Identification of hypothermia-inducing neurons in the preoptic area and activation of them by isoflurane anesthesia and central injection of adenosine.

Hibernation and torpor are not passive responses caused by external temperature drops and fasting but are active brain functions that lower body temperature. A population of neurons in the preoptic area was recently identified as such active torpor-regulating neurons. We hypothesized that the other hypothermia-inducing maneuvers would also activate these neurons. To test our hypothesis, we first refined the previous observations, examined the brain regions explicitly activated during the falling phase of body temperature using c-Fos expression, and confirmed the preoptic area. Next, we observed long-lasting hypothermia by reactivating torpor-tagged Gq-expressing neurons using the activity tagging and DREADD systems. Finally, we found that about 40-60% of torpor-tagged neurons were activated by succeeding isoflurane anesthesia and by icv administration of an adenosine A1 agonist. Isoflurane-induced and central adenosine-induced hypothermia is, at least in part, an active process mediated by the torpor-regulating neurons in the preoptic area.

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.

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.

Possible Use of Phytochemicals for Recovery from COVID-19-Induced Anosmia and Ageusia.

The year 2020 became the year of the outbreak of coronavirus, SARS-CoV-2, which escalated into a worldwide pandemic and continued into 2021. One of the unique symptoms of the SARS-CoV-2 disease, COVID-19, is the loss of chemical senses, i.e., smell and taste. Smell training is one of the methods used in facilitating recovery of the olfactory sense, and it uses essential oils of lemon, rose, clove, and eucalyptus. These essential oils were not selected based on their chemical constituents. Although scientific studies have shown that they improve recovery, there may be better combinations for facilitating recovery. Many phytochemicals have bioactive properties with anti-inflammatory and anti-viral effects. In this review, we describe the chemical compounds with anti- inflammatory and anti-viral effects, and we list the plants that contain these chemical compounds. We expand the review from terpenes to the less volatile flavonoids in order to propose a combination of essential oils and diets that can be used to develop a new taste training method, as there has been no taste training so far. Finally, we discuss the possible use of these in clinical settings.

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.

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.

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.

Linalool Odor-Induced Anxiolytic Effects in Mice.

In folk medicine, it has long been believed that odorous compounds derived from plant extracts can have anxiolytic effects. Among them, linalool, one of the terpene alcohols in lavender extracts, has been reported to have the anxiolytic effects. However, the anxiolytic nature of the linalool odor itself as well as its potential action through the olfactory system has not been thoroughly examined. In this study, we examined the anxiolytic effects of linalool odor with light/dark box test and with elevated plus maze (EPM), and found that linalool odor has an anxiolytic effect without motor impairment in mice. The effect was not observed in anosmic mice, indicating that it was triggered by olfactory input evoked by linalool odor. Furthermore, the effect was antagonized by flumazenil, indicating that the linalool odor-induced anxiolytic effect was mediated by γ-aminobutyric acid (GABA)ergic transmission via benzodiazepine (BDZ)-responsive GABAA receptors. These results provide information about the potential central neuronal mechanisms underlying the odor-induced anxiolytic effects and the foundation for exploring clinical application of linalool odor in anxiety treatments.

Application of calibrated forceps for assessing mechanical nociception with high time resolution in mice.

In order to investigate the basic physiological mechanisms of pain and the anti-nociceptive effects of analgesics, development of pain assays in mice is critical due to the advances of genetic manipulation techniques. The von Frey hairs/Semmes-Weinstein monofilaments test (von Frey test) has long been applied to examine mechanical nociception in mice. Though the von Frey test is a well-established and standardized method, it is inappropriate to assess a rapid change in the nociceptive threshold because voluntary resting/sleeping states are necessary to examine the response. In this study, we assessed the effectiveness of calibrated forceps to determine the mechanical nociceptive threshold in mice. Repeated daily measurements of the threshold over 5 days indicated that the device obtained stable and reliable values. Furthermore, repeated measurements with 5 minute intervals revealed that the device detected the rapid change of the threshold induced by remifentanil, a short-acting µ-receptor agonist. These results indicate that the calibrated forceps are well-suited for measuring the mechanical nociceptive threshold in mice, and are useful in assessing the effects of short-acting analgesics on mechanical nociception.

Odour-induced analgesia mediated by hypothalamic orexin neurons in mice.

