C. difficile intoxicates neurons and pericytes to drive neurogenic inflammation.

Clostridioides difficile infection (CDI) is a major cause of healthcare-associated gastrointestinal infections1,2. The exaggerated colonic inflammation caused by C. difficile toxins such as toxin B (TcdB) damages tissues and promotes C. difficile colonization3-6, but how TcdB causes inflammation is unclear. Here we report that TcdB induces neurogenic inflammation by targeting gut-innervating afferent neurons and pericytes through receptors, including the Frizzled receptors (FZD1, FZD2 and FZD7) in neurons and chondroitin sulfate proteoglycan 4 (CSPG4) in pericytes. TcdB stimulates the secretion of the neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) from neurons and pro-inflammatory cytokines from pericytes. Targeted delivery of the TcdB enzymatic domain, through fusion with a detoxified diphtheria toxin, into peptidergic sensory neurons that express exogeneous diphtheria toxin receptor (an approach we term toxogenetics) is sufficient to induce neurogenic inflammation and recapitulates major colonic histopathology associated with CDI. Conversely, mice lacking SP, CGRP or the SP receptor (neurokinin 1 receptor) show reduced pathology in both models of caecal TcdB injection and CDI. Blocking SP or CGRP signalling reduces tissue damage and C. difficile burden in mice infected with a standard C. difficile strain or with hypervirulent strains expressing the TcdB2 variant. Thus, targeting neurogenic inflammation provides a host-oriented therapeutic approach for treating CDI.

Urothelium-derived prostanoids enhance contractility of urinary bladder smooth muscle and stimulate bladder afferent nerve activity in the mouse.

The transitional epithelial cells (urothelium) that line the lumen of the urinary bladder form a barrier between potentially harmful pathogens, toxins, and other bladder contents and the inner layers of the bladder wall. The urothelium, however, is not simply a passive barrier, as it can produce signaling factors, such as ATP, nitric oxide, prostaglandins, and other prostanoids, that can modulate bladder function. We investigated whether substances produced by the urothelium could directly modulate the contractility of the underlying urinary bladder smooth muscle. Force was measured in isolated strips of mouse urinary bladder with the urothelium intact or denuded. Bladder strips developed spontaneous tone and phasic contractions. In urothelium-intact strips, basal tone, as well as the frequency and amplitude of phasic contractions, were 25%, 32%, and 338% higher than in urothelium-denuded strips, respectively. Basal tone and phasic contractility in urothelium-intact bladder strips were abolished by the cyclooxygenase (COX) inhibitor indomethacin (10 µM) or the voltage-dependent Ca2+ channel blocker diltiazem (50 µM), whereas blocking neuronal sodium channels with tetrodotoxin (1 µM) had no effect. These results suggest that prostanoids produced in the urothelium enhance smooth muscle tone and phasic contractions by activating voltage-dependent Ca2+ channels in the underlying bladder smooth muscle. We went on to demonstrate that blocking COX inhibits the generation of transient pressure events in isolated pressurized bladders and greatly attenuates the afferent nerve activity during bladder filling, suggesting that urothelial prostanoids may also play a role in sensory nerve signaling.NEW & NOTEWORTHY This paper provides evidence for the role of urothelial-derived prostanoids in maintaining tone in the urinary bladder during bladder filling, not only underscoring the role of the urothelium as more than a barrier but also contributing to active regulation of the urinary bladder. Furthermore, cyclooxygenase products greatly augment sensory nerve activity generated by bladder afferents during bladder filling and thus may play a role in perception of bladder fullness.

Identifying vagal bronchopulmonary afferents mediating cough response to inhaled sulfur dioxide in mice.

Sulfur dioxide (SO2), a common environmental and industrial air pollutant, possesses a potent effect in eliciting cough reflex, but the primary type of airway sensory receptors involved in its tussive action has not been clearly identified. This study was carried out to determine the relative roles of three major types of vagal bronchopulmonary afferents [slowly adapting receptors (SARs), rapidly adapting receptors (RARs), and C-fibers] in regulating the cough response to inhaled SO2. Our results showed that inhalation of SO2 (300 or 600 ppm for 8 min) evoked an abrupt and intense stimulatory effect on bronchopulmonary C-fibers, which continued for the entire duration of inhalation challenge and returned toward the baseline in 1-2 min after resuming room air-breathing in anesthetized and mechanically ventilated mice. In stark contrast, the same SO2 inhalation challenge generated a distinct and consistent inhibitory effect on both SARs and phasic RARs; their phasic discharges synchronized with respiratory cycles during the baseline (breathing room air) began to decline progressively within 1-3 min after the onset of SO2 inhalation, ceased completely before termination of the 8-min inhalation challenge, and then slowly returned toward the baseline after >40 min. In a parallel study in awake mice, inhalation of SO2 at the same concentration and duration as that in the nerve recording experiments evoked cough responses in a pattern and time course similar to that observed in the C-fiber responses. Based on these results, we concluded that stimulation of vagal bronchopulmonary C-fibers is primarily responsible for triggering the cough response to inhaled SO2.NEW & NOTEWORTHY This study demonstrated that inhalation of a high concentration of sulfur dioxide, an irritant gas and common air pollutant, completely and reversibly inhibited the neural activities of both slowly adapting receptor and rapidly adapting receptor, two major types of mechanoreceptors in the lungs with their activities conducted by myelinated fibers. Furthermore, the results of this study suggested that stimulation of vagal bronchopulmonary C-fibers is primarily responsible for triggering the cough reflex responses to inhaled sulfur dioxide.

