IL-6/gp130 signaling in CD4+ T cells drives the pathogenesis of pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is characterized by stenosis and occlusions of small pulmonary arteries, leading to elevated pulmonary arterial pressure and right heart failure. Although accumulating evidence shows the importance of interleukin (IL)-6 in the pathogenesis of PAH, the target cells of IL-6 are poorly understood. Using mice harboring the floxed allele of gp130, a subunit of the IL-6 receptor, we found substantial Cre recombination in all hematopoietic cell lineages from the primitive hematopoietic stem cell level in SM22α-Cre mice. We also revealed that a CD4+ cell-specific gp130 deletion ameliorated the phenotype of hypoxia-induced pulmonary hypertension in mice. Disruption of IL-6 signaling via deletion of gp130 in CD4+ T cells inhibited phosphorylation of signal transducer and activator of transcription 3 (STAT3) and suppressed the hypoxia-induced increase in T helper 17 cells. To further examine the role of IL-6/gp130 signaling in more severe PH models, we developed Il6 knockout (KO) rats using the CRISPR/Cas9 system and showed that IL-6 deficiency could improve the pathophysiology in hypoxia-, monocrotaline-, and Sugen5416/hypoxia (SuHx)-induced rat PH models. Phosphorylation of STAT3 in CD4+ cells was also observed around the vascular lesions in the lungs of the SuHx rat model, but not in Il6 KO rats. Blockade of IL-6 signaling had an additive effect on conventional PAH therapeutics, such as endothelin receptor antagonist (macitentan) and soluble guanylyl cyclase stimulator (BAY41-2272). These findings suggest that IL-6/gp130 signaling in CD4+ cells plays a critical role in the pathogenesis of PAH.

Gut dysbiosis is associated with aortic aneurysm formation and progression in Takayasu arteritis.

BACKGROUND: Takayasu arteritis (TAK) is an autoimmune large vessel vasculitis that affects the aorta and its major branches, eventually leading to the development of aortic aneurysm and vascular stenosis or occlusion. This retrospective and prospective study aimed to investigate whether the gut dysbiosis exists in patients with TAK and to identify specific gut microorganisms related to aortic aneurysm formation/progression in TAK. METHODS: We analysed the faecal microbiome of 76 patients with TAK and 56 healthy controls (HCs) using 16S ribosomal RNA sequencing. We examined the relationship between the composition of the gut microbiota and clinical parameters. RESULTS: The patients with TAK showed an altered gut microbiota with a higher abundance of oral-derived bacteria, such as Streptococcus and Campylobacter, regardless of the disease activity, than HCs. This increase was significantly associated with the administration of a proton pump inhibitor used for preventing gastric ulcers in patients treated with aspirin and glucocorticoids. Among patients taking a proton pump inhibitor, Campylobacter was more frequently detected in those who underwent vascular surgeries and endovascular therapy for aortic dilatation than in those who did not. Among the genus of Campylobacter, Campylobacter gracilis in the gut microbiome was significantly associated with clinical events related to aortic aneurysm formation/worsening in patients with TAK. In a prospective analysis, patients with a gut microbiome positive for Campylobacter were significantly more likely to require interventions for aortic dilatation than those who were negative for Campylobacter. Furthermore, patients with TAK who were positive for C. gracilis by polymerase chain reaction showed a tendency to have severe aortic aneurysms. CONCLUSIONS: A specific increase in oral-derived Campylobacter in the gut may be a novel predictor of aortic aneurysm formation/progression in patients with TAK.

