in the modulation of the sleep-wake cycle in vertebrates. We have evaluated the possible role of the EGFR signaling pathway in the molecular regulation of sleep. Understanding the molecular mechanisms that underlie sleep-wake regulation will provide critical insight into the fundamental regulatory functions of the brain. New findings of sleep-regulatory pathways may provide new drug targets and approaches for the treatment of sleep-related diseases.

Efficiency of Orexin-A for Inflammatory Flare and Mucosal Healing in Experimental Colitis: Comparison with the Anti-TNF Alpha Infliximab.

Inflammatory bowel diseases are chronic inflammation of the intestinal mucosa characterized by relapsing-remitting cycle periods of variable duration. Infliximab (IFX) was the first monoclonal antibody used for the treatment of Crohn's disease and ulcerative colitis (UC). High variability between treated patients and loss of IFX efficiency over time support the further development of drug therapy. An innovative approach has been suggested based on the presence of orexin receptor (OX1R) in the inflamed human epithelium of UC patients. In that context, the aim of this study was to compare, in a mouse model of chemically induced colitis, the efficacy of IFX compared to the hypothalamic peptide orexin-A (OxA). C57BL/6 mice received 3.5% dextran sodium sulfate (DSS) in drinking water for 5 days. Since the inflammatory flare was maximal at day 7, IFX or OxA was administered based on a curative perspective at that time for 4 days using intraperitoneal injection. Treatment with OxA promoted mucosal healing and decreased colonic myeloperoxidase activity, circulating concentrations of lipopolysaccharide-binding protein, IL-6 and tumor necrosis factor alpha (TNFα) and decreased expression of genes encoding cytokines in colonic tissues with better efficacy than IFX allowing for more rapid re-epithelization. This study demonstrates the comparable anti-inflammatory properties of OxA and IFX and shows that OxA is efficient in promoting mucosal healing, suggesting that OxA treatment is a promising new biotherapy.

Orexin A Suppresses the Expression of Exosomal PD-L1 in Colon Cancer and Promotes T Cell Activity by Inhibiting JAK2/STAT3 Signaling Pathway.

BACKGROUND: Colon cancer, ranked third in cancer related mortality, is the most common malignant cancer of digestive tract. Though immune checkpoint inhibitors show promising efficacy in colon cancer, a rather high unresponsive rate and recurrence rate requires further elucidation of the underlying regulatory mechanism of cancer-related immunity. AIMS: To study the regulatory function of Orexin A in the expression of exosomal PD-L1 and T cell activity. METHODS: Orthotopic colon cancer transplantation mice model were established to study the cancer growth and immune infiltration between Orexin A treated group and untreated group. In vitro studies using mouse CT-26 and human HCT-116 colon cancer cell model studied the effect of Orexin A on cellular and exosomal PD-L1 expression. Co-culturing Jurkat cells with exosomes delivered by cancer cells treated with Orexin A, PD-L1 knockdown and PBS studied different effects on T cell. Comparing Orexin A with WP1066, a JAK2/STAT3 inhibitor verified the mechanism of these changes. RESULTS: The growth rate of orthotopic transplanted colon cancer was slower in Orexin A treated group, with lower PD-L1 expression and higher immune infiltration. Orexin A could inhibit cellular and exosomal PD-L1 expression. The decreased expression of PD-L1 in exosomes could promote the activity of Jurkat cells secreting higher level of IFN-γ and IL-2. Orexin A showed a similar effect like WP1066 which proved JAK2/STAT3 signaling pathway was its downstream signaling pathway. CONCLUSIONS: Orexin A could suppress the expression of exosomal PD-L1 in colon cancer cells and promote T cells activity by inhibiting JAK2/STAT3 signaling pathway.

The premonitory phase of migraine is due to hypothalamic dysfunction: revisiting the evidence.