Nitroxyl Delivered by Angeli's Salt Causes Short-Lasting Activation Followed by Long-Lasting Deactivation of Meningeal Afferents in Models of Headache Generation.

The role of TRPA1 receptor channels in meningeal nociception underlying the generation of headaches is still unclear. Activating as well as inhibitory effects of TRPA1 agonists have been reported in animal models of headache. The aim of the present study was to clarify the effect of the TRPA1 agonist nitroxyl (HNO) delivered by Angeli's salt in two rodent models of meningeal nociception. Single fibre recordings were performed using half-skull preparations of mice (C57BL/6) in vitro. Angeli's salt solution (AS, 300 µM) caused short-lasting vigorous increases in neuronal activity of primary meningeal afferents, followed by deactivation and desensitisation. These effects were similar in TRPA1 knockout and even more pronounced in TRPA1/TRPV1 double-knockout mice in comparison to wild-type mice. The activity of spinal trigeminal neurons with afferent input from the dura mater was recorded in vivo in anesthetised rats. AS (300 µM) or the TRPA1 agonist acrolein (100 and 300 µM) was applied to the exposed dura mater. AS caused no significant changes in spontaneous activity, while the mechanically evoked activity was reduced after acrolein application. These results do not confirm the assumption that activation of trigeminal TRPA1 receptor channels triggers the generation of headaches or contributes to its aggravation. Instead, there is evidence that TRPA1 activation may have an inhibitory function in the nociceptive trigeminal system.

Developmentally Transient CB1Rs on Cerebellar Afferents Suppress Afferent Input, Downstream Synaptic Excitation, and Signaling to Migrating Neurons.

The endocannabinoid system plays important roles in brain development, but mechanistic studies have focused on neuronal differentiation, migration, and synaptogenesis, with less attention to transcellular interactions that coordinate neurodevelopmental processes across developing neural networks. We determined that, in the developing rodent cerebellar cortex (of both sexes), there is a transient window when the dominant brain cannabinoid receptor, CB1R, is expressed on afferent terminals instead of output neuron Purkinje cell synapses that dominate the adult cerebellum. Activation of these afferent CB1Rs suppresses synaptic transmission onto developing granule cells, and consequently also suppresses excitation of downstream neurons in the developing cortical network, including nonsynaptic, migrating neurons. Application of a CB1R antagonist during afferent stimulation trains and depolarizing voltage steps caused a significant, sustained potentiation of synaptic amplitude. Our data demonstrate that transiently expressed afferent CB1Rs regulate afferent synaptic strength during synaptogenesis, which enables coordinated dampening of transcortical developmental signals.SIGNIFICANCE STATEMENT The endogenous cannabinoid system plays diverse roles in brain development, which, combined with the rapidly changing legal and medical status of cannabis-related compounds, makes understanding how exogenous cannabinoids affect brain development an important biomedical objective. The cerebellum is a key brain region in a variety of neurodevelopmental disorders, and the adult cerebellum has one of the highest expression levels of CB1R, but little is known about CB1R in the developing cerebellum. Here we report a developmentally distinct expression and function of CB1R in the cerebellum, in which endogenous or exogenous activation of CB1Rs modifies afferent synaptic strength and coordinated downstream network signaling. These findings have implications for recreational and medical use of exogenous cannabinoids by pregnant and breastfeeding women.

Neurogenic substance P-influences on action potential production in afferent neurons of the kidney?

