Nonpeptidergic neurons suppress mast cells via glutamate to maintain skin homeostasis.

Cutaneous mast cells mediate numerous skin inflammatory processes and have anatomical and functional associations with sensory afferent neurons. We reveal that epidermal nerve endings from a subset of sensory nonpeptidergic neurons expressing MrgprD are reduced by the absence of Langerhans cells. Loss of epidermal innervation or ablation of MrgprD-expressing neurons increased expression of a mast cell gene module, including the activating receptor, Mrgprb2, resulting in increased mast cell degranulation and cutaneous inflammation in multiple disease models. Agonism of MrgprD-expressing neurons reduced expression of module genes and suppressed mast cell responses. MrgprD-expressing neurons released glutamate which was increased by MrgprD agonism. Inhibiting glutamate release or glutamate receptor binding yielded hyperresponsive mast cells with a genomic state similar to that in mice lacking MrgprD-expressing neurons. These data demonstrate that MrgprD-expressing neurons suppress mast cell hyperresponsiveness and skin inflammation via glutamate release, thereby revealing an unexpected neuroimmune mechanism maintaining cutaneous immune homeostasis.

Cutaneous TRPV1+ Neurons Trigger Protective Innate Type 17 Anticipatory Immunity.

Cutaneous TRPV1+ neurons directly sense noxious stimuli, inflammatory cytokines, and pathogen-associated molecules and are required for innate immunity against some skin pathogens. Important unanswered questions are whether TRPV1+ neuron activation in isolation is sufficient to initiate innate immune responses and what is the biological function for TRPV1+ neuron-initiated immune responses. We used TRPV1-Ai32 optogenetic mice and cutaneous light stimulation to activate cutaneous neurons in the absence of tissue damage or pathogen-associated products. We found that TRPV1+ neuron activation was sufficient to elicit a local type 17 immune response that augmented host defense to C. albicans and S. aureus. Moreover, local neuron activation elicited type 17 responses and augmented host defense at adjacent, unstimulated skin through a nerve reflex arc. These data show the sufficiency of TRPV1+ neuron activation for host defense and demonstrate the existence of functional anticipatory innate immunity at sites adjacent to infection that depends on antidromic neuron activation.

Sympathetic Input to Multiple Cell Types in Mouse and Human Colon Produces Region-Specific Responses.

BACKGROUND & AIMS: The colon is innervated by intrinsic and extrinsic neurons that coordinate functions necessary for digestive health. Sympathetic input suppresses colon motility by acting on intrinsic myenteric neurons, but the extent of sympathetic-induced changes on large-scale network activity in myenteric circuits has not been determined. Compounding the complexity of sympathetic function, there is evidence that sympathetic transmitters can regulate activity in non-neuronal cells (such as enteric glia and innate immune cells). METHODS: We performed anatomical tracing, immunohistochemistry, optogenetic (GCaMP calcium imaging, channelrhodopsin), and colon motility studies in mice and single-cell RNA sequencing in human colon to investigate how sympathetic postganglionic neurons modulate colon function. RESULTS: Individual neurons in each sympathetic prevertebral ganglion innervated the proximal or distal colon, with processes closely opposed to multiple cell types. Calcium imaging in semi-intact mouse colon preparations revealed changes in spontaneous and evoked neural activity, as well as activation of non-neuronal cells, induced by sympathetic nerve stimulation. The overall pattern of response to sympathetic stimulation was unique to the proximal or distal colon. Region-specific changes in cellular activity correlated with motility patterns produced by electrical and optogenetic stimulation of sympathetic pathways. Pharmacology experiments (mouse) and RNA sequencing (human) indicated that appropriate receptors were expressed on different cell types to account for the responses to sympathetic stimulation. Regional differences in expression of α-1 adrenoceptors in human colon emphasize the translational relevance of our mouse findings. CONCLUSIONS: Sympathetic neurons differentially regulate activity of neurons and non-neuronal cells in proximal and distal colon to promote distinct changes in motility patterns, likely reflecting the distinct roles played by these 2 regions.