OBJECTIVE: To critically appraise the evidence for and against premonitory symptoms in migraine being due to hypothalamic dysfunction. DISCUSSION: Some premonitory symptoms (e.g. fatigue, mood changes, yawning, and food craving) are associated with the physiologic effects of neurotransmitters such as orexins, neuropeptide Y, and dopamine; all of which are expressed in hypothalamic neurons. In rodents, electrophysiologic recordings have shown that these neurotransmitters modulate nociceptive transmission at the level of second-order neurons in the trigeminocervical complex (TCC). Additional insights have been gained from neuroimaging studies that report hypothalamic activation during the premonitory phase of migraine. However, the available evidence is limited by methodologic issues, inconsistent reporting, and a lack of adherence to ICHD definitions of premonitory symptoms (or prodromes) in human experimental studies. CONCLUSIONS: The current trend to accept that premonitory symptoms are due to hypothalamic dysfunction might be premature. More rigorously designed studies are needed to ascertain whether the neurobiologic basis of premonitory symptoms is due to hypothalamic dysfunction or rather reflects modulatory input to the trigeminovascular system from several cortical and subcortical areas. On a final note, the available epidemiologic data raises questions as to whether the existence of premonitory symptoms and even more so a distinct premonitory phase is a true migraine phenomenon. Video recording of the debate held at the 1st International Conference on Advances in Migraine Sciences (ICAMS 2022, Copenhagen, Denmark) is available at: https://www.youtube.com/watch?v=d4Y2x0Hr4Q8 .

Circadian actions of orexins on the retinorecipient lateral geniculate complex in rat.

KEY POINTS: Rhythmic processes in living organisms are controlled by biological clocks. The orexinergic system of the lateral hypothalamus carries circadian information to provide arousal for the brain during the active phase. Here, we show that orexins exert an excitatory action in three parts of the lateral geniculate nucleus (LGN), in particular upon directly retinorecipient neurons in the non-image forming visual structures. We provide evidence for the high nocturnal levels of orexins with stable circadian expression of predominant orexin receptor 2 in the LGN. Our data additionally establish the convergence of orexinergic and pituitary adenylate cyclase (PAC)-activating peptide/PAC1 receptor systems (used by melanopsin-expressing retinal ganglion cells), which directly regulates responses to the retinal input. These results help us better understand circadian orexinergic control over the non-image forming subcortical visual system, forming the animal's preparedness for the behaviourally active night. ABSTRACT: The orexinergic system of the lateral hypothalamus is tightly interlinked with the master circadian clock and displays daily variation in activity to provide arousal-related excitation for the plethora of brain structures in a circadian manner. Here, using a combination of electrophysiological, optogenetic, histological, molecular and neuronal tracing methods, we explore a particular link between orexinergic and visual systems in rat. The results of the present study demonstrate that orexinergic fibre density at the area of subcortical visual system exerts a clear day to night variability, reaching a maximum at behaviourally active night. We also show pronounced electrophysiological activations of neurons in the lateral geniculate nucleus by orexin A through 24 h, via identified distinct orexin receptors, with the ventrolateral geniculate displaying a daily cycle of responsiveness. In addition, for the first time, we provide a direct evidence for orexins to act on retinorecipient neurons with a high convergence of orexinergic and putatively retinal pituitary adenylate cyclase (PAC)-activating peptide/PAC1 receptor systems. Altogether, the present study ties orexins to non-image forming visual structures with implications for circadian orexinergic modulation of neurons, which process information on ambient light levels.

Orexins (hypocretins): The intersection between homeostatic and hedonic feeding.

Orexins are hypothalamic neuropeptides originally discovered to play a role in the regulation of feeding behaviour. The broad connections of orexin neurons to mesocorticolimbic circuitry suggest they may play a role in mediating reward-related behaviour beyond homeostatic feeding. Here, we review the role of orexin in a variety of eating-related behaviour, with a focus on reward and motivation, and the neural circuits driving these effects. One emerging finding is the involvement of orexins in hedonic and appetitive behaviour towards palatable food, in addition to their role in homeostatic feeding. This review discusses the brain circuitry and possible mechanisms underlying the role of orexins in these behaviours. Overall, there is a marked bias in the literature towards studies involving male subjects. As such, future work needs to be done to involve female subjects. In summary, orexins play an important role in driving motivation for high salient rewards such as highly palatable food and may serve as the intersection between homeostatic and hedonic feeding.

Orexin Modulation of VTA Dopamine Neuron Activity: Relevance to Schizophrenia.