We recently showed that a substance P (SP)-dependent sympatho-inhibitory mechanism via afferent renal nerves is impaired in mesangioproliferative nephritis. Therefore, we tested the hypothesis that SP released from renal afferents inhibits the action potential (AP) production in their dorsal root ganglion (DRG) neurons. Cultured DRG neurons (Th11-L2) were investigated in current clamp mode to assess AP generation during both TRPV1 stimulation by protons (pH 6) and current injections with and without exposure to SP (0.5 µmol) or CGRP (0.5 µmol). Neurons were classified as tonic (sustained AP generation) or phasic (≤ 4 APs) upon current injection; voltage clamp experiments were performed for the investigation of TRPV1-mediated inward currents due to proton stimulation. Superfusion of renal neurons with protons and SP increased the number of action potentials in tonic neurons (9.6 ± 5 APs/10 s vs. 16.9 ± 6.1 APs/10 s, P < 0.05, mean ± SD, n = 7), while current injections with SP decreased it (15.2 ± 6 APs/600 ms vs. 10.2 ± 8 APs/600 ms, P < 0.05, mean ± SD, n = 29). Addition of SP significantly reduced acid-induced TRPV1-mediated currents in renal tonic neurons (- 518 ± 743 pA due to pH 6 superfusion vs. - 82 ± 50 pA due to pH 6 with SP superfusion). In conclusion, SP increased action potential production via a TRPV1-dependent mechanism in acid-sensitive renal neurons. On the other hand, current injection in the presence of SP led to decreased action potential production. Thus, the peptide SP modulates signaling pathways in renal neurons in an unexpected manner leading to both stimulation and inhibition of renal neuronal activity in different (e.g., acidic) environmental contexts.

CB2 cannabinoid receptor agonist selectively inhibits the mechanosensitivity of mucosal afferents in the guinea pig bladder.

Bladder afferents play a pivotal role in bladder function such as urine storage and micturition as well as conscious sensations such as urgency and pain. Endocannabinoids are ligands of cannabinoid 1 and 2 (CB1 and CB2) receptors but can influence the activity of a variety of G protein-coupled receptors as well as ligand-gated and voltage-gated channels. It is still not known which classes of bladder afferents are influenced by CB1 and CB2 receptor agonists. This study aimed to determine the role of CB2 receptors in two major classes of afferents in the guinea pig bladder: mucosal and muscular-mucosal. The mechanosensitivity of these two classes was determined by an ex vivo extracellular electrophysiological recording technique. A stable analog of endocannabinoid anandamide, methanandamide (mAEA), potentiated the mechanosensitivity of mucosal bladder afferents in response to stroking. In the presence of a transient receptor potential vanilloid 1 antagonist (capsazepine), the effect of mAEA switched from excitatory to inhibitory. A selective CB2 receptor agonist, 4-quinolone-3-carboxyamide (4Q3C), significantly inhibited the mechanosensitivity of mucosal bladder afferents to stroking. In the presence of a CB2 receptor antagonist, the inhibitory effect of 4Q3C was lost. mAEA and 4Q3C did not affect responses to stretch and/or mucosal stroking of muscular-mucosal afferents. Our findings revealed that agonists of CB2 receptors selectively inhibited the mechanosensitivity of capsaicin-sensitive mucosal bladder afferents but not muscular-mucosal afferents. This may have important implications for understanding of the role of endocannabinoids in modulating bladder function and sensation in health and diseases.NEW & NOTEWORTHY This article describes, for the first time, to our knowledge, the direct inhibitory effect of cannabinoid 2 receptor agonists on guinea pig mucosal bladder afferents. The cannabinoid 2 receptor is involved in pain and inflammation, suggesting that this may be a viable target for treatment of bladder disorders such as cystitis.

Nicotinic receptor modulation of primary afferent excitability with selective regulation of Aδ-mediated spinal actions.

Somatosensory input strength can be modulated by primary afferent depolarization (PAD) generated predominantly via presynaptic GABAA receptors on afferent terminals. We investigated whether ionotropic nicotinic acetylcholine receptors (nAChRs) also provide modulatory actions, focusing on myelinated afferent excitability in in vitro murine spinal cord nerve-attached models. Primary afferent stimulation-evoked synaptic transmission was recorded in the deep dorsal horn as extracellular field potentials (EFPs), whereas concurrently recorded dorsal root potentials (DRPs) were used as an indirect measure of PAD. Changes in afferent membrane excitability were simultaneously measured as direct current (DC)-shifts in membrane polarization recorded in dorsal roots or peripheral nerves. The broad nAChR antagonist d-tubocurarine (d-TC) selectively and strongly depressed Aδ-evoked synaptic EFPs (36% of control) coincident with similarly depressed A-fiber DRP (43% of control), whereas afferent electrical excitability remained unchanged. In comparison, acetylcholine (ACh) and the nAChR agonists, epibatidine and nicotine, reduced afferent excitability by generating coincident depolarizing DC-shifts in peripheral axons and intraspinally. Progressive depolarization corresponded temporally with the emergence of spontaneous axonal spiking and reductions in the DRP and all afferent-evoked synaptic actions (31%-37% of control). Loss of evoked response was long-lasting, independent of DC repolarization, and likely due to mechanisms initiated by spontaneous C-fiber activity. DC-shifts were blocked with d-TC but not GABAA receptor blockers and retained after tetrodotoxin block of voltage-gated Na+ channels. Notably, actions tested were comparable between three mouse strains, in rat, and when performed in different labs. Thus, nAChRs can regulate afferent excitability via two distinct mechanisms: by central Aδ-afferent actions, and by transient extrasynaptic axonal activation of high-threshold primary afferents.NEW & NOTEWORTHY Primary afferents express many nicotinic ACh receptor (nAChR) subtypes but whether activation is linked to presynaptic inhibition, facilitation, or more complex and selective activity modulation is unknown. Recordings of afferent-evoked responses in the lumbar spinal cord identified two nAChR-mediated modulatory actions: 1) selective control of Aδ afferent transmission and 2) robust changes in axonal excitability initiated via extrasynaptic shifts in DC polarization. This work broadens the diversity of presynaptic modulation of primary afferents by nAChRs.

