Steroid therapy is widely used, but it frequently causes severe sleep disorders as a side effect. The underlying neurobiological mechanisms remain poorly understood, leading to a lack of clear evidence-based empirical drug selection. The aims of this study are: (1) to establish a mouse model of steroid-induced sleep disorder evoked by repeated dexamethasone administration, (2) to investigate the role of the orexin system in this disorder using neurophysiological techniques, and (3) to compare the efficacy of a benzodiazepine agonist (brotizolam) and a dual orexin receptor antagonist (suvorexant) in the model. Male C57BL/6J mice received intraperitoneal injections of dexamethasone (30 mg/kg) for five days before sleep architecture assessment by electroencephalography and electromyography. Orexin neuron activity was analyzed using fiber photometry in mice (Orexin-tTA) expressing GCaMP6 specifically in orexin neurons. Chronic dexamethasone administration in mice significantly increased wakefulness and reduced non-rapid eye movement sleep during the light-phase sleep period, and mimicked key features of clinical sleep disorders. Importantly, in vivo fiber photometry provided the first direct evidence that these states are driven by hyperactivity of orexin neurons during the arousal-promoting transition phase. Both brotizolam and suvorexant effectively reversed dexamethasone-induced sleep disruption and normalized sleep architecture. These findings indicate that steroid-induced sleep disturbance is mediated by hyperactivity of the orexinergic system. Furthermore, we show that both a benzodiazepine and a targeted orexin antagonist are effective in this model, providing a preclinical evidence base for the pharmacological management of this common and severe side effect.

Tissue clearing has been widely used for fluorescence imaging of fixed tissues, but its application to live tissues has been limited by toxicity. Here we develop minimally invasive optical clearing media for fluorescence imaging of live mammalian tissues. Light scattering is minimized by adding spherical polymers with low osmolarity to the extracellular medium. A clearing medium containing bovine serum albumin (SeeDB-Live) is compatible with live cells, enabling structural and functional imaging of live tissues, such as spheroids, organoids, acute brain slices and the mouse brains in vivo. SeeDB-Live minimally affects neuronal electrophysiological properties and sensory responses in vivo, and facilitates fluorescence imaging of deep cortical layers in live animals without detectable toxicity to neurons or behavior. We further demonstrate its utility to epifluorescence voltage imaging in acute brain slices and in vivo preparations. Thus, SeeDB-Live expands both the depth and modality range of fluorescence imaging in live mammalian tissues.

Functional imaging of neural structures at the base of the cranium, including the trigeminal ganglion (TG), is technically challenging due to limited optical access. The TG-the largest sensory ganglion in the head-houses primary afferent neurons that relay information from the teeth, oral cavity, and face, yet investigation of somatosensory processing at the population level has remained limited. Here, we present a surgical procedure for an optical-window preparation that enables direct optical access to the TG. The ganglion is exposed by a large temporal craniotomy with removal of overlying tissue, and a glass cuboid is then placed in direct contact with the TG to suppress motion while maintaining the cranial cavity as a closed compartment without continuous perfusion. This preparation allows reliable visualization and recording of individual TG neurons during controlled stimulation of diverse facial and intraoral sites. Our approach provides a practical platform to map peripheral sensory representations within the TG and to investigate mechanisms underlying dental sensation, orofacial pain, and trigeminal circuit function. Key features • Establishes stable optical access to the mouse trigeminal ganglion using a hemispherectomy-based glass cuboid cranial window preparation. • Reduces motion artifacts through direct cuboid-TG contact, enabling robust single-neuron calcium imaging. • Provides a large field of view of the whole TG with fluorescence microscopy hardware and supports imaging in vivo.

