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.

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.

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.

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.

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.

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.

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.

Targeting C-fibers for peripheral acting anti-tussive drugs.

Activation of vagal C-fibers is likely involved in some types of pathological coughing, especially coughing that is associated with airway inflammation. This is because stimulation of vagal C-fibers leads to strong urge to cough sensations, and because C-fiber terminals can be strongly activated by mediators associated with airway inflammation. The most direct manner in which a given mediator can activate a C-fiber terminal is through interacting with its receptor expressed in the terminal membrane. The agonist-receptor interaction then must lead to the opening (or potentially closing) of ion channels that lead to a membrane depolarization. This depolarization is referred to as a generator potential. If, and only if, the generator potential reaches the voltage necessary to activate voltage-gated sodium channels, action potentials are initiated and conducted to the central terminals within the CNS. Therefore, there are three target areas to block the inflammatory mediator induced activation of C-fiber terminals. First, at the level of the mediator-receptor interaction, secondly at the level of the generator potential, and third at the level of the voltage-gated sodium channels. Here we provide a brief overview of each of these therapeutic strategies.

Sphingosine-1-phosphate activates mouse vagal airway afferent C-fibres via S1PR3 receptors.

KEY POINTS: Sphingosine-1-phosphate (S1P) strongly activates mouse vagal C-fibres in the airways. Airway-specific nodose and jugular C-fibre neurons express mRNA coding for the S1P receptor S1PR3. S1P activation of nodose C-fibres is inhibited by a S1PR3 antagonist. S1P activation of nodose C-fibres does not occur in S1PR3 knockout mice. ABSTRACT: We evaluated the effect of sphingosine-1-phosphate (S1P), a lipid that is elevated during airway inflammatory conditions like asthma, for its ability to stimulate vagal afferent C-fibres in mouse lungs. Single cell RT-PCR on lung-specific vagal afferent neurons revealed that both TRPV1-expressing and TRPV1-non-expressing nodose neurons express mRNA coding for the S1P receptor S1PR3. TRPV1-expressing airway-specific jugular ganglion neurons also express S1PR3 mRNA. S1PR1 and S1PR2 mRNAs were also found to be expressed but only in a limited subset (32% and 22%, respectively) of airway-specific vagal sensory neurons; whereas S1PR4 and S1PR5 were rarely expressed. We used large scale two-photon imaging of the nodose ganglia from our ex vivo preparation isolated from Pirt-Cre;R26-GCaMP6s transgenic mice, which allows for simultaneous monitoring of calcium transients in ∼1000 neuronal cell bodies in the ganglia during tracheal perfusion with S1P (10 µM). We found that S1P in the lungs strongly activated 81.5% of nodose fibres, 70% of which were also activated by capsaicin. Single fibre electrophysiological recordings confirmed that S1P evoked action potential (AP) generation in a concentration-dependent manner (0.1-10 µM). Action potential generation by S1P in nodose C-fibres was effectively inhibited by the S1PR3 antagonist TY 52156 (10 µM). Finally, in S1PR3 knockout mice, S1P was not able to activate any of the airway nodose C-fibres analysed. These results support the hypothesis that S1P may play a role in evoking C-fibre-mediated airway sensations and reflexes that are associated with airway inflammatory diseases.

KCNQ/M-channels regulate mouse vagal bronchopulmonary C-fiber excitability and cough sensitivity.

Increased airway vagal sensory C-fiber activity contributes to the symptoms of inflammatory airway diseases. The KCNQ/Kv7/M-channel is a well-known determinant of neuronal excitability, yet whether it regulates the activity of vagal bronchopulmonary C-fibers and airway reflex sensitivity remains unknown. Here we addressed this issue using single-cell RT-PCR, patch clamp technique, extracellular recording of single vagal nerve fibers innervating the mouse lungs, and telemetric recording of cough in free-moving mice. Single-cell mRNA analysis and biophysical properties of M-current (IM) suggest that KCNQ3/Kv7.3 is the major M-channel subunit in mouse nodose neurons. The M-channel opener retigabine negatively shifted the voltage-dependent activation of IM, leading to membrane hyperpolarization, increased rheobase, and suppression of both evoked and spontaneous action potential (AP) firing in nodose neurons in an M-channel inhibitor XE991-sensitive manner. Retigabine also markedly suppressed the α,ß-methylene ATP-induced AP firing in nodose C-fiber terminals innervating the mouse lungs, and coughing evoked by irritant gases in awake mice. In conclusion, KCNQ/M-channels play a role in regulating the excitability of vagal airway C-fibers at both the cell soma and nerve terminals. Drugs that open M-channels in airway sensory afferents may relieve the sufferings associated with pulmonary inflammatory diseases such as chronic coughing.

