Electrophysiological characterization of a CaV3.1 calcium channel mutation linked to trigeminal neuralgia.

Trigeminal neuralgia is a rare and debilitating disorder that affects one or more branches of the trigeminal nerve, leading to severe pain attacks and a poor quality of life. It has been reported that the CaV3.1 T-type calcium channel may play an important role in trigeminal pain and a recent study identified a new missense mutation in the CACNA1G gene that encodes the pore forming α1 subunit of the CaV3.1 calcium channel. The mutation leads to a substitution of an Arginine (R) by a Glutamine (Q) at position 706 in the I-II linker region of the channel. Here, we used whole-cell voltage-clamp recordings to evaluate the biophysical properties of CaV3.1 wild-type and R706Q mutant channels expressed in tsA-201 cells. Our data indicate an increase in current density in the R706Q mutant, leading to a gain-of-function effect, without changes in the voltage for half activation. Moreover, voltage clamp using an action potential waveform protocol revealed an increase in the tail current at the repolarization phase in the R706Q mutant. No changes were observed in the voltage-dependence of inactivation. However, the R706Q mutant displayed a faster recovery from inactivation. Hence, the gain-of-function effects in the R706Q CaV3.1 mutant have the propensity to impact pain transmission in the trigeminal system, consistent with a contribution to trigeminal neuralgia pathophysiology.

Regulation of CaV3.2 channels by the receptor for activated C kinase 1 (Rack-1).

This study describes the interaction between CaV3.2 calcium channels and the receptor for activated C kinase 1 (Rack-1), a scaffold protein which has recently been implicated in neuropathic pain. The coexpression of CaV3.2 and Rack-1 in tsA-201 cells led to a reduction in the magnitude of whole-cell CaV3.2 currents and CaV3.2 channel expression at the plasma membrane. Co-immunoprecipitations from transfected cells show the formation of a molecular protein complex between Cav3.2 channels and Rack-1. We determined that the interaction of Rack-1 occurs at the intracellular II-III loop and the C-terminus of the channel. Finally, the coexpression of PKCßII abolished the effect of Rack-1 on current densities. Altogether, our findings show that Rack-1 regulates CaV3.2-mediated calcium entry in a PKC-dependent manner.

Discovery of Michael acceptor containing 1,4-dihydropyridines as first covalent inhibitors of L-/T-type calcium channels.

1,4-Dihydropyridines (DHPs) are an important class of blockers targeting different calcium channel subtypes and have great therapeutic value against cardiovascular and neurophysiologic conditions. Here, we present the design of DHP-based hexahydroquinoline derivatives as either selective or covalent inhibitors of calcium channels. These compounds were synthesized via a modified Hantzsch reaction under microwave irradiation and characterized by IR, 1H NMR, 13C NMR and mass spectra. Additionally, the proposed structure of HM12 was resolved by single crystal X-ray analysis. The abilities of the target compounds to block both L- and T-type calcium channels were evaluated by utilizing the whole-cell patch clamp technique. Our results identified covalent inhibitors of calcium channels for the first time, which could be achieved by introducing a Michael acceptor group into the ester side chain of the compounds. The proposed covalent binding between the compounds and the cysteine amino acid (Cys1492) within the DHP binding pocket of L-type calcium channel was supported by docking and pharmacophore analysis as well as a glutathione reactivity assay.

A serotonergic deficit in the dorsal periaqueductal gray matter may underpin enhanced panic-like behavior in diabetic rats.

It is known that diabetic (DBT) animals present dysregulation on the serotonergic system in several brain areas associated with anxiety-like responses. The aim of this study was to investigate the involvement of 5-HT1A receptors on dorsal periaqueductal gray (dPAG) in the behavioral response related to panic disorder in type-1 DBT animals. For this, the escape response by electric stimulation (ES) of dPAG in DBT and normoglycemic (NGL) animals was assessed. Both NGL and DBT animals were exposed to an open-field test (OFT) 28 days after DBT confirmation. The current threshold to induce escape behavior in DBT animals was reduced compared with NGL animals. No impairment in locomotor activity was observed when DBT animals were compared with NGL animals. An intra-dPAG injection of the 5-HT1A receptor agonist (±)-8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) increased the [INCREMENT] threshold in both DBT and NGL, suggesting a panicolytic-like effect. DBT animals presented a more pronounced panicolytic-like response compared with NGL as a higher [INCREMENT] threshold was observed after 8-OH-DPAT treatment, which could be a consequence of the increased expression of the 5-HT1A receptor in the dPAG from DBT animals. Our results are in line with the proposal that a deficiency in serotonergic modulation of the dPAG is involved in triggering the panic attack and the 5-HT1A receptors might be essential for the panicolytic-like response.