Neuronally expressed PDL1, not PD1, suppresses acute nociception.

PDL1 is a protein that induces immunosuppression by binding to PD1 expressed on immune cells. In line with historical studies, we found that membrane-bound PD1 expression was largely restricted to immune cells; PD1 was not detectable at either the mRNA or protein level in peripheral neurons using single neuron qPCR, immunolabeling and flow cytometry. However, we observed widespread expression of PDL1 in both sensory and sympathetic neurons that could have important implications for patients receiving immunotherapies targeting this pathway that include unexpected autonomic and sensory related effects. While signaling pathways downstream of PD1 are well established, little to no information is available regarding the intracellular signaling downstream of membrane-bound PDL1 (also known as reverse signaling). Here, we administered soluble PD1 to engage neuronally expressed PDL1 and found that PD1 significantly reduced nocifensive behaviors evoked by algogenic capsaicin. We used calcium imaging to examine the underlying neural mechanism of this reduction and found that exogenous PD1 diminished TRPV1-dependent calcium transients in dissociated sensory neurons. Furthermore, we observed a reduction in membrane expression of TRPV1 following administration of PD1. Exogenous PD1 had no effect on pain-related behaviors in sensory neuron specific PDL1 knockout mice. These data indicate that neuronal PDL1 activation is sufficient to modulate sensitivity to noxious stimuli and as such, may be an important homeostatic mechanism for regulating acute nociception.

Unique Molecular Characteristics of Visceral Afferents Arising from Different Levels of the Neuraxis: Location of Afferent Somata Predicts Function and Stimulus Detection Modalities.

Viscera receive innervation from sensory ganglia located adjacent to multiple levels of the brainstem and spinal cord. Here we examined whether molecular profiling could be used to identify functional clusters of colon afferents from thoracolumbar (TL), lumbosacral (LS), and nodose ganglia (NG) in male and female mice. Profiling of TL and LS bladder afferents was also performed. Visceral afferents were back-labeled using retrograde tracers injected into proximal and distal regions of colon or bladder, followed by single-cell qRT-PCR and analysis via an automated hierarchical clustering method. Genes were chosen for assay (32 for bladder; 48 for colon) based on their established role in stimulus detection, regulation of sensitivity/function, or neuroimmune interaction. A total of 132 colon afferents (from NG, TL, and LS ganglia) and 128 bladder afferents (from TL and LS ganglia) were analyzed. Retrograde labeling from the colon showed that NG and TL afferents innervate proximal and distal regions of the colon, whereas 98% of LS afferents only project to distal regions. There were clusters of colon and bladder afferents, defined by mRNA profiling, that localized to either TL or LS ganglia. Mixed TL/LS clustering also was found. In addition, transcriptionally, NG colon afferents were almost completely segregated from colon TL and LS neurons. Furthermore, colon and bladder afferents expressed genes at similar levels, although different gene combinations defined the clusters. These results indicate that genes implicated in both homeostatic regulation and conscious sensations are found at all anatomic levels, suggesting that afferents from different portions of the neuraxis have overlapping functions.SIGNIFICANCE STATEMENT Visceral organs are innervated by sensory neurons whose cell bodies are located in multiple ganglia associated with the brainstem and spinal cord. For the colon, this overlapping innervation is proposed to facilitate visceral sensation and homeostasis, where sensation and pain are mediated by spinal afferents and fear and anxiety (the affective aspects of visceral pain) are the domain of nodose afferents. The transcriptomic analysis performed here reveals that genes implicated in both homeostatic regulation and pain are found in afferents across all ganglia types, suggesting that conscious sensation and homeostatic regulation are the result of convergence, and not segregation, of sensory input.

Optogenetic activation of the distal colon epithelium engages enteric nervous system circuits to initiate motility patterns.