BACKGROUND: The hippocampus is a region consistently implicated in schizophrenia and has been advanced as a therapeutic target for positive, negative, and cognitive deficits associated with the disease. Recently, we reported that the paraventricular nucleus of the thalamus (PVT) works in concert with the ventral hippocampus to regulate dopamine system function; however, the PVT has yet to be investigated as target for the treatment of the disease. Given the dense expression of orexin receptors in the thalamus, we believe these to be a possible target for pharmacological regulation of PVT activity. METHODS: Here we used the methylazoxymethanol acetate (MAM) rodent model, which displays pathological alterations consistent with schizophrenia to determine whether orexin receptor blockade can restore ventral tegmental area dopamine system function. We measured dopamine neuron population activity, using in vivo electrophysiology, following administration of the dual orexin antagonist, TCS 1102 (both intraperitoneal and intracranial into the PVT in MAM- and saline-treated rats), and orexin A and B peptides (intracranial into the PVT in naïve rats). RESULTS: Aberrant dopamine system function in MAM-treated rats was normalized by the systemic administration of TCS 1102. To investigate the potential site of action, the orexin peptides A and B were administered directly into the PVT, where they significantly increased ventral tegmental area dopamine neuron population activity in control rats. In addition, the direct administration of TCS 1102 into the PVT reproduced the beneficial effects seen with the systemic administration in MAM-treated rats. CONCLUSION: Taken together, these data suggest the orexin system may represent a novel site of therapeutic intervention for psychosis via an action in the PVT.

Orexin B affects the transcriptome of incubated in vitro porcine endometrial explants from the early-implantation period.

This study determined the effect of orexin B (OXB) on the porcine endometrial transcriptome during the embryo attachment phase. Microarray analyses of gene ontology (GO), biological pathways, networks and differentially expressed genes (DEG) were performed. Orexin B influenced the expression of 887 genes (fold change > 1.2; p < .05): 620 genes were up-regulated, and 267 were down-regulated. The analysis of the relationship between DEG revealed that OXB interacts with genes linked with processes such as cell hormone binding, regulation of hormone levels, lipid transport, steroid metabolic processes, the apoptotic signalling pathway and the acute inflammatory response, which are pivotal for reproductive success. Orexin B played a bivalent role in the early-pregnant uterus by limiting the pregnancy outcome, promoting embryo development, suppressing the immune system and, consequently, preventing embryo rejection. These findings suggest that OXB could be responsible for the proper course of gestation by adapting litter size to the metabolic status of the maternal organism.

Migraine and Sleep-An Unexplained Association?

Migraine and sleep disorders are common chronic diseases in the general population, with significant negative social and economic impacts. The association between both of these phenomena has been observed by clinicians for years and is confirmed by many epidemiological studies. Despite this, the nature of this relationship is still not fully understood. In recent years, there has been rapid progress in understanding the common anatomical structures of and pathogenetic mechanism between sleep and migraine. Based on a literature review, the authors present the current view on this topic as well as ongoing research in this field, with reference to the key points of the biochemical and neurophysiological processes responsible for both these disorders. In the future, a better understanding of these mechanisms will significantly expand the range of treatment options.

Orexins/Hypocretins and Cancer: A Neuropeptide as Emerging Target.

Over 20 years ago, orexin neuropeptides (Orexin-A/hypocretin-1 and Orexin-B/hypocretins-2) produced from the same precursor in hypothalamus were identified. These two neurotransmitters and their receptors (OX1R and OX1R), present in the central and peripheral nervous system, play a major role in wakefulness but also in drug addiction, food consumption, homeostasis, hormone secretion, reproductive function, lipolysis and blood pressure regulation. With respect to these biological functions, orexins were involved in various pathologies encompassing narcolepsy, neurodegenerative diseases, chronic inflammations, metabolic syndrome and cancers. The expression of OX1R in various cancers including colon, pancreas and prostate cancers associated with its ability to induce a proapoptotic activity in tumor cells, suggested that the orexins/OX1R system could have a promising therapeutic role. The present review summarizes the relationship between cancers and orexins/OX1R system as an emerging target.

