A Novel Flp Reporter Mouse Shows That TRPA1 Expression Is Largely Limited to Sensory Neuron Subsets.

Transient receptor potential ankyrin 1 (TRPA1) is a polymodal cation channel that is activated by electrophilic irritants, oxidative stress, cold temperature, and GPCR signaling. TRPA1 expression has been primarily identified in subsets of nociceptive sensory afferents and is considered a target for future analgesics. Nevertheless, TRPA1 has been implicated in other cell types including keratinocytes, epithelium, enterochromaffin cells, endothelium, astrocytes, and CNS neurons. Here, we developed a knock-in mouse that expresses the recombinase FlpO in TRPA1-expressing cells. We crossed the TRPA1Flp mouse with the R26ai65f mouse that expresses tdTomato in a Flp-sensitive manner. We found tdTomato expression correlated well with TRPA1 mRNA expression and sensitivity to TRPA1 agonists in subsets of TRPV1 (transient receptor potential vanilloid receptor type 1)-expressing neurons in the vagal ganglia and dorsal root ganglia (DRGs), although tdTomato expression efficiency was limited in DRG. We observed tdTomato-expressing afferent fibers centrally (in the medulla and spinal cord) and peripherally in the esophagus, gut, airways, bladder, and skin. Furthermore, chemogenetic activation of TRPA1-expressing nerves in the paw evoked flinching behavior. tdTomato expression was very limited in other cell types. We found tdTomato in subepithelial cells in the gut mucosa but not in enterochromaffin cells. tdTomato was also observed in supporting cells within the cochlea, but not in hair cells. Lastly, tdTomato was occasionally observed in neurons in the somatomotor cortex and the piriform area, but not in astrocytes or vascular endothelium. Thus, this novel mouse strain may be useful for mapping and manipulating TRPA1-expressing cells and deciphering the role of TRPA1 in physiological and pathophysiological processes.

A Comparative Analysis of Chikungunya in Kidney Transplant Recipients Compared With the General Population: A Multicenter, Retrospective, Cohort Study From India.

OBJECTIVES: Chikungunya is an arboviral illness, with patients presenting with fever, arthralgias, and myalgias. Outbreaks have occurred in tropical regions, and the virus is now endemic to many tropics, including South Asia, with India contributing a large part of the global burden. The presentation and long-term effects on transplant recipients are largely unknown. MATERIALS AND METHODS: In this retrospective analytical study, we compared chikungunya infection in 44 kidney transplant recipients from multiple centers in India and 34 patients from the general population. Data were collected from medical records and patient recall. RESULTS: Differences in presentation were remarkable between the 2 groups, with significantly lower incidence of musculoskeletal symptoms on presentation in transplant recipients compared with the general population. The incidence of acute graft dysfunction was 17.08% in transplant recipients, with return to baseline at the end of 1 month. Acute symptomatology resolved in transplant recipients within 1 month, and insignificant chronic symptoms were reported after 3 months. CONCLUSIONS: Chikungunya in kidney transplant recipients is markedly different from that of the general population, with significantly lower incidence of musculoskeletal symptoms such as arthralgias. The infection caused acute graft dysfunction, but no long-term sequelae were shown at the end of 1 year.

Ginger Constituent 6-Shogaol Attenuates Vincristine-Induced Activation of Mouse Gastroesophageal Vagal Afferent C-Fibers.