Digestive functions of the colon depend on sensory-motor reflexes in the enteric nervous system (ENS), initiated by intrinsic primary afferent neurons (IPANs). IPAN terminals project to the mucosal layer of the colon, allowing communication with epithelial cells comprising the colon lining. The chemical nature and functional significance of this epithelial-neural communication in regard to secretion and colon motility are of high interest. Colon epithelial cells can produce and release neuroactive substances such as ATP and 5-hydroxytryptamine (5-HT), which can activate receptors on adjacent nerve fibers, including IPAN subtypes. In this study, we examined if stimulation of epithelial cells alone is sufficient to activate neural circuits that control colon motility. Optogenetics and calcium imaging were used in ex vivo preparations of the mouse colon to selectively stimulate the colon epithelium, measure changes in motility, and record activity of neurons within the myenteric plexus. Light-mediated activation of epithelial cells lining the distal, but not proximal, colon caused local contractions and increased the rate of colonic migrating motor complexes. Epithelial-evoked local contractions in the distal colon were reduced by both ATP and 5-HT receptor antagonists. Our findings indicate that colon epithelial cells likely use purinergic and serotonergic signaling to initiate activity in myenteric neurons, produce local contractions, and facilitate large-scale coordination of ENS activity responsible for whole colon motility patterns.NEW & NOTEWORTHY Using an all-optical approach to measure real-time cell-to-cell communication responsible for colon functions, we show that selective optogenetic stimulation of distal colon epithelium produced activity in myenteric neurons, as measured with red genetically encoded calcium indicators. The epithelial-induced neural response led to local contractions, mediated by both purinergic and serotonergic signaling, and facilitated colonic motor complexes that propagate from proximal to distal colon.

Extrinsic Primary Afferent Neurons Link Visceral Pain to Colon Motility Through a Spinal Reflex in Mice.

BACKGROUND & AIMS: Proper colon function requires signals from extrinsic primary afferent neurons (ExPANs) located in spinal ganglia. Most ExPANs express the vanilloid receptor TRPV1, and a dense plexus of TRPV1-positive fibers is found around myenteric neurons. Capsaicin, a TRPV1 agonist, can initiate activity in myenteric neurons and produce muscle contraction. ExPANs might therefore form motility-regulating synapses onto myenteric neurons. ExPANs mediate visceral pain, and myenteric neurons mediate colon motility, so we investigated communication between ExPANs and myenteric neurons and the circuits by which ExPANs modulate colon function. METHODS: In live mice and colon tissues that express a transgene encoding the calcium indicator GCaMP, we visualized levels of activity in myenteric neurons during smooth muscle contractions induced by application of capsaicin, direct colon stimulation, stimulation of ExPANs, or stimulation of preganglionic parasympathetic neuron (PPN) axons. To localize central targets of ExPANs, we optogenetically activated TRPV1-expressing ExPANs in live mice and then quantified Fos immunoreactivity to identify activated spinal neurons. RESULTS: Focal electrical stimulation of mouse colon produced phased-locked calcium signals in myenteric neurons and produced colon contractions. Stimulation of the L6 ventral root, which contains PPN axons, also produced myenteric activation and contractions that were comparable to those of direct colon stimulation. Surprisingly, capsaicin application to the isolated L6 dorsal root ganglia, which produced robust calcium signals in neurons throughout the ganglion, did not activate myenteric neurons. Electrical activation of the ganglia, which activated even more neurons than capsaicin, did not produce myenteric activation or contractions unless the spinal cord was intact, indicating that a complete afferent-to-efferent (PPN) circuit was necessary for ExPANs to regulate myenteric neurons. In TRPV1-channel rhodopsin-2 mice, light activation of ExPANs induced a pain-like visceromotor response and expression of Fos in spinal PPN neurons. CONCLUSIONS: In mice, ExPANs regulate myenteric neuron activity and smooth muscle contraction via a parasympathetic spinal circuit, linking sensation and pain to motility.

Optogenetic Activation of Colon Epithelium of the Mouse Produces High-Frequency Bursting in Extrinsic Colon Afferents and Engages Visceromotor Responses.

