Accepted

T-type calcium channels activate in response to subthreshold membrane depolarizations and represent an important source of Ca influx near the resting membrane potential. These channels regulate neuronal excitability and have been linked to pain. For this reason, T-type calcium channels are suitable molecular targets for the development of new non-opioid analgesics. Our previous work identified an analogue of benzimidazolonepiperidine, 5bk, that preferentially inhibited Ca3.2 channels and reversed mechanical allodynia. In this study, we synthesized and screened a small library of 47 compounds derived from 5bk. We found several compounds that inhibited the Ca influx in DRG neurons of all sizes. After separating the enantiomers of each active compound, we found two compounds, 3-25-R and 3-14-3-S, that potently inhibited the Ca influx. Whole-cell patch clamp recordings from small- to medium-sized DRG neurons revealed that both compounds decreased total Ca. Application of 3-14-3-S (but not 3-25-R) blocked transiently expressed Ca3.1-3.3 channels with a similar IC value. 3-14-3-S decreased T-type, but not N-type, Ca currents in DRG neurons. Furthermore, intrathecal delivery of 3-14-3-S relieved tonic, neuropathic, and inflammatory pain in preclinical models. 3-14-3-S did not exhibit any activity against G protein-coupled opioid receptors. Preliminary docking studies also suggest that 3-14-3-S can bind to the central pore domain of T-type channels. Together, our chemical characterization and functional and behavioral data identify a novel T-type calcium channel blocker with in vivo efficacy in experimental models of tonic, neuropathic, and inflammatory pain.

Specialized cutaneous Schwann cells (SCs), termed nociceptive SCs, were recently discovered. Their function is not fully understood, but they are believed not only to support peripheral axons in mouse skin by forming a mesh-like neural-glio networking structure in subepidermal area, but also contributing to transduction of mechanical sensation and neuropathic pain. Diabetic neuropathy (DPN) is one of the most common complication of diabetes, however, the mechanisms behind painful and painless DPN remain unclear. Using a mouse model of DPN, we want to investigate if there are quantitative differences in nociceptive SC density between the condition of hyperglycemia-induced sensory abnormalities and control condition and at which stage in the disease the damage occurs. Here, we developed a set of counting rules for nociceptive SCs based on immunofluorescent staining, and applied the method to quantify the density of nociceptive SCs in control mice (n=10), mice with nociceptive hypersensitivity at early diabetic stage (n=5), and mice with sensory hyposensitivity at late diabetic stage (n=5) in the Streptozotocin (STZ) model of type 1 diabetes. Nociceptive SCs were identified as S100/Sox10/DAPI cells abutting to peripheral nerves, with the somas located within 25µm depth in the subepidermal area and outside glands and large fiber bundles. Hypersensitive diabetic mice had decreased nociceptive SC density, despite having normal epidermal nerve fiber density, compared with age-matched control mice (P=0.023). In contrast, there was a reduction in intraepidermal nerve fiber density but no difference in nociceptive SC density between hyposensitive diabetic mice and the age-matched control mice. This study provides a detailed description of how to identify and quantify nociceptive SC and demonstrates that nociceptive SC density declines before nerve fiber deterioration, which supports previous observations that nociceptive SCs are critical for maintenance of cutaneous sensory nerves.

Varicella zoster virus (VZV) is responsible for chronic pain. VZV injection has similarities to herpes zoster "shingles" pain in humans. In this study orofacial pain was induced by injecting male rats with the human varicella zoster virus (VZV). The amygdala and parabrachial have been implicated to control affective/motivational orofacial pain. Recently our lab reported neurexin 3α (Nrxn3α) is expressed in the central amygdala and parabrachial. GABAergic neurons descend from the central amygdala to the lateral parabrachial region and Nrxn3α is important for presynaptic GABA release. Thus, we hypothesized that lateral parabrachial neuronal activity and orofacial pain are controlled by Nrxn3α within the central amygdala. To test the hypothesis Nrxn3 expression was knocked down (i.e., using shRNA) in the central amygdala and GABA release and neuronal activity were quantitated in the parabrachial concomitant with measurement of the VZV induced pain response. Results revealed that attenuating Nrxn3 expression within the amygdala reduces GABA release in the parabrachial and increases neuronal activity within the lateral parabrachial region. Attenuating Nrxn3 expression also increases VZV associated orofacial pain. Activating GABAergic neurons within the central amygdala with opsins increase GABA release in the parabrachial and reduced the pain response after Nrxn3 shRNA treatment. These results are consistent with the idea that Nrxn3 within the central amygdala controls VZV associated pain by regulating GABA release in the lateral parabrachial that then controls the activity of ascending pain neurons.

Cold sensation is initiated in the periphery by a specialized population of cold-sensitive neurons, referred to as cold receptors, who transmit decreases in temperature with sub-degree resolution using a diverse assortment of ion channels and receptors. It is largely accepted that normal cold signaling is initiated through activation of transient receptor potential melastatin 8 (TRPM8) expressing neurons. Conversely, the mechanisms underlying cold-induced pain signaling are not as well defined. Interestingly, mounting evidence demonstrates functional interplay between cold signaling and other somatic sensations, such as itch and warmth; thus, cold-sensing pathways also engage in sensory crosstalk and population coding mechanisms. In this review, we will discuss recent advances in our understanding of cold sensation and address major gaps in knowledge that require more investigation.