Somatotopy serves as a fundamental principle underlying sensory information processing, traditionally emphasized in the study of the cerebral cortex. However, little effort has been directed towards unraveling the spatial organization characterizing the earlier stages of somatosensory pathways. In this study, we developed a novel methodology to visualize individual neurons within the trigeminal ganglion-a crucial cluster of cell bodies of sensory neurons innervating the face. Our investigations revealed a reliable sensory response to stimulation of the lower incisor or lip within this ganglion. The responsive neurons were confined to a specific portion of the trigeminal ganglion, consistent with innervation of the lower oral cavity by the mandibular nerve (V3). Contrary to our expectations, we did not observe a discernible map differentiating the lower tooth and the lip. Instead, the spatial representation of the tooth and lip within this portion of trigeminal ganglion exhibited intermingling with no clear border between tooth-responding and lip-responding neurons. These findings contrast with earlier studies that identified tooth and lip responding regions in the somatosensory cortex. Our study sheds light on the complex spatial organization of sensory processing in the trigeminal system, highlighting the need for further research to elucidate the underlying mechanisms and implications for sensory perception and clinical interventions.

The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) and its specific receptor (PAC1R) are widely present in the central nervous system (CNS), and PACAP/PAC1R signaling has been implicated in anxiety-related behaviors. The locus coeruleus (LC), with its extensive noradrenergic (NA) projections throughout the CNS, is also implicated in anxiety. Although the LC exhibits a high expression of PAC1R, the precise role of PACAP/PAC1R signaling in the LC's involvement in anxiety remains unclear. Histochemical analysis confirmed high levels of PAC1R mRNA in the LC and showed that PAC1R gene transcripts were highly localized to NA neurons. Targeted deletion of PAC1R from these cells led to a hyperactive/low anxiety phenotype in the open field and elevated-plus maze tests. Retrograde neurocircuit tracing indicated PACAP neurons from the anterior insular cortex (aIC) and a few other regions projected axons to the LC. The selective activation of PACAP neurons in the aIC led to significantly increased anxiety behavior without a change in overall locomotor activity. Moreover, shRNA PACAP knockdown in the aIC in wild-type mice led to a selective decrease in anxiety. The present results identify an aIC to LC neurocircuit controlling anxiety that critically requires PACAP/PAC1R signaling.

Significance The initiation of goal-directed actions is a complex process involving the medial prefrontal cortex and dopaminergic inputs through the mesocortical pathway. However, it is unclear what information the mesocortical pathway conveys and how it impacts action initiation. In this study, we unveiled the indispensable role of mesocortical axon terminals in encoding the execution of movements in self-initiated actions. Aim To investigate the role of mesocortical axon terminals in encoding the execution of movements in self-initiated actions. Approach We designed a lever-press task in which mice internally determine the timing of the press, receiving a larger reward for longer waiting periods. Results Our study revealed that self-initiated actions depend on dopaminergic signaling mediated by D2 receptors, whereas sensory-triggered lever-press actions do not involve D2 signaling. Microprism-mediated two-photon calcium imaging further demonstrated ramping activity in mesocortical axon terminals approximately 0.5 s before the self-initiated lever press. Remarkably, the ramping patterns remained consistent whether the mice responded to cues immediately for a smaller reward or held their response for a larger reward. Conclusions We conclude that mesocortical dopamine axon terminals encode the timing of self-initiated actions, shedding light on a crucial aspect of the intricate neural mechanisms governing goal-directed behavior.

Midbrain dopamine neurons impact neural processing in the prefrontal cortex (PFC) through mesocortical projections. However, the signals conveyed by dopamine projections to the PFC remain unclear, particularly at the single-axon level. Here, we investigated dopaminergic axonal activity in the medial PFC (mPFC) during reward and aversive processing. By optimizing microprism-mediated two-photon calcium imaging of dopamine axon terminals, we found diverse activity in dopamine axons responsive to both reward and aversive stimuli. Some axons exhibited a preference for reward, while others favored aversive stimuli, and there was a strong bias for the latter at the population level. Long-term longitudinal imaging revealed that the preference was maintained in reward- and aversive-preferring axons throughout classical conditioning in which rewarding and aversive stimuli were paired with preceding auditory cues. However, as mice learned to discriminate reward or aversive cues, a cue activity preference gradually developed only in aversive-preferring axons. We inferred the trial-by-trial cue discrimination based on machine learning using anticipatory licking or facial expressions, and found that successful discrimination was accompanied by sharper selectivity for the aversive cue in aversive-preferring axons. Our findings indicate that a group of mesocortical dopamine axons encodes aversive-related signals, which are modulated by both classical conditioning across days and trial-by-trial discrimination within a day.