Regnase-1 Prevents Pulmonary Arterial Hypertension Through mRNA Degradation of Interleukin-6 and Platelet-Derived Growth Factor in Alveolar Macrophages.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a type of pulmonary hypertension (PH) characterized by obliterative pulmonary vascular remodeling, resulting in right-sided heart failure. Although the pathogenesis of PAH is not fully understood, inflammatory responses and cytokines have been shown to be associated with PAH, in particular, with connective tissue disease-PAH. In this sense, Regnase-1, an RNase that regulates mRNAs encoding genes related to immune reactions, was investigated in relation to the pathogenesis of PH. METHODS: We first examined the expression levels of ZC3H12A (encoding Regnase-1) in peripheral blood mononuclear cells from patients with PH classified under various types of PH, searching for an association between the ZC3H12A expression and clinical features. We then generated mice lacking Regnase-1 in myeloid cells, including alveolar macrophages, and examined right ventricular systolic pressures and histological changes in the lung. We further performed a comprehensive analysis of the transcriptome of alveolar macrophages and pulmonary arteries to identify genes regulated by Regnase-1 in alveolar macrophages. RESULTS: ZC3H12A expression in peripheral blood mononuclear cells was inversely correlated with the prognosis and severity of disease in patients with PH, in particular, in connective tissue disease-PAH. The critical role of Regnase-1 in controlling PAH was also reinforced by the analysis of mice lacking Regnase-1 in alveolar macrophages. These mice spontaneously developed severe PAH, characterized by the elevated right ventricular systolic pressures and irreversible pulmonary vascular remodeling, which recapitulated the pathology of patients with PAH. Transcriptomic analysis of alveolar macrophages and pulmonary arteries of these PAH mice revealed that Il6, Il1b, and Pdgfa/b are potential targets of Regnase-1 in alveolar macrophages in the regulation of PAH. The inhibition of IL-6 (interleukin-6) by an anti-IL-6 receptor antibody or platelet-derived growth factor by imatinib but not IL-1ß (interleukin-1ß) by anakinra, ameliorated the pathogenesis of PAH. CONCLUSIONS: Regnase-1 maintains lung innate immune homeostasis through the control of IL-6 and platelet-derived growth factor in alveolar macrophages, thereby suppressing the development of PAH in mice. Furthermore, the decreased expression of Regnase-1 in various types of PH implies its involvement in PH pathogenesis and may serve as a disease biomarker, and a therapeutic target for PH as well.

Aryl hydrocarbon receptor is essential for the pathogenesis of pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a devastating disease characterized by arteriopathy in the small to medium-sized distal pulmonary arteries, often accompanied by infiltration of inflammatory cells. Aryl hydrocarbon receptor (AHR), a nuclear receptor/transcription factor, detoxifies xenobiotics and regulates the differentiation and function of various immune cells. However, the role of AHR in the pathogenesis of PAH is largely unknown. Here, we explore the role of AHR in the pathogenesis of PAH. AHR agonistic activity in serum was significantly higher in PAH patients than in healthy volunteers and was associated with poor prognosis of PAH. Sprague-Dawley rats treated with the potent endogenous AHR agonist, 6-formylindolo[3,2-b]carbazole, in combination with hypoxia develop severe pulmonary hypertension (PH) with plexiform-like lesions, whereas Sprague-Dawley rats treated with the potent vascular endothelial growth factor receptor 2 inhibitors did not. Ahr-knockout (Ahr-/- ) rats generated using the CRISPR/Cas9 system did not develop PH in the SU5416/hypoxia model. A diet containing Qing-Dai, a Chinese herbal drug, in combination with hypoxia led to development of PH in Ahr+/+ rats, but not in Ahr-/- rats. RNA-seq analysis, chromatin immunoprecipitation (ChIP)-seq analysis, immunohistochemical analysis, and bone marrow transplantation experiments show that activation of several inflammatory signaling pathways was up-regulated in endothelial cells and peripheral blood mononuclear cells, which led to infiltration of CD4+ IL-21+ T cells and MRC1+ macrophages into vascular lesions in an AHR-dependent manner. Taken together, AHR plays crucial roles in the development and progression of PAH, and the AHR-signaling pathway represents a promising therapeutic target for PAH.

Pristane/Hypoxia (PriHx) Mouse as a Novel Model of Pulmonary Hypertension Reflecting Inflammation and Fibrosis.