Impact of anesthesia, sex, and circadian cycle on renal afferent nerve sensitivity.

Elevated renal afferent nerve (ARNA) activity or dysfunctional reno-renal reflexes via altered ARNA sensitivity contribute to hypertension and chronic kidney disease. These nerves contain mechano- and chemosensitive fibers that respond to ischemia, changes in intrarenal pressures, and chemokines. Most studies have utilized various anesthetized preparations and exclusively male animals to characterize ARNA responses. Therefore, this study assessed the impact of anesthesia, sex, and circadian period on ARNA responses and sensitivity. Multifiber ARNA recordings were performed in male and female Sprague-Dawley rats (250-400 g) and compared across decerebrate versus Inactin, isoflurane, and urethane anesthesia groups. Intrarenal artery infusion of capsaicin (0.1-50.0 µM, 0.05 mL) produced concentration-dependent increases in ARNA; however, the ARNA sensitivity was significantly greater in decerebrate versus Inactin, isoflurane, and urethane groups. Increases in renal pelvic pressure (0-30 mmHg, 30 s) produced pressure-dependent increases in ARNA; however, ARNA sensitivity was again greater in decerebrate and Inactin groups versus isoflurane and urethane. Acute renal artery occlusion (30 s) increased ARNA, but responses did not differ across groups. Analysis of ARNA responses to increased pelvic pressure between male and female rats revealed significant sex differences only in isoflurane and urethane groups. ARNA responses to intrarenal capsaicin infusion were significantly blunted at nighttime versus daytime; however, ARNA responses to increased pelvic pressure or renal artery occlusion were not different between daytime and nighttime. These results demonstrate that ARNA sensitivity is greatest in decerebrate and Inactin-anesthetized groups but was not consistently influenced by sex.NEW & NOTEWORTHY We determined the impact of anesthesia, sex, and circadian cycle on renal afferent nerve (ARNA) sensitivity to chemical and mechanical stimuli. ARNA sensitivity to renal capsaicin infusion was greatest in decerebrate > Inactin > urethane or isoflurane groups. Elevated renal pelvic pressure significantly increased ARNA; decerebrate and Inactin groups exhibited the greatest ARNA sensitivity. Sex differences in renal afferent responses were not consistently observed. Circadian cycle altered chemosensory but not mechanosensory responses.

Acute high-intensity exercise and skeletal muscle mitochondrial respiratory function: role of metabolic perturbation.

Recently it was documented that fatiguing, high-intensity exercise resulted in a significant attenuation in maximal skeletal muscle mitochondrial respiratory capacity, potentially due to the intramuscular metabolic perturbation elicited by such intense exercise. With the utilization of intrathecal fentanyl to attenuate afferent feedback from group III/IV muscle afferents, permitting increased muscle activation and greater intramuscular metabolic disturbance, this study aimed to better elucidate the role of metabolic perturbation on mitochondrial respiratory function. Eight young, healthy males performed high-intensity cycle exercise in control (CTRL) and fentanyl-treated (FENT) conditions. Liquid chromatography-mass spectrometry and high-resolution respirometry were used to assess metabolites and mitochondrial respiratory function, respectively, pre- and postexercise in muscle biopsies from the vastus lateralis. Compared with CTRL, FENT yielded a significantly greater exercise-induced metabolic perturbation (PCr: -67% vs. -82%, Pi: 353% vs. 534%, pH: -0.22 vs. -0.31, lactate: 820% vs. 1,160%). Somewhat surprisingly, despite this greater metabolic perturbation in FENT compared with CTRL, with the only exception of respiratory control ratio (RCR) (-3% and -36%) for which the impact of FENT was significantly greater, the degree of attenuated mitochondrial respiratory capacity postexercise was not different between CTRL and FENT, respectively, as assessed by maximal respiratory flux through complex I (-15% and -33%), complex II (-36% and -23%), complex I + II (-31% and -20%), and state 3CI+CII control ratio (-24% and -39%). Although a basement effect cannot be ruled out, this failure of an augmented metabolic perturbation to extensively further attenuate mitochondrial function questions the direct role of high-intensity exercise-induced metabolite accumulation in this postexercise response.

