ß-Caryophyllene Inhibits Monoacylglycerol Lipase Activity and Increases 2-Arachidonoyl Glycerol Levels In Vivo: A New Mechanism of Endocannabinoid-Mediated Analgesia?

The mechanisms of ß-caryophyllene (BCP)-induced analgesia are not well studied. Here, we tested the efficacy of BCP in an acute postsurgical pain model and evaluated its effect on the endocannabinoid system. Rats were treated with vehicle and 10, 25, 50, and 75 mg/kg BCP. Paw withdrawal responses to mechanical stimuli were evaluated using an electronic von Frey anesthesiometer. Endocannabinoids, including 2-arachidonoylglycerol (2-AG), were also evaluated in plasma and tissues using high-performance liquid chromatography-tandem mass spectrometry. Monoacylglycerol lipase (MAGL) activity was evaluated in vitro as well as ex vivo. We observed a dose-dependent and time-dependent alleviation of hyperalgesia in incised paws up to 85% of the baseline value at 30 minutes after administration of BCP. We also observed dose-dependent increases in the 2-AG levels of about threefold after administration of BCP as compared with vehicle controls. Incubations of spinal cord tissue homogenates from BCP-treated rats with isotope-labeled 2-arachidonoylglycerol-d8 revealed a reduced formation of the isotope-labeled MAGL product 2-AG-d8 as compared with vehicle controls, indicating MAGL enzyme inhibition. In vitro MAGL enzyme activity assessment using 2-AG as the substrate revealed an IC50 of 15.8 µM for MAGL inhibition using BCP. These data showed that BCP inhibits MAGL activity in vitro and in vivo, causing 2-AG levels to rise. Since the endocannabinoid 2-AG is a CB1 and CB2 receptor agonist, we propose that 2-AG-mediated cannabinoid receptor activation contributes to BCP's mechanism of analgesia. SIGNIFICANCE STATEMENT: ß-Caryophyllene (BCP) consumption is relatively safe and is approved by the Food and Drug Administration as a flavoring agent, which can be used in cosmetic and food additives. BCP is a potent anti-inflammatory agent that showed substantial antihyperalgesic properties in this study of acute pain suggesting that BCP might be an alternative to opioids. This study shows an additive mechanism (monoacylglycerol lipase inhibition) by which BCP might indirectly alter CB1 and CB2 receptor activity and exhibit its pharmacological properties.

The Role of Neuroactive Steroids in Analgesia and Anesthesia: An Interesting Comeback?

Published evidence over the past few decades suggests that general anesthetics could be neurotoxins especially when administered at the extremes of age. The reported pathology is not only at the morphological level when examined in very young and aged brains, given that, importantly, newly developing evidence suggests a variety of behavioral impairments. Since anesthesia is unavoidable in certain clinical settings, we should consider the development of new anesthetics. A promising and safe solution could be a new family of anesthetics referred to as neuroactive steroids. In this review, we summarize the currently available evidence regarding their anesthetic and analgesic properties.

Distinct excitability of thalamocortical neurons correlates with the presence of cerebellar afferents.

Thalamocortical (TC) neurons within the ventrolateral thalamus (VL) receive projections from the cerebellum and the basal ganglia (BG) to facilitate motor and non-motor functions. Tonic and rebound firing patterns in response to excitatory cerebellar and inhibitory BG inputs, respectively, are a canonical feature of TC neurons and plays a key role in signal processing. The intrinsic excitability of TC neurons has a strong influence on how they respond to synaptic inputs, however, it is unknown whether their afferents influence their firing properties. Understanding the input-specific firing patterns could shed light into movement disorders with cerebellar or BG involvement. Here, we used whole-cell electrophysiology in brain slices from C57BL/6 mice to investigate the firing of TC neurons with optogenetic confirmation of cerebellar or BG afferents. TC neurons with cerebellar afferents exhibited higher tonic and rebound firing rates than those with BG afferents. This increased firing was associated with faster action potential depolarization kinetics and a smaller afterhyperpolarization potential. We also found differences in the passive membrane properties and sag currents during hyperpolarization. Despite higher rebound firing in TC neurons with cerebellar afferents, there were no differences in T-type calcium channel function compared to those with BG inputs. These data suggest input-specific differences in sodium and SK, but not T-type calcium channels, impact firing properties in TC populations. Altogether, we showed that the pronounced divergence observed in TC neuron firing properties correlate with its heterogeneous anatomical connectivity, which could signify a distinct signal integration and processing by these neurons. Keypoints: Thalamocortical neurons in the VL with cerebellar afferents have higher intrinsic tonic and rebound firing properties than those with basal ganglia afferents.Membrane resistance and action potential depolarization slope were different based on the presence of cerebellar afferents.Despite elevated rebound burst firing, T-type mediated currents did not correlate with increased firing in neurons with cerebellar afferents.