Chemotherapeutic agent-induced nausea and vomiting are the severe adverse effects that are induced by their stimulations on the peripheral and/or central emetic nerve pathways. Even though ginger has been widely used as an herbal medicine to treat emesis, mechanisms underlying its neuronal actions are still less clear. The present study aimed to determine the chemotherapeutic agent vincristine-induced effect on gastroesophageal vagal afferent nerve endings and the potential inhibitory role of ginger constituent 6-shogaol on such response. Two-photon neuron imaging studies were performed in ex vivo gastroesophageal-vagal preparations from Pirt-GCaMP6 transgenic mice. Vincristine was applied to the gastroesophageal vagal afferent nerve endings, and the evoked calcium influxes in their intact nodose ganglion neuron somas were recorded. The responsive nodose neuron population was first characterized, and the inhibitory effects of 5-HT3 antagonist palonosetron, TRPA1 antagonist HC-030031, and ginger constituent 6-shogaol were then determined. Vincristine application at gastroesophageal vagal afferent nerve endings elicited intensive calcium influxes in a sub-population of vagal ganglion neurons. These neurons were characterized by their positive responses to P2X2/3 receptor agonist α,ß-methylene ATP and TRPA1 agonist cinnamaldehyde, suggesting their nociceptive placodal nodose C-fiber neuron lineages. Pretreatment with TRPA1 selective blocker HC-030031 inhibited vincristine-induced calcium influxes in gastroesophageal nodose C-fiber neurons, indicating that TRPA1 played a functional role in mediating vincristine-induced activation response. Such inhibitory effect was comparable to that from 5-HT3 receptor antagonist palonosetron. Alternatively, pretreatment with ginger constituent 6-shogaol significantly attenuated vincristine-induced activation response. The present study provides new evidence that chemotherapeutic agent vincristine directly activates vagal nodose nociceptive C-fiber neurons at their peripheral nerve endings in the upper gastrointestinal tract. This activation response requires both TRPA1 and 5-HT3 receptors and can be attenuated by ginger constituent 6-shogaol.

Qualitative and Quantitative Evaluation of Donor Corneal Tissue by Slit Lamp and Specular Microscopy.

Background In India, donor eye collection and promotion of eye banking are insufficient to meet the needs. By adequately evaluating donor corneas, eye banks can maximize the number of viable corneas for transplantation. This study evaluated donor corneal tissue based on age, lens status, and cause of death by their morphology and endothelial cell count via slit lamp and specular microscopy. Methods We conducted a prospective observational study of all eye bank donor corneas indicated for eye donation at a tertiary hospital and research center in Western Maharashtra between September 2019 to December 2021. We evaluated the corneoscleral discs by slit-lamp microscopy specular microscopy. We analyzed donor corneas quantitatively and qualitatively and graded them accordingly. We also collected blood samples for serological testing and the donor's behavioral and family medical histories. Results We collected 94 eyes from 47 donors; the mean age of the donor population was 48.2 years, and most donors were aged 41 to 80 years. Thirty-one donors (65.96%) were male, and 16 were female (34.04%. For preservation, we used Cornisol (Aurolab, Madurai, India) in 36 cases (77%) and McCarey-Kaufman medium in 11 cases (23%). We found a mean endothelial cell density (ECD) of 2214.40/mm2, with hexagonality of 53.05%, and a coefficient of variation of 38.01. Further, we observed that ECD and hexagonality of cells in phakic donors were significantly greater than that of pseudophakic (PP) donors. Moreover, ECD and hexagonality significantly decreased in donors with the chronic disease compared to those who had a sudden, unexpected death. Conclusion Corneal grafts from younger donors, phakic donors, and donors who experienced an acute cause of death were qualitatively and quantitatively significantly better than those of older donors, PP donors, and donors who experienced sudden, unexpected death. Therefore, eye bank specular examination can improve tissue utilization and transplantation success. Therefore, we strongly recommend that eye bank personnel evaluate their donor tissue with a specular microscope to enhance the quality of eye care.

Investigating Pore-Opening of Hydrogel Foams at the Scale of Freestanding Thin Films.

Controlling the pore connectivity of polymer foams is key for most of their applications, ranging from liquid uptake, mechanics, and acoustic/thermal insulation to tissue engineering. Despite their importance, the scientific phenomena governing the pore-opening processes remain poorly understood, requiring tedious trial-and-error procedures for property optimization. This lack of understanding is partly explained by the high complexity of the different interrelated, multiscale processes which take place as the foam transforms from an initially fluid foam into a solid foam. To progress in this field, this work takes inspiration from long-standing research on liquid foams and thin films to develop model experiments in a microfluidic "Thin Film Pressure Balance." These experiments allow the investigation of isolated thin films under well-controlled environmental conditions reproducing those arising within a foam undergoing cross-linking and drying. Using the example of alginate hydrogel films, the evolution of isolated thin films undergoing gelation and drying is correlated with the evolution of the rheological properties of the same alginate solution in bulk. The overall approach is introduced and a first set of results is presented to propose a starting point for the phenomenological description of the different types of pore-opening processes and the classification of the resulting pore-opening types.