Protective Effect of TRPM8 against Indomethacin-Induced Small Intestinal Injury via the Release of Calcitonin Gene-Related Peptide in Mice.

Transient receptor potential melastatin 8 (TRPM8) is a non-selective cation channel activated by mild cooling and chemical agents including menthol. Nonsteroidal anti-inflammatory drugs have antipyretic, analgesic effects, and they can cause stomach and small intestinal injury. The current study investigated the role of TRPM8 in the pathogenesis of indomethacin-induced small intestinal injury. In male TRPM8-deficient (TRPM8KO) and wild-type (WT) mice, intestinal injury was induced via the subcutaneous administration of indomethacin. In addition, the effect of WS-12, a specific TRPM8 agonist, was examined in TRPM8KO and WT mice with indomethacin-induced intestinal injury. TRPM8KO mice had a significantly higher intestinal ulcerogenic response to indomethacin than WT mice. The repeated administration of WS-12 significantly attenuated the severity of intestinal injury in WT mice. However, this response was abrogated in TRPM8KO mice. Furthermore, in TRPM8-enhanced green fluorescent protein (EGFP) transgenic mice, which express EGFP under the direction of TRPM8 promoter, the EGFP signals in the indomethacin-treated intestinal mucosa were upregulated. Further, the EGFP signals were commonly found in calcitonin gene-related peptide (CGRP)-positive sensory afferent neurons and partly colocalized with substance P (SP)-positive neurons in the small intestine. The intestinal CGRP-positive neurons were significantly upregulated after the administration of indomethacin in WT mice. Nevertheless, this response was abrogated in TRPM8KO mice. In contrast, indomethacin increased the expression of intestinal SP-positive neurons in not only WT mice but also TRPM8KO mice. Thus, TRPM8 has a protective effect against indomethacin-induced small intestinal injury. This response may be mediated by the upregulation of CGRP, rather than SP.

Role of Lateral Hypothalamus in Acupuncture Inhibition of Cocaine Psychomotor Activity.

Acupuncture modulates the mesolimbic dopamine (DA) system; an area implicated in drug abuse. However, the mechanism by which peripheral sensory afferents, during acupuncture stimulation, modulate this system needs further investigation. The lateral hypothalamus (LH) has been implicated in reward processing and addictive behaviors. To investigate the role of the LH in mediating acupuncture effects, we evaluated the role of LH and spinohypothalamic neurons on cocaine-induced psychomotor activity and NAc DA release. Systemic injection of cocaine increased locomotor activity and 50 kHz ultrasonic vocalizations (USVs), which were attenuated by mechanical stimulation of needles inserted into HT7 but neither ST36 nor LI5. The acupuncture effects were blocked by chemical lesions of the LH or mimicked by activation of LH neurons. Single-unit extracellular recordings showed excitation of LH and spinohypothalamic neurons following acupuncture. Our results suggest that acupuncture recruits the LH to suppress the mesolimbic DA system and psychomotor responses following cocaine injection.

17ß-Estradiol Exacerbated Experimental Occlusal Interference-Induced Chronic Masseter Hyperalgesia by Increasing the Neuronal Excitability and TRPV1 Function of Trigeminal Ganglion in Ovariectomized Rats.

Pain symptoms in temporomandibular disorders (TMD) predominantly affect reproductive women, suggesting that estrogen regulates pain perception. However, how estrogen contributes to chronic TMD pain remains largely unclear. In the present study, we performed behavioral tests, electrophysiology, Western blot and immunofluorescence to investigate the role and underlying mechanisms of estrogen in dental experimental occlusal interference (EOI)-induced chronic masseter mechanical hyperalgesia in rats. We found that long-term 17ß-estradiol (E2) replacement exacerbated EOI-induced masseter hyperalgesia in a dose-dependent manner in ovariectomized (OVX) rats. Whole-cell patch-clamp recordings demonstrated that E2 (100 nM) treatment enhanced the excitability of isolated trigeminal ganglion (TG) neurons in OVX and OVX EOI rats, and EOI increased the functional expression of transient receptor potential vanilloid-1 (TRPV1). In addition, E2 replacement upregulated the protein expression of TRPV1 in EOI-treated OVX rats. Importantly, intraganglionic administration of the TRPV1 antagonist AMG-9810 strongly attenuated the facilitatory effect of E2 on EOI-induced masseter mechanical sensitivity. These results demonstrate that E2 exacerbated EOI-induced chronic masseter mechanical hyperalgesia by increasing TG neuronal excitability and TRPV1 function. Our study helps to elucidate the E2 actions in chronic myogenic TMD pain and may provide new therapeutic targets for relieving estrogen-sensitive pain.

TRPM3 Is Expressed in Afferent Bladder Neurons and Is Upregulated during Bladder Inflammation.