Various folk remedies employ certain odorous compounds with analgesic effects. In fact, linalool, a monoterpene alcohol found in lavender extracts, has been found to attenuate pain responses via subcutaneous, intraperitoneal, intrathecal, and oral administration. However, the analgesic effects of odorous compounds mediated by olfaction have not been thoroughly examined. We performed behavioural pain tests under odourant vapour exposure in mice. Among six odourant molecules examined, linalool significantly increased the pain threshold and attenuated pain behaviours. Olfactory bulb or epithelium lesion removed these effects, indicating that olfactory sensory input triggered the effects. Furthermore, immunohistochemical analysis revealed that linalool activated hypothalamic orexin neurons, one of the key mediators for pain processing. Formalin tests in orexin neuron-ablated and orexin peptide-deficient mice showed orexinergic transmission was essential for linalool odour-induced analgesia. Together, these findings reveal central analgesic circuits triggered by olfactory input in the mammalian brain and support a potential therapeutic approach for treating pain with linalool odour stimulation.

Vagal afferent activation induces salivation and swallowing-like events in anesthetized rats.

The aim of this study was to clarify the effect of vagal afferent activation on salivation and swallowing-like events. Salivation is part of a reflex induced by stimulation of the oral area during feeding or chewing. Recently, we reported that nausea induced by gastroesophageal reflux (GER) activation produced salivation and swallowing in humans. Here, we investigated the ability of visceral sensation to enhance salivation and swallowing in rodents in order to inform the mechanism of GER-mediated stomatognathic activation. First, we administered LiCl to anesthetized male rats to induce nausea. LiCl significantly increased salivation and increased the activity of the vagal afferent nerve. Next, we simultaneously recorded salivation and swallowing using an electrode attached to the mylohyoid muscle during vagal afferent stimulation in a physiological range of frequencies. Vagal afferent stimulation significantly increased salivation and swallowing-like events in a frequency-dependent manner. A muscle relaxant, vecuronium bromide, diminished the swallowing-like response but did not affect salivation. These results indicate that visceral sensation induces salivation and swallowing-like events in anesthetized rodents through vagal afferent activation.

Nasal TRPA1 mediates irritant-induced bradypnea in mice.

Transient receptor potential ankyrin 1 (TRPA1), a member of the TRP superfamily, exists in sensory neurons such as trigeminal neurons innervating the nasal cavity and vagal neurons innervating the trachea and the lung. Although TRPA1 has been proposed as an irritant receptor that, when stimulated, triggers bradypnea, precise locations of the receptors responsible have not been elucidated. Here, we examined the relative importance of TRPA1 located in the upper airway (nasal) and the lower airway (trachea/lungs) in urethane-anesthetized mice. To stimulate the upper and lower airways separately, two cannulas were inserted through a hole made in the trachea just caudal to the thyroid cartilage, one into the nasal cavity and the second into the lower trachea. A vapor of one of the TRPA1-agonists, allyl isothiocyanate (AITC), was introduced by placing a piece of cotton paper soaked with AITC solution into the airline. AITC decreased the respiratory frequency when applied to the upper airway (ca -30%) but not to the lower airway (ca -5%). No response was observed in TRPA1 knockout mice. Contribution of the olfactory nerve seemed minimal because olfactory bulbectomized wild-type mice showed a similar response to that of the intact mice. AITC-induced bradypnea seemed to be mediated, at least in part, by the trigeminal nerve because trigeminal ganglion neurons were activated by AITC as revealed by an increase in the phosphorylated form of extracellular signal-regulated kinase in the neurons. These data clearly show that trigeminal TRPA1 in the nasal cavity play an essential role in irritant-induced bradypnea.

TRPA1 detects environmental chemicals and induces avoidance behavior and arousal from sleep.

Detecting threats and escaping before serious confrontations are important for animals to avoid danger and death. Transient receptor potential ankyrin 1 (TRPA1), a member of the TRP superfamily, is expressed in a subset of sensory neurons and mediates nociception evoked by pungent chemicals. Using behavioral testing, we found that TRPA1 knockout mice failed to avoid entering a chamber filled with vapor of formalin, allyl isothiocyanate, and acrolein. The avoidance behavior was blocked by nasal but not subcutaneous administration of a blocker to TRPA1. We also found that TRPA1 knockout mice did not wake when exposed to formalin during sleep. Additionally, the spinal trigeminal nucleus, the first relay neurons of the trigeminal system, showed massive expression of c-Fos after a brief (3 min) exposure to formalin vapor. TRPA1 seems to be a sentinel for environmental chemicals and induces avoidance behaviors and waking by way of the trigeminal system.