Ocular Surface Diseases in Patients With Diabetes.

Purpose Diabetes is a major cause of ocular morbidity as multiple mechanisms play a role in inducing inflammatory changes in the eye. Diabetic retinopathy is the most common complication and is well-documented. However, in the era of modern medicine, attention is also being focused on ocular surface changes in diabetes. Therefore, this study aimed to determine the association between diabetes and ocular surface diseases. Materials and Methods This is a cross-sectional study examining 320 eyes of 160 patients with diabetes who were grouped according to their duration of diabetes. The symptoms were evaluated using the ocular surface disease index (OSDI) questionnaire. Their recent hemoglobin (Hb) A1c value was recorded. Their external or internal hordeolum, blepharitis, meibomian gland dysfunction, and corneal sensitivity were also evaluated. The tear film was examined using tests, such as Schirmer's test, tear film breakup time (TBUT), tear film meniscus height (TFMH), fluorescein stain, and rose bengal stain. The results were correlated with the duration and control of diabetes. Results The mean age of the study population was 56.60 years comprising 56% (n=89) females and 44% (n=71) males. The mean OSDI scores were 7.9 ± 3.55 and 57 ± 19.22 in patients without dry eye and with severe dry eye, respectively. The study observed OSDI scores were consistently high with diabetes severity. About 67% (n=24) of patients with HbA1c of >8% had dry eyes. Dry eye was found in 68% (n=59) of patients with the duration of diabetes being >10 years. About 23.7% (n=38) had blepharitis, whereas only 4% (n=7) had external or internal hordeolum and 44% (n=86) had different grades of meibomian gland dysfunction. Corneal sensitivity was abnormal in only 12% (n=12) of patients. About 55% (n=86) of patients had varying degrees of dry eye. A statistically significant correlation was found between the severity of dry eye and TBUT, TFMH values, and grades of corneal staining (P < 0.0001). Conclusion This study observed that the incidence of dry eyes was found to be higher when patients had uncontrolled diabetes and diabetes for a longer period. The OSDI scoring system is an important diagnostic tool while examining patients with dry eye. In an ophthalmology clinic, patients with diabetes should always be evaluated for any ocular surface changes when being screened for diabetic retinopathy, and proper guidelines should be implemented to detect changes in the ocular surface system as early as possible so that any long-term complications such as infectious or neurotrophic keratitis may be avoided at an early stage.

KV 1/D-type potassium channels inhibit the excitability of bronchopulmonary vagal afferent nerves.

The KV 1/D-type potassium current (ID ) is an important determinant of neuronal excitability. This study explored whether and how ID channels regulate the activation of bronchopulmonary vagal afferent nerves. The single-neuron RT-PCR assay revealed that nearly all mouse bronchopulmonary nodose neurons expressed the transcripts of α-dendrotoxin (α-DTX)-sensitive, ID channel-forming KV 1.1, KV 1.2 and/or KV 1.6 α-subunits, with the expression of KV 1.6 being most prevalent. Patch-clamp recordings showed that ID , defined as the α-DTX-sensitive K+ current, activated at voltages slightly more negative than the resting membrane potential in lung-specific nodose neurons and displayed little inactivation at subthreshold voltages. Inhibition of ID channels by α-DTX depolarized the lung-specific nodose neurons and caused an increase in input resistance, decrease in rheobase, as well as increase in action potential number and firing frequency in response to suprathreshold current steps. Application of α-DTX to the lungs via trachea in the mouse ex vivo vagally innervated trachea-lungs preparation led to action potential discharges in nearly half of bronchopulmonary nodose afferent nerve fibres, including nodose C-fibres, as detected by the two-photon microscopic Ca2+ imaging technique and extracellular electrophysiological recordings. In conclusion, ID channels act as a critical brake on the activation of bronchopulmonary vagal afferent nerves by stabilizing the membrane potential, counterbalancing the subthreshold depolarization and promoting the adaptation of action potential firings. Down-regulation of ID channels, as occurs in various inflammatory diseases, may contribute to the enhanced C-fibre activity in airway diseases that are associated with excessive coughing, dyspnoea, and reflex bronchospasm and secretions. KEY POINTS: The α-dendrotoxin (α-DTX)-sensitive D-type K+ current (ID ) is an important determinant of neuronal excitability. Nearly all bronchopulmonary nodose afferent neurons in the mouse express ID and the transcripts of α-DTX-sensitive, ID channel-forming KV 1.1, KV 1.2 and/or KV 1.6 α-subunits. Inhibition of ID channels by α-DTX depolarizes the bronchopulmonary nodose neurons, reduces the minimal depolarizing current needed to evoke an action potential (AP) and increases AP number and AP firing frequency in response to suprathreshold stimulations. Application of α-DTX to the lungs ex vivo elicits AP discharges in about half of bronchopulmonary nodose C-fibre terminals. Our novel finding that ID channels act as a critical brake on the activation of bronchopulmonary vagal afferent nerves suggests that their down-regulation, as occurs in various inflammatory diseases, may contribute to the enhanced C-fibre activity in airway inflammation associated with excessive respiratory symptoms.