The cation channel TRPM3 is activated by heat and the neurosteroid pregnenolone sulfate. TRPM3 is expressed on sensory neurons innervating the skin, where together with TRPV1 and TRPA1, it functions as one of three redundant sensors of acute heat. Moreover, functional upregulation of TRPM3 during inflammation contributes to heat hyperalgesia. The role of TRPM3 in sensory neurons innervating internal organs such as the bladder is currently unclear. Here, using retrograde labeling and single-molecule fluorescent RNA in situ hybridization, we demonstrate expression of mRNA encoding TRPM3 in a large subset of dorsal root ganglion (DRG) neurons innervating the mouse bladder, and confirm TRPM3 channel functionality in these neurons using Fura-2-based calcium imaging. After induction of cystitis by injection of cyclophosphamide, we observed a robust increase of the functional responses to agonists of TRPM3, TRPV1, and TRPA1 in bladder-innervating DRG neurons. Cystometry and voided spot analysis in control and cyclophosphamide-treated animals did not reveal differences between wild type and TRPM3-deficient mice, indicating that TRPM3 is not critical for normal voiding. We conclude that TRPM3 is functionally expressed in a large proportion of sensory bladder afferent, but its role in bladder sensation remains to be established.

Tetrodotoxin-Sensitive Sodium Channels Mediate Action Potential Firing and Excitability in Menthol-Sensitive Vglut3-Lineage Sensory Neurons.

Small-diameter vesicular glutamate transporter 3-lineage (Vglut3lineage) dorsal root ganglion (DRG) neurons play an important role in mechanosensation and thermal hypersensitivity; however, little is known about their intrinsic electrical properties. We therefore set out to investigate mechanisms of excitability within this population. Calcium microfluorimetry analysis of male and female mouse DRG neurons demonstrated that the cooling compound menthol selectively activates a subset of Vglut3lineage neurons. Whole-cell recordings showed that small-diameter Vglut3lineage DRG neurons fire menthol-evoked action potentials and exhibited robust, transient receptor potential melastatin 8 (TRPM8)-dependent discharges at room temperature. This heightened excitability was confirmed by current-clamp and action potential phase-plot analyses, which showed menthol-sensitive Vglut3lineage neurons to have more depolarized membrane potentials, lower firing thresholds, and higher evoked firing frequencies compared with menthol-insensitive Vglut3lineage neurons. A biophysical analysis revealed voltage-gated sodium channel (NaV) currents in menthol-sensitive Vglut3lineage neurons were resistant to entry into slow inactivation compared with menthol-insensitive neurons. Multiplex in situ hybridization showed similar distributions of tetrodotoxin (TTX)-sensitive NaV transcripts between TRPM8-positive and -negative Vglut3lineage neurons; however, NaV1.8 transcripts, which encode TTX-resistant channels, were more prevalent in TRPM8-negative neurons. Conversely, pharmacological analyses identified distinct functional contributions of NaV subunits, with NaV1.1 driving firing in menthol-sensitive neurons, whereas other small-diameter Vglut3lineage neurons rely primarily on TTX-resistant NaV channels. Additionally, when NaV1.1 channels were blocked, the remaining NaV current readily entered into slow inactivation in menthol-sensitive Vglut3lineage neurons. Thus, these data demonstrate that TTX-sensitive NaVs drive action potential firing in menthol-sensitive sensory neurons and contribute to their heightened excitability.SIGNIFICANCE STATEMENT Somatosensory neurons encode various sensory modalities including thermoreception, mechanoreception, nociception, and itch. This report identifies a previously unknown requirement for tetrodotoxin-sensitive sodium channels in action potential firing in a discrete subpopulation of small-diameter sensory neurons that are activated by the cooling agent menthol. Together, our results provide a mechanistic understanding of factors that control intrinsic excitability in functionally distinct subsets of peripheral neurons. Furthermore, as menthol has been used for centuries as an analgesic and anti-pruritic, these findings support the viability of NaV1.1 as a therapeutic target for sensory disorders.

Muscarinic Inhibition of Hypoglossal Motoneurons: Possible Implications for Upper Airway Muscle Hypotonia during REM Sleep.

