Orexin neurons play contrasting roles in itch and pain neural processing via projecting to the periaqueductal gray.

Pain and itch are recognized as antagonistically regulated sensations; pain suppresses itch, whilst pain inhibition enhances itch. The neural mechanisms at the central nervous system (CNS) underlying these pain-itch interactions still need to be explored. Here, we revealed the contrasting role of orexin-producing neurons (ORX neurons) in the lateral hypothalamus (LH), which suppresses pain while enhancing itch neural processing, by applying optogenetics to the acute pruritus and pain model. We also revealed that the circuit of ORX neurons from LH to periaqueductal gray regions served in the contrasting modulation of itch and pain processing using optogenetic terminal inhibition techniques. Additionally, by using an atopic dermatitis model, we confirmed the involvement of ORX neurons in regulating chronic itch processing, which could lead to a novel therapeutic target for persistent pruritus in clinical settings. Our findings provide new insight into the mechanism of antagonistic regulation between pain and itch in the CNS.

Activation of neurons in the insular cortex and lateral hypothalamus during food anticipatory period caused by food restriction in mice.

Mice fed a single meal daily at a fixed time display food anticipatory activity (FAA). It has been reported that the insular cortex (IC) plays an essential role in food anticipation, and lateral hypothalamus (LH) regulates the expression of FAA. However, how these areas contribute to FAA production is still unclear. Thus, we examined the temporal and spatial activation pattern of neurons in the IC and LH during the food anticipation period to determine their role in FAA establishment. We observed an increase of c-Fos-positive neurons in the IC and LH, including orexin neurons of male adult C57BL/6 mice. These neurons were gradually activated from the 1st day to 15th day of restricted feeding. The activation of these brain regions, however, peaked at a distinct point in the food restriction procedure. These results suggest that the IC and LH are differently involved in the neural network for FAA production.

The physiological response during optogenetic-based cardiac pacing in awake freely moving mice.

There are several methods to control a heart rate, such as electrical stimulation and drug administration. However, these methods may be invasive or affect other organs. Recently, an optogenetic-based cardiac pacing method has enabled us to stimulate the cardiac muscle in non-contact. In many previous studies, the pacing was applied ex vivo or in anesthetized animals. Therefore, the physiologic response of animals during optogenetic pacing remains unclear. Here, we established a method of optogenetic-based cardiac pacing in awake, freely moving mice and simultaneously measured electrocardiogram, blood pressure, and respiration. As a result, light-induced myocardial contraction produces blood flow and indirectly affects the respiration rhythm. Additionally, light illumination enabled heart rate recovery in bradycardic mice. These findings may be employed for further research that relates a heartbeat state to animal behavior. Together, this method may drive the development of less invasive pacemakers without pacing leads.

TRPA1 channel in the airway underlies protection against airborne threats by modulating respiration and behaviour.

Transient receptor potential ankyrin 1 (TRPA1), a member of the TRP superfamily of cation channels, is broadly expressed in sensory neural pathways, including the trigeminal neurons innervating the nasal cavity and vagal neurons innervating the trachea and the lung. TRPA1 acts as a detector of various irritant chemicals as well as hypoxia and hyperoxia. For the past 15 years, we have characterised its role in respiratory and behavioural modulation in vivo using Trpa1 knockout (KO) mice and wild-type (WT) littermates. Trpa1 KO mice failed to detect, wake up from sleeping, and escape from formalin vapour and a mild hypoxic (15% O2 ) environment. Respiratory augmentation induced by mild hypoxia was absent in either Trpa1 KO mice or WT mice treated with a TRPA1 antagonist. Irritant gas introduced into the nasal cavity inhibited respiratory responses in WT mice but not in the KO mice. The effect of TRPA1 on the olfactory system seemed minimal because olfactory bulbectomized WT mice reacted similarly to the intact mice. Immunohistological analyses using a cellar activation marker, the phosphorylated form of extracellular signal-regulated kinase, confirmed activation of trigeminal neurons in WT mice but not in Trpa1 KO mice in response to irritant chemicals and mild hypoxia. These data collectively show that TRPA1 is necessary for multiple chemical-induced protective responses in respiration and behaviour. We propose that TRPA1 channels in the airway may play a sentinel role for environmental threats and prevent incoming damage.

Activation of the rostral nucleus accumbens shell by optogenetics induces cataplexy-like behavior in orexin neuron-ablated mice.