Mapping of the Sensory Innervation of the Mouse Lung by Specific Vagal and Dorsal Root Ganglion Neuronal Subsets.

The airways are densely innervated by sensory afferent nerves, whose activation regulates respiration and triggers defensive reflexes (e.g., cough, bronchospasm). Airway innervation is heterogeneous, and distinct afferent subsets have distinct functional responses. However, little is known of the innervation patterns of subsets within the lung. A neuroanatomical map is critical for understanding afferent activation under physiological and pathophysiological conditions. Here, we quantified the innervation of the mouse lung by vagal and dorsal root ganglion (DRG) sensory subsets defined by the expression of Pirt (all afferents), 5HT3 (vagal nodose afferents), Tac1 (tachykinergic afferents), and transient receptor potential vanilloid 1 channel (TRPV1; defensive/nociceptive afferents) using Cre-mediated reporter expression. We found that vagal afferents innervate almost all conducting airways and project into the alveolar region, whereas DRG afferents only innervate large airways. Of the two vagal ganglia, only nodose afferents project into the alveolar region, but both nodose and jugular afferents innervate conducting airways throughout the lung. Many afferents that project into the alveolar region express TRPV1. Few DRG afferents expressed TRPV1. Approximately 25% of blood vessels were innervated by vagal afferents (many were Tac1+). Approximately 10% of blood vessels had DRG afferents (some were Tac1+), but this was restricted to large vessels. Lastly, innervation of neuroepithelial bodies (NEBs) correlated with the cell number within the bodies. In conclusion, functionally distinct sensory subsets have distinct innervation patterns within the conducting airways, alveoli and blood vessels. Physiologic (e.g., stretch) and pathophysiological (e.g., inflammation, edema) stimuli likely vary throughout these regions. Our data provide a neuroanatomical basis for understanding afferent responses in vivo.

Impact of COVID-19-associated Mucormycosis in Kidney Transplant Recipients: A Multicenter Cohort Study.

BACKGROUND: COVID-19-associated mucormycosis (CAM) is a recently emerging entity. There is a lack of reports of CAM in organ transplant recipients. METHODS: We conducted a multicenter (n = 18) retrospective research in India during November 2020 to July 2021. The purpose of this study was to explore the clinical spectrum, outcome and risk factors for mortality of CAM in kidney transplant recipients (KTRs). RESULTS: The incidence of CAM was 4.4% (61/1382 COVID-19-positive KTRs) with 26.2% mortality. The median age of the cohort was 45 (38-54) y. Twenty (32%) were not hospitalized and 14 (22.9%) were on room air during COVID-19. The proportion of postdischarge CAM was 59.1%, while concurrent CAM was reported in 40.9%. The presentation of CAM was 91.8% rhino-orbital-cerebral mucormycosis and 8.2% pulmonary with 19.6% and 100% mortality, respectively. In the univariable analysis, older age, obesity, difficulty of breathing, high-flow oxygen requirement, and delay in starting therapy were significantly associated with mortality. In the multivariable logistic regression analysis, patients requiring high-flow oxygen therapy [odds ratio (95% confidence interval) = 9.3 (1.6-51); P = 0.01] and obesity [odds ratio (95% confidence interval) = 5.2 (1-28); P = 0.05] was associated with mortality. The median follow-up of the study was 60 (35-60) d. CONCLUSIONS: We describe the largest case series of CAM in KTRs. Morality in pulmonary CAM is extremely high. Severe COVID-19 pose extra risk for the development of CAM and associated mortality. Our report will help in better understanding the conundrum and management of CAM.

