Transcriptomic Profiling in Mice With CB1 receptor Deletion in Primary Sensory Neurons Suggests New Analgesic Targets for Neuropathic Pain

Type 1 and type 2 cannabinoid receptors (CB1 and CB2, respectively) mediate cannabinoid-induced analgesia. Loss of endogenous CB1 is associated with hyperalgesia. However, the downstream targets affected by ablation of CB1 in primary sensory neurons remain unknown. In the present study, we hypothesized that conditional knockout of CB1 in primary sensory neurons (CB1cKO) alters downstream gene expression in the dorsal root ganglion (DRG) and that targeting these pathways alleviates neuropathic pain. We found that CB1cKO in primary sensory neurons induced by tamoxifen in adult Advillin-Cre:CB1-floxed mice showed persistent hyperalgesia. Transcriptome/RNA sequencing analysis of the DRG indicated that differentially expressed genes were enriched in energy regulation and complement and coagulation cascades at the early phase of CB1cKO, whereas pain regulation and nerve conduction pathways were affected at the late phase of CB1cKO. Chronic constriction injury in mice induced neuropathic pain and changed transcriptome expression in the DRG of CB1cKO mice, and differentially expressed genes were mainly associated with inflammatory and immune-related pathways. Nerve injury caused a much larger increase in CB2 expression in the DRG in CB1cKO than in wildtype mice. Interfering with downstream target genes of CB1, such as antagonizing CB2, inhibited activation of astrocytes, reduced neuroinflammation, and alleviated neuropathic pain. Our results demonstrate that CB1 in primary sensory neurons functions as an endogenous analgesic mediator. CB2 expression is regulated by CB1 and may be targeted for the treatment of neuropathic pain.

Imaging sensory transmission and neuronal plasticity in primary sensory neurons with a positively tuned voltage indicator

Detection of somatosensory inputs requires conversion of external stimuli into electrical signals by activation of primary sensory neurons. The mechanisms by which heterogeneous primary sensory neurons encode different somatosensory inputs remains unclear. In vivo dorsal root ganglia (DRG) imaging using genetically-encoded Ca2+ indicators (GECIs) is currently the best technique for this purpose mapping neuronal function in DRG circuits by providing an unprecedented spatial and populational resolution. It permits the simultaneous imaging of >1800 neurons/DRG in live mice. However, this approach is not ideal given that Ca2+ is a second messenger and has inherently slow response kinetics. In contrast, genetically-encoded voltage indicators (GEVIs) have the potential to track voltage changes in multiple neurons in real time but often lack the brightness and dynamic range required for in vivo use. Here, we used soma-targeted ASAP4.4-Kv, a novel positively tuned GEVI, to dissect the temporal dynamics of noxious and non-noxious neuronal signals during mechanical, thermal, or chemical stimulation in DRG neurons of live mice. ASAP4.4-Kv is sufficiently bright and fast enough to optically characterize individual neuron coding dynamics. Notably, using ASAP4.4-Kv, we uncovered cell-to-cell electrical synchronization between adjacent DRG neurons and robust dynamic transformations in sensory coding following tissue injury. Finally, we found that a combination of GEVI and GECI imaging empowered in vivo optical studies of sensory signal processing and integration mechanisms with optimal spatiotemporal analysis.

C/EBPβ Participates in Nerve Trauma-Induced TLR7 Upregulation in Primary Sensory Neurons