l -Lactate is increasingly appreciated as a key metabolite and signaling molecule in mammals. However, investigations of the inter- and intra-cellular dynamics of l -lactate are currently hampered by the limited selection and performance of l -lactate-specific genetically encoded biosensors. Here we now report a spectrally and functionally orthogonal pair of high-performance genetically encoded biosensors: a green fluorescent extracellular l -lactate biosensor, designated eLACCO2.1, and a red fluorescent intracellular l -lactate biosensor, designated R-iLACCO1. eLACCO2.1 exhibits excellent membrane localization and robust fluorescence response. To the best of our knowledge, R-iLACCO1 and its affinity variants exhibit larger fluorescence responses than any previously reported intracellular l -lactate biosensor. We demonstrate spectrally and spatially multiplexed imaging of l -lactate dynamics by coexpression of eLACCO2.1 and R-iLACCO1 in cultured cells, and in vivo imaging of extracellular and intracellular l -lactate dynamics in mice.

Midbrain dopamine neurons impact neural processing in the prefrontal cortex (PFC) through mesocortical projections. However, the signals conveyed by dopamine projections to the PFC remain unclear, particularly at the single-axon level. Here, we investigated dopaminergic axonal activity in the medial PFC (mPFC) during reward and aversive processing. By optimizing microprism-mediated two-photon calcium imaging of dopamine axon terminals, we found diverse responses in dopamine axons, with some preferring reward and others preferring aversive stimuli, with a strong bias for the latter at the population level. Long-term longitudinal imaging revealed that the preference was maintained in reward- and aversive-preferring axons throughout classical conditioning in which rewarding and aversive stimuli were paired with preceding auditory cues. However, as mice learned to discriminate reward or aversive cues, a cue activity preference gradually developed only in aversive-preferring axons, becoming more selective for aversive processing. We inferred the trial-by-trial cue discrimination based on machine learning using anticipatory licking or facial expressions, and found that successful discrimination was accompanied by sharper selectivity for the aversive cue in aversive-preferring axons. Our findings implicate mesocortical dopamine axon activity in the encoding of aversive processing that is modulated by both classical conditioning across days and trial-by-trial discrimination within a day. Impact statement Two-photon calcium imaging revealed that many mesocortical dopamine axons show enhanced selectivity for aversive cue processing during classical conditioning.

Emotion affects physical reaction by the autonomic nervous system in the top-down process. However, there are few reported whether physical reactions, such as increased heart rate, might induce emotional changes like anxiety or fear responses. Here, we have investigated emotional behavior by controlling heart rate directly by a drug administration or an optogenetic-based cardiac pacing system in freely moving mice. First, the heart rate was controlled by ivabradine, an inhibitor of the hyperpolarization-activated cyclic nucleotide-gated channels. We found that ivabradine evoked bradycardia and partially reduced anxiety-like behavior. Second, heart rate was controlled by the optical cardiac pacing in freely moving mice. In many previous studies, the pacing was applied ex vivo or in anesthetized animals. Therefore, we developed the optical cardiac pacing system in awake, freely moving mice and simultaneously measured electrocardiograms. We found that Optically increased heart rate by using this system potentially enhanced anxiety-like behavior. These results suggested that emotional states are partially driven by heart rate in the bottom-up process. In conclusion, the brain function and the feedback from the physical reactions must be considered together to understand the mechanisms of expressed emotion.

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.

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 β-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.