Neonatal exposure to a neuroactive steroid alters low-frequency oscillations in the subiculum.

Preclinical studies have established that neonatal exposure to contemporary sedative/hypnotic drugs causes neurotoxicity in the developing rodent and primate brains. Our group recently reported that novel neuroactive steroid (3ß,5ß,17ß)-3-hydroxyandrostane-17-carbonitrile (3ß-OH) induced effective hypnosis in both neonatal and adult rodents but did not cause significant neurotoxicity in vulnerable brain regions such as subiculum, an output region of hippocampal formation particularly sensitive to commonly used sedatives/hypnotics. Despite significant emphasis on patho-morphological changes, little is known about long-term effects on subicular neurophysiology after neonatal exposure to neuroactive steroids. Hence, we explored the lasting effects of neonatal exposure to 3ß-OH on sleep macrostructure as well as subicular neuronal oscillations in vivo and synaptic plasticity ex vivo in adolescent rats. At postnatal day 7, we exposed rat pups to either 10 mg/kg of 3ß-OH over a period of 12 h or to volume-matched cyclodextrin vehicle. At weaning age, a cohort of rats was implanted with a cortical electroencephalogram (EEG) and subicular depth electrodes. At postnatal day 30-33, we performed in vivo assessment of sleep macrostructure (divided into wake, non-rapid eye movement, and rapid eye movement sleep) and power spectra in cortex and subiculum. In a second cohort of 3ß-OH exposed animals, we conducted ex vivo studies of long-term potentiation (LTP) in adolescent rats. Overall, we found that neonatal exposure to 3ß-OH decreased subicular delta and sigma oscillations during non-rapid eye movement sleep without altering sleep macrostructure. Furthermore, we observed no significant changes in subicular synaptic plasticity. Interestingly, our previous study found that neonatal exposure to ketamine increased subicular gamma oscillations during non-rapid eye movement sleep and profoundly suppressed subicular LTP in adolescent rats. Together these results suggest that exposure to different sedative/hypnotic agents during a critical period of brain development may induce distinct functional changes in subiculum circuitry that may persist into adolescent age.

Sex-specific hypnotic effects of the neuroactive steroid (3ß,5ß,17ß)-3-hydroxyandrostane-17-carbonitrile are mediated by peripheral metabolism into an active hypnotic steroid.

BACKGROUND: The novel synthetic neuroactive steroid (3ß,5ß,17ß)-3-hydroxyandrostane-17-carbonitrile (3ß-OH) blocks T-type calcium channels but does not directly modulate neuronal γ-aminobutyric acid type A (GABAA) currents like other anaesthetic neurosteroids. As 3ß-OH has sex-specific hypnotic effects in adult rats, we studied the mechanism contributing to sex differences in its effects. METHODS: We used a combination of behavioural loss of righting reflex, neuroendocrine, pharmacokinetic, in vitro patch-clamp electrophysiology, and in vivo electrophysiological approaches in wild-type mice and in genetic knockouts of the CaV3.1 T-type calcium channel isoform to study the mechanisms by which 3ß-OH and its metabolite produces sex-specific hypnotic effects. RESULTS: Adult male mice were less sensitive to the hypnotic effects of 3ß-OH compared with female mice, and these differences appeared during development. Adult males had higher 3ß-OH brain concentrations despite being less sensitive to its hypnotic effects. Females metabolised 3ß-OH into the active GABAA receptor positive allosteric modulator (3α,5ß,17ß)-3-hydroxyandrostane-17-carbonitrile (3α-OH) to a greater extent than males. The 3α-OH metabolite has T-channel blocking properties with sex-specific hypnotic and pharmacokinetic effects. Sex-dependent suppression of the cortical electroencephalogram is more pronounced with 3α-OH compared with 3ß-OH. CONCLUSIONS: The sex-specific differences in the hypnotic effect of 3ß-OH in mice are attributable to differences in its peripheral metabolism into the more potent hypnotic metabolite 3α-OH.