Contribution of tetrodotoxin-sensitive, voltage-gated sodium channels (NaV1) to action potential discharge from mouse esophageal tension mechanoreceptors.

Action potentials depend on voltage-gated sodium channels (NaV1s), which have nine α subtypes. NaV1 inhibition is a target for pathologies involving excitable cells such as pain. However, because NaV1 subtypes are widely expressed, inhibitors may inhibit regulatory sensory systems. Here, we investigated specific NaV1s and their inhibition in mouse esophageal mechanoreceptors-non-nociceptive vagal sensory afferents that are stimulated by low threshold mechanical distension, which regulate esophageal motility. Using single fiber electrophysiology, we found mechanoreceptor responses to esophageal distension were abolished by tetrodotoxin. Single-cell RT-PCR revealed that esophageal-labeled TRPV1-negative vagal neurons expressed multiple tetrodotoxin-sensitive NaV1s: NaV1.7 (almost all neurons) and NaV1.1, NaV1.2, and NaV1.6 (in ∼50% of neurons). Inhibition of NaV1.7, using PF-05089771, had a small inhibitory effect on mechanoreceptor responses to distension. Inhibition of NaV1.1 and NaV1.6, using ICA-121341, had a similar small inhibitory effect. The combination of PF-05089771 and ICA-121341 inhibited but did not eliminate mechanoreceptor responses. Inhibition of NaV1.2, NaV1.6, and NaV1.7 using LSN-3049227 inhibited but did not eliminate mechanoreceptor responses. Thus, all four tetrodotoxin-sensitive NaV1s contribute to action potential initiation from esophageal mechanoreceptors terminals. This is different to those NaV1s necessary for vagal action potential conduction, as demonstrated using GCaMP6s imaging of esophageal vagal neurons during electrical stimulation. Tetrodotoxin-sensitive conduction was abolished in many esophageal neurons by PF-05089771 alone, indicating a critical role of NaV1.7. In summary, multiple NaV1 subtypes contribute to electrical signaling in esophageal mechanoreceptors. Thus, inhibition of individual NaV1s would likely have minimal effect on afferent regulation of esophageal motility.

Recent Advances in Microwave Assisted Extraction of Bioactive Compounds from Complex Herbal Samples: A Review.

Microwaves are utilized for extraction of Phytoconstituents from complex herbal sample as a result of incredible research. Conventional extraction strategies are tedious and need more solvents and are no more relevant for thermal sensitive plant components. This review emphasize on the working and significance of microwave extraction technology in herbal research and medical field. The extraction step must be more yielding; quick, particular, not more solvent consuming, ensuring stability of thermolabile components and these features are available with microwave extraction method. In this nonconventional technology heat is created utilizing microwave energy. The important parameters that influence extraction efficiency are solvent properties, volume, duration of exposure, microwave control, system attributes, temperature and application were discussed in this article. The microwave assisted extraction, as green technology is contrasted with other extraction technique. This review is intended to discuss this green extraction technique along with its critical parameters for extracting bioactive compounds from complex plant matrices.

Acute activation of bronchopulmonary vagal nociceptors by type I interferons.