Proper function of pharyngeal dilator muscles, including the genioglossus muscle of the tongue, is required to maintain upper airway patency. During sleep, the activity of these muscles is suppressed, and as a result individuals with obstructive sleep apnea experience repeated episodes of upper airway closure when they are asleep, in particular during rapid-eye-movement (REM) sleep. Blocking cholinergic transmission in the hypoglossal motor nucleus (MoXII) restores REM sleep genioglossus activity, highlighting the importance of cholinergic transmission in the inhibition of hypoglossal motor neurons (HMNs) during REM sleep. Glutamatergic afferent input from neurons in the parahypoglossal (PH) region to the HMNs is critical for MoXII respiratory motor output. We hypothesized that state-dependent cholinergic regulation may be mediated by this pathway. Here we studied the effects of cholinergic transmission in HMNs in adult male and female mice using patch-clamp recordings in brain slices. Using channelrhodopsin-2-assisted circuit mapping, we first demonstrated that PH glutamatergic neurons directly and robustly activate HMNs (PHGlut → HMNs). We then show that carbachol consistently depresses this input and that this effect is presynaptic. Additionally, carbachol directly affects HMNs by a variable combination of muscarinic-mediated excitatory and inhibitory responses. Altogether, our results suggest that cholinergic signaling impairs upper airway dilator muscle activity by suppressing glutamatergic input from PH premotoneurons to HMNs and by directly inhibiting HMNs. Our findings highlight the complexity of cholinergic control of HMNs at both the presynaptic and postsynaptic levels and provide a possible mechanism for REM sleep suppression of upper airway muscle activity.SIGNIFICANCE STATEMENT Individuals with obstructive sleep apnea can breathe adequately when awake but experience repeated episodes of upper airway closure when asleep, in particular during REM sleep. Similar to skeletal postural muscles, pharyngeal dilator muscles responsible for maintaining an open upper airway become hypotonic during REM sleep. Unlike spinal motoneurons controlling postural muscles that are inhibited by glycinergic transmission during REM sleep, hypoglossal motoneurons that control the upper airway muscles are inhibited in REM sleep by the combination of monoaminergic disfacilitation and cholinergic inhibition. In this study, we demonstrated how cholinergic signaling inhibits hypoglossal motoneurons through presynaptic and postsynaptic muscarinic receptors. Our results provide a potential mechanism for upper airway hypotonia during REM sleep.

Contributing mechanisms underlying desensitization of cholecystokinin-induced activation of primary nodose ganglia neurons.

Cholecystokinin (CCK) is a gut-derived peptide that potently promotes satiety and facilitates gastric function in part by activating G protein-coupled CCK1 receptors on primary vagal afferent neurons. CCK signaling is dynamic and rapidly desensitizes, due to decreases in either receptor function and the resulting signal cascade, ion channel effectors, or both. Here we report a decay-time analytical approach using fluorescent calcium imaging that relates peak and steady-state calcium responses in dissociated vagal afferent neurons, enabling discrimination between receptor and ion channel effector functions. We found desensitization of CCK-induced activation was predictable, consistent across cells, and strongly concentration dependent. The decay-time constant (tau) was inversely proportional to CCK concentration, apparently reflecting the extent of receptor activation. To test this possibility, we directly manipulated the ion channel effector(s) with either decreased bath calcium or the broad-spectrum pore blocker ruthenium red. Conductance inhibition diminished the magnitude of the CCK responses without altering decay kinetics, confirming changes in tau reflect changes in receptor function selectively. Next, we investigated the contributions of the PKC and PKA signaling cascades on the magnitude and decay-time constants of CCK calcium responses. While inhibition of either PKC or PKA increased CCK calcium response magnitude, only general PKC inhibition significantly decreased the decay-time constant. These findings suggest that PKC alters CCK receptor signaling dynamics, while PKA alters the ion channel effector of the CCK response. This analytical approach should prove useful in understanding receptor/effector changes underlying acute desensitization of G-protein coupled signaling and provide insight into CCK receptor dynamics.

Afferent renal innervation in anti-Thy1.1 nephritis in rats.

Afferent renal nerves exhibit a dual function controlling central sympathetic outflow via afferent electrical activity and influencing intrarenal immunological processes by releasing peptides such as calcitonin gene-related peptide (CGRP). We tested the hypothesis that increased afferent and efferent renal nerve activity occur with augmented release of CGRP in anti-Thy1.1 nephritis, in which enhanced CGRP release exacerbates inflammation. Nephritis was induced in Sprague-Dawley rats by intravenous injection of OX-7 antibody (1.75 mg/kg), and animals were investigated neurophysiologically, electrophysiologically, and pathomorphologically 6 days later. Nephritic rats exhibited proteinuria (169.3 ± 10.2 mg/24 h) with increased efferent renal nerve activity (14.7 ± 0.9 bursts/s vs. control 11.5 ± 0.9 bursts/s, n = 11, P < 0.05). However, afferent renal nerve activity (in spikes/s) decreased in nephritis (8.0 ± 1.8 Hz vs. control 27.4 ± 4.1 Hz, n = 11, P < 0.05). In patch-clamp recordings, neurons with renal afferents from nephritic rats showed a lower frequency of high activity following electrical stimulation (43.4% vs. 66.4% in controls, P < 0.05). In vitro assays showed that renal tissue from nephritic rats exhibited increased CGRP release via spontaneous (14 ± 3 pg/mL vs. 6.8 ± 2.8 pg/ml in controls, n = 7, P < 0.05) and stimulated mechanisms. In nephritic animals, marked infiltration of macrophages in the interstitium (26 ± 4 cells/mm2) and glomeruli (3.7 ± 0.6 cells/glomerular cross-section) occurred. Pretreatment with the CGRP receptor antagonist CGRP8-37 reduced proteinuria, infiltration, and proliferation. In nephritic rats, it can be speculated that afferent renal nerves lose their ability to properly control efferent sympathetic nerve activity while influencing renal inflammation through increased CGRP release.