CaV3.1 T-type calcium channels are important for spatial memory processing in the dorsal subiculum.

The dorsal subiculum (dSub) is one of the key structures responsible for the formation of hippocampal memory traces but the contribution of individual ionic currents to its cognitive function is not well studied. Although we recently reported that low-voltage-activated T-type calcium channels (T-channels) are crucial for the burst firing pattern regulation in the dSub pyramidal neurons, their potential role in learning and memory remains unclear. Here we used in vivo local field potential recordings and miniscope calcium imaging in freely behaving mice coupled with pharmacological and genetic tools to address this gap in knowledge. We show that the CaV3.1 isoform of T-channels is critically involved in controlling neuronal activity in the dSub in vivo. Altering neuronal excitability by inhibiting T-channel activity markedly affects calcium dynamics, synaptic plasticity, neuronal oscillations and phase-amplitude coupling in the dSub, thereby disrupting spatial learning. These results provide an important causative link between the CaV3.1 channels, burst firing of dSub neurons and memory formation, thus further supporting the notion that changes in neuronal excitability regulate memory processing. We posit that subicular CaV3.1 T-channels could be a promising novel drug target for cognitive disorders.

Opioid-induced hyperalgesia: Are thalamic T-type calcium channels treatment targets?

Opioid-induced hyperalgesia (OIH) is a state of paradoxically enhanced pain transmission, termed nociceptive sensitization, described to occur in both humans and animals after repeated administration of opioid drugs, including rapidly acting remifentanil. However, molecular mechanisms of OIH remain understudied. In this issue of the JCI, Yan Jin and colleagues provided strong evidence that hyperexcitable thalamocortical networks drive remifentanil-induced hyperalgesia in a rodent model of postsurgical pain. Furthermore, the authors specifically identified an important role of the CaV3.1 isoform of low-voltage-activated or T-type calcium channels (T-channels) in this process. Further experiments are needed to determine whether thalamic T channels could serve as targets for the treatment of OIH.

Neonatal ketamine exposure impairs infrapyramidal bundle pruning and causes lasting increase in excitatory synaptic transmission in hippocampal CA3 neurons.

Preclinical models demonstrate that nearly all anesthetics cause widespread neuroapoptosis in the developing brains of infant rodents and non-human primates. Anesthesia-induced developmental apoptosis is succeeded by prolonged neuropathology in the surviving neurons and lasting cognitive impairments, suggesting that anesthetics interfere with the normal developmental trajectory of the brain. However, little is known about effects of anesthetics on stereotyped axonal pruning, an important developmental algorithm that sculpts neural circuits for proper function. Here, we proposed that neonatal ketamine exposure may interfere with stereotyped axonal pruning of the infrapyramidal bundle (IPB) of the hippocampal mossy fiber system and that impaired pruning may be associated with alterations in the synaptic transmission of CA3 neurons. To test this hypothesis, we injected postnatal day 7 (PND7) mouse pups with ketamine or vehicle over 6 h and then studied them at different developmental stages corresponding to IPB pruning (PND20-40). Immunohistochemistry with synaptoporin (a marker of mossy fibers) revealed that in juvenile mice treated with ketamine at PND7, but not in vehicle-treated controls, positive IPB fibers extended farther into the stratum pyramidale of CA3 region. Furthermore, immunofluorescent double labeling for synaptoporin and PSD-95 strongly suggested that the unpruned IPB caused by neonatal ketamine exposure makes functional synapses. Importantly, patch-clamp electrophysiology for miniature excitatory postsynaptic currents (mEPSCs) in acute brain slices ex vivo revealed increased frequency and amplitudes of mEPSCs in hippocampal CA3 neurons in ketamine-treated groups when compared to vehicle controls. We conclude that neonatal ketamine exposure interferes with normal neural circuit development and that this interference leads to lasting increase in excitatory synaptic transmission in hippocampus.

