Deletion of Tsc2 in nociceptors reduces target innervation, ion channel expression, and sensitivity to heat

AbstractThe mechanistic target of rapamycin complex 1 (mTORC1) is known to regulate cellular growth pathways, and its genetic activation is sufficient to enhance regenerative axon growth following injury to the central or peripheral nervous systems. However, excess mTORC1 activation may promote innervation defects, and mTORC1 activity mediates injury-induced hypersensitivity, reducing enthusiasm for the pathway as a therapeutic target. While mTORC1 activity is required for full expression of some pain modalities, the effects of pathway activation on nociceptor phenotypes and sensory behaviors are currently unknown. To address this, we genetically activated mTORC1 in mouse peripheral sensory neurons by conditional deletion of its negative regulator Tuberous Sclerosis Complex 2 (Tsc2). Consistent with the well-known role of mTORC1 in regulating cell size, soma size and axon diameter of C-nociceptors were increased in Tsc2-deleted mice. Glabrous skin and spinal cord innervation by C-fiber neurons were also disrupted. Transcriptional profiling of nociceptors enriched by fluorescence-associated cell sorting (FACS) revealed downregulation of multiple classes of ion channels as well as reduced expression of markers for peptidergic nociceptors in Tsc2-deleted mice. In addition to these changes in innervation and gene expression, Tsc2-deleted mice exhibited reduced noxious heat sensitivity and decreased injury-induced cold hypersensitivity, but normal baseline sensitivity to cold and mechanical stimuli. Together, these data show that excess mTORC1 activity in sensory neurons produces changes in gene expression, neuron morphology and sensory behavior.</jats:p

Differential regulation of bladder pain and voiding function by sensory afferent populations revealed by selective optogenetic activation

Bladder-innervating primary sensory neurons mediate reflex-driven bladder function under normal conditions, and contribute to debilitating bladder pain and/or overactivity in pathological states. The goal of this study was to examine the respective roles of defined subtypes of afferent neurons in bladder sensation and function in vivo via direct optogenetic activation. To accomplish this goal, we generated transgenic lines that express a Channelrhodopsin-2-eYFP fusion protein (ChR2-eYFP) in two distinct populations of sensory neurons: TRPV1-lineage neurons (Trpv1Cre;Ai32, the majority of nociceptors) and Nav1.8+ neurons (Scn10aCre;Ai32, nociceptors and some mechanosensitive fibers). In spinal cord, eYFP+ fibers in Trpv1Cre;Ai32 mice were observed predominantly in dorsal horn (DH) laminae I-II, while in Scn10aCre;Ai32 mice they extended throughout the DH, including a dense projection to lamina X. Fiber density correlated with number of retrogradely-labeled eYFP+ dorsal root ganglion neurons (82.2% Scn10aCre;Ai32 vs. 62% Trpv1Cre;Ai32) and degree of DH excitatory synaptic transmission. Photostimulation of peripheral afferent terminals significantly increased visceromotor responses to noxious bladder distension (30–50 mmHg) in both transgenic lines, and to non-noxious distension (20 mmHg) in Scn10aCre;Ai32 mice. Depolarization of ChR2+ afferents in Scn10aCre;Ai32 mice produced low- and high-amplitude bladder contractions respectively in 53% and 27% of stimulation trials, and frequency of high-amplitude contractions increased to 60% after engagement of low threshold (LT) mechanoreceptors by bladder filling. In Trpv1Cre;Ai32 mice, low-amplitude contractions occurred in 27% of trials before bladder filling, which was pre-requisite for light-evoked high-amplitude contractions (observed in 53.3% of trials). Potential explanations for these observations include physiological differences in the thresholds of stimulated fibers and their connectivity to spinal circuits

Divergent modulation of nociception by glutamatergic and GABAergic neuronal subpopulations in the periaqueductal gray