Epithelial cells of the colon provide a vital interface between the internal environment (lumen of the colon) and colon parenchyma. To examine epithelial-neuronal signaling at this interface, we analyzed mice in which channelrhodopsin (ChR2) was targeted to either TRPV1-positive afferents or to villin-expressing colon epithelial cells. Expression of a ChR2-EYFP fusion protein was directed to either primary sensory neurons or to colon epithelial cells by crossing Ai32 mice with TRPV1-Cre or villin-Cre mice, respectively. An ex vivo preparation of the colon was used for single-fiber analysis of colon sensory afferents of the pelvic nerve. Afferents were characterized using previously described criteria as mucosal, muscular, muscular-mucosal, or serosal and then tested for blue light-induced activation. Light activation of colon epithelial cells produced robust firing of action potentials, similar to that elicited by physiologic stimulation (e.g., circumferential stretch), in 50.5% of colon afferents of mice homozygous for ChR2 expression. Light-induced activity could be reduced or abolished in most fibers using a cocktail of purinergic receptor blockers suggesting ATP release by the epithelium contributed to generation of sensory neuron action potentials. Using electromyographic recording of visceromotor responses we found that light stimulation of the colon epithelium evoked behavioral responses in Vil-ChR2 mice that was similar to that seen with balloon distension of the colon. These ex vivo and in vivo data indicate that light stimulation of colon epithelial cells alone, without added mechanical or chemical stimuli, can directly activate colon afferents and elicit behavioral responses.SIGNIFICANCE STATEMENT Abdominal pain that accompanies inflammatory diseases of the bowel is particularly vexing because it can occur without obvious changes in the structure or inflammatory condition of the colon. Pain reflects abnormal sensory neuron activity that may be controlled in part by release of substances from lining epithelial cells. In support of this mechanism we determined that blue-light stimulation of channelrhodopsin-expressing colon epithelial cells could evoke action potential firing in sensory neurons and produce changes in measures of behavioral sensitivity. Thus, activity of colon epithelial cells alone, without added mechanical or chemical stimuli, is sufficient to activate pain-sensing neurons.

Ablation of sensory neurons in a genetic model of pancreatic ductal adenocarcinoma slows initiation and progression of cancer.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by an exuberant inflammatory desmoplastic response. The PDAC microenvironment is complex, containing both pro- and antitumorigenic elements, and remains to be fully characterized. Here, we show that sensory neurons, an under-studied cohort of the pancreas tumor stroma, play a significant role in the initiation and progression of the early stages of PDAC. Using a well-established autochthonous model of PDAC (PKC), we show that inflammation and neuronal damage in the peripheral and central nervous system (CNS) occurs as early as the pancreatic intraepithelial neoplasia (PanIN) 2 stage. Also at the PanIN2 stage, pancreas acinar-derived cells frequently invade along sensory neurons into the spinal cord and migrate caudally to the lower thoracic and upper lumbar regions. Sensory neuron ablation by neonatal capsaicin injection prevented perineural invasion (PNI), astrocyte activation, and neuronal damage, suggesting that sensory neurons convey inflammatory signals from Kras-induced pancreatic neoplasia to the CNS. Neuron ablation in PKC mice also significantly delayed PanIN formation and ultimately prolonged survival compared with vehicle-treated controls (median survival, 7.8 vs. 4.5 mo; P = 0.001). These data establish a reciprocal signaling loop between the pancreas and nervous system, including the CNS, that supports inflammation associated with oncogenic Kras-induced neoplasia. Thus, pancreatic sensory neurons comprise an important stromal cell population that supports the initiation and progression of PDAC and may represent a potential target for prevention in high-risk populations.

Cutaneous neurturin overexpression alters mechanical, thermal, and cold responsiveness in physiologically identified primary afferents.