KEY POINTS: Type I interferon receptors are expressed by the majority of vagal C-fibre neurons innervating the respiratory tract Interferon alpha and beta acutely and directly activate vagal C-fibers in the airways. The interferon-induced activation of C-fibers occurs secondary to stimulation of type 1 interferon receptors Type 1 interferons may contribute to the symptoms as well as the spread of respiratory viral infections by causing coughing and other defensive reflexes associated with vagal C-fibre activation ABSTRACT: We evaluated the ability of type I interferons to acutely activate airway vagal afferent nerve terminals in mouse lungs. Using single cell RT-PCR of lung-specific vagal neurons we found that IFNAR1 and IFNAR2 were expressed in 70% of the TRPV1-positive neurons (a marker for vagal C-fibre neurons) and 44% of TRPV1-negative neurons. We employed an ex vivo vagal innervated mouse trachea-lung preparation to evaluate the effect of interferons in directly activating airway nerves. Utilizing 2-photon microscopy of the nodose ganglion neurons from Pirt-Cre;R26-GCaMP6s mice we found that applying IFNα or IFNß to the lungs acutely activated the majority of vagal afferent nerve terminals. When the type 1 interferon receptor, IFNAR1, was blocked with a blocking antibody the response to IFNß was largely inhibited. The type 2 interferon, IFNγ, also activated airway nerves and this was not inhibited by the IFNAR1 blocking antibody. The Janus kinase inhibitor GLPG0634 (1 µm) virtually abolished the nerve activation caused by IFNß. Consistent with the activation of vagal afferent C-fibers, infusing IFNß into the mouse trachea led to defensive breathing reflexes including apneas and gasping. These reflexes were prevented by pretreatment with an IFN type-1 receptor blocking antibody. Finally, using whole cell patch-clamp electrophysiology of lung-specific neurons we found that IFNß (1000 U ml-1 ) directly depolarized the membrane potential of isolated nodose neurons, in some cases beyond to action potential threshold. This acute non-genomic activation of vagal sensory nerve terminals by interferons may contribute to the incessant coughing that is a hallmark of respiratory viral infections.

Neuroendocrine Mechanisms Governing Sex Differences in Hyperalgesic Priming Involve Prolactin Receptor Sensory Neuron Signaling.

Many clinical and preclinical studies report higher prevalence and severity of chronic pain in females. We used hyperalgesic priming with interleukin 6 (IL-6) priming and PGE2 as a second stimulus as a model for pain chronicity. Intraplantar IL-6 induced hypersensitivity was similar in magnitude and duration in both males and females, while both paw and intrathecal PGE2 hypersensitivity was more persistent in females. This difference in PGE2 response was dependent on both circulating estrogen and translation regulation signaling in the spinal cord. In males, the duration of hypersensitivity was regulated by testosterone. Since the prolactin receptor (Prlr) is regulated by reproductive hormones and is female-selectively activated in sensory neurons, we evaluated whether Prlr signaling contributes to hyperalgesic priming. Using ΔPRL, a competitive Prlr antagonist, and a mouse line with ablated Prlr in the Nav1.8 sensory neuronal population, we show that Prlr in sensory neurons is necessary for the development of hyperalgesic priming in female, but not male, mice. Overall, sex-specific mechanisms in the initiation and maintenance of chronic pain are regulated by the neuroendocrine system and, specifically, sensory neuronal Prlr signaling.SIGNIFICANCE STATEMENT Females are more likely to experience chronic pain than males, but the mechanisms that underlie this sex difference are not completely understood. Here, we demonstrate that the duration of mechanical hypersensitivity is dependent on circulating sex hormones in mice, where estrogen caused an extension of sensitivity and testosterone was responsible for a decrease in the duration of the hyperalgesic priming model of chronic pain. Additionally, we demonstrated that prolactin receptor expression in Nav1.8+ neurons was necessary for hyperalgesic priming in female, but not male, mice. Our work demonstrates a female-specific mechanism for the promotion of chronic pain involving the neuroendrocrine system and mediated by sensory neuronal prolactin receptor.

Development of a Mouse Reporter Strain for the Purinergic P2X2 Receptor.

The ATP-sensitive P2X2 ionotropic receptor plays a critical role in a number of signal processes including taste and hearing, carotid body detection of hypoxia, the exercise pressor reflex and sensory transduction of mechanical stimuli in the airways and bladder. Elucidation of the role of P2X2 has been hindered by the lack of selective tools. In particular, detection of P2X2 using established pharmacological and biochemical techniques yields dramatically different expression patterns, particularly in the peripheral and central nervous systems. Here, we have developed a knock-in P2X2-cre mouse, which we crossed with a cre-sensitive tdTomato reporter mouse to determine P2X2 expression. P2X2 was found in more than 80% of nodose vagal afferent neurons, but not in jugular vagal afferent neurons. Reporter expression correlated in vagal neurons with sensitivity to α,ß methylene ATP (αßmATP). P2X2 was expressed in 75% of petrosal afferents, but only 12% and 4% of dorsal root ganglia (DRG) and trigeminal afferents, respectively. P2X2 expression was limited to very few cell types systemically. Together with the central terminals of P2X2-expressing afferents, reporter expression in the CNS was mainly found in brainstem neurons projecting mossy fibers to the cerebellum, with little expression in the hippocampus or cortex. The structure of peripheral terminals of P2X2-expressing afferents was demonstrated in the tongue (taste buds), carotid body, trachea and esophagus. P2X2 was observed in hair cells and support cells in the cochlear, but not in spiral afferent neurons. This mouse strain provides a novel approach to the identification and manipulation of P2X2-expressing cell types.