Cataplexy is one of the symptoms of type 1 narcolepsy, characterized by a sudden loss of muscle tone. It can be seen as a behavioral index of salience, predominantly positive emotion, since it is triggered by laughter in humans and palatable foods in mice. In our previous study using chemogenetic techniques in narcoleptic mice (orexin neuron-ablated mice), we found that the rostral nucleus accumbens (NAc) shell is needed for chocolate-induced cataplexy. In this study, we investigated whether a short-lasting stimulation/inhibition of the NAc by optogenetics led to a similar result. Photo-illumination to the NAc in the channel rhodopsin-expressing mice showed a higher incidence (34.9 ± 5.1%) of cataplexy-like behavior than the control mice (17.8 ± 3.1%, P = 0.0056). Meanwhile, inactivation with archaerhodopsin did not affect incidence. The episode duration of cataplexy-like behavior was not affected by activation or inactivation. Immunohistochemical analysis revealed that photo-illumination activated channel rhodopsin-expressing NAc shell neurons. Thus, activation of the NAc, whether transient (light stimulation) or longer-lasting (chemical stimulation in our previous study), facilitates cataplexy-like behaviors and contributes to the induction but not maintenance in them. On the other hand, our study's result from optogenetic inhibition of the NAc (no effect) was different from chemogenetic inhibition (reduction of cataplexy-like behavior) in our previous study. We propose that the initiation of cataplexy-like behavior is facilitated by activation of the NAc, while NAc-independent mechanisms determine the termination of the behavior.

The opposite roles of orexin neurons in pain and itch neural processing.

Pain and itch are antagonistically regulated sensations; pain suppresses itch, and inhibition of pain enhances itch. Understanding the central neural circuit of antagonistic regulation between pain and itch is required to develop new therapeutics better to manage these two feelings in a clinical situation. However, evidence of the neural mechanism underlying the pain-itch interaction in the central nervous system (CNS) is still insufficient. To pave the way for this research area, our laboratory has focused on orexin (ORX) producing neurons in the hypothalamus, which is known as a master switch that induces various defense responses when animals face a stressful environment. This review article summarized the previous evidence and our latest findings to argue the neural regulation between pain and itch and the bidirectional roles of ORX neurons in processing these two sensations. i.e., pain relief and itch exacerbation. Further, we discussed the possible neural circuit mechanism for the opposite controlling of pain and itch by ORX neurons. Focusing on the roles of ORX neurons would provide a new perspective to understand the antagonistic regulation of pain and itch in CNS.

Positive memory increases cataplexy-like behaviors in narcolepsy mice as revealed using conditioned place preference test.

BACKGROUND: Cataplexy is a loss of muscle tone that can lead to postural collapse, disturbing the daily life of narcolepsy patients; it is often triggered by positive emotions such as laughter in human patients. Narcolepsy model mice also show cataplexy, and its incidence increases in response to positive emotion-inducing stimuli such as chocolate and female courtship. Although such observation indicates a positive emotion-related nature of cataplexy in narcolepsy mice, they also show cataplexy without any apparent triggering stimulus ~ (spontaneous cataplexy). Therefore, we hypothesized that some spontaneous cataplexy in narcoleptic mice might indicate the remembering of happy moments. RESULTS: To test our hypothesis, we did a conditioned place preference test on orexin/hypocretin neuron-ablated (ORX-AB) mice, one of the animal models of human narcolepsy, and counted the number of cataplexy-like behaviors. ORX-AB mice successfully remembered the chocolate-associated chamber, and the number of cataplexy-like behaviors significantly increased in the chocolate-associated chamber but not in the control chamber. In addition, ORX-AB mice remembered the aversive odor-associated chamber and avoided entering without affecting the number of cataplexy-like behaviors. Finally, similar activation of the nucleus accumbens, a positive emotion-related nucleus, was observed during both spontaneous and chocolate-induced cataplexy behaviors. CONCLUSIONS: These results support our hypothesis and will promote the usefulness of a narcolepsy mice model in emotion research and serve as a basis for a better understanding of cataplexy in narcolepsy patients.

Hypothalamic orexinergic neurons modulate pain and itch in an opposite way: pain relief and itch exacerbation.

Pain and itch are recognized as antagonistic sensations; pain suppresses itch and inhibition of pain generates itch. There is still a lack of evidence about the neural mechanism of the interaction between pain and itch in the central nervous system. In this study, we focused on the orexin (ORX) neurons in the lateral hypothalamus (LH), which mediate various "defense responses" when animals confront stressors. We found that the scratching behaviors induced by the pruritogen were significantly suppressed in ORX-neuron-ablated (ORX-abl) mice. The exaggerated pain behavior and attenuated itch behavior observed in ORX-abl mice indicated that ORX neurons modulate pain and itch in an opposite way, i.e., pain relief and itch exacerbation. In addition, most of the ORX neurons responded to both pain and itch input. Our results suggest that ORX neurons inversely regulate pain- and itch-related behaviors, which could be understood as a defense response to cope with stress environment.