Nerve trauma-induced toll-like receptor 7 (TLR7) expression level increase in primary sensory neurons in the damaged dorsal root ganglion (DRG) avails to neuropathic pain, but the reason is still unknown. In the current study, we showed that unilateral lumbar 4 (L4) spinal nerve ligation (SNL) upregulated CCAAT/enhancer-binding protein-β (C/EBPβ) expression in ipsilateral L4 DRG. Preventing this elevation attenuated the SNL-induced upregulation of TLR7 in the ipsilateral L4 DRG and inhibited cold/thermal hyperalgesia and mechanical allodynia. Mimicking nerve trauma-induced C/EBPβ upregulation generated an elevated level of TLR7 in injected DRG, augmented responses to cold/thermal/mechanical stimuli while causing ipsilateral spontaneous pain with no SNL. Mechanistically, SNL upregulated binding of increased C/EBPβ to Tlr7 promoter in ipsilateral L4 DRG. Accorded that C/EBPβ could trigger the activation of Tlr7 promoter and co-expressed with Tlr7 mRNA in individual DRG neurons, our findings strongly suggest the role of C/EBPβ in nerve trauma-mediated TLR7 upregulation in damaged primary sensory neurons.

Sensory Neuron Expressed TRPC3 Mediates Acute and Chronic Itch

Background: Chronic pruritus is a prominent symptom of allergic contact dermatitis (ACD) and represent a huge unmet health problem. However, its underlying cellular and molecular mechanisms remain largely unexplored. TRPC3 is highly expressed in primary sensory neurons and has been implicated in peripheral sensitization induced by proinflammatory mediators. However, the role of TRPC3 in acute and chronic itch is still not well defined. Methods: RNAscope in situ hybridization and immunohistochemical staining were performed on mouse trigeminal ganglion (TG) neurons. Fura-2 calcium imaging was used to characterize the function of TRPC3 in dissociated TG neurons. In native mice, the TRPC3 agonist and pruritogens were subcutaneously injected to the cheek and nape of the neck of mice, respectively. Site directed scratching and/or wiping behaviors were video recorded. Contact hypersensitivity (CHS) model was induced in mouse ears by topical application of SADBE or DNCB. Spontaneous scratching behaviors were recorded by video monitoring. Global and conditional Trpc3 knockout mice were employed to determine the contribution of TRPC3 to acute and chronic itch. The mRNA expression levels of Trpc3 and proinflammatory cytokines were assayed by quantitative real-time PCR. H&E. staining was used for the evaluation of the thickness of mouse ears. Flow cytometry was performed to assess immune cell infiltration in mouse ear tissues. Results: Among mouse TG neurons, RNAscope assay revealed that Trpc3 mRNA was predominantly expressed in nonpeptidergic small diameter neurons. Moreover, Trpc3 mRNA signal was present in the majority of itch sensing neurons. TRPC3 agonism induced TG neuronal activation and acute nonhistaminergic itch- and pain-like behaviors in naïve mice. In addition, genetic deletion of Trpc3 attenuated acute itch evoked by certain common nonhistaminergic pruritogens, including endothelin-1 and SLIGRL-NH2. In a murine model of CHS, Trpc3 mRNA expression level and function were upregulated in the TG following CHS. Pharmacological inhibition and global knockout of Trpc3 significantly alleviated spontaneous scratching behaviors without affecting concurrent cutaneous inflammation in the CHS model. Furthermore, conditional deletion of Trpc3 in primary sensory neurons but not in keratinocytes produced similar antipruritic effects in this model. Conclusions: These findings suggest that TRPC3 expressed in primary sensory neurons may contribute to acute and chronic itch via a histamine independent mechanism and that targeting neuronal TRPC3 might benefit the treatment of chronic itch associated with ACD and other inflammatory skin disorders.

The Calcium Channel α2δ1 Subunit: Interactional Targets in Primary Sensory Neurons and Role in Neuropathic Pain

Neuropathic pain is mainly triggered after nerve injury and associated with plasticity of the nociceptive pathway in primary sensory neurons. Currently, the treatment remains a challenge. In order to identify specific therapeutic targets, it is necessary to clarify the underlying mechanisms of neuropathic pain. It is well established that primary sensory neuron sensitization (peripheral sensitization) is one of the main components of neuropathic pain. Calcium channels act as key mediators in peripheral sensitization. As the target of gabapentin, the calcium channel subunit α2δ1 (Cavα2δ1) is a potential entry point in neuropathic pain research. Numerous studies have demonstrated that the upstream and downstream targets of Cavα2δ1 of the peripheral primary neurons, including thrombospondins, N-methyl-D-aspartate receptors, transient receptor potential ankyrin 1 (TRPA1), transient receptor potential vanilloid family 1 (TRPV1), and protein kinase C (PKC), are involved in neuropathic pain. Thus, we reviewed and discussed the role of Cavα2δ1 and the associated signaling axis in neuropathic pain conditions.