Mapping of Sensory Nerve Subsets within the Vagal Ganglia and the Brainstem Using Reporter Mice for Pirt, TRPV1, 5-HT3, and Tac1 Expression.

Vagal afferent sensory nerves, originating in jugular and nodose ganglia, are composed of functionally distinct subsets whose activation evokes distinct thoracic and abdominal reflex responses. We used Cre-expressing mouse strains to identify specific vagal afferent populations and map their central projections within the brainstem. We show that Pirt is expressed in virtually all vagal afferents; whereas, 5-HT3 is expressed only in nodose neurons, with little expression in jugular neurons. Transient receptor potential vanilloid 1 (TRPV1), the capsaicin receptor, is expressed in a subset of small nodose and jugular neurons. Tac1, the gene for tachykinins, is expressed predominantly in jugular neurons, some of which also express TRPV1. Vagal fibers project centrally to the nucleus tractus solitarius (nTS), paratrigeminal complex, area postrema, and to a limited extent the dorsal motor nucleus of the vagus. nTS subnuclei preferentially receive projections by specific afferent subsets, with TRPV1+ fibers terminating in medial and dorsal regions predominantly caudal of obex, whereas TRPV1- fibers terminate in ventral and lateral regions throughout the rostral-caudal aspect of the medulla. Many vagal Tac1+ afferents (mostly derived from the jugular ganglion) terminate in the nTS. The paratrigeminal complex was the target of multiple vagal afferent subsets. Importantly, lung-specific TRPV1+ and Tac1+ afferent terminations were restricted to the caudal medial nTS, with no innervation of other medulla regions. In summary, this study identifies the specific medulla regions innervated by vagal afferent subsets. The distinct terminations provide a neuroanatomic substrate for the diverse range of reflexes initiated by vagal afferent activation.

Sensitization of small-diameter sensory neurons is controlled by TRPV1 and TRPA1 association.

Unique features of sensory neuron subtypes are manifest by their distinct physiological and pathophysiological functions. Using patch-clamp electrophysiology, Ca2+ imaging, calcitonin gene-related peptide release assay from tissues, protein biochemistry approaches, and behavioral physiology on pain models, this study demonstrates the diversity of sensory neuron pathophysiology is due in part to subtype-dependent sensitization of TRPV1 and TRPA1. Differential sensitization is influenced by distinct expression of inflammatory mediators, such as prostaglandin E2 (PGE2), bradykinin (BK), and nerve growth factor (NGF) as well as multiple kinases, including protein kinase A (PKA) and C (PKC). However, the co-expression and interaction of TRPA1 with TRPV1 proved to be the most critical for differential sensitization of sensory neurons. We identified N- and C-terminal domains on TRPV1 responsible for TRPA1-TRPV1 (A1-V1) complex formation. Ablation of A1-V1 complex with dominant-negative peptides against these domains substantially reduced the sensitization of TRPA1, as well as BK- and CFA-induced hypersensitivity. These data indicate that often occurring TRP channel complexes regulate diversity in neuronal sensitization and may provide a therapeutic target for many neuroinflammatory pain conditions.

Prolactin Regulates Pain Responses via a Female-Selective Nociceptor-Specific Mechanism.