Reestablishment of the hyperglycemia to the normal levels seems not to be essential to the anxiolytic-like effect induced by insulin.

Diabetes is a chronic metabolic disease accompanied by several comorbidities, including neuropsychiatric conditions. Since the hyperglycemia appears to be the primary factor involved in diabetic conditions, we examined the effect of insulin treatment in diabetic rats on behavioral responses related to anxiety and aversive memory extinction. For this, normoglycemic (NGL) or streptozotocin-diabetic (DBT) rats were submitted to the elevated T maze (ETM) and the contextual conditioned fear (CCF) tests. Therefore, animals were subjected to the prolonged treatment with insulin (6 IU/day, s.c.) to investigate the effect of the treatment on distinct behaviors. When anxiety-like responses such as the inhibitory avoidance (IA) on the ETM and the time of freezing in the first session of the CCF test were evaluated, our data showed a more pronounced anxiogenic-like behavior in DBT animals when compared to NGL ones. In addition, an increased freezing time was observed in DBT animals exposed to the CCF test (sessions 2 and 3) when compared to the NGL group, suggestive of an impairment in the extinction of aversive memory. Insulin treatment induced an anxiolytic-like effect when IA and freezing time (session 1) was evaluated, but did not alter the impaired extinction of aversive memory (sessions 2 and 3). To better understand the involvement of a rigorous control of glycaemia, we also investigated the effect of a lower dose of insulin (3 IU/day, s.c.), unable to reestablish the hyperglycemia to the normal levels, on the same behavioral parameters. Our data show that independent of the dose of insulin, the same effects were observed when animals were evaluated in the ETM and CCF tests. However, only the highest dose of insulin was able to reduce the hyperglycemia to the normal levels. To conclude, our data suggest that a severe glycemic control by insulin treatment seems to be important, but not essential in improving diabetes-induced anxiety.

Voltage-gated Calcium Channels as Potential Therapeutic Targets in Migraine.

Migraine is a complex and highly incapacitating neurological disorder that affects around 15% of the general population with greater incidence in women, often at the most productive age of life. Migraine physiopathology is still not fully understood, but it involves multiple mediators and events in the trigeminovascular system and the central nervous system. The identification of calcitonin gene-related peptide as a key mediator in migraine physiopathology has led to the development of effective and highly selective antimigraine therapies. However, this treatment is neither accessible nor effective for all migraine sufferers. Thus, a better understanding of migraine mechanisms and the identification of potential targets are still clearly warranted. Voltage-gated calcium channels (VGCCs) are widely distributed in the trigeminovascular system, and there is accumulating evidence of their contribution to the mechanisms associated with headache pain. Several drugs used in migraine abortive or prophylactic treatment target VGCCs, which probably contributes to their analgesic effect. This review aims to summarize the current evidence of VGGC contribution to migraine physiopathology and to discuss how current pharmacological options for migraine treatment interfere with VGGC function. PERSPECTIVE: Calcitonin gene-related peptide (CGRP) represents a major migraine mediator, but few studies have investigated the relationship between CGRP and VGCCs. CGRP release is calcium channel-dependent and VGGCs are key players in familial migraine. Further studies are needed to determine whether VGCCs are suitable molecular targets for treating migraine.

Pronociceptive role of spinal Cav2.3 (R-type) calcium channels in a mouse model of postoperative pain.