Activity of putative orexin neurons during cataplexy.

It is unclear why orexin-deficient animals, but not wild-type mice, show cataplexy. The current hypothesis predicts simultaneous excitation of cataplexy-inhibiting orexin neurons and cataplexy-inducing amygdala neurons. To test this hypothesis, we measured the activity of putative orexin neurons in orexin-knockout mice during cataplexy episodes using fiber photometry. We created two animal models of orexin-knockout mice with a GCaMP6 fluorescent indicator expressed in putative orexin neurons. We first prepared orexin-knockout mice crossed with transgenic mice carrying a tetracycline-controlled transactivator transgene under the control of the orexin promoter. TetO-GCaMP6 was then introduced into mice via an adeno-associated virus injection or natural crossing. The resulting two models showed restricted expression of GCaMP6 in the hypothalamus, where orexin neurons should be located, and showed excitation to an intruder stress that was similar to that observed in orexin-intact mice in our previous study. The activity of these putative orexin neurons increased immediately before the onset of cataplexy-like behavior but decreased (approximately - 20% of the baseline) during the cataplexy-like episode. We propose that the activity of orexin neurons during cataplexy is moderately inhibited by an unknown mechanism. The absence of cataplexy in wild-type mice may be explained by basal or residual activity-induced orexin release, and emotional stimulus-induced counter activation of orexin neurons may not be necessary. This study will serve as a basis for better treatment of cataplexy in narcolepsy patients.

Multifaceted roles of orexin neurons in mediating methamphetamine-induced changes in body temperature and heart rate.

Methamphetamine (METH), which is used to improve the alertness of narcoleptic patients, elicits autonomic physiological responses such as increases in body temperature, blood pressure and heart rate. We have shown that orexin synthesizing neurons, which have an important role in maintaining wakefulness, greatly contribute to the regulation of cardiovascular and thermoregulatory function. This regulation is partly mediated by glutamatergic as well as orexinergic signalling from the orexin neurons. These signals may also be involved in the autonomic response elicited by METH. This study aimed to determine if loss of either orexin or glutamate in orexin neurons would affect METH-induced changes in heart rate and body temperature. We used transgenic mice in which the vesicular glutamate transporter 2 gene was disrupted selectively in orexin-producing neurons (ORX;vGT2-KO), prepro-orexin knockout mice (ORX-KO), and control wild type mice (WT). We measured body temperature, heart rate and locomotor activity with a pre-implanted telemetry probe and compared the effect of METH (0.5, 2 and 5 mg/kg i.p.) on these parameters between these three groups. A low dose of METH induced hyperthermia and tachycardia responses in ORX;vGT2-KO mice, which were significant compared to ORX-KO and WT mice. The highest dose of METH induced hypothermia and bradycardia in ORX-KO mice, however, it induced hyperthermia in both WT and ORX;vGT2-KO mice. These results suggest that glutamate and orexin from orexin neurons have differential roles in mediating METH-induced changes in body temperature and heart rate.

Involvement of A5/A7 noradrenergic neurons and B2 serotonergic neurons in nociceptive processing: a fiber photometry study.

In the central nervous system, the A6 noradrenaline (NA) and the B3 serotonin (5-HT) cell groups are well-recognized players in the descending antinociceptive system, while other NA/5-HT cell groups are not well characterized. A5/A7 NA and B2 5-HT cells project to the spinal horn and form descending pathways. We recorded G-CaMP6 green fluorescence signal intensities in the A5/A7 NA and the B2 5-HT cell groups of awake mice in response to acute tail pinch stimuli, acute heat stimuli, and in the context of a non-noxious control test, using fiber photometry with a calcium imaging system. We first introduced G-CaMP6 in the A5/A7 NA or B2 5-HT neuronal soma, using transgenic mice carrying the tetracycline-controlled transactivator transgene under the control of either a dopamine ß-hydroxylase or a tryptophan hydroxylase-2 promoters and by the site-specific injection of adeno-associated virus (AAV-TetO(3G)-G-CaMP6). After confirming the specific expression patterns of G-CaMP6, we recorded G-CaMP6 green fluorescence signals in these sites in awake mice in response to acute nociceptive stimuli. G-CaMP6 fluorescence intensity in the A5, A7, and B2 cell groups was rapidly increased in response to acute nociceptive stimuli and soon after, it returned to baseline fluorescence intensity. This was not observed in the non-noxious control test. The results indicate that acute nociceptive stimuli rapidly increase the activities of A5/A7 NA or B2 5-HT neurons but the non-noxious stimuli do not. The present study suggests that A5/A7 NA or B2 5-HT neurons play important roles in nociceptive processing in the central nervous system. We suggest that A5/A7/B2 neurons may be new therapeutic targets. All performed procedures were approved by the Institutional Animal Use Committee of Kagoshima University (MD17105) on February 22, 2018.

