Alpha lipoic acid attenuates evoked and spontaneous pain following surgical skin incision in rats.

Our previous studies have implicated CaV3.2 isoform of T-type Ca2+ channels (T-channels) in the development of postsurgical pain. We have also previously established that different T-channel antagonists can alleviate in vivo postsurgical pain. Here we investigated the analgesic potential of another T-channel blocker and endogenous antioxidant molecule, α-lipoic acid (ALA), in a postsurgical pain model in rats. Our in vivo results suggest that single and repetitive intraperitoneal injections of ALA after surgery or preemptively, significantly reduced evoked mechanical hyperalgesia following surgical paw incision. Furthermore, repeated preemptive systemic injections of ALA effectively alleviated spontaneous postsurgical pain as determined by dynamic weight-bearing testing. We expect that our preclinical study may lead to further investigation of analgesic properties and mechanisms of analgesic action of ALA in patients undergoing surgery.

Glycosylation of CaV3.2 Channels Contributes to the Hyperalgesia in Peripheral Neuropathy of Type 1 Diabetes.

Our previous studies implicated glycosylation of the CaV3.2 isoform of T-type Ca2+ channels (T-channels) in the development of Type 2 painful peripheral diabetic neuropathy (PDN). Here we investigated biophysical mechanisms underlying the modulation of recombinant CaV3.2 channel by de-glycosylation enzymes such as neuraminidase (NEU) and PNGase-F (PNG), as well as their behavioral and biochemical effects in painful PDN Type 1. In our in vitro study we used whole-cell recordings of current-voltage relationships to confirm that CaV3.2 current densities were decreased ~2-fold after de-glycosylation. Furthermore, de-glycosylation induced a significant depolarizing shift in the steady-state relationships for activation and inactivation while producing little effects on the kinetics of current deactivation and recovery from inactivation. PDN was induced in vivo by injections of streptozotocin (STZ) in adult female C57Bl/6j wild type (WT) mice, adult female Sprague Dawley rats and CaV3.2 knock-out (KO mice). Either NEU or vehicle (saline) were locally injected into the right hind paws or intrathecally. We found that injections of NEU, but not vehicle, completely reversed thermal and mechanical hyperalgesia in diabetic WT rats and mice. In contrast, NEU did not alter baseline thermal and mechanical sensitivity in the CaV3.2 KO mice which also failed to develop painful PDN. Finally, we used biochemical methods with gel-shift analysis to directly demonstrate that N-terminal fragments of native CaV3.2 channels in the dorsal root ganglia (DRG) are glycosylated in both healthy and diabetic animals. Our results demonstrate that in sensory neurons glycosylation-induced alterations in CaV3.2 channels in vivo directly enhance diabetic hyperalgesia, and that glycosylation inhibitors can be used to ameliorate painful symptoms in Type 1 diabetes. We expect that our studies may lead to a better understanding of the molecular mechanisms underlying painful PDN in an effort to facilitate the discovery of novel treatments for this intractable disease.

Novel neuroactive steroid with hypnotic and T-type calcium channel blocking properties exerts effective analgesia in a rodent model of post-surgical pain.

BACKGROUND AND PURPOSE: Neuroactive steroid (3ß,5ß,17ß)-3-hydroxyandrostane-17-carbonitrile (3ß-OH) is a novel hypnotic and voltage-dependent blocker of T-type calcium channels. Here, we examine its potential analgesic effects and adjuvant anaesthetic properties using a post-surgical pain model in rodents. EXPERIMENTAL APPROACH: Analgesic properties of 3ß-OH were investigated in thermal and mechanical nociceptive tests in sham or surgically incised rats and mice, with drug injected either systemically (intraperitoneal) or locally via intrathecal or intraplantar routes. Hypnotic properties of 3ß-OH and its use as an adjuvant anaesthetic in combination with isoflurane were investigated using behavioural experiments and in vivo EEG recordings in adolescent rats. KEY RESULTS: A combination of 1% isoflurane with 3ß-OH (60 mg·kg-1 , i.p.) induced suppression of cortical EEG and stronger thermal and mechanical anti-hyperalgesia during 3 days post-surgery, when compared to isoflurane alone and isoflurane with morphine. 3ß-OH exerted prominent enantioselective thermal and mechanical antinociception in healthy rats and reduced T-channel-dependent excitability of primary sensory neurons. Intrathecal injection of 3ß-OH alleviated mechanical hyperalgesia, while repeated intraplantar application alleviated both thermal and mechanical hyperalgesia in the rats after incision. Using mouse genetics, we found that CaV 3.2 T-calcium channels are important for anti-hyperalgesic effect of 3ß-OH and are contributing to its hypnotic effect. CONCLUSION AND IMPLICATIONS: Our study identifies 3ß-OH as a novel analgesic for surgical procedures. 3ß-OH can be used to reduce T-channel-dependent excitability of peripheral sensory neurons as an adjuvant for induction and maintenance of general anaesthesia while improving analgesia and lowering the amount of volatile anaesthetic needed for surgery.