Neurotrophic factors play an important role in the regulation of functional properties of sensory neurons under normal and pathological conditions. The GDNF family member neurturin is one such factor that has been linked to modulating responsiveness to peripheral stimuli. Neurturin binds to the GFRα2 receptor, a receptor found primarily in isolectin B4-expressing polymodal cutaneous nociceptors. Previous work has shown that knockout of GFRα2 alters heat, but not mechanical, responses in dissociated sensory neurons and reduces pain-related behaviors during the second phase of the formalin test. Research has also shown that overexpression of neurturin in basal keratinocytes increases behavioral responsiveness to mechanical stimulation and innocuous cooling of the skin without affecting noxious heat responses. Here we directly examined the impact of neurturin overexpression on cutaneous afferent function. We compared physiological responses of individual sensory neurons to mechanical and thermal stimulation of the skin, using an ex vivo skin-nerve-dorsal root ganglion-spinal cord preparation produced from neurturin-overexpressing (NRTN/OE) mice and wild-type littermate controls. We found that neurturin overexpression increases responsiveness to innocuous mechanical stimuli in A-fiber nociceptors, alters thermal responses in the polymodal subpopulation of C-fiber sensory neurons, and changes the relative numbers of mechanically sensitive but thermally insensitive C-fiber afferents. These results demonstrate the potential roles of different functional groups of sensory neurons in the behavioral changes observed in mice overexpressing cutaneous neurturin and highlight the importance of neurturin in regulating cutaneous afferent response properties.NEW & NOTEWORTHY GDNF family neurotrophic factors regulate the development and function of primary sensory neurons. Of these, neurturin has been shown to modulate mechanical and cooling sensitivity behaviorally. Here we show that overexpression of neurturin in basal keratinocytes regulates mechanical responsiveness in A-fiber primary sensory neurons while increasing the overall numbers of cold-sensing units. Results demonstrate a crucial role for cutaneous neurturin in modulating responsiveness to peripheral stimuli at the level of the primary afferent.

Mechanism, assessment and management of pain in chronic pancreatitis: Recommendations of a multidisciplinary study group.

DESCRIPTION: Pain in patients with chronic pancreatitis (CP) remains the primary clinical complaint and source of poor quality of life. However, clear guidance on evaluation and treatment is lacking. METHODS: Pancreatic Pain working groups reviewed information on pain mechanisms, clinical pain assessment and pain treatment in CP. Levels of evidence were assigned using the Oxford system, and consensus was based on GRADE. A consensus meeting was held during PancreasFest 2012 with substantial post-meeting discussion, debate, and manuscript refinement. RESULTS: Twelve discussion questions and proposed guidance statements were presented. Conference participates concluded: Disease Mechanism: Pain etiology is multifactorial, but data are lacking to effectively link symptoms with pathologic feature and molecular subtypes. Assessment of Pain: Pain should be assessed at each clinical visit, but evidence to support an optimal approach to assessing pain character, frequency and severity is lacking. MANAGEMENT: There was general agreement on the roles for endoscopic and surgical therapies, but less agreement on optimal patient selection for medical, psychological, endoscopic, surgical and other therapies. CONCLUSIONS: Progress is occurring in pain biology and treatment options, but pain in patients with CP remains a major problem that is inadequately understood, measured and managed. The growing body of information needs to be translated into more effective clinical care.

TRPV1 and TRPA1 antagonists prevent the transition of acute to chronic inflammation and pain in chronic pancreatitis.

Visceral afferents expressing transient receptor potential (TRP) channels TRPV1 and TRPA1 are thought to be required for neurogenic inflammation and development of inflammatory hyperalgesia. Using a mouse model of chronic pancreatitis (CP) produced by repeated episodes (twice weekly) of caerulein-induced AP (AP), we studied the involvement of these TRP channels in pancreatic inflammation and pain-related behaviors. Antagonists of the two TRP channels were administered at different times to block the neurogenic component of AP. Six bouts of AP (over 3 wks) increased pancreatic inflammation and pain-related behaviors, produced fibrosis and sprouting of pancreatic nerve fibers, and increased TRPV1 and TRPA1 gene transcripts and a nociceptive marker, pERK, in pancreas afferent somata. Treatment with TRP antagonists, when initiated before week 3, decreased pancreatic inflammation and pain-related behaviors and also blocked the development of histopathological changes in the pancreas and upregulation of TRPV1, TRPA1, and pERK in pancreatic afferents. Continued treatment with TRP antagonists blocked the development of CP and pain behaviors even when mice were challenged with seven more weeks of twice weekly caerulein. When started after week 3, however, treatment with TRP antagonists was ineffective in blocking the transition from AP to CP and the emergence of pain behaviors. These results suggest: (1) an important role for neurogenic inflammation in pancreatitis and pain-related behaviors, (2) that there is a transition from AP to CP, after which TRP channel antagonism is ineffective, and thus (3) that early intervention with TRP channel antagonists may attenuate the transition to and development of CP effectively.