BACKGROUND: Pulmonary arterial hypertension (PAH), particularly connective tissue disease-associated PAH (CTD-PAH), is a progressive disease and novel therapeutic agents based on the specific molecular pathogenesis are desired. In the pathogenesis of CTD-PAH, inflammation, immune cell abnormality, and fibrosis play important roles. However, the existing mouse pulmonary hypertension (PH) models do not reflect these features enough. The relationship between inflammation and hypoxia is still unclear.Methods and Results:Intraperitoneal administration of pristane, a kind of mineral oil, and exposure to chronic hypoxia were combined, and this model is referred to as pristane/hypoxia (PriHx) mice. Hemodynamic and histological analyses showed that the PriHx mice showed a more severe phenotype of PH than pristane or hypoxia alone. Immunohistological and flow cytometric analyses revealed infiltration of immune cells, including hemosiderin-laden macrophages and activated CD4+helper T lymphocytes in the lungs of PriHx mice. Pristane administration exacerbated lung fibrosis and elevated the expression of fibrosis-related genes. Inflammation-related genes such asIl6andCxcl2were also upregulated in the lungs of PriHx mice, and interleukin (IL)-6 blockade by monoclonal anti-IL-6 receptor antibody MR16-1 ameliorated PH of PriHx mice. CONCLUSIONS: A PriHx model, a novel mouse model of PH reflecting the pathological features of CTD-PAH, was developed through a combination of pristane administration and exposure to chronic hypoxia.

Molecular Characterization of Striated Muscle-Specific Gab1 Isoform as a Critical Signal Transducer for Neuregulin-1/ErbB Signaling in Cardiomyocytes.

Grb2-associated binder (Gab) docking proteins regulate signals downstream of a variety of growth factors and receptor tyrosine kinases. Neuregulin-1 (NRG-1), a member of epidermal growth factor family, plays a critical role for cardiomyocyte proliferation and prevention of heart failure via ErbB receptors. We previously reported that Gab1 and Gab2 in the myocardium are essential for maintenance of myocardial function in the postnatal heart via transmission of NRG-1/ErbB-signaling through analysis of Gab1/Gab2 cardiomyocyte-specific double knockout mice. In that study, we also found that there is an unknown high-molecular weight (high-MW) Gab1 isoform (120 kDa) expressed exclusively in the heart, in addition to the ubiquitously expressed low-MW (100 kDa) Gab1. However, the high-MW Gab1 has been molecularly ill-defined to date. Here, we identified the high-MW Gab1 as a striated muscle-specific isoform. The high-MW Gab1 has an extra exon encoding 27 amino acid residues between the already-known 3rd and 4th exons of the ubiquitously expressed low-MW Gab1. Expression analysis by RT-PCR and immunostaining with the antibody specific for the high-MW Gab1 demonstrate that the high-MW Gab1 isoform is exclusively expressed in striated muscle including heart and skeletal muscle. The ratio of high-MW Gab1/ total Gab1 mRNAs increased along with heart development. The high-MW Gab1 isoform in heart underwent tyrosine-phosphorylation exclusively after intravenous administration of NRG-1, among several growth factors. Adenovirus-mediated overexpression of the high-MW Gab1 induces more sustained activation of AKT after stimulation with NRG-1 in cardiomyocytes compared with that of ß-galactosidase. On the contrary, siRNA-mediated knockdown of the high-MW Gab1 significantly attenuated AKT activation after stimulation with NRG-1 in cardiomyocytes. Taken together, these findings suggest that the striated muscle-specific high-MW isoform of Gab1 has a crucial role for NRG-1/ErbB signaling in cardiomyocytes.

The Possible Role of TASK Channels in Rank-Ordered Recruitment of Motoneurons in the Dorsolateral Part of the Trigeminal Motor Nucleus.

Because a rank-ordered recruitment of motor units occurs during isometric contraction of jaw-closing muscles, jaw-closing motoneurons (MNs) may be recruited in a manner dependent on their soma sizes or input resistances (IRs). In the dorsolateral part of the trigeminal motor nucleus (dl-TMN) in rats, MNs abundantly express TWIK (two-pore domain weak inwardly rectifying K channel)-related acid-sensitive-K(+) channel (TASK)-1 and TASK3 channels, which determine the IR and resting membrane potential. Here we examined how TASK channels are involved in IR-dependent activation/recruitment of MNs in the rat dl-TMN by using multiple methods. The real-time PCR study revealed that single large MNs (>35 µm) expressed TASK1 and TASK3 mRNAs more abundantly compared with single small MNs (15-20 µm). The immunohistochemistry revealed that TASK1 and TASK3 channels were complementarily distributed in somata and dendrites of MNs, respectively. The density of TASK1 channels seemed to increase with a decrease in soma diameter while there were inverse relationships between the soma size of MNs and IR, resting membrane potential, or spike threshold. Dual whole-cell recordings obtained from smaller and larger MNs revealed that the recruitment of MNs depends on their IRs in response to repetitive stimulation of the presumed Ia afferents. 8-Bromoguanosine-cGMP decreased IRs in small MNs, while it hardly changed those in large MNs, and subsequently decreased the difference in spike-onset latency between the smaller and larger MNs, causing a synchronous activation of MNs. These results suggest that TASK channels play critical roles in rank-ordered recruitment of MNs in the dl-TMN.

