Neuromedin B receptor stimulation of Cav3.2 T-type Ca2+ channels in primary sensory neurons mediates peripheral pain hypersensitivity.

Background: Neuromedin B (Nmb) is implicated in the regulation of nociception of sensory neurons. However, the underlying cellular and molecular mechanisms remain unknown. Methods: Using patch clamp recording, western blot analysis, immunofluorescent labelling, enzyme-linked immunosorbent assays, adenovirus-mediated shRNA knockdown and animal behaviour tests, we studied the effects of Nmb on the sensory neuronal excitability and peripheral pain sensitivity mediated by Cav3.2 T-type channels. Results: Nmb reversibly and concentration-dependently increased T-type channel currents (IT) in small-sized trigeminal ganglion (TG) neurons through the activation of neuromedin B receptor (NmbR). This NmbR-mediated IT response was Gq protein-coupled, but independent of protein kinase C activity. Either intracellular application of the QEHA peptide or shRNA-mediated knockdown of Gß abolished the NmbR-induced IT response. Inhibition of protein kinase A (PKA) or AMP-activated protein kinase (AMPK) completely abolished the Nmb-induced IT response. Analysis of phospho-AMPK (p-AMPK) revealed that Nmb significantly activated AMPK, while AMPK inhibition prevented the Nmb-induced increase in PKA activity. In a heterologous expression system, activation of NmbR significantly enhanced the Cav3.2 channel currents, while the Cav3.1 and Cav3.3 channel currents remained unaffected. Nmb induced TG neuronal hyperexcitability and concomitantly induced mechanical and thermal hypersensitivity, both of which were attenuated by T-type channel blockade. Moreover, blockade of NmbR signalling prevented mechanical hypersensitivity in a mouse model of complete Freund's adjuvant-induced inflammatory pain, and this effect was attenuated by siRNA knockdown of Cav3.2. Conclusions: Our study reveals a novel mechanism by which NmbR stimulates Cav3.2 channels through a Gßγ-dependent AMPK/PKA pathway. In mouse models, this mechanism appears to drive the hyperexcitability of TG neurons and induce pain hypersensitivity.

The Transcription Factor Shox2 Shapes Neuron Firing Properties and Suppresses Seizures by Regulation of Key Ion Channels in Thalamocortical Neurons.

Thalamocortical neurons (TCNs) play a critical role in the maintenance of thalamocortical oscillations, dysregulation of which can result in certain types of seizures. Precise control over firing rates of TCNs is foundational to these oscillations, yet the transcriptional mechanisms that constrain these firing rates remain elusive. We hypothesized that Shox2 is a transcriptional regulator of ion channels important for TCN function and that loss of Shox2 alters firing frequency and activity, ultimately perturbing thalamocortical oscillations into an epilepsy-prone state. In this study, we used RNA sequencing and quantitative PCR of control and Shox2 knockout mice to determine Shox2-affected genes and revealed a network of ion channel genes important for neuronal firing properties. Protein regulation was confirmed by Western blotting, and electrophysiological recordings showed that Shox2 KO impacted the firing properties of a subpopulation of TCNs. Computational modeling showed that disruption of these conductances in a manner similar to Shox2's effects modulated frequency of oscillations and could convert sleep spindles to near spike and wave activity, which are a hallmark for absence epilepsy. Finally, Shox2 KO mice were more susceptible to pilocarpine-induced seizures. Overall, these results reveal Shox2 as a transcription factor important for TCN function in adult mouse thalamus.

Systemic administration of α-lipoic acid suppresses excitability of nociceptive wide-dynamic range neurons in rat spinal trigeminal nucleus caudalis.

Although a modulatory role has been reported for α-lipoic acid (LA) on T-type Ca2+ channels in the nervous system, the acute effects of LA in vivo, particularly on nociceptive transmission in the trigeminal system, remain to be determined. The aim of the present study was to investigate whether acute intravenous LA administration to rats attenuates the excitability of wide dynamic range (WDR) spinal trigeminal nucleus caudalis (SpVc) neurons in response to nociceptive and non-nociceptive mechanical stimulation in vivo. Extracellular single unit recordings were made from seventeen SpVc neurons in response to orofacial mechanical stimulation of pentobarbital-anesthetized rats. Responses to both non-noxious and noxious mechanical stimuli were analyzed in the present study. The mean firing frequency of SpVc WDR neurons in response to both non-noxious and noxious mechanical stimuli was significantly and dose-dependently inhibited by LA (1-100 mM, i.v.) and maximum inhibition of the discharge frequency of both non-noxious and noxious mechanical stimuli was seen within 5 min. These inhibitory effects lasted for approximately 10 min. These results suggest that acute intravenous LA administration suppresses trigeminal sensory transmission, including nociception, via possibly blocking T-type Ca2+ channels. LA may be used as a therapeutic agent for the treatment of trigeminal nociceptive pain.

Polaprezinc attenuates cyclophosphamide-induced cystitis and related bladder pain in mice.

Cav3.2 T-type Ca(2+) channels targeted by H2S, a gasotransmitter, participate in cyclophosphamide-induced cystitis and bladder pain. Given that zinc selectively inhibits Cav3.2 among T-channel isoforms and also exhibits antioxidant activity, we examined whether polaprezinc (zinc-l-carnosine), a medicine for peptic ulcer treatment and zinc supplementation, reveals preventive or therapeutic effects on bladder inflammation and/or pain in the mouse with cyclophosphamide-induced cystitis, a model for interstitial cystitis. Systemic administration of cyclophosphamide caused cystitis-related symptoms including increased bladder weight and vascular permeability, and histological signs of bladder edema, accompanied by bladder pain-like nociceptive behavior/referred hyperalgesia. All these symptoms were significantly attenuated by oral preadministration of polaprezinc at 400 mg/kg. The same dose of polaprezinc also prevented the increased malondialdehyde level, an indicator of lipid peroxidation, and protein upregulation of cystathionine-γ-lyase, an H2S-generating enzyme, but not occludin, a tight junction-related membrane protein, in the bladder tissue of cyclophosphamide-treated mice. Oral posttreatment with polaprezinc at 30-100 mg/kg reversed the nociceptive behavior/referred hyperalgesia in a dose-dependent manner without affecting the increased bladder weight. Together, our data show that zinc supplementation with polaprezinc prevents the cyclophosphamide-induced cystitis probably through the antioxidant activity, and, like T-channel blockers, reverses the established cystitis-related bladder pain in mice, suggesting novel therapeutic usefulness of polaprezinc.