From the Cover: Neurons in the anterior olfactory nucleus pars externa detect right or left localization of odor sources.

Rodents can localize odor sources by comparing odor inputs to the right and left nostrils. However, the neuronal circuits underlying such odor localization are not known. We recorded neurons in the anterior olfactory nucleus (AON) while administering odors to the ipsilateral or contralateral (ipsi- or contra-) nostril. Neurons in the AON pars externa (AONpE) showed respiration phase-locked excitatory spike responses to ipsinostril-only stimulation with a category of odorants, and inhibitory responses to contranostril-only stimulation with the same odorants. Simultaneous odor stimulation of the ipsi- and contranostrils elicited significantly smaller responses than ipsinostril-only stimulation, indicating that AONpE neurons subtract the contranostril odor inputs from ipsinostril odor inputs. An ipsilateral odor source induced larger responses than a centrally located source, whereas an odor source at the contralateral position elicited inhibitory responses. These results indicate that individual AONpE neurons can distinguish the right or left position of an odor source by referencing signals from the two nostrils.

Odor-induced persistent discharge of mitral cells in the mouse olfactory bulb.

Short-term retention of sensory information in the form of persistent activity of central neurons plays a key role in transforming a brief sensory stimulation into longer-lasting brain responses. The olfactory system uses this transformation for various functional purposes, but the underlying neuronal mechanisms remain elusive. Here, we recorded odor-evoked, single-unit spike responses of mitral and tufted (M/T) cells in the mouse olfactory bulb (OB) under urethane anesthesia and examined the neuronal mechanisms of the persistent discharge (PD) of M/T cells that outlasts the odor stimulus for tens of seconds. The properties of the persistent afterdischarge that occurred after odor stimulation were distinct from those of odor-induced immediate spike responses in terms of the magnitude, odorant specificity, and odorant concentration-response relationship. This suggests that neuronal mechanisms other than prolonged input from olfactory sensory neurons are involved in generating these afterdischarges. Metabotropic glutamate receptor 1 (mGluR1) is expressed in the dendrites of M/T cells and is thought to participate in intraglomerular interactions among M/T cells. In OBs lacking mGluR1, or treated locally with an mGluR1-selective antagonist, the duration of the odor-induced spike responses was significantly lower than that in control OBs, indicating that mGluR1 within the bulbar neuronal circuits participates in the PD generation. These results suggest that neuronal circuits in the OB can actively prolong the odor-induced spike activity of bulbar output neurons and thus transform a brief odor input into longer-lasting activity in the central olfactory system.

Compensatory rapid switching of binasal inputs in the olfactory cortex.

Odors are inhaled through the nostrils into two segregated nasal passages and detected by sensory neurons in the bilateral olfactory epithelia. Airflow through the two nasal passages is usually asymmetrical because of alternating changes in nasal mucosal congestion. Here we show that neurons in the anterior olfactory nucleus (AON) of the adult rat olfactory cortex are ordinarily dominated by ipsi-nasal inputs and that binasal neurons in the AON respond to ipsilateral and contralateral nasal inputs with nearly equivalent odorant category selectivity. Deprivation of ipsilateral nasal inputs by unilateral nostril obstruction greatly enhanced the response to contralateral odor stimulation, in a reversible manner, in approximately 33% of AON neurons within only several minutes. In 27% of AON neurons that showed spike responses induced by the inspiration of room air, ipsilateral nasal obstruction initially suppressed respiration phase-locked spike discharges and, several minutes later, induced respiration phase-locked discharges with longer delays between inspiration and response. Recordings from AON neurons in rats with anterior commissure (AC) transection indicated that the resumed respiration phase-locked discharges with longer delays were mediated by the contralateral pathway via the AC. The ipsi-nasal occlusion-induced switching of nasal inputs to individual AON neurons shows that a subset of AON neurons in the adult rat has neuronal mechanisms for rapid nostril dominance plasticity, which may enable both right and left olfactory cortices to preserve their responsiveness to the external odor world, despite reciprocal changes in nasal airflow.

Behavioral state regulation of dendrodendritic synaptic inhibition in the olfactory bulb.