The Discovery of Suvorexant, the First Orexin Receptor Drug for Insomnia.

Historically, pharmacological therapies have used mechanisms such as γ-aminobutyric acid A (GABAA) receptor potentiation to drive sleep through broad suppression of central nervous system activity. With the discovery of orexin signaling loss as the etiology underlying narcolepsy, a disorder associated with hypersomnolence, orexin antagonism emerged as an alternative approach to attenuate orexin-induced wakefulness more selectively. Dual orexin receptor antagonists (DORAs) block the activity of orexin 1 and 2 receptors to both reduce the threshold to transition into sleep and attenuate orexin-mediated arousal. Among DORAs evaluated clinically, suvorexant has pharmacokinetic properties engineered for a plasma half-life appropriate for rapid sleep onset and maintenance at low to moderate doses. Unlike GABAA receptor modulators, DORAs promote both non-rapid eye movement (NREM) and REM sleep, do not disrupt sleep stage-specific quantitative electroencephalogram spectral profiles, and allow somnolence indistinct from normal sleep. The preservation of cognitive performance and the ability to arouse to salient stimuli after DORA administration suggest further advantages over historical therapies.

Orexin A alleviates neuroinflammation via OXR2/CaMKKß/AMPK signaling pathway after ICH in mice.

BACKGROUND: Orexins are two neuropeptides (orexin A, OXA; orexin B, OXB) secreted mainly from the lateral hypothalamus, which exert a wide range of physiological effects by activating two types of receptors (orexin receptor 1, OXR1; orexin receptor 2, OXR2). OXA has equal affinity for OXR1 and OXR2, whereas OXB binds preferentially to OXR2. OXA rapidly crosses the blood-brain barrier by simple diffusion. Many studies have reported OXA's protective effect on neurological diseases via regulating inflammatory response which is also a fundamental pathological process in intracerebral hemorrhage (ICH). However, neuroprotective mechanisms of OXA have not been explored in ICH. METHODS: ICH models were established using stereotactic injection of autologous arterial blood into the right basal ganglia of male CD-1 mice. Exogenous OXA was administered intranasally; CaMKKß inhibitor (STO-609), OXR1 antagonist (SB-334867), and OXR2 antagonist (JNJ-10397049) were administered intraperitoneally. Neurobehavioral tests, hematoma volume, and brain water content were evaluated after ICH. Western blot and ELISA were utilized to evaluate downstream mechanisms. RESULTS: OXA, OXR1, and OXR2 were expressed moderately in microglia and astrocytes and abundantly in neurons. Expression of OXA decreased whereas OXR1 and OXR2 increased after ICH. OXA treatment significantly improved not only short-term but also long-term neurofunctional outcomes and reduced brain edema in ipsilateral hemisphere. OXA administration upregulated p-CaMKKß, p-AMPK, and anti-inflammatory cytokines while downregulated p-NFκB and pro-inflammatory cytokines after ICH; this effect was reversed by STO-609 or JNJ-10397049 but not SB-334867. CONCLUSIONS: OXA improved neurofunctional outcomes and mitigated brain edema after ICH, possibly through alleviating neuroinflammation via OXR2/CaMKKß/AMPK pathway.

Effect of ambient temperature on sleep breathing phenotype in mice: the role of orexins.

The loss of orexinergic neurons, which release orexins, results in narcolepsy. Orexins participate in the regulation of many physiological functions, and their role as wake-promoting molecules has been widely described. Less is known about the involvement of orexins in body temperature and respiratory regulation. The aim of this study was to investigate if orexin peptides modulate respiratory regulation as a function of ambient temperature (Ta) during different sleep stages. Respiratory phenotype of male orexin knockout (KO-ORX, N=9) and wild-type (WT, N=8) mice was studied at thermoneutrality (Ta=30°C) or during mild cold exposure (Ta=20°C) inside a whole-body plethysmography chamber. The states of wakefulness (W), non-rapid eye movement sleep (NREMS) and rapid eye movement sleep (REMS) were scored non-invasively, using a previously validated technique. In both WT and KO-ORX mice, Ta strongly and significantly affected ventilatory period and minute ventilation values during NREMS and REMS; moreover, the occurrence rate of sleep apneas in NREMS was significantly reduced at Ta=20°C compared with Ta=30°C. Overall, there were no differences in respiratory regulation during sleep between WT and KO-ORX mice, except for sigh occurrence rate, which was significantly increased at Ta=20°C compared with Ta=30°C in WT mice, but not in KO-ORX mice. These results do not support a main role for orexin peptides in the temperature-dependent modulation of respiratory regulation during sleep. However, we showed that the occurrence rate of sleep apneas critically depends on Ta, without any significant effect of orexin peptides.