BACKGROUND: More than 80% of patients may experience acute pain after a surgical procedure, and this is often refractory to pharmacological intervention. The identification of new targets to treat postoperative pain is necessary. There is an association of polymorphisms in the Cav2.3 gene with postoperative pain and opioid consumption. Our study aimed to identify Cav2.3 as a potential target to treat postoperative pain and to reduce opioid-related side effects. EXPERIMENTAL APPROACH: A plantar incision model was established in adult male and female C57BL/6 mice. Cav2.3 expression was detected by qPCR and suppressed by siRNA treatment. The antinociceptive efficacy and safety of a Cav2.3 blocker-alone or together with morphine-was also assessed after surgery. KEY RESULTS: Paw incision in female and male mice caused acute nociception and increased Cav2.3 mRNA expression in the spinal cord but not in the incised tissue. Intrathecal treatment with siRNA against Cav2.3, but not with a scrambled siRNA, prevented the development of surgery-induced nociception in both male and female mice, with female mice experiencing long-lasting effects. High doses of i.t. SNX-482, a Cav2.3 channel blocker, or morphine injected alone, reversed postoperative nociception but also induced side effects. A combination of lower doses of morphine and SNX-482 mediated a long-lasting reversal of postsurgical pain in female and male mice. CONCLUSION: Our results demonstrate that Cav2.3 has a pronociceptive role in the induction of postoperative pain, indicating that it is a potential target for the development of therapeutic approaches for the treatment of postoperative pain.

Pannexin-1 channel inhibition alleviates opioid withdrawal in rodents by modulating locus coeruleus to spinal cord circuitry.

Opioid withdrawal is a liability of chronic opioid use and misuse, impacting people who use prescription or illicit opioids. Hyperactive autonomic output underlies many of the aversive withdrawal symptoms that make it difficult to discontinue chronic opioid use. The locus coeruleus (LC) is an important autonomic centre within the brain with a poorly defined role in opioid withdrawal. We show here that pannexin-1 (Panx1) channels expressed on microglia critically modulate LC activity during opioid withdrawal. Within the LC, we found that spinally projecting tyrosine hydroxylase (TH)-positive neurons (LCspinal) are hyperexcitable during morphine withdrawal, elevating cerebrospinal fluid (CSF) levels of norepinephrine. Pharmacological and chemogenetic silencing of LCspinal neurons or genetic ablation of Panx1 in microglia blunted CSF NE release, reduced LC neuron hyperexcitability, and concomitantly decreased opioid withdrawal behaviours in mice. Using probenecid as an initial lead compound, we designed a compound (EG-2184) with greater potency in blocking Panx1. Treatment with EG-2184 significantly reduced both the physical signs and conditioned place aversion caused by opioid withdrawal in mice, as well as suppressed cue-induced reinstatement of opioid seeking in rats. Together, these findings demonstrate that microglial Panx1 channels modulate LC noradrenergic circuitry during opioid withdrawal and reinstatement. Blocking Panx1 to dampen LC hyperexcitability may therefore provide a therapeutic strategy for alleviating the physical and aversive components of opioid withdrawal.

Inhibitory insula-ACC projections modulate affective but not sensory aspects of neuropathic pain.

The insula and anterior cingulate cortex (ACC) are brain regions that undergo structural and functional reorganization in neuropathic pain states. Here, we aimed to study inhibitory parvalbumin positive (PV+) posterior insula (pIC) to posterior ACC (pACC) projections, and to evaluate the effects of direct optogenetic manipulation of such projections on mechanical nociception and spontaneous ongoing pain in mice with Spared Nerve Injury (SNI). CTB488 tract-tracing in male PVCrexAi9 mice revealed a small proportion of PV+ projections from the pIC to the pACC. Electrophysiological analysis confirmed the existence of synaptic inputs into the pACC by pIC GABAergic cells. Optogenetic stimulation of these pathways did not change mechanical nociception, but induced conditioned place preference behavior responses. Our results suggest the presence of inhibitory projections between the pIC and the pACC which are able to selectively modulate affective aspects of neuropathic pain.

Ca V 3.2 calcium channels contribute to trigeminal neuralgia.