Neurturin overexpression in skin enhances expression of TRPM8 in cutaneous sensory neurons and leads to behavioral sensitivity to cool and menthol.

Neurturin (NRTN) is a member of the glial cell line-derived neurotrophic factor family of ligands that exerts its actions via Ret tyrosine kinase and GFRα2. Expression of the Ret-GFRα2 coreceptor complex is primarily restricted to the peripheral nervous system and is selectively expressed by sensory neurons that bind the isolectin B(4) (IB(4)). To determine how target-derived NRTN affects sensory neuron properties, transgenic mice that overexpress NRTN in keratinocytes (NRTN-OE mice) were analyzed. Overexpression of NRTN increased the density of PGP9.5-positive, but not calcitonin gene-related peptide-positive, free nerve endings in footpad epidermis. GFRα2-immunopositive somata were hypertrophied in NRTN-OE mice. Electron microscopic analysis further revealed hypertrophy of unmyelinated sensory axons and a subset of myelinated axons. Overexpression of NRTN increased the relative level of mRNAs encoding GFRα2 and Ret, the ATP receptor P2X(3) (found in IB(4)-positive, GFRα2-expressing sensory neurons), the acid-sensing ion channel 2a, and transient receptor potential cation channel subfamily member M8 (TRPM8) in sensory ganglia. Behavioral testing of NRTN-OE mice revealed an increased sensitivity to mechanical stimuli in glabrous skin of the hindpaw. NRTN-OE mice also displayed increased behavioral sensitivity to cool temperature (17°C-20°C) and oral sensitivity to menthol. The increase in cool and menthol sensitivity correlated with a significant increase in TRPM8 expression and the percentage of menthol-responsive cutaneous sensory neurons. These data indicate that the expression level of NRTN in the skin modulates gene expression in cutaneous sensory afferents and behavioral sensitivity to thermal, chemical, and mechanical stimuli.

Artemin growth factor increases nicotinic cholinergic receptor subunit expression and activity in nociceptive sensory neurons.

BACKGROUND: Artemin (Artn), a member of the glial cell line-derived growth factor (GDNF) family, supports the development and function of a subpopulation of peptidergic, TRPV1-positive sensory neurons. Artn (enovin, neublastin) is elevated in inflamed tissue and its injection in skin causes transient thermal hyperalgesia. A genome wide expression analysis of trigeminal ganglia of mice that overexpress Artn in the skin (ART-OE mice) showed elevation in nicotinic acetylcholine receptor (nAChR) subunits, suggesting these ion channels contribute to Artn-induced sensitivity. Here we have used gene expression, immunolabeling, patch clamp electrophysiology and behavioral testing assays to investigate the link between Artn, nicotinic subunit expression and thermal hypersensitivity. RESULTS: Reverse transcriptase-PCR validation showed increased levels of mRNAs encoding the nAChR subunits α3 (13.3-fold), ß3 (4-fold) and ß4 (7.7-fold) in trigeminal ganglia and α3 (4-fold) and ß4 (2.8-fold) in dorsal root ganglia (DRG) of ART-OE mice. Sensory ganglia of ART-OE mice had increased immunoreactivity for nAChRα3 and exhibited increased overlap in labeling with GFRα3-positive neurons. Patch clamp analysis of back-labeled cutaneous afferents showed that while the majority of nicotine-evoked currents in DRG neurons had biophysical and pharmacological properties of α7-subunit containing nAChRs, the Artn-induced increase in α3 and ß4 subunits resulted in functional channels. Behavioral analysis of ART-OE and wildtype mice showed that Artn-induced thermal hyperalgesia can be blocked by mecamylamine or hexamethonium. Complete Freund's adjuvant (CFA) inflammation of paw skin, which causes an increase in Artn in the skin, also increased the level of nAChR mRNAs in DRG. Finally, the increase in nAChRs transcription was not dependent on the Artn-induced increase in TRPV1 or TRPA1 in ART-OE mice since nAChRs were elevated in ganglia of TRPV1/TRPA1 double knockout mice. CONCLUSIONS: These findings suggest that Artn regulates the expression and composition of nAChRs in GFRα3 nociceptors and that these changes contribute to the thermal hypersensitivity that develops in response to Artn injection and perhaps to inflammation.