Characteristics of sensory neuronal groups in CGRP-cre-ER reporter mice: Comparison to Nav1.8-cre, TRPV1-cre and TRPV1-GFP mouse lines.

Peptidergic sensory neurons play a critical role in nociceptive pathways. To precisely define the function and plasticity of sensory neurons in detail, new tools such as transgenic mouse models are needed. We employed electrophysiology and immunohistochemistry to characterize in detail dorsal root ganglion (DRG) neurons expressing an inducible CGRPcre-ER (CGRP-cre+); and compared them to DRG neurons expressing Nav1.8cre (Nav1.8-cre+), TRPV1cre (TRPV1-cre+) and TRPV1-GFP (V1-GFP+). Tamoxifen effectively induced CGRPcre-ER production in DRG. ≈87% of CGRPcre-ER-expressing neurons were co-labeled CGRP antibody. Three small and two medium-large-sized (5HT3a+/NPY2R- and NPY2R+) neuronal groups with unique electrophysiological profiles were CGRP-cre+. Nav1.8-cre+ neurons were detected in all CGRP-cre+ groups, as well as in 5 additional neuronal groups: MrgprD+/TRPA1-, MrgprD+/TRPA1+, TRPV1+/CGRP-, vGLUT3+ and ≈30% of trkC+ neurons. Differences between TRPV1cre and Nav1.8cre reporters were that unlike TRPV1-cre+, Nav1.8-cre+ expression was detected in non-nociceptive vGLUT3+ and trkC+ populations. Many TRPV1-cre+ neurons did not respond to capsaicin. In contrast, V1-GFP+ neurons were in 4 groups, each of which was capsaicin-sensitive. Finally, none of the analyzed reporter lines showed cre-recombination in trkB+, calbindin+, 70% of trkC+ or parvalbumin+ neurons, which together encompassed ≈20% of Nav1.8-cre- DRG neurons. The data presented here increases our knowledge of peptidergic sensory neuron characteristics, while showing the efficiency and specificity manipulation of peptidergic neurons by the CGRPcre-ER reporter. We also demonstrate that manipulation of all C- and A-nociceptors is better achieved with TRPV1-cre reporter. Finally, the described approach for detailed characterization of sensory neuronal groups can be applied to a variety of reporter mice.

Effects of local and spinal administrations of mu-opioids on postoperative pain in aged versus adult mice.

Introduction: Suboptimal management of postoperative pain leads to increased risk of chronic opioid therapy, especially in elderly patients. Objectives: Although this age-dependent phenomenon has been observed clinically, basic mechanisms including baseline nociception, postoperative hypersensitivity, and mu-opioid efficiency in aged animals have never been evaluated. Methods: We tested these criteria using incision model on adult (3­6 months) and aged (24 months) mice to assess translatability of postoperative animal studies to clinical observations. Results: Thermal and mechanical testing revealed lower baseline nociception in aged vs adult mice, while behavioral assays after hind paw plantar incision showed similar hypersensitivity levels for both age groups. Efficiency of local and spinal mu-opioid injections on postoperative pain was assessed next. DAMGO, a pure mu-opioid, was effective in reducing postoperative hypersensitivity in aged and adult mice, although adult mice displayed increased sensitivity to higher doses (50 µg local; 1­15 µg spinal). Buprenorphine, a mixed mu-opioid agonist, produced dose-dependent antihypersensitivity with adult mice more sensitive to lower doses (0.1 µg local; 0.02 µg spinal), and aged mice more sensitive to higher doses (1, 10 µg local; 0.1, 1 µg spinal). Finally, exploratory locomotor activity was used to evaluate the suppression of incision-induced spontaneous pain by DAMGO. Spinal and systemic (intraperitoneal) DAMGO inhibited ongoing pain more in adults compared with aged mice. Conclusion: As in humans, baseline nociception was lower in aged vs adult mice, while postoperative hypersensitivity magnitudes were comparable between groups. Unlike in humans, adult mice were more sensitive to mu-opioids, although higher doses of mixed mu-opioids were more effective for postoperative antihypersensitivity in aged mice.