Tmem45b is essential for inflammation and tissue injury-induced persistent mechanical hyperalgesia

Persistent mechanical hyperalgesia, associated with peripheral inflammation and tissue injury, impairs patient’s quality of life and daily activity. However, its molecular mechanism and treatment are yet to be deciphered. Herein, we report that Tmeme45b is expressed in a subset of unmyelinated primary sensory neurons and plays an essential role in inflammation and tissue injury-induced mechanical hyperalgesia. Our findings provide new insights into the mechanisms and the treatment of mechanical hyperalgesia.

Continuous, multidimensional coding of 3D complex tactile stimuli by primary sensory neurons of the vibrissal system

Across all sensory modalities, first-stage sensory neurons are an information bottleneck: they must convey all information available for an animal to perceive and act in its environment. Our understanding of coding properties of primary sensory neurons in the auditory and visual systems has been aided by the use of increasingly complex, naturalistic stimulus sets. By comparison, encoding properties of primary somatosensory afferents are poorly understood. Here, we use the rodent whisker system to examine how tactile information is represented in primary sensory neurons of the trigeminal ganglion (Vg). Vg neurons have long been thought to segregate into functional classes associated with separate streams of information processing. However, this view is based on Vg responses to restricted stimulus sets which potentially underreport the coding capabilities of these neurons. In contrast, the current study records Vg responses to complex three-dimensional (3D) stimulation while quantifying the complete 3D whisker shape and mechanics, thereby beginning to reveal their full representational capabilities. The results show that individual Vg neurons simultaneously represent multiple mechanical features of a stimulus, do not preferentially encode principal components of the stimuli, and represent continuous and tiled variations of all available mechanical information. These results directly contrast with proposed codes in which subpopulations of Vg neurons encode select stimulus features. Instead, individual Vg neurons likely overcome the information bottleneck by encoding large regions of a complex sensory space. This proposed tiled and multidimensional representation at the Vg directly constrains the computations performed by more central neurons of the vibrissotrigeminal pathway.

Tetrodotoxin, a Potential Drug for Neuropathic and Cancer Pain Relief?

Tetrodotoxin (TTX) is a potent neurotoxin found mainly in puffer fish and other marine and terrestrial animals. TTX blocks voltage-gated sodium channels (VGSCs) which are typically classified as TTX-sensitive or TTX-resistant channels. VGSCs play a key role in pain signaling and some TTX-sensitive VGSCs are highly expressed by adult primary sensory neurons. During pathological pain conditions, such as neuropathic pain, upregulation of some TTX-sensitive VGSCs, including the massive re-expression of the embryonic VGSC subtype NaV1.3 in adult primary sensory neurons, contribute to painful hypersensitization. In addition, people with loss-of-function mutations in the VGSC subtype NaV1.7 present congenital insensitive to pain. TTX displays a prominent analgesic effect in several models of neuropathic pain in rodents. According to this promising preclinical evidence, TTX is currently under clinical development for chemo-therapy-induced neuropathic pain and cancer-related pain. This review focuses primarily on the preclinical and clinical evidence that support a potential analgesic role for TTX in these pain states. In addition, we also analyze the main toxic effects that this neurotoxin produces when it is administered at therapeutic doses, and the therapeutic potential to alleviate neuropathic pain of other natural toxins that selectively block TTX-sensitive VGSCs.