Cannabidiol activation of vagal afferent neurons requires TRPA1.

Vagal afferent neurons abundantly express excitatory transient receptor potential (TRP) channels, which strongly influence afferent signaling. Cannabinoids have been identified as direct agonists of TRP channels, including TRPA1 and TRPV1, suggesting that exogenous cannabinoids may influence vagal signaling via TRP channel activation. The diverse therapeutic effects of electrical vagus nerve stimulation also result from administration of the nonpsychotropic cannabinoid, cannabidiol (CBD); however, the direct effects of CBD on vagal afferent signaling remain unknown. We investigated actions of CBD on vagal afferent neurons, using calcium imaging and electrophysiology. CBD produced strong excitatory effects in neurons expressing TRPA1. CBD responses were prevented by removal of bath calcium, ruthenium red, and the TRPA1 antagonist A967079, but not the TRPV1 antagonist SB366791, suggesting an essential role for TRPA1. These pharmacological experiments were confirmed using genetic knockouts where TRPA1 KO mice lacked CBD responses, whereas TRPV1 knockout (KO) mice exhibited CBD-induced activation. We also characterized CBD-provoked inward currents at resting potentials in vagal afferents expressing TRPA1 that were absent in TRPA1 KO mice, but persisted in TRPV1 KO mice. CBD also inhibited voltage-activated sodium conductances in A-fiber, but not in C-fiber afferents. To simulate adaptation, resulting from chronic cannabis use, we administered cannabis extract vapor daily for 3 wk. Cannabis exposure reduced the magnitude of CBD responses, likely due to a loss of TRPA1 signaling. Together, these findings detail a novel excitatory action of CBD at vagal afferent neurons, which requires TRPA1 and may contribute to the vagal mimetic effects of CBD and adaptation following chronic cannabis use.NEW & NOTEWORTHY CBD usage has increased with its legalization. The clinical efficacy of CBD has been demonstrated for conditions including some forms of epilepsy, depression, and anxiety that are also treatable by vagus nerve stimulation. We found CBD exhibited direct excitatory effects on vagal afferent neurons that required TRPA1, were augmented by TRPV1, and attenuated following chronic cannabis vapor exposure. These effects may contribute to vagal mimetic effects of CBD and adaptation after chronic cannabis use.

Reorganization of motor modules for standing reactive balance recovery following pyridoxine-induced large-fiber peripheral sensory neuropathy in cats.

Task-level goals such as maintaining standing balance are achieved through coordinated muscle activity. Consistent and individualized groupings of synchronously activated muscles can be estimated from muscle recordings in terms of motor modules or muscle synergies, independent of their temporal activation. The structure of motor modules can change with motor training, neurological disorders, and rehabilitation, but the central and peripheral mechanisms underlying motor module structure remain unclear. To assess the role of peripheral somatosensory input on motor module structure, we evaluated changes in the structure of motor modules for reactive balance recovery following pyridoxine-induced large-fiber peripheral somatosensory neuropathy in previously collected data in four adult cats. Somatosensory fiber loss, quantified by postmortem histology, varied from mild to severe across cats. Reactive balance recovery was assessed using multidirectional translational support-surface perturbations over days to weeks throughout initial impairment and subsequent recovery of balance ability. Motor modules within each cat were quantified by non-negative matrix factorization and compared in structure over time. All cats exhibited changes in the structure of motor modules for reactive balance recovery after somatosensory loss, providing evidence that somatosensory inputs influence motor module structure. The impact of the somatosensory disturbance on the structure of motor modules in well-trained adult cats indicates that somatosensory mechanisms contribute to motor module structure, and therefore may contribute to some of the pathological changes in motor module structure in neurological disorders. These results further suggest that somatosensory nerves could be targeted during rehabilitation to influence pathological motor modules for rehabilitation.NEW & NOTEWORTHY Stable motor modules for reactive balance recovery in well-trained adult cats were disrupted following pyridoxine-induced peripheral somatosensory neuropathy, suggesting somatosensory inputs contribute to motor module structure. Furthermore, the motor module structure continued to change as the animals regained the ability to maintain standing balance, but the modules generally did not recover pre-pyridoxine patterns. These results suggest changes in somatosensory input and subsequent learning may contribute to changes in motor module structure in pathological conditions.