Influence of hypophysectomy, ovariectomy and gonadectomy on postoperative hypersensitivity in rats.

Surgical procedures lead to profound and sustained (up to 1-2 weeks) activation of the pituitary gland, resulting in changes in endocrine function. Questions remain on whether activation of the pituitary influences the threshold and development time-course of postoperative pain. To address these questions, we evaluated postoperative hypersensitivity in female and male rats with ablated pituitary and gonadal hormone productions via hypophysectomy, ovariectomy and gonadectomy, respectively. Plantar incision, a model of acute postoperative pain, or sham operation was performed on rat hind paws. Hypophysectomy, ovariectomy and gonadectomy were achieved by surgical disconnection of pituitary, ovaries and testicles, respectively. Postoperative thermal and mechanical hypersensitivity were monitored for 7 days post incision. Hypophysectomy on female and male rats produced statistically similar thermal and mechanical postoperative hypersensitivity thresholds and time-courses as compared to intact estrous female and male rats. Moreover, ovariectomy and gonadectomy did not significantly change postoperative hypersensitivity observed in control female and male animals. Our experiments demonstrate that hypophysectomy, ovariectomy and gonadectomy do not significantly impact postoperative hypersensitivity observed in normal female and male animals. These data suggest that surgery-induced changes in the endocrine system via activation of pituitary and subsequently gonadal tissues have little impact on the threshold and development of postoperative pain in female and male rats.

Effect of a Novel, Orally Active Matrix Metalloproteinase-2 and -9 Inhibitor in Spinal and Trigeminal Rat Models of Neuropathic Pain.

AIMS: To study the effects of a novel matrix metalloproteinase-2 (MMP-2) and MMP-9 inhibitor, AQU-118, on mechanical allodynia in the spinal nerve ligation (SNL) model of neuropathic pain and the chronic constriction injury of the infraorbital nerve (CCI-IoN) model of neuropathic orofacial pain. METHODS: Five groups of SNL rats were given daily oral doses of AQU-118 (5, 10, 20 mg/kg), gabapentin (100 mg/kg), or vehicle (0.5% methylcellulose) and then paw withdrawal threshold was measured with von Frey filaments (VF). Three groups of CCI-IoN rats were given daily oral doses of either AQU-118 (40 mg/kg), gabapentin (100 mg/kg), or vehicle (0.5% methylcellulose) and then mechanical allodynia was measured with facial VF and non-reflex-based orofacial stimulation test (OFST) assay. Naïve rats were also tested for the effect of AQU-118 (40 mg/kg) on basal sensitivity to mechanical stimulation/locomotive activity. RESULTS: Mechanical allodynia in SNL rats was attenuated by gabapentin (100 mg/kg) and AQU-118 (in a dose-dependent manner). Mechanical allodynia in CCI-IoN rats was also attenuated (in an equipotent manner) by both AQU-118 (40 mg/ kg) and gabapentin (100 mg/kg) as measured by both facial VF and OFST assay. Upon cessation of either AQU-118 or gabapentin, VF-related responses in both models and OFST assay times reverted to levels observed in vehicle-treated rats. No statistically significant change was observed in locomotive activity/paw withdrawal threshold by AQU-118 (40 mg/kg) in naïve rats. CONCLUSION: The results demonstrated that oral AQU-118 attenuates mechanical allodynia in both neuropathic pain models and with efficacies that mirror gabapentin at the 40 mg/kg dose used in the CCI-IoN model but without effect on basal sensitivity to mechanical stimulation/locomotive activity. These findings support a possible role for MMP-2/-9 in the etiology of neuropathic pain and also suggest that inhibition strategies represent a viable treatment option.