Orexins and stress.

The neuropeptides orexins are important in regulating the neurobiological systems that respond to stressful stimuli. Furthermore, orexins are known to play a role many of the phenotypes associated with stress-related mental illness such as changes in cognition, sleep-wake states, and appetite. Interestingly, orexins are altered in stress-related psychiatric disorders such as Major Depressive Disorder and Anxiety Disorders. Thus, orexins may be a potential target for treatment of these disorders. In this review, we will focus on what is known about the role of orexins in acute and repeated stress, in stress-induced phenotypes relevant to psychiatric illness in preclinical models, and in stress-related psychiatric illness in humans. We will also briefly discuss how orexins may contribute to sex differences in the stress response and subsequent phenotypes relevant to mental health, as many stress-related psychiatric disorders are twice as prevalent in women.

Orexin A depolarises rat intergeniculate leaflet neurons through non-selective cation channels.

Orexins/hypocretins are hypothalamic neuropeptides that have a variety of functions, including maintenance of arousal, control over the sleep/wake cycle, reward and feeding. Accumulating evidence links orexins to the time-keeping system with a documented action in the master clock-the suprachiasmatic nucleus. The intergeniculate leaflet (IGL) is a thalamic structure with the well-known function of collecting photic and non-photic cues to adjust the rhythm of the suprachiasmatic nucleus to changing environmental conditions. The IGL consists of GABAergic neurons that are intrinsically active, even in slice preparations. Our previous studies revealed the excitatory postsynaptic effects of orexins on single IGL neurons, even though the ionic mechanism underlying this effect remained elusive. Therefore, in this study, we used patch clamp electrophysiology to identify the ions and distinct ion channels responsible for the observed depolarisations. The major finding of this article is that the orexin A-evoked depolarisation of IGL neurons depends on non-selective cation channels, implicating the orexinergic tone in establishing the basal firing rate in these cells. The data presented here strengthen the mutual connections between the time-keeping and orexinergic systems.

Orexins increase the firing activity of nigral dopaminergic neurons and participate in motor control in rats.

Orexin is a member of neuropeptides which is involved in the central motor control. The substantia nigra pars compacta (SNc) is an important nucleus participating in motor control under both physiological and pathological conditions. Morphological studies reveal that orexinergic neurons located in lateral hypothalamus innervate the SNc. Both orexin-1 receptors (OX1 R) and orexin-2 receptors (OX2 R) are expressed in the SNc. To investigate the effects of orexins on SNc, single unit in vivo extracellular recordings and behavioral tests were performed in this study. Micro-pressure administration of orexin A and orexin B significantly increased the spontaneous firing rate of nigral DAergic neurons by 65.87 ± 7.73% and 90.49 ± 17.83%, respectively. The excitatory effects of orexin A on nigral DAergic neurons were mainly mediated by OX1 R, while OX2 R were involved in the increase in firing rate induced by orexin B. Selectively blocking OX1 R and OX2 R significantly decreased the firing rate of nigral DAergic neurons by 36.77 ± 6.26% and 32.04 ± 6.12%, respectively, which suggested that endogenous orexins modulated the spontaneous firing activity of nigral DAergic neurons. Finally, both elevated body swing test and haloperidol-induced postural behavioral test showed that unilateral microinjection of orexin A and orexin B induced significantly contralateral-biased swing and deflection behavior. Meanwhile, the specific OX1 R and OX2 R antagonists produced opposite effects. The present electrophysiological and behavioral studies suggested that orexins increased the firing activity of nigral DAergic neurons and participated in central motor control. Open Practices Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/.