The Etv1 transcription factor activity-dependently downregulates a set of genes controlling cell growth and differentiation in maturing cerebellar granule cells.

In the early postnatal period, cerebellar granule cells exhibit an activity-dependent downregulation of a set of immaturation genes involved in cell growth and migration and are shifted to establishment of a mature network formation. Through the use of a granule cell culture and both pharmacological and RNA interference (siRNA) analyses, the present investigation revealed that the downregulation of these immaturation genes is controlled by strikingly unified signaling mechanisms that operate sequentially through the stimulation of AMPA and NMDA receptors, tetrodotoxin-sensitive Na(+) channels and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). This signaling cascade induces the Etv1 transcription factor, and knockdown of Etv1 by a siRNA technique prevented this activity-dependent downregulation of immaturation genes. Thus, taken into consideration the mechanism that controls the upregulation of maturation genes involved in synaptic formation, these results indicate that Etv1 orchestrates the activity-dependent regulation of both maturation and immaturation genes in developing granule cells and plays a key role in specifying the identity of mature granule cells in the cerebellum.

Gene regulation via excitation and BDNF is mediated by induction and phosphorylation of the Etv1 transcription factor in cerebellar granule cells.

In maturing postnatal cerebellar granule cells, the Etv1/Er81 transcription factor is induced by sequential activity-dependent mechanisms through stimulation of AMPA and NMDA receptors, voltage-dependent Nav1.2 Na(+) channels, and voltage-dependent Ca(2+) channels. Etv1 then up-regulates a battery of maturation genes involved in the cerebellar circuitry. In this process, BDNF is also induced and participates in the up-regulation of these maturation genes. Using cultures of granule cells, we addressed how the activity-dependent and BDNF signaling mechanisms converge on the regulation of the representative NR2C NMDA receptor and Tiam1 maturation genes. BDNF up-regulated both the NR2C and Tiam1 genes via the TrkB-Erk cascade and this up-regulation was blocked not only by inhibition of the activity-dependent signaling mechanisms but also by suppression of Etv1 expression with Etv1 siRNA. Importantly, Etv1 was selectively phosphorylated by Erk1/2 in the BDNF signaling cascade, and the inhibition of this phosphorylation abrogated the BDNF-induced up-regulation of the NR2C and Tiam1 genes. The luciferase reporter assays in combination with mutations of MEK and Etv1 indicated that the Erk-mediated, phosphorylated Etv1 interacted with the Ets motifs of the NR2C promoter sequence and that phosphorylation at both serine 94 and a cluster of threonines and a serine (Thr139, Thr143, and Ser146) of Etv1 was indispensable for the BDNF-mediated activation of the NR2C promoter activity. This study demonstrates that the NR2C and Tiam1 maturation genes are synergistically controlled by the activity-dependent induction of Etv1 and its phosphorylation by the BDNF signaling cascade.

The Etv1/Er81 transcription factor orchestrates activity-dependent gene regulation in the terminal maturation program of cerebellar granule cells.