Aversive emotion rapidly activates orexin neurons and increases heart rate in freely moving mice.

The perifornical area of the hypothalamus has been known as the center for the defense response, or fight-or-flight response, which is characterized by a concomitant rise in arterial blood pressure, heart rate, and respiratory frequency. It is well established that orexin neurons, which are located in this region, play a critical role in this response. In this study, we further examined this role by recording orexin neuronal activity and heart rate in freely moving mice using an original dual-channel fiber photometry system in vivo. Analysis of orexin neuron activity in relation to autonomic responses to aversive stimuli revealed a rapid increase in neuronal activity just prior to changes in heart rate. In addition, we examined whether orexin neurons would be activated by a conditioned neutral sound that was previously associated with aversive stimulus. We show that the memory of the aversive stimulus activated orexin neurons and increased heart rate. Our data suggest that orexin neurons are a key component linking aversive emotions to autonomic defense response. Our data also suggest that targeting orexin neurons may enable treatment of psychiatric disorders associated with chronic stress and traumatic memories.

Linalool odor-induced analgesia is triggered by TRPA1-independent pathway in mice.

We had recently reported that linalool odor exposure induced significant analgesic effects in mice and that the effects were disappeared in olfactory-deprived mice in which the olfactory epithelium was damaged, thus indicating that the effects were triggered by chemical senses evoked by linalool odor exposure. However, the peripheral neuronal mechanisms, including linalool receptors that contribute toward triggering the linalool odor-induced analgesia, still remain unexplored. In vitro studies have shown that the transient receptor potential ankyrin 1 (TRPA1) responded to linalool, thus raising the possibility that TRPA1 expressed on the trigeminal nerve terminal detects linalool odor inhaled into the nostril and triggers the analgesic effects. To address this hypothesis, we measured the behavioral pain threshold for noxious mechanical stimulation in TRPA1-deficient mice. In contrast to our expectation, we found a significant increase in the threshold after linalool odor exposure in TRPA1-deficient mice, indicating the analgesic effects of linalool odor even in TRPA1-deficient mice. Furthermore, intranasal application of TRPA1 selective antagonist did not alter the analgesic effect of linalool odor. These results showed that the linalool odor-induced analgesia was triggered by a TRPA1-independent pathway in mice.

Orexinergic descending inhibitory pathway mediates linalool odor-induced analgesia in mice.

Linalool odor exposure induces an analgesic effect in mice. This effect disappeared in the anosmic model mice, indicating that olfactory input evoked by linalool odor triggered this effect. Furthermore, hypothalamic orexinergic neurons play a pivotal role in this effect. However, the neuronal circuit mechanisms underlying this effect have not been fully addressed. In this study, we focused on the descending orexinergic projection to the spinal cord and examined whether this pathway contributes to the effect. We assessed the effect of intrathecal administration of orexin receptor antagonists on linalool odor-induced analgesia in the tail capsaicin test. We found that the selective orexin type 1 receptor antagonist, but not the selective orexin type 2 receptor antagonist, prevented the odor-induced analgesic effect. Furthermore, immunohistochemical analyses of c-Fos expression induced by the capsaicin test revealed that neuronal activity of spinal cord neurons was suppressed by linalool odor exposure, which was prevented by intrathecal administration of the orexin 1 receptor antagonist. These results indicate that linalool odor exposure drives the orexinergic descending pathway and suppresses nociceptive information flow at the spinal level.

Orexin (hypocretin) participates in central autonomic regulation during fight-or-flight response.