ABSTRACT: Trigeminal neuralgia (TN) is a rare but debilitating disorder characterized by excruciating facial pain, with a higher incidence in women. Recent studies demonstrated that TN patients present mutations in the gene encoding the Ca V 3.2 T-type calcium channel, an important player in peripheral pain pathways. We characterize the role of Ca V 3.2 channels in TN at 2 levels. First, we examined the biophysical properties of CACNA1H variants found in TN patients. Second, we investigated the role of Ca V 3.2 in an animal model of trigeminal neuropathic pain. Whole-cell patch-clamp recordings from 4 different mutants expressed in tsA-201 cells (E286K in the pore loop of domain I, H526Y, G563R, and P566T in the domain I-II linker) identified a loss of function in activation in the E286K mutation and gain of function in the G563R and P566T mutations. Moreover, a loss of function in inactivation was observed with the E286K and H526Y mutations. Cell surface biotinylation revealed no difference in channel trafficking among the variants. The G563R mutant also caused a gain of function in the firing properties of transfected trigeminal ganglion neurons. In female and male mice, constriction of the infraorbital nerve induced facial thermal heat hyperalgesia. Block of T-type channels with Z944 resulted in antihyperalgesia. The effect of Z944 was absent in Ca V 3.2 -/- mice, indicating that Ca V 3.2 is the molecular target of the antihyperalgesic Z944 effect. Finally, enzyme-linked immunosorbent assay analysis revealed increased Ca V 3.2 channel expression in the spinal trigeminal subnucleus caudalis. Altogether, the present study demonstrates an important role of Ca V 3.2 channels in trigeminal pain.

Electrophysiological and computational analysis of Cav3.2 channel variants associated with familial trigeminal neuralgia.

Trigeminal neuralgia (TN) is a rare form of chronic neuropathic pain characterized by spontaneous or elicited paroxysms of electric shock-like or stabbing pain in a region of the face. While most cases occur in a sporadic manner and are accompanied by intracranial vascular compression of the trigeminal nerve root, alteration of ion channels has emerged as a potential exacerbating factor. Recently, whole exome sequencing analysis of familial TN patients identified 19 rare variants in the gene CACNA1H encoding for Cav3.2T-type calcium channels. An initial analysis of 4 of these variants pointed to a pathogenic role. In this study, we assessed the electrophysiological properties of 13 additional TN-associated Cav3.2 variants expressed in tsA-201 cells. Our data indicate that 6 out of the 13 variants analyzed display alteration of their gating properties as evidenced by a hyperpolarizing shift of their voltage dependence of activation and/or inactivation resulting in an enhanced window current supported by Cav3.2 channels. An additional variant enhanced the recovery from inactivation. Simulation of neuronal electrical membrane potential using a computational model of reticular thalamic neuron suggests that TN-associated Cav3.2 variants could enhance neuronal excitability. Altogether, the present study adds to the notion that ion channel polymorphisms could contribute to the etiology of some cases of TN and further support a role for Cav3.2 channels.

A Synthetically Accessible Small-Molecule Inhibitor of USP5-Cav3.2 Calcium Channel Interactions with Analgesic Properties.

Cav3.2 calcium channels are important mediators of nociceptive signaling in the primary afferent pain pathway, and their expression is increased in various rodent models of chronic pain. Previous work from our laboratory has shown that this is in part mediated by an aberrant expression of deubiquitinase USP5, which associates with these channels and increases their stability. Here, we report on a novel bioactive rhodanine compound (II-1), which was identified in compound library screens. II-1 inhibits biochemical interactions between USP5 and the Cav3.2 domain III-IV linker in a dose-dependent manner, without affecting the enzymatic activity of USP5. Molecular docking analysis reveals two potential binding pockets at the USP5-Cav3.2 interface that are distinct from the binding site of the deubiquitinase inhibitor WP1130 (a.k.a. degrasyn). With an understanding of the ability of some rhodanines to produce false positives in high-throughput screening, we have conducted several orthogonal assays to confirm the validity of this hit, including in vivo experiments. Intrathecal delivery of II-1 inhibited both phases of formalin-induced nocifensive behaviors in mice, as well as abolished thermal hyperalgesia induced by the delivery of complete Freund's adjuvant (CFA) to the hind paw. The latter effects were abolished in Cav3.2 null mice, thus confirming that Cav3.2 is required for the action of II-1. II-1 also mediated a robust inhibition of mechanical allodynia induced by injury to the sciatic nerve. Altogether, our data uncover a novel class of analgesics─well suited to rapid structure-activity relationship studies─that target the Cav3.2/USP5 interface.