The ventrolateral periaqueductal gray (vlPAG) constitutes a major descending pain modulatory system and is a crucial site for opioid-induced analgesia. A number of previous studies have demonstrated that glutamate and GABA play critical opposing roles in nociceptive processing in the vlPAG. It has been suggested that glutamatergic neurotransmission exerts antinociceptive effects, whereas GABAergic neurotransmission exert pronociceptive effects on pain transmission, through descending pathways. The inability to exclusively manipulate subpopulations of neurons in the PAG has prevented direct testing of this hypothesis. Here, we demonstrate the different contributions of genetically defined glutamatergic and GABAergic vlPAG neurons in nociceptive processing by employing cell type-specific chemogenetic approaches in mice. Global chemogenetic manipulation of vlPAG neuronal activity suggests that vlPAG neural circuits exert tonic suppression of nociception, consistent with previous pharmacological and electrophysiological studies. However, selective modulation of GABAergic or glutamatergic neurons demonstrates an inverse regulation of nociceptive behaviors by these cell populations. Selective chemogenetic activation of glutamatergic neurons, or inhibition of GABAergic neurons, in vlPAG suppresses nociception. In contrast, inhibition of glutamatergic neurons, or activation of GABAergic neurons, in vlPAG facilitates nociception. Our findings provide direct experimental support for a model in which excitatory and inhibitory neurons in the PAG bidirectionally modulate nociception

A TRPV4-dependent neuroimmune axis in the spinal cord promotes neuropathic pain

Microglia, resident macrophages of the CNS, are essential to brain development, homeostasis, and disease. Microglial activation and proliferation are hallmarks of many CNS diseases, including neuropathic pain. However, molecular mechanisms that govern the spinal neuroimmune axis in the setting of neuropathic pain remain incompletely understood. Here, we show that genetic ablation or pharmacological blockade of transient receptor potential vanilloid type 4 (TRPV4) markedly attenuated neuropathic pain-like behaviors in a mouse model of spared nerve injury. Mechanistically, microglia-expressed TRPV4 mediated microglial activation and proliferation and promoted functional and structural plasticity of excitatory spinal neurons through release of lipocalin-2. Our results suggest that microglial TRPV4 channels reside at the center of the neuroimmune axis in the spinal cord, which transforms peripheral nerve injury into central sensitization and neuropathic pain, thereby identifying TRPV4 as a potential new target for the treatment of chronic pain

Optogenetic induction of colonic motility in mice

Background & Aims: Strategies are needed to increase gastrointestinal transit without systemic pharmacologic agents. We investigated whether optogenetics, focal application of light to control enteric nervous system excitability, could be used to evoke propagating contractions and increase colonic transit in mice. Methods: We generated transgenic mice with Cre-mediated expression of light-sensitive channelrhodopsin-2 (CHR2) in calretinin neurons (CAL-ChR2 Cre+ mice); Cre– littermates served as controls. Colonic myenteric neurons were analyzed by immunohistochemistry, patch clamp, and calcium imaging studies. Motility was assessed by mechanical, electrophysiological, and video recording in vitro and by fecal output in vivo. Results: In isolated colons, focal light stimulation of calretinin enteric neurons evoked classic polarized motor reflexes (50/58 stimulations), followed by premature anterograde propagating contractions (39/58 stimulations). Light stimulation could evoke motility from sites along the entire colon. These effects were prevented by neural blockade with tetrodotoxin (n = 2), and did not occur in control mice (n = 5). Light stimulation of proximal colon increased the proportion of natural fecal pellets expelled over 15 minutes in vitro (75 ± 17% vs 32 ± 8% for controls) (P <.05). In vivo, activation of wireless light-emitting diodes implanted onto the colon wall significantly increased hourly fecal pellet output in conscious, freely moving mice (4.17 ± 0.4 vs 1.3 ± 0.3 in controls) (P<.001). Conclusions: In studies of mice, we found that focal activation of a subset of enteric neurons can increase motility of the entire colon in vitro, and fecal output in vivo. Optogenetic control of enteric neurons might therefore be used to modify gut motility.This work was supported by grants from the National Institutes of Health (NIH), R01GM101218, R01DK103901 (to HH), Washington University School of Medicine Digestive Disease Research Core Center (NIDDK P30 DK052574), The Center for the Study of Itch of Department of Anesthesiology at Washington University School of Medicine (to HH) and NIH SPARC Award (NIBIB U18EB021793) and NIH Director’s Transformative Research Award (TR01 NS081707) to RWG. TJH was supported by a grant (#1127140) from the NH&MRC of Australia to NJS. Urology Care Foundation Research Scholars Program and Kailash Kedia Research Scholar Award to VKS