We have previously shown that pituitary adenylate cyclase-activating polypeptide (PACAP) in the ventromedial hypothalamus (VMH) enhances feeding during the dark cycle and after fasting, and inhibits feeding during the light cycle. On the other hand, galanin is highly expressed in the hypothalamus and has been reported to be involved in feeding regulation. In this study, we investigated the involvement of the VMH-PACAP to the dorsomedial hypothalamus (DMH)-galanin signaling in the regulation of feeding. Galanin expression in the hypothalamus was significantly increased with fasting, but this increment was canceled in PACAP-knockout (KO) mice. Furthermore, overexpression of PACAP in the VMH increased the expression of galanin, while knockdown (KD) of PACAP in the VMH decreased the expression of galanin, indicating that the expression of galanin in the hypothalamus might be regulated by PACAP in the VMH. Therefore, we expressed the synaptophysin-EGFP fusion protein (SypEGFP) in PACAP neurons in the VMH and visualized the neural projection to the hypothalamic region where galanin was highly expressed. A strong synaptophysin-EGFP signal was observed in the DMH, indicating that PACAP-expressing cells of the VMH projected to the DMH. Furthermore, galanin immunostaining in the DMH showed that galanin expression was weak in PACAP-KO mice. When galanin in the DMH was knocked down, food intake during the dark cycle and after fasting was decreased, and food intake during the light cycle was increased, as in PACAP-KO mice. These results indicated that galanin in the DMH may regulate the feeding downstream of PACAP in the VMH.

Both pituitary adenylate cyclase-activating polypeptides (PACAP) and noradrenaline are known to be involved in anxiety and fear memory. However, the crosstalk of these substances has not been clarified previously. Here we investigated the PACAP action in the locus coeruleus (LC) noradrenergic system based on the mice. To evaluate the significance of PACAP on the mice behavior, we used the PACAP-knockout (-/-) mice.

We have previously shown that PACAP in the ventromedial hypothalamic nucleus (VMH) enhances feeding at night and after fasting, but inhibits feeding during daytime. On the other hand, the neuropeptide galanin is also shown to be highly expressed in the hypothalamus and involved in feeding regulation. In this study, we investigated the possible involvement of VMH-PACAP in the dorsomedial hypothalamic nucleus (DMH)-galanin signaling for mouse feeding behavior. The expression of galanin in the hypothalamus was significantly increased by fasting, but this increase was cancelled in PACAP knockout mice. Furthermore, overexpression of PACAP in the VMH increased the expression of galanin, while knockdown of PACAP in the VMH decreased the expression of galanin, indicating that the expression of galanin in the hypothalamus might be regulated by PACAP in the VMH. Therefore, we expressed synaptophysin-EGFP chimeric protein in PACAP neurons in the VMH, and visualized the neural projections to the hypothalamic region where galanin was highly expressed. Strong EGFP signal was observed in the DMH, suggesting that PACAP-expressing neurons in the VMH projected to the DMH. Furthermore, in the DMH, immunostaining of galanin showed that galanin expression increased with fasting, but this was not observed in PACAP knockout mice. When galanin in the DMH was knocked down by shRNA treatment, food intake at night and after fasting was decreased, whereas food intake during daytime was increased, as shown in the PACAP knockout mice. These results suggested that VMH-PACAP may regulate mouse feeding behavior through DMH-galanin.

Lactate was initially thought of as a fatigue substance. In recent years, however, lactate not only functions as an energy carrier and contributes to ATP production, but also its role as a signal transmitter has been attracting attention due to the identification of lactate receptors. Lactate is synthesized from glucose and glycogen through the glycolytic system. The central nervous system is a major organ of glucose metabolism and is rich in glycogen. Therefore, this review summarizes the recent findings on the contribution of lactate to the pathophysiology of the central nervous system.