Prolactin receptor in regulation of neuronal excitability and channels.

Prolactin (PRL) activates PRL receptor isoforms to exert regulation of specific neuronal circuitries, and to control numerous physiological and clinically-relevant functions including; maternal behavior, energy balance and food intake, stress and trauma responses, anxiety, neurogenesis, migraine and pain. PRL controls these critical functions by regulating receptor potential thresholds, neuronal excitability and/or neurotransmission efficiency. PRL also influences neuronal functions via activation of certain neurons, resulting in Ca(2+) influx and/or electrical firing with subsequent release of neurotransmitters. Although PRL was identified almost a century ago, very little specific information is known about how PRL regulates neuronal functions. Nevertheless, important initial steps have recently been made including the identification of PRL-induced transient signaling pathways in neurons and the modulation of neuronal transient receptor potential (TRP) and Ca(2+) -dependent K(+) channels by PRL. In this review, we summarize current knowledge and recent progress in understanding the regulation of neuronal excitability and channels by PRL.

Mechanisms of transient signaling via short and long prolactin receptor isoforms in female and male sensory neurons.

Prolactin (PRL) regulates activity of nociceptors and causes hyperalgesia in pain conditions. PRL enhances nociceptive responses by rapidly modulating channels in nociceptors. The molecular mechanisms underlying PRL-induced transient signaling in neurons are not well understood. Here we use a variety of cell biology and pharmacological approaches to show that PRL transiently enhanced capsaicin-evoked responses involve protein kinase C ε (PKCε) or phosphatidylinositol 3-kinase (PI3K) pathways in female rat trigeminal (TG) neurons. We next reconstituted PRL-induced signaling in a heterologous expression system and TG neurons from PRL receptor (PRLR)-null mutant mice by expressing rat PRLR-long isoform (PRLR-L), PRLR-short isoform (PRLR-S), or a mix of both. Results show that PRLR-S, but not PRLR-L, is capable of mediating PRL-induced transient enhancement of capsaicin responses in both male and female TG neurons. However, co-expression of PRLR-L with PRLR-S (1:1 ratio) leads to the inhibition of the transient PRL actions. Co-expression of PRLR-L deletion mutants with PRLR-S indicated that the cytoplasmic site adjacent to the trans-membrane domain of PRLR-L was responsible for inhibitory effects of PRLR-L. Furthermore, in situ hybridization and immunohistochemistry data indicate that in normal conditions, PRLR-L is expressed mainly in glia with little expression in rat sensory neurons (3-5%) and human nerves. The predominant PRLR form in TG neurons/nerves from rats and humans is PRLR-S. Altogether, PRL-induced transient signaling in sensory neurons is governed by PI3K or PKCε, mediated via the PRLR-S isoform, and transient effects mediated by PRLR-S are inhibited by presence of PRLR-L in these cells.

Prolactin regulates TRPV1, TRPA1, and TRPM8 in sensory neurons in a sex-dependent manner: Contribution of prolactin receptor to inflammatory pain.

Prolactin (PRL) is a hormone produced in the anterior pituitary but also synthesized extrapituitary where it can influence diverse cellular processes, including inflammatory responses. Females experience greater pain in certain inflammatory conditions, but the contribution of the PRL system to sex-dependent inflammatory pain is unknown. We found that PRL regulates transient receptor potential (TRP) channels in a sex-dependent manner in sensory neurons. At >20 ng/ml, PRL sensitizes TRPV1 in female, but not male, neurons. This effect is mediated by PRL receptor (PRL-R). Likewise, TRPA1 and TRPM8 were sensitized by 100 ng/ml PRL only in female neurons. We showed that complete Freund adjuvant (CFA) upregulated PRL levels in the inflamed paw of both male and female rats, but levels were higher in females. In contrast, CFA did not change mRNA levels of long and short PRL-R in the dorsal root ganglion or spinal cord. Analysis of PRL and PRL-R knockout (KO) mice demonstrated that basal responses to cold stimuli were only altered in females, and with no significant effects on heat and mechanical responses in both sexes. CFA-induced heat and cold hyperalgesia were not changed in PRL and PRL-R KO compared with wild-type (WT) males, whereas significant reduction of heat and cold post-CFA hyperalgesia was detected in PRL and PRL-R KO females. Attenuation of CFA-induced mechanical allodynia was observed in both PRL and PRL-R KO females and males. Thermal hyperalgesia in PRL KO females was restored by administration of PRL into hindpaws. Overall, we demonstrate a sex-dependent regulation of peripheral inflammatory hyperalgesia by the PRL system.