PDX1, a transcription factor essential for organ differentiation, regulates SERCA-dependent Ca2+ homeostasis in sensory neurons.

Pancreatic and duodenal homeobox 1 (PDX1) is a transcription factor required for the development and differentiation of the pancreas. Previous studies indicated that PDX1 expression was restricted to the gastrointestinal tract. Using a cre-dependent reporter, we observed PDX1-dependent expression of tdtomato (PDX1-tom) in a subpopulation of sensory nerves. Many of these PDX1-tom afferents expressed the neurofilament 200 protein and projected to the skin. Tdtomato-labeled terminals were associated with hair follicles in the form of longitudinal and circumferential lanceolate endings suggesting a role in tactile and proprioceptive perception. To begin to examine the functional significance of PDX1 in afferents, we used Fura-2 imaging to examine calcium (Ca2+) handling under naïve and nerve injury conditions. Neuropathic injury is associated with increased intracellular Ca2+ signaling that in part results from dysregulation of the sarco/endoplasmic reticulum calcium transport ATPase (SERCA). Here we demonstrate that under naïve conditions, PDX1 regulates expression of the SERCA2B isoform in sensory neurons. In response to infraorbital nerve injury, a significant reduction of PDX1 and SERCA2B expression and dysregulation of Ca2+ handling occurs in PDX1-tom trigeminal ganglia neurons. The identification of PDX1 expression in the somatosensory system and its regulation of SERCA2B and Ca2+ handling provide a new mechanism to explain pathological changes in primary afferents that may contribute to pain associated with nerve injury.

TRPV1 and TRPA1 function and modulation are target tissue dependent.

The nerve growth factor (NGF) and glial cell line-derived neurotrophic factor (GDNF) families of growth factors regulate the sensitivity of sensory neurons. The ion channels transient receptor potential vanilloid 1 (TRPV1) and transient receptor potential channel, subfamily A, member 1 (TRPA1), are necessary for development of inflammatory hypersensitivity and are functionally potentiated by growth factors. We have shown previously that inflamed skin exhibits rapid increases in artemin mRNA with slower, smaller increases in NGF mRNA. Here, using mice, we show that, in inflamed colon, mRNA for both growth factors increased with a pattern distinct from that seen in skin. Differences were also seen in the pattern of TRPV1 and TRPA1 mRNA expression in DRG innervating inflamed skin and colon. Growth factors potentiated capsaicin (a specific TRPV1 agonist) and mustard oil (a specific TRPA1 agonist) behavioral responses in vivo, raising the question as to how these growth factors affect individual afferents. Because individual tissues are innervated by afferents with unique properties, we investigated modulation of TRPV1 and TRPA1 in identified afferents projecting to muscle, skin, and colon. Muscle and colon afferents are twice as likely as skin afferents to express functional TRPV1 and TRPA1. TRPV1 and TRPA1 responses were potentiated by growth factors in all afferent types, but compared with skin afferents, muscle afferents were twice as likely to exhibit NGF-induced potentiation and one-half as likely to exhibit artemin-induced potentiation of TRPV1. Furthermore, skin afferents showed no GDNF-induced potentiation of TRPA1, but 43% of muscle and 38% of colon afferents exhibited GDNF-induced potentiation. These results show that interpretation of afferent homeostatic mechanisms must incorporate properties that are specific to the target tissue.