Many clinical and preclinical studies report an increased prevalence and severity of chronic pain among females. Here, we identify a sex-hormone-controlled target and mechanism that regulates dimorphic pain responses. Prolactin (PRL), which is involved in many physiologic functions, induces female-specific hyperalgesia. A PRL receptor (Prlr) antagonist in the hind paw or spinal cord substantially reduced hyperalgesia in inflammatory models. This effect was mimicked by sensory neuronal ablation of Prlr. Although Prlr mRNA is expressed equally in female and male peptidergic nociceptors and central terminals, Prlr protein was found only in females and PRL-induced excitability was detected only in female DRG neurons. PRL-induced excitability was reproduced in male Prlr+ neurons after prolonged treatment with estradiol but was prevented with addition of a translation inhibitor. We propose a novel mechanism for female-selective regulation of pain responses, which is mediated by Prlr signaling in sensory neurons via sex-dependent control of Prlr mRNA translation.

Prolactin receptor expression in mouse dorsal root ganglia neuronal subtypes is sex-dependent.

Sensory neurones exhibit sex-dependent responsiveness to prolactin (PRL). This could contribute to sexual dimorphism in pathological pain conditions. The present study aimed to determine the mechanisms underlying sex-dependent PRL sensitivity in sensory neurones. A quantitative reverse transcriptase-polymerase chain reaction shows that prolactin receptor (Prlr) long and short isoform mRNAs are expressed at comparable levels in female and male mouse dorsal root ganglia (DRG). In Prlrcre/+ ;Rosa26LSL-tDTomato/+ reporter mice, percentages of Prlr+ sensory neurones in female and male DRG are also similar. Characterisation of Prlr+ DRG neurones using immunohistochemistry and electrophysiology revealed that Prlr+ DRG neurones are mainly peptidergic nociceptors in females and males. However, sensory neurone type-dependent expression of Prlr is sex dimorphic. Thus, Prlr+ populations fell into three small- and two medium-large-sized sensory neuronal groups. Prlr+ DRG neurones are predominantly medium-large sized in males and are proportionally more comprised of small-sized sensory neurones in females. Specifically, Prlr+ /IB4+ /CGRP+ neurones are four- to five-fold higher in numbers in female DRG. By contrast, Prlr+ /IB4- /CGRP+ /5HT3a+ /NPYR2- are predominant in male DRG. Prlr+ /IB4- /CGRP- , Prlr+ /IB4- /CGRP+ and Prlr+ /IB4- /CGRP+ /NPYR2+ neurones are evenly encountered in female and male DRG. These differences were confirmed using an independently generated single-cell sequencing dataset. Overall, we propose a novel mechanism by which sensory neurone type-dependent expression of Prlr could explain the unique sex dimorphism in responsiveness of nociceptors to PRL.

Effects of ginger constituent 6-shogaol on gastroesophageal vagal afferent C-fibers.

BACKGROUND: Ginger has been used as an herbal medicine worldwide to relieve nausea/vomiting and gastrointestinal discomfort, but the cellular and molecular mechanisms of its neuronal action remain unclear. The present study aimed to determine the effects of ginger constituent 6-shogaol on gastroesophageal vagal nodose C-fibers. METHODS: Extracellular single-unit recording and two-photon nodose neuron imaging were performed, respectively, in ex vivo gastroesophageal-vagal preparations from wild type and Pirt-GCaMP6 transgenic mice. The action potential discharge or calcium influx evoked by mechanical distension and chemical perfusions applied to the gastroesophageal vagal afferent nerve endings were recorded, respectively, at their intact neuronal cell soma in vagal nodose ganglia. The effects of 6-shogaol on nodose C-fiber neurons were then compared and determined. KEY RESULTS: Gastroesophageal application of 6-shogaol-elicited intensive calcium influxes in nodose neurons and evoked robust action potential discharges in most studied nodose C-fibers. Such activation effects were followed by a desensitized response to the second application of 6-shogaol. However, action potential discharges evoked by esophageal mechanical distension, after 6-shogaol perfusion, did not significantly change. Pretreatment with TRPA1 selective blocker HC-030031 inhibited 6-shogaol-induced action potential discharges in gastric and esophageal nodose C-fiber neurons, suggesting that TRPA1 played a role in mediating 6-shogaol-induced activation response. CONCLUSION AND INFERENCES: This study provides evidence that ginger constituent 6-shogaol directly activates vagal afferent C-fiber peripheral gastrointestinal endings. This activation leads to desensitization to subsequent application of 6-shogaol but not subsequent esophageal mechanical distension. Further investigation is required to establish a possible contribution in its anti-emetic effects.