The role of voltage-gated calcium channels in the mechanisms of anesthesia and perioperative analgesia.

PURPOSE OF REVIEW: A family of neuronal voltage-gated calcium channels (VGCCs) have received only recently a significant consideration regarding the mechanisms of anesthesia because VGCC inhibition may be important in anesthetic action by decreasing neuronal excitability and presynaptic excitatory transmission. The T-type VGCCs channels (T-channels), although rarely involved in synaptic neurotransmitter release, play an important role in controlling neuronal excitability and in generating spontaneous oscillatory bursting of groups of neurons in the thalamus thought to be involved in regulating the state of arousal and sleep. Furthermore, these channels are important regulators of neuronal excitability in pain pathway. This review will provide an overview of historic perspective and the recent literature on the role of VGCCs and T-channel inhibition in particular in the mechanisms of action of anesthetics and analgesics. RECENT FINDINGS: Recent research in the field of novel mechanisms of hypnotic action of anesthetics revealed significant contribution of the Ca V 3.1 isoform of T-channels expressed in the thalamus. Furthermore, perioperative analgesia can be achieved by targeting Ca V 3.2 isoform of these channels that is abundantly expressed in pain pathways. SUMMARY: The review summarizes current knowledge regarding the contribution of T-channels in hypnosis and analgesia. Further preclinical and clinical studies are needed to validate their potential for developing novel anesthetics and new perioperative pain therapies.

Further Evidence that Inhibition of Neuronal Voltage-Gated Calcium Channels Contributes to the Hypnotic Effect of Neurosteroid Analogue, 3ß-OH.

We recently reported that a neurosteroid analogue with T-channel-blocking properties (3ß,5ß,17ß)-3-hydroxyandrostane-17-carbonitrile (3ß-OH), induced hypnosis in rat pups without triggering neuronal apoptosis. Furthermore, we found that the inhibition of the CaV3.1 isoform of T-channels contributes to the hypnotic properties of 3ß-OH in adult mice. However, the specific mechanisms underlying the role of other subtypes of voltage-gated calcium channels in thalamocortical excitability and oscillations in vivo during 3ß-OH-induced hypnosis are largely unknown. Here, we used patch-clamp recordings from acute brain slices, in vivo electroencephalogram (EEG) recordings, and mouse genetics with wild-type (WT) and CaV2.3 knock-out (KO) mice to further investigate the molecular mechanisms of neurosteroid-induced hypnosis. Our voltage-clamp recordings showed that 3ß-OH inhibited recombinant CaV2.3 currents. In subsequent current-clamp recordings in thalamic slices ex vivo, we found that selective CaV2.3 channel blocker (SNX-482) inhibited stimulated tonic firing and increased the threshold for rebound burst firing in WT animals. Additionally, in thalamic slices we found that 3ß-OH inhibited spike-firing more profoundly in WT than in mutant mice. Furthermore, 3ß-OH reduced bursting frequencies in WT but not mutant animals. In ensuing in vivo experiments, we found that intra-peritoneal injections of 3ß-OH were less effective in inducing LORR in the mutant mice than in the WT mice, with expected sex differences. Furthermore, the reduction in total α, ß, and low γ EEG power was more profound in WT than in CaV2.3 KO females over time, while at 60 min after injections of 3ß-OH, the increase in relative ß power was higher in mutant females. In addition, 3ß-OH depressed EEG power more strongly in the male WT than in the mutant mice and significantly increased the relative δ power oscillations in WT male mice in comparison to the mutant male animals. Our results demonstrate for the first time the importance of the CaV2.3 subtype of voltage-gated calcium channels in thalamocortical excitability and the oscillations that underlie neurosteroid-induced hypnosis.

The Mechanisms of Plasticity of Nociceptive Ion Channels in Painful Diabetic Neuropathy.