An orbitofrontal cortex to midbrain projection modulates hypersensitivity after peripheral nerve injury.

Neuropathic pain is a debilitating condition that is often refractory to treatment. The network of neural substrates for pain transmission and control within the brain is complex and remains poorly understood. Through a combination of neuronal tracing, optogenetics, chemogenetics, electrophysiological recordings, and behavioral assessment, we demonstrate that activation of layer 5 pyramidal neurons in the ventrolateral orbitofrontal cortex (vlOFC) attenuates mechanical and thermal hypersensitivity and cold allodynia in mice with neuropathic pain induced by spared nerve injury (SNI). These vlOFC output neurons project to the posterior ventrolateral periaqueductal gray (vlPAG) region and receive inputs from the ventromedial thalamus (VM). Specific optogenetic and chemogenetic activation of the vlOFC-vlPAG and the VM-vlOFC circuits inhibits hypersensitivity associated with neuropathy. Thus, we reveal a modulatory role of the vlOFC and its projections to the vlPAG circuit in the processing of hypersensitive nociception.

A CACNA1A variant associated with trigeminal neuralgia alters the gating of Cav2.1 channels.

A novel missense mutation in the CACNA1A gene that encodes the pore forming α1 subunit of the CaV2.1 voltage-gated calcium channel was identified in a patient with trigeminal neuralgia. This mutation leads to a substitution of proline 2455 by histidine (P2455H) in the distal C-terminus region of the channel. Due to the well characterized role of this channel in neurotransmitter release, our aim was to characterize the biophysical properties of the P2455H variant in heterologously expressed CaV2.1 channels. Whole-cell patch clamp recordings of wild type and mutant CaV2.1 channels expressed in tsA-201 cells reveal that the mutation mediates a depolarizing shift in the voltage-dependence of activation and inactivation. Moreover, the P2455H mutant strongly reduced calcium-dependent inactivation of the channel that is consistent with an overall gain of function. Hence, the P2455H CaV2.1 missense mutation alters the gating properties of the channel, suggesting that associated changes in CaV2.1-dependent synaptic communication in the trigeminal system may contribute to the development of trigeminal neuralgia.

Dopamine Inputs from the Ventral Tegmental Area into the Medial Prefrontal Cortex Modulate Neuropathic Pain-Associated Behaviors in Mice.

The medial prefrontal cortex (mPFC) is a brain region involved in the affective components of pain and undergoes plasticity during the development of chronic pain. Dopamine (DA) is a key neuromodulator in the mesocortical circuit and modulates working memory and aversion. Although DA inputs into the mPFC are known to modulate plasticity, whether and how these inputs affect pain remains incompletely understood. By using optogenetics, we find that phasic activation of DA inputs from the ventral tegmental area (VTA) into the mPFC reduce mechanical hypersensitivity during neuropathic pain states. Mice with neuropathic pain exhibit a preference for contexts paired with photostimulation of DA terminals in the mPFC. Fiber photometry-based calcium imaging reveals that DA increases the activity of mPFC neurons projecting to the ventrolateral periaqueductal gray (vlPAG). Together, our findings indicate an important role of mPFC DA signaling in pain modulation.

Posttraumatic stress disorder-type behaviors in streptozotocin-induced diabetic rats can be prevented by prolonged treatment with vitamin E.