Novel neurosteroid hypnotic blocks T-type calcium channel-dependent rebound burst firing and suppresses long-term potentiation in the rat subiculum.

BACKGROUND: Hypnotics and general anaesthetics impair memory by altering hippocampal synaptic plasticity. We recently reported on a neurosteroid analogue with potent hypnotic activity [(3ß,5ß,17ß)-3-hydroxyandrostane-17-carbonitrile; 3ß-OH], which does not cause developmental neurotoxicity in rat pups. Here, we investigated the effects of 3ß-OH on neuronal excitability in the subiculum, the major output structure of the hippocampal formation, and synaptic plasticity at two key hippocampal synapses in juvenile rats. METHODS: Biophysical properties of isolated T-type calcium currents (T-currents) in the rat subiculum were investigated using acute slice preparations. Subicular T-type calcium channel (T-channel) subtype mRNA expression was compared using qRT-PCR. Using electrophysiological recordings, we examined the effects of 3ß-OH and an endogenous neuroactive steroid, allopregnanolone (Allo), on T-currents and burst firing properties of subicular neurones, and on the long-term potentiation (LTP) in CA3-CA1 and CA1-subiculum pathways. RESULTS: Biophysical and molecular studies confirmed that CaV3.1 channels represent the dominant T-channel isoform in the subiculum of juvenile rats. 3ß-OH and Allo inhibited rebound burst firing by decreasing the amplitude of T-currents in a voltage-dependent manner with similar potency, with 30-80% inhibition. Both neurosteroids suppressed LTP at the CA1-subiculum, but not at the CA3-CA1 Schaffer collateral synapse. CONCLUSIONS: Neurosteroid effects on T-channels modulate hippocampal output and provide possible molecular mechanisms for the amnestic action of the novel hypnotic 3ß-OH. Effects on T-channels in the subiculum provide a novel target for amnestic effects of hypnotics.

Inhibition of multiple voltage-gated calcium channels may contribute to spinally mediated analgesia by epipregnanolone in a rat model of surgical paw incision.

Voltage-activated calcium channels play an important role in excitability of sensory nociceptive neurons in acute and chronic pain models. We have previously shown that low-voltage-activated calcium channels, or T-type channels (T-channels), increase excitability of sensory neurons after surgical incision in rats. We have also found that endogenous 5ß-reduced neuroactive steroid epipregnanolone [(3ß,5ß)-3-hydroxypregnan-20-one] blocked isolated T-currents in dorsal root ganglion (DRG) cells in vitro, and reduced nociceptive behavior in vivo, after local intraplantar application into the foot pads of heathy rats and mice. Here, we investigated if epipregnanolone exerts an antinociceptive effect after intrathecal (i.t.) application in healthy rats, as well as an antihyperalgesic effect in a postsurgical pain model. We also studied if this endogenous neurosteroid blocks currents originating from high voltage-activated (HVA) calcium channels in rat sensory neurons. In in vivo studies, we found that epipregnanolone alleviated thermal and mechanical nociception in healthy rats after i.t. administration without affecting their sensory-motor abilities. Furthermore, epipregnanolone effectively reduced mechanical hyperalgesia after i.t application in rats after surgery. In subsequent in vitro studies, we found that epipregnanolone blocked isolated HVA currents in nociceptive sensory neurons with an IC50 of 3.3 µM in a G-protein-dependent fashion. We conclude that neurosteroids that have combined inhibitory effects on T-type and HVA calcium currents may be suitable for development of novel pain therapies during the perioperative period.

The role of T-type calcium channels in the subiculum: to burst or not to burst?

KEY POINTS: Pharmacological, molecular and genetic data indicate a prominent role of low-voltage-activated T-type calcium channels (T-channels) in the firing activity of both pyramidal and inhibitory interneurons in the subiculum. Pharmacological inhibition of T-channels switched burst firing with lower depolarizing stimuli to regular spiking, and fully abolished hyperpolarization-induced burst firing. Our molecular studies showed that CaV 3.1 is the most abundantly expressed isoform of T-channels in the rat subiculum. Consistent with this finding, both regular-spiking and burst firing patterns were profoundly depressed in the mouse with global deletion of CaV 3.1 isoform of T-channels. Selective inhibition of T-channels and global deletion of CaV 3.1 channels completely suppressed development of long-term potentiation (LTP) in the CA1-subiculum, but not in the CA3-CA1 pathway. ABSTRACT: Several studies suggest that voltage-gated calcium currents are involved in generating high frequency burst firing in the subiculum, but the exact nature of these currents remains unknown. Here, we used selective pharmacology, molecular and genetic approaches to implicate Cav3.1-containing T-channels in subicular burst firing, in contrast to several previous reports discounting T-channels as major contributors to subicular neuron physiology. Furthermore, pharmacological antagonism of T-channels, as well as global deletion of CaV3.1 isoform, completely suppressed development of long-term potentiation (LTP) in the CA1-subiculum, but not in the CA3-CA1 pathway. Our results indicate that excitability and synaptic plasticity of subicular neurons relies heavily on T-channels. Hence, T-channels may be a promising new drug target for different cognitive deficits.