Treating pain in patients suffering from small fiber neuropathies still represents a therapeutic challenge for health care providers and drug developers worldwide. Unfortunately, none of the currently available treatments can completely reverse symptoms of either gain or loss of peripheral nerve sensation. Therefore, there is a clear need for novel mechanism-based therapies for peripheral diabetic neuropathy (PDN) that would improve treatment of this serious condition. In this review, we summarize the current knowledge on the mechanisms and causes of peripheral sensory neurons damage in diabetes. In particular, we focused on the subsets of voltage-gated sodium channels, TRP family of ion channels and a CaV3.2 isoform of T-type voltage-gated calcium channels. However, even though their potential is well-validated in multiple rodent models of painful PDN, clinical trials with specific pharmacological blockers of these channels have failed to exhibit therapeutic efficacy. We argue that understanding the development of diabetes and causal relationship between hyperglycemia, glycosylation, and other post-translational modifications may lead to the development of novel therapeutics that would efficiently alleviate painful PDN by targeting disease-specific mechanisms rather than individual nociceptive ion channels.

L-cysteine modulates visceral nociception mediated by the CaV2.3 R-type calcium channels.

CaV2.3 channels are subthreshold voltage-gated calcium channels that play crucial roles in neurotransmitter release and regulation of membrane excitability, yet modulation of these channels with endogenous molecules and their role in pain processing is not well studied. Here, we hypothesized that an endogenous amino acid l-cysteine could be a modulator of these channels and may affect pain processing in mice. To test this hypothesis, we employed conventional patch-clamp technique in the whole-cell configuration using recombinant CaV2.3 subunit stably expressed in human embryonic kidney (HEK-293) cells. We found in our in vitro experiments that l-cysteine facilitated gating and increased the amplitudes of recombinant CaV2.3 currents likely by chelating trace metals that tonically inhibit the channel. In addition, we took advantage of mouse genetics in vivo using the acetic acid visceral pain model that was performed on wildtype and homozygous Cacna1e knockout male littermates. In ensuing in vivo experiments, we found that l-cysteine administered both subcutaneously and intraperitoneally evoked more prominent pain responses in the wildtype mice, while the effect was completely abolished in knockout mice. Conversely, intrathecal administration of l-cysteine lowered visceral pain response in the wildtype mice, and again the effect was completely abolished in the knockout mice. Our study strongly suggests that l-cysteine-mediated modulation of CaV2.3 channels plays an important role in visceral pain processing. Furthermore, our data are consistent with the contrasting roles of CaV2.3 channels in mediating visceral nociception in the peripheral and central pain pathways.

General Anesthesia and the Young Brain: The Importance of Novel Strategies with Alternate Mechanisms of Action.

Over the past three decades, we have been grappling with rapidly accumulating evidence that general anesthetics (GAs) may not be as innocuous for the young brain as we previously believed. The growing realization comes from hundreds of animal studies in numerous species, from nematodes to higher mammals. These studies argue that early exposure to commonly used GAs causes widespread apoptotic neurodegeneration in brain regions critical to cognition and socio-emotional development, kills a substantial number of neurons in the young brain, and, importantly, results in lasting disturbances in neuronal synaptic communication within the remaining neuronal networks. Notably, these outcomes are often associated with long-term impairments in multiple cognitive-affective domains. Not only do preclinical studies clearly demonstrate GA-induced neurotoxicity when the exposures occur in early life, but there is a growing body of clinical literature reporting similar cognitive-affective abnormalities in young children who require GAs. The need to consider alternative GAs led us to focus on synthetic neuroactive steroid analogues that have emerged as effective hypnotics, and analgesics that are apparently devoid of neurotoxic effects and long-term cognitive impairments. This would suggest that certain steroid analogues with different cellular targets and mechanisms of action may be safe alternatives to currently used GAs. Herein we summarize our current knowledge of neuroactive steroids as promising novel GAs.

Thalamic T-Type Calcium Channels as Targets for Hypnotics and General Anesthetics.