Anxiety and stress disorders, such as posttraumatic stress disorder (PTSD) have been described as debilitating comorbidities of diabetes. In the present study, we aimed to investigate anxiety-like behavior and the extinction and generalization of aversive memories in fear conditioning using a streptozotocin-induced model of diabetes (DBT). Moreover, considering that DBT animals present increased oxidative stress in brain areas related to anxiety and memory, we aimed to evaluate the effect of prolonged treatment with antioxidant vitamin E on behavioral parameters of anxiety and fear memory and on the diabetic condition. It was observed that DBT animals showed a deficiency in extinguishing the aversive memory in a fear conditioning test, along with a generalization of the fear memory. They also present a more pronounced anxiety-like behavior in the elevated plus maze test. VIT E treatment (300 mg/kg, p.o.) was not able to reduce hyperglycemia; however, it was able to block the anxiogenic-like behavior, also improving the deficit in the extinction of the aversive memory as well as blocking the generalization of such memory in a different context. Taken together, our data suggest that DBT animals are prone to extinction deficits and generalization of fear memories, behaviors which are observed in models of PTSD. Lastly, prolonged VIT E supplementation may be effective in the treatment of anxiety, extinction deficit and generalization of fear memories induced by the diabetic condition.

Comparison of antinociceptive effects of plain lidocaine versus lidocaine complexed with hydroxypropyl-ß-cyclodextrin in animal models of acute and persistent orofacial pain.

Herein, it was investigated whether a complex of lidocaine with 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD) would present a better antinociceptive profile in vivo when compared with plain lidocaine in models of orofacial pain. Plain lidocaine (LDC) and complexed lidocaine (LDC:HP-ß-CD) were initially evaluated in vitro to determine the release rate of the two formulations. Subsequently, the effect of both formulations was evaluated in independent groups of rats submitted to the orofacial formalin test, induction of facial heat hyperalgesia by capsaicin and carrageenan, and induction of facial heat and mechanical hyperalgesia by constriction of the infraorbital nerve. LDC:HP-ß-CD led to a reduction in the lidocaine release assessed in the in vitro release assay compared to plain LDC. Both formulations presented an antinociceptive effect in all models, but LDC:HP-ß-CD showed a better effect in the second phase of the formalin response, in carrageenan-induced heat hyperalgesia, and in the heat hyperalgesia associated to infraorbital nerve constriction. Our results show that complexation improved in vivo antinociceptive effects of LDC, but further studies are necessary to elucidate what properties contribute to the better effect of the complexed formulation on this models and/or what characteristics of the pain model facilitate the action of the complexed formulation.

Blockade of endothelin receptors reduces tumor-induced ongoing pain and evoked hypersensitivity in a rat model of facial carcinoma induced pain.

Pain reported by patients with head and neck cancer is characterized as persistent pain with mechanical allodynia. Pain management is inadequate for many patients, highlighting the need for improved therapies. We examined the hypothesis that the mixed endothelin ETA and ETB receptor antagonist, bosentan, reduces tumor-induced ongoing pain and evoked hypersensitivity in a rat model of facial cancer pain. Facial cancer was induced by inoculating a suspension of Walker-256 cells into the rat's right vibrissal pad. Tumor-bearing rats developed heat and tactile hypersensitivity along with increased spontaneous grooming behavior. Systemic morphine (2.5mg/kg, s.c.) blocked tumor-induced thermal and tactile hypersensitivity, with a lower dose (0.625mg/kg, s.c.) effective only against thermal hypersensitivity. Systemic bosentan blocked tumor-induced thermal hypersensitivity only at a high (300mg/kg, p.o.) dose, but failed to modify tactile hypersensitivity. Co-administration of the low doses of bosentan and morphine resulted in improved reduction of the tumor-induced heat and tactile hypersensitivity compared to either dose alone. Bosentan (100mg/kg, p.o.) reduced spontaneous grooming and induced conditioned place preference (CPP) selectively in tumor-bearing rats, suggesting that bosentan reduces tumor-induced ongoing pain at a lower dose than required to block tumor-induced hypersensitivity. This study provides evidence that endothelins may mediate tumor-induced ongoing pain and thermal hypersensitivity. In addition, bosentan enhanced morphine's effects on blocking tumor-induced heat and tactile hypersensitivity indicating that endothelin antagonists may be beneficial therapeutic targets that can be used to manage cancer-induced facial pain with opioid-sparing effects.