Our daily life does not only involve a calm resting state but is rather full of perturbations that induce active states such as moving, eating, and communicating. During such active conditions, cardiorespiratory regulation should be adjusted according to bodily demand, which differs from that during the resting state, by modulating or resetting the operating point. To explore neural mechanisms in the state-dependent adjustment of central autonomic regulation, my research group has recently focused on the fight-or-flight response because the stressor induces not only cognitive, emotional, and behavioral changes but also autonomic changes. In this brief review, I will summarize our discovery using orexin knockout mice and orexin neuron-ablated mice for the possible contribution of orexin, a hypothalamic neuropeptide, to the state-dependent adjustment of the central autonomic regulation. In addition, I will introduce some recent discovery using optogenetic manipulation of the orexin and related systems. The diversity of synaptic control of the cardiovascular and respiratory neurons appears necessary for animals to adapt themselves to ever-changing life circumstances and behavioral states. The orexin system is likely to function as one of the essential modulators for coordinating the circuits controlling autonomic functions and behaviors.

Sexual excitation induces courtship ultrasonic vocalizations and cataplexy-like behavior in orexin neuron-ablated male mice.

Cataplexy is triggered by laughter in humans and palatable food in mice. To further evaluate mice's cataplexy, we examined courtship behavior in orexin neuron-ablated mice (ORX-AB), one of the animal models of narcolepsy/cataplexy. Wild-type female mice were placed into the home cage of male ORX-AB and cataplexy-like behavior was observed along with ultrasonic vocalizations (USVs), also known as the "love song". ORX-AB with a female encounter showed cataplexy-like behavior both during the dark and light periods, whereas ORX-AB with chocolate predominantly showed it during the dark period. During the light period observation, more than 85% of cataplexy-like bouts were preceded by USVs. A strong positive correlation was observed between the number of USVs and cataplexy-like bouts. Cataplexy-like behavior in narcoleptic mice is a good behavioral measure to study the brain mechanisms behind positive emotion because they can be induced by different kinds of positive stimuli, including chocolate and female courtship.

Transient Receptor Potential Ankyrin 1 Mediates Hypoxic Responses in Mice.

Transient receptor potential ankyrin 1 (TRPA1) is a non-selective cation channel that is broadly expressed in sensory pathways, such as the trigeminal and vagus nerves. It is capable of detecting various irritants in inspired gasses and is activated during hypoxia. In this study, the role of TRPA1 in hypoxia-induced behavioral, respiratory, and cardiovascular responses was examined through four lines of experiments using TRPA1 knockout (KO) mice and wild type (WT) littermates. First, KO mice showed significantly attenuated avoidance behavior in response to a low (15%) oxygen environment. Second, the wake-up response to a hypoxic ramp (from 21 to 10% O2 in 40 s) was measured using EEG electrodes. WT mice woke up within 30 s when oxygen was at 13-14%, but KO mice did not wake up until oxygen levels reached 10%. Histological analysis confirmed that mild (13% O2) hypoxia resulted in an attenuation of trigeminal neuronal activation in KO mice. Third, the ventilatory response to hypoxia was measured with whole body plethysmography. KO mice showed attenuated responses to mild hypoxia (15% O2) but not severe hypoxia (10% O2). Similar responses were observed in WT mice treated with the TRPA1 blocker, AP-18. These data clearly show that TRPA1 is necessary for multiple mild hypoxia (13-15% O2)-induced physiological responses. We propose that TRPA1 channels in the sensory pathways innervating the airway can detect hypoxic environments and prevent systemic and/or cellular hypoxia from occurring.

Transcriptomic Evaluation of Pulmonary Fibrosis-Related Genes: Utilization of Transgenic Mice with Modifying p38 Signal in the Lungs.

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrosing lung disease that is caused by the dysregulation of alveolar epithelial type II cells (AEC II). The mechanisms involved in the progression of IPF remain incompletely understood, although the immune response accompanied by p38 mitogen-activated protein kinase (MAPK) activation may contribute to some of them. This study aimed to examine the association of p38 activity in the lungs with bleomycin (BLM)-induced pulmonary fibrosis and its transcriptomic profiling. Accordingly, we evaluated BLM-induced pulmonary fibrosis during an active fibrosis phase in three genotypes of mice carrying stepwise variations in intrinsic p38 activity in the AEC II and performed RNA sequencing of their lungs. Stepwise elevation of p38 signaling in the lungs of the three genotypes was correlated with increased severity of BLM-induced pulmonary fibrosis exhibiting reduced static compliance and higher collagen content. Transcriptome analysis of these lung samples also showed that the enhanced p38 signaling in the lungs was associated with increased transcription of the genes driving the p38 MAPK pathway and differentially expressed genes elicited by BLM, including those related to fibrosis as well as the immune system. Our findings underscore the significance of p38 MAPK in the progression of pulmonary fibrosis.