In the postnatal period, cerebellar granule cells express a set of the maturation gene battery in an activity-dependent manner and establish synaptic function in the cerebellar circuitry. Using primary cultures combined with specific inhibition of signaling cascades, the present investigation revealed that the expression of the maturation genes, including the NMDA glutamate receptor NR2C and GABA(A) receptor GABA(A)Rα6 genes, is controlled by strikingly unified signaling mechanisms that operate sequentially through stimulation of AMPA and NMDA receptors, Na(+) channels [voltage-gated Na channel type II (Nav1.2)], and voltage-dependent Ca(2+) channels. This signaling then induces the Ets variant gene 1 (Etv1/Er81) transcription factor of the ETS family in an activity-dependent manner. Consistent with the culture study, the ChIP assay indicated that Etv1 up-regulates the maturation genes in a developmentally regulated manner. This activation, as revealed by the luciferase assay, occurrs by interacting with the Etv1-interacting motifs present in the promoter region. Importantly, in vivo knockdown of Etv1 by DNA electroporation in the developing cerebellum prevents the up-regulation of the maturation genes but has no effects on preceding developmental processes occurring in the granule cells. Etv1 thus orchestrates the activity-dependent gene regulation in the terminal maturation program and specifies the identity of cerebellar granule cells.

Protein kinase G dynamically modulates TASK1-mediated leak K+ currents in cholinergic neurons of the basal forebrain.

Leak K(+) conductance generated by TASK1/3 channels is crucial for neuronal excitability. However, endogenous modulators activating TASK channels in neurons remained unknown. We previously reported that in the presumed cholinergic neurons of the basal forebrain (BF), activation of NO-cGMP-PKG (protein kinase G) pathway enhanced the TASK1-like leak K(+) current (I-K(leak)). As 8-Br-cGMP enhanced the I-K(leak) mainly at pH 7.3 as if changing the I-K(leak) from TASK1-like to TASK3-like current, such an enhancement of the I-K(leak) would result either from an enhancement of hidden TASK3 component or from an acidic shift in the pH sensitivity profile of TASK1 component. In view of the report that protonation of TASK channel decreases its open probability, the present study was designed to examine whether the activation of PKG increases the conductance of TASK1 channels by reducing their binding affinity for H(+), i.e., by increasing K(d) for protonation, or not. We here demonstrate that PKG activation and inhibition respectively upregulate and downregulate TASK1 channels heterologously expressed in PKG-loaded HEK293 cells at physiological pH, by causing shifts in the K(d) in the acidic and basic directions, respectively. Such PKG modulations of TASK1 channels were largely abolished by mutating pH sensor H98. In the BF neurons that were identified to express ChAT and TASK1 channels, similar dynamic modulations of TASK1-like pH sensitivity of I-K(leak) were caused by PKG. It is strongly suggested that PKG activation and inhibition dynamically modulate TASK1 currents at physiological pH by bidirectionally changing K(d) values for protonation of the extracellular pH sensors of TASK1 channels in cholinergic BF neurons.

Abnormal behavior in a chromosome-engineered mouse model for human 15q11-13 duplication seen in autism.

Substantial evidence suggests that chromosomal abnormalities contribute to the risk of autism. The duplication of human chromosome 15q11-13 is known to be the most frequent cytogenetic abnormality in autism. We have modeled this genetic change in mice by using chromosome engineering to generate a 6.3 Mb duplication of the conserved linkage group on mouse chromosome 7. Mice with a paternal duplication display poor social interaction, behavioral inflexibility, abnormal ultrasonic vocalizations, and correlates of anxiety. An increased MBII52 snoRNA within the duplicated region, affecting the serotonin 2c receptor (5-HT2cR), correlates with altered intracellular Ca(2+) responses elicited by a 5-HT2cR agonist in neurons of mice with a paternal duplication. This chromosome-engineered mouse model for autism seems to replicate various aspects of human autistic phenotypes and validates the relevance of the human chromosome abnormality. This model will facilitate forward genetics of developmental brain disorders and serve as an invaluable tool for therapeutic development.

Role of calcineurin signaling in membrane potential-regulated maturation of cerebellar granule cells.