Chronic alteration in phosphatidylinositol 4,5-biphosphate levels regulates capsaicin and mustard oil responses.

There is an agreement that acute (in minutes) hydrolysis and accumulation of phosphatidylinositol 4,5-bisphosphate (PIP(2) ) modulate TRPV1 and TRPA1 activities. Because inflammation results in PIP(2) depletion, persisting for long periods (hours to days) in pain models and in the clinic, we examined whether chronic depletion and accumulation of PIP(2) affect capsaicin (CAP) and mustard oil (MO) responses. In addition, we wanted to evaluate whether the effects of PIP(2) depend on TRPV1 and TRPA1 coexpression and whether the PIP(2) actions vary in expression cells vs. sensory neurons. Chronic PIP(2) production was stimulated by overexpression of phosphatidylinositol-4-phosphate-5-kinase, and PIP(2) -specific phospholipid 5'-phosphatase was selected to reduce plasma membrane levels of PIP(2) . Our results demonstrate that CAP (100 nM) responses and receptor tachyphylaxis are not significantly influenced by chronic changes in PIP(2) levels in wild-type (WT) or TRPA1 null-mutant sensory neurons as well as CHO cells expressing TRPV1 alone or with TRPA1. However, low concentrations of CAP (20 nM) produced a higher response after PIP(2) depletion in cells containing TRPV1 alone but not TRPV1 together with TRPA1. MO (25 µM) responses were also not affected by PIP(2) in WT sensory neurons and cells coexpressing TRPA1 and TRPV1. In contrast, PIP(2) reduction leads to pronounced tachyphylaxis to MO in cells with both channels. Chronic effect of PIP(2) on TRPA1 activity depends on presence of the TRPV1 channel and cell type (CHO vs. sensory neurons). In summary, chronic alterations in PIP(2) levels regulate magnitude of CAP and MO responses as well as MO tachyphylaxis. This regulation depends on coexpression profile of TRPA1 and TRPV1 and cell type.

In vitro sarcoma cells release a lipophilic substance that activates the pain transduction system via TRPV1.

BACKGROUND: Despite success in treating many forms of cancer, pain associated with malignancy remains a serious clinical issue with a poorly understood etiology. This study determined if certain sarcoma cell lines produced a soluble factor that activates the TRPV1 ion channel expressed on nociceptive sensory neurons, thereby activating a major pain transduction system. MATERIALS AND METHODS: Trigeminal ganglia were harvested from rats and cultured. A rhabdomyosarcoma (CRL1598) and osteosarcoma (CRL 1543) cell line were grown to 75% confluency. Conditioned media (CM) was collected after 24 h of exposure and subjected to reverse phase chromatography. Neuronal activation in the presence of CM was measured using iCGRP RIA and calcium imaging after treatment with vehicle or I-RTX, a potent TRPV1 antagonist. Data were analyzed by ANOVA/Bonferroni or t test. RESULTS: The rhabdomyosarcoma CM produced a 4-fold increase in iCGRP release compared with control media (P < 0.001). The osteosarcoma cell line CM produced a 7-fold increase in iCGRP release compared with control media (P < 0.001). This evoked iCGRP release was via TRPV1 activation since the effect was blocked by the antagonist I-RTX. The application of rhabdomyosarcoma CM produced about a 4-fold increase in [Ca(2+)]I levels (P < 0.001), and this effect was blocked by pretreatment with the TRPV1 antagonist, I-RTX. CONCLUSIONS: We have shown that certain sarcoma cell lines produce a soluble, lipophilic factor that activates the peripheral nociceptor transduction system via TRPV1 activation, thereby contributing to cancer pain. Further investigations are needed to develop tumor-specific analgesics that do not produce unwanted or harmful side-effects.