Fractalkine is one of the CX3C chemokine family, and it is widely expressed in the brain including the hypothalamus. In the brain, fractalkine is expressed in neurons and binds to a CX3C chemokine receptor 1 (CX3CR1) in microglia. The hypothalamus regulates energy homeostasis of which dysregulation is associated with obesity. Therefore, we examined whether fractalkine-CX3CR1 signalling involved in regulating food intake and hypothalamic inflammation associated with obesity pathogenesis. In the present study, fractalkine significantly reduced food intake induced by several experimental stimuli and significantly increased brain-derived neurotrophic factor (BDNF) mRNA expression in the hypothalamus. Moreover, tyrosine receptor kinase B (TrkB) antagonist impaired fractalkine-induced anorexigenic actions. In addition, compared with wild-type mice, CX3CR1-deficient mice showed a significant increase in food intake and a significant decrease in BDNF mRNA expression in the hypothalamus. Mice fed a high-fat diet (HFD) for 16 weeks showed hypothalamic inflammation and reduced fractalkine mRNA expression in the hypothalamus. Intracerebroventricular administration of fractalkine significantly suppressed HFD-induced hypothalamic inflammation in mice. HFD intake for 4 weeks caused hypothalamic inflammation in CX3CR1-deficient mice, but not in wild-type mice. These findings suggest that fractalkine-CX3CR1 signalling induces anorexigenic actions via activation of the BDNF-TrkB pathway and suppresses HFD-induced hypothalamic inflammation in mice.

Recent evidence has suggested that pituitary adenylate cyclase-activating polypeptide (PACAP) has critical roles in central and peripheral pathways, such as spino-parabrachio-amygdaloid and hypothalamic-pituitary-adrenal pathways, mediating stress-related negative emotional behaviors. Although it is well established that there is a great degree of comorbidity of chronic pain and negative emotional behaviors, the cellular mechanism underlying chronic pain and anxiety/depression interaction still remains to be elucidated. Here, we evaluated possible involvement of PACAP signaling in the development of anxiety- and depression-like behaviors after peripheral nerve injury in mice. We observed that spinal nerve ligation (SNL) induced anxiety- and depression-like behaviors lasting for at least 3 weeks in wild-type (PACAP +/+) mice. However, the development of SNL-induced anxiety- and depression-like behaviors was almost completely abrogated in PACAP -/- mice. Furthermore, we found that selective overexpression of PACAP by the infection of adeno-associated virus in the hypothalamic paraventricular nucleus (PVN), but not neighboring ventromedial hypothalamus, region resulted in the induction of anxiety-like behavior. In contrast, siRNA-mediated knockdown of PVN PACAP attenuated the development of SNL-induced anxiety- but not depressive-like behavior. Our data support that PVN PACAP signaling is involved in an important mechanism underlying the anxiety-like behaviors in peripheral neuropathic pain condition.

Brain glycogen metabolism is known to be involved in the learning and memory processes. Protein targeting to glycogen (PTG) is a crucial molecule for glycogenesis, and its expression level is shown to be increased in the dorsal hippocampus during fear memory acquisition and recall, suggesting that PTG may contribute to the memory process. However, its detailed role in the dorsal hippocampus remains unclear. Therefore, we knocked down the expression of PTG in the dorsal hippocampus and attempted to analyze its function behaviorally. PTG expression was found to be enriched in astrocytes. Furthermore, short hairpin RNA against PTG suppressed the expression of PTG in astrocytes. Mice with knockdown of PTG in the dorsal hippocampus showed suppressed alternation behavior in the Y-maze test and reduced memory recall at the first hour after acquisition in the passive avoidance test. Knockdown of mouse dorsal hippocampal astrocyte-specific PTG also impaired working memory in the Y-maze test. GluR1, GluR2, and NR2a subunits expressions were significantly down-regulated in the dorsal hippocampus of mice in which PTG was knocked down. These results indicate that PTG in the dorsal hippocampal astrocytes may contribute to working and short-term memories by maintaining the expression of glutamate receptor subunits.