A potential role for stress-induced microbial alterations in IgA-associated irritable bowel syndrome with diarrhea

Stress is a known trigger for flares of inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS); however, this process is not well understood. Here, we find that restraint stress in mice leads to signs of diarrhea, fecal dysbiosis, and a barrier defect via the opening of goblet-cell associated passages. Notably, stress increases host immunity to gut bacteria as assessed by immunoglobulin A (IgA)-bound gut bacteria. Stress-induced microbial changes are necessary and sufficient to elicit these effects. Moreover, similar to mice, many diarrhea-predominant IBS (IBS-D) patients from two cohorts display increased antibacterial immunity as assessed by IgA-bound fecal bacteria. This antibacterial IgA response in IBS-D correlates with somatic symptom severity and was distinct from healthy controls or IBD patients. These findings suggest that stress may play an important role in patients with IgA-associated IBS-D by disrupting the intestinal microbial community that alters gastrointestinal function and host immunity to commensal bacteria

Optogenetic silencing of nociceptive primary afferents reduces evoked and ongoing bladder pain

Abstract Patients with interstitial cystitis/bladder pain syndrome (IC/BPS) suffer from chronic pain that severely affects quality of life. Although the underlying pathophysiology is not well understood, inhibition of bladder sensory afferents temporarily relieves pain. Here, we explored the possibility that optogenetic inhibition of nociceptive sensory afferents could be used to modulate bladder pain. The light-activated inhibitory proton pump Archaerhodopsin (Arch) was expressed under control of the sensory neuron-specific sodium channel (sns) gene to selectively silence these neurons. Optically silencing nociceptive sensory afferents significantly blunted the evoked visceromotor response to bladder distension and led to small but significant changes in bladder function. To study of the role of nociceptive sensory afferents in freely behaving mice, we developed a fully implantable, flexible, wirelessly powered optoelectronic system for the long-term manipulation of bladder afferent expressed opsins. We found that optogenetic inhibition of nociceptive sensory afferents reduced both ongoing pain and evoked cutaneous hypersensitivity in the context of cystitis, but had no effect in uninjured, naïve mice. These results suggest that selective optogenetic silencing of nociceptive bladder afferents may represent a potential future therapeutic strategy for the treatment of bladder pain

A photoswitchable GPCR-based opsin for presynaptic inhibition

Optical manipulations of genetically defined cell types have generated significant insights into the dynamics of neural circuits. While optogenetic activation has been relatively straightforward, rapid and reversible synaptic inhibition has proven more elusive. Here, we leveraged the natural ability of inhibitory presynaptic GPCRs to suppress synaptic transmission and characterize parapinopsin (PPO) as a GPCR-based opsin for terminal inhibition. PPO is a photoswitchable opsin that couples to Gi/o signaling cascades and is rapidly activated by pulsed blue light, switched off with amber light, and effective for repeated, prolonged, and reversible inhibition. PPO rapidly and reversibly inhibits glutamate, GABA, and dopamine release at presynaptic terminals. Furthermore, PPO alters reward behaviors in a time-locked and reversible manner in vivo. These results demonstrate that PPO fills a significant gap in the neuroscience toolkit for rapid and reversible synaptic inhibition and has broad utility for spatiotemporal control of inhibitory GPCR signaling cascades