Behavioral states regulate how information is processed in local neuronal circuits. Here, we asked whether dendrodendritic synaptic interactions in the olfactory bulb vary with brain and behavioral states. To examine the state-dependent change of the dendrodendritic synaptic transmission, we monitored changes in field potential responses in the olfactory bulb of urethane-anesthetized and freely behaving rats. In urethane-anesthetized rats, granule-to-mitral dendrodendritic synaptic inhibition was larger and longer when slow waves were present in the electroencephalogram (slow-wave state) than during the fast-wave state. The state-dependent alternating change in the granule-to-mitral inhibition was regulated by the cholinergic system. In addition, the frequency of the spontaneous oscillatory activity of local field potentials and periodic discharges of mitral cells in the olfactory bulb shifted in synchrony with shifts in the neocortical brain state. Freely behaving rats showed multilevel changes in dendrodendritic synaptic inhibition that corresponded to diverse behavioral states; the inhibition was the largest during slow-wave sleep state, and successively smaller during light sleep, awake immobility, and awake moving states. These results provide evidence that behavioral state-dependent global changes in cholinergic tone modulate dendrodendritic synaptic inhibition and the information processing mode in the olfactory bulb.

State-dependent sensory gating in olfactory cortex.

Sensory systems show behavioral state-dependent gating of information flow that largely depends on the thalamus. Here we examined whether the state-dependent gating occurs in the central olfactory pathway that lacks a thalamic relay. In urethane-anesthetized rats, neocortical EEG showed a periodical alternation between two states: a slow-wave state (SWS) characterized by large and slow waves and a fast-wave state (FWS) characterized by faster waves. Single-unit recordings from olfactory cortex neurons showed robust spike responses to adequate odorants during FWS, whereas they showed only weak responses during SWS. The state-dependent change in odorant-evoked responses was observed in a majority of olfactory cortex neurons, but in only a small percentage of olfactory bulb neurons. These findings demonstrate a powerful state-dependent gating of odor information in the olfactory cortex that works in synchrony with the gating of other sensory systems. They suggest a state-dependent switchover of signal processing modes in the olfactory cortex.

Role of thalamic phospholipase C[beta]4 mediated by metabotropic glutamate receptor type 1 in inflammatory pain.

Phospholipase C (PLC) beta4, one of the four isoforms of PLCbetas, is the sole isoform expressed in the mouse ventral posterolateral thalamic nucleus (VPL), a key station in pain processing. The mouse thalamus also has been shown to express a high level of metabotropic glutamate receptor type 1 (mGluR1), which stimulates PLCbetas through activation of Galphaq/11 protein. It is therefore expected that the thalamic mGluR1-PLCbeta4 cascade may play a functional role in nociceptive transmission. To test this hypothesis, we first studied behavioral responses to various nociceptive stimuli in PLCbeta4 knock-out mice. We performed the formalin test and found no difference in the pain behavior in the first phase of the formalin test, which is attributed to acute nociception, between PLCbeta4 knock-out and wild-type mice. Consistent with this result, acute pain responses in the hot plate and tail flick tests were also unaffected in the PLCbeta4 knock-out mice. However, the nociceptive behavior in the second phase of the formalin test, resulting from the tissue inflammation, was attenuated in PLCbeta4 knock-out mice. In the dorsal horn of the spinal cord where PLCbeta1 and PLCbeta4 mRNAs are expressed, no difference was found between the wild-type and knock-out mice in the number of Fos-like immunoreactive neurons, which represent neuronal activity in the second phase in the formalin test. Thus, it is unlikely that spinal PLCbeta4 is involved in the formalin-induced inflammatory pain. Next, we found that pretreatment with PLC inhibitors, mGluR1 antagonists, or both, by either intracerebroventricular or intrathalamic injection, attenuated the formalin-induced pain behavior in the second phase in wild-type mice. Furthermore, activation of mGluR1 at the VPL enhanced pain behavior in the second phase in the wild-type mice. In contrast, PLCbeta4 knock-out mice did not show such enhancement, indicating that mGluR1 is connected to PLCbeta4 in the VPL. Finally, in parallel with the behavioral results, we showed in an electrophysiological study that the time course of firing discharges in VPL corresponds well to that of pain behavior in the formalin test in both wild-type and PLCbeta4 knock-out mice. These findings indicate that the thalamic mGluR1-PLCbeta4 cascade is indispensable for the formalin-induced inflammatory pain by regulating the response of VPL neurons.