Synergistic role of TRPV1 and TRPA1 in pancreatic pain and inflammation.

BACKGROUND & AIMS: The transient receptor potential (TRP) channels TRPV1 and TRPA1 have each been associated with regulation of efferent properties of primary afferent neurons that initiate neurogenic inflammation and are required for the development of inflammatory hyperalgesia. To evaluate the role of these channels in producing pain during pancreatic inflammation, we studied pancreatic nodose ganglion (NG) and dorsal root ganglion (DRG) sensory neurons (identified by content of retrograde tracer) and behavioral outcomes in a mouse model of acute pancreatitis. METHODS: Pancreatic inflammation was induced by 8 hourly injections of cerulein (50 µg/kg). The extent of inflammation, pancreatic neuron TRP channel expression and function and excitability, and pain-related behaviors were evaluated over the course of the following week. RESULTS: Histology and myeloperoxidase activity confirmed pancreatic inflammation that was associated with increased excitability and messenger RNA expression of the TRP channels in NG and DRG pancreatic neurons. Calcium imaging of pancreatic NG and DRG neurons from mice given cerulein revealed increased responses to TRP agonists. TRPV1 and TRPA1 antagonists attenuated cerulein-induced pain behaviors and pancreatic inflammation; they had a synergistic effect. CONCLUSIONS: Pancreatic inflammation significantly increased the expression and functional properties of TRPV1 and TRPA1, as well as the excitability of pancreatic sensory neurons in vagal and spinal pathways. TRP channel antagonists acted synergistically to reverse pancreatic inflammation and associated pain behaviors; reagents that target interactions between these channels might be developed to reduce pain in patients with acute pancreatitis.

Sox11 modulates brain-derived neurotrophic factor expression in an exon promoter-specific manner.

Sox11 is a high-mobility group (HMG)-containing transcription factor that is significantly elevated in peripheral neurons in response to nerve injury. In vitro and in vivo studies support a central role for Sox11 in adult neuron growth and survival following injury. Brain-derived neurotrophic factor (BDNF) is a pleiotropic growth factor that has effects on neuronal survival, differentiation, synaptic plasticity, and regeneration. BDNF transcription is elevated in the dorsal root ganglia (DRG) following nerve injury in parallel with Sox11, allowing for the possible regulation by Sox11. To begin to assess the possible influence of Sox11, we used reverse transcriptase PCR assays to determine the relative expression of the nine (I-IXa) noncoding exons and one coding exon (exon IX) of the BDNF gene after sciatic nerve axotomy in the mouse. Exons with upstream promoter regions containing the Sox binding motif 5'-AACAAAG-3' (I, IV, VII, and VIII) were increased at 1 or 3 days following axotomy. Exons 1 and IV showed the greatest increase, and only exon 1 remained elevated at 3 days. Luciferase assays showed that Sox11 could activate the most highly regulated exons, I and IV, and that this activation was reduced by mutation of putative Sox binding sites. Exon expression in injured DRG neurons had some overlap with Neuro2a cells that overexpress Sox11, showing elevation in exon IV and VII transcripts. These findings indicate cell type and contextual specificity of Sox11 in modulation of BDNF transcription.

Pain in Inflammatory Bowel Disease: Optogenetic Strategies for Study of Neural-Epithelial Signaling.

Abdominal pain is common in patients with active inflammation of the colon but can persist even in its absence, suggesting other mechanisms of pain signaling. Recent findings suggest colon epithelial cells are direct regulators of pain-sensing neurons. Optogenetic activation of epithelial cells evoked nerve firing and pain-like behaviors. Inhibition of epithelial cells caused the opposite effect, reducing responses to colon distension and inflammatory hypersensitivity. Thus, epithelial cells alone can regulate the activation of pain circuits. Future goals are to define the anatomical and cellular mechanisms that underlie epithelial-neural pain signaling and how it is altered in response to colon inflammation.