At the early postnatal period, cerebellar granule cells proliferate, differentiate, migrate, and finally form refined synaptic connections with mossy fibers. During this period, the resting membrane potential of immature granule cells is relatively depolarized, but it becomes hyperpolarized in mature cells. This investigation was conducted to examine the role of this alteration in membrane potential and its downstream signaling mechanism in development and maturation of granule cells. Experiments were designed to precisely characterize the ontogenic processes of developing granule cells by combining organotypic cerebellar cultures with the specific expression of EGFP (enhanced green fluorescent protein) in granule cells by use of DNA transfection. Multiple approaches using morphology, electrophysiology, and immunohistochemistry demonstrated that granule cells developed and matured at the physiological KCl concentration in organotypic cultures in a temporally regulated manner. We addressed how persistent membrane depolarization influences the developmental and maturation processes of granule cells by depolarizing organotypic cultures with high KCl. Depolarization preserved the developmental processes of granule cells up to the stage of formation of immature dendrites but prevented the maturation processes for synaptic formation by granule cells. Importantly, this blockade of the terminal maturation of granule cells was reversed by inactivation of calcineurin with its specific inhibitor. This investigation has demonstrated that alteration of the membrane potential and its downstream calcineurin signaling play a pivotal role in triggering the maturation program for the synaptic organization of postnatally developing granule cells.

Dual regulation of NR2B and NR2C expression by NMDA receptor activation in mouse cerebellar granule cell cultures.

In the developing cerebellum, switching of the subunit composition of NMDA receptors occurs in granule cells from NR2B-containing receptors to NR2C-containing ones. We investigated the mechanisms underlying switching of NR2B and NR2C subunit composition in primary cultures of mouse granule cells at the physiological KCl concentration (5 mM). Granule cells extensively extended their neuritic processes 48 h after having been cultured in serum-free medium containing 5 mM KCl. Consistent with this morphological change, NR2B mRNA and NR2C mRNA were down- and up-regulated, respectively, in the granule cells. This dual regulation of the two mRNAs was abrogated by blocking excitation of granule cells with TTX. This neuronal activity-dependent regulation of NR2B and NR2C mRNAs was abolished by the addition of selective antagonists of AMPA receptors and NMDA receptors. Furthermore, the dual regulation of NR2B and NR2C mRNAs in TTX-treated cells was restored by the addition of NMDA in the presence of the AMPA receptor antagonist, but not by that of AMPA in the presence of the NMDA receptor antagonist. Importantly, the NMDA receptor activation drove the NR2B/NR2C switching of NMDA receptors in the cell-surface membrane of granule cells. This investigation demonstrates that stimulation of NMDA receptors in conjunction with the AMPA receptor-mediated excitation of granule cells plays a key role in functional subunit switching of NMDA receptors in maturing granule cells at the physiological KCl concentration.

Membrane potential-regulated Ca2+ signalling in development and maturation of mammalian cerebellar granule cells.

In many developing neuronal cell types, the resting membrane potential is relatively depolarized, then gradually hyperpolarizes during the early postnatal period. The regulatory roles of membrane potential changes in neuronal development and maturation have been extensively studied in developing cerebellar granule cells, using primary culture under depolarizing and non-depolarizing conditions in combination with in vivo analysis. Depolarization enhances calcium entry via voltage-sensitive Ca2+ channels (VSCCs) and activates Ca2+-calmodulin-dependent protein kinase (CaMK) and calcineurin phophatase (CaN). The activation of CaN induces many genes encoding extracellular and intracellular signalling molecules implicated in granule cell development. The inactivation of CaN in turn up-regulates many other genes characteristic of mature granule cells, including NR2C NMDA receptor and GABAAalpha1 and alpha6 receptors. The induction of NR2C also requires CaMK-up-regulated brain-derived neurotrophic factor (BDNF), indicating a convergence of signalling mechanism of the CaMK and CaN cascades. The inactivation of CaN maintains the phosphorylated and sumoylated form of a transcriptional myocyte enhances factor 2A (MEF2A) regulator. This form of MEF2A acts as a transcriptional repressor and is essential for the dendritic morphogenesis of differentiated granule cells. Collectively, the membrane potential change and the resulting Ca2+ signalling play a pivotal role in development and maturation of neuronal cells.

TRPM8 protein localization in trigeminal ganglion and taste papillae.

TRPM8 is a TRP family cation channel which can be activated by cold stimuli or l-menthol. However, TRPM8 protein localization of nerve terminals in sensory organs remains unknown. Here we generated an antibody against TRPM8 and analyzed TRPM8 protein localization in trigeminal ganglia (TG) and in sensory nerve fibers in the tongue. TRPM8 immunoreactivity was detected in a subset of neurons with a small diameter in TG and in nerve fibers in the tongue. TRPM8-immunoreactive nerve fibers were rich in fungiform papillae, but sparse in foliate and circumvallate papillae. The TRPM8-immunoreactive nerve fibers reached the outer epithelial layer in each papilla, while no TRPM8-immunoreactive nerve fibers penetrated into taste buds. Double labeling analysis revealed that TRPM8 immunoreactivity was co-expressed with a part of TRPV1 or CGRP-immunoreactive neurons in TG. However, TRPM8 immunoreactivity was not observed in TRPV1- or CGRP-positive nerve fibers in fungiform, foliate, and circumvallate papillae. These results suggest that TRPM8 protein is present in sensory lingual nerve fibers mainly projected from TG and might work as cold and l-menthol receptors on tongue.

Noxious heat receptors present in cold-sensory cells in rats.

Noxious heat above approximately 45 degrees C applied on cold spots evokes a paradoxical cold sensation by activating cold fibers. It remains unresolved whether cold receptors respond to heat as well, or whether noxious-heat receptors and cold receptors coexist in the same fiber. Recently, noxious heat receptors (TRPV1) and cold receptors (TRPM8) have been cloned. It is controversial, however, whether TRPV1 and TRPM8 coexist in the same sensory neuron. Here, we investigate colocalization of these receptors in dorsal root ganglion (DRG) of rats. TRPV1 was expressed in 29% of TRPM8-positive cells in DRG sections. In Ca2+ imaging, noxious heat excited many of the cold-sensitive cells in culture. In a whole-cell current-clamp mode, noxious heat, capsaicin, cooling and menthol all evoked receptor potentials and impulses in a subset of DRG neurons. This colocalization of TRPV1 and TRPM8 in a DRG neuron may be the basis for the paradoxical cold sensation.

Primary cold-sensitive neurons in acutely dissociated cells of rat hypothalamus.

Local warming or cooling of the preoptic and anterior hypothalamus (PO/AH) areas evokes various thermoregulatory responses in mammals. We have hypothesized that warm- and cold-sensitive neurons recorded in the PO/AH are multiple thermostats that regulate the core temperature against heat and cold, respectively. However, since the proportion of cold-sensitive neurons is low, it is still controversial whether primary cold-sensitive neurons exist in the PO/AH. To answer this question, we investigated cold-sensitive neurons with Ca(2+) imaging in acutely dissociated PO/AH cells. Their threshold temperatures were 27.3+/-0.44 degrees C (mean+/-SEM, n=55). In extracellular recordings cooling evoked discharges in these cold-sensitive neurons. We conclude that primary cold-sensitive neurons with low threshold temperatures exist in PO/AH.

Ionic basis of cold receptors acting as thermostats.

When temperature (T) of skin decreases stepwise, cold fibers evoke transient afferent discharges, inducing cold sensation and heat-gain responses. Hence we have proposed that cold receptors at distal ends of cold fibers are thermostats to regulate skin T against cold. Here, with patch-clamp techniques, we studied the ionic basis of cold receptors in cultured dorsal root ganglion (DRG) neurons of rats, as a model of nerve endings. Cells that increased cytosolic Ca(2+) level in response to moderate cooling were identified as neurons with cold receptors. In whole-cell current-clamp recordings of these cells, in response to cooling, cold receptors evoked a dynamic receptor potential (RP), eliciting impulses briefly. In voltage-clamp recordings (-60 mV), step cooling induced an inward cold current (I(cold)) with inactivation, underlying the dynamic RP. Ca(2+) ions that entered into cells from extracellular side induced the inactivation. Analysis of the reversal potential implied that I(cold) was nonselective cation current with high Ca(2+) permeability. Threshold temperatures of cooling-induced Ca(2+) response and I(cold) were different primarily among cells. In outside-out patches, when T decreased, single nonselective cation channels became active at a critical T. This implies that a cold receptor is an ion channel and acts as the smallest thermostat. Because these thermal properties were consistent with that in cold fibers, we conclude that the same cold receptors exist at nerve endings and generate afferent impulses for cold sensation and heat-gain behaviors in response to cold.