General anesthetics mainly act by modulating synaptic inhibition on the one hand (the potentiation of GABA transmission) or synaptic excitation on the other (the inhibition of NMDA receptors), but they can also have effects on numerous other proteins, receptors, and channels. The effects of general anesthetics on ion channels have been the subject of research since the publication of reports of direct actions of these drugs on ion channel proteins. In particular, there is considerable interest in T-type voltage-gated calcium channels that are abundantly expressed in the thalamus, where they control patterns of cellular excitability and thalamocortical oscillations during awake and sleep states. Here, we summarized and discussed our recent studies focused on the CaV3.1 isoform of T-channels in the nonspecific thalamus (intralaminar and midline nuclei), which acts as a key hub through which natural sleep and general anesthesia are initiated. We used mouse genetics and in vivo and ex vivo electrophysiology to study the role of thalamic T-channels in hypnosis induced by a standard general anesthetic, isoflurane, as well as novel neuroactive steroids. From the results of this study, we conclude that CaV3.1 channels contribute to thalamocortical oscillations during anesthetic-induced hypnosis, particularly the slow-frequency range of δ oscillations (0.5-4 Hz), by generating "window current" that contributes to the resting membrane potential. We posit that the role of the thalamic CaV3.1 isoform of T-channels in the effects of various classes of general anesthetics warrants consideration.

Synthetic neuroactive steroids as new sedatives and anaesthetics: Back to the future.

Since the 1990s, there has been waning interest in researching general anaesthetics (anaesthetics). Although currently used anaesthetics are mostly safe and effective, they are not without fault. In paediatric populations and neonatal animal models, they are associated with learning impairments and neurotoxicity. In an effort to research safer anaesthetics, we have gone back to re-examine neuroactive steroids as anaesthetics. Neuroactive steroids are steroids that have direct, local effects in the central nervous system. Since the discovery of their anaesthetic effects, neuroactive steroids have been consistently used in human or veterinary clinics as preferred anaesthetic agents. Although briefly abandoned for clinical use due to unwanted vehicle side effects, there has since been renewed interest in their therapeutic value. Neuroactive steroids are safe sedative/hypnotic and anaesthetic agents across various animal species. Importantly, unlike traditional anaesthetics, they do not cause extensive neurotoxicity in the developing rodent brain. Similar to traditional anaesthetics, neuroactive steroids are modulators of synaptic and extrasynaptic γ-aminobutyric acid type A (GABAA ) receptors and their interactions at the GABAA receptor are stereo- and enantioselective. Recent work has also shown that these agents act on other ion channels, such as high- and low-voltage-activated calcium channels. Through these mechanisms of action, neuroactive steroids modulate neuronal excitability, which results in characteristic burst suppression of the electroencephalogram, and a surgical plane of anaesthesia. However, in addition to their interactions with voltage and ligand gated ions channels, neuroactive steroids interact with membrane bound metabotropic receptors and xenobiotic receptors to facilitate signaling of prosurvival, antiapoptotic pathways. These pathways play a role in their neuroprotective effects in neuronal injury and may also prevent extensive apoptosis in the developing brain during anaesthesia. The current review explores the history of neuroactive steroids as anaesthetics in humans and animal models, their diverse mechanisms of action, and their neuroprotective properties.

Neonatal Isoflurane Does Not Affect Sleep Architecture and Minimally Alters Neuronal Beta Oscillations in Adolescent Rats.

General anesthetics are neurotoxic to the developing rodent and primate brains leading to neurocognitive and socio-affective impairment later in life. In addition, sleep patterns are important predictors of cognitive outcomes. Yet, little is known about how anesthetics affect sleep-wake behaviors and their corresponding oscillations. Here we examine how neonatal general anesthesia affects sleep and wake behavior and associated neuronal oscillations. We exposed male and female rat pups to either 6 h of continuous isoflurane or sham anesthesia (compressed air) at the peak of their brain development (postnatal day 7). One cohort of animals was used to examine neurotoxic insult 2 h post-anesthesia exposure. At weaning age, a second cohort of rats was implanted with cortical electroencephalogram electrodes and allowed to recover. During adolescence, we measured sleep architecture (divided into wake, non-rapid eye movement, and rapid eye movement sleep) and electroencephalogram power spectra over a 24 h period. We found that exposure to neonatal isoflurane caused extensive neurotoxicity but did not disrupt sleep architecture in adolescent rats. However, these animals had a small but significant reduction in beta oscillations, specifically in the 12-20 Hz beta 1 range, associated with wake behavior. Furthermore, beta oscillations play a critical role in cortical development, cognitive processing, and homeostatic sleep drive. We speculate that dysregulation of beta oscillations may be implicated in cognitive and socio-affective outcomes associated with neonatal anesthesia.

Painful diabetic neuropathy leads to functional CaV3.2 expression and spontaneous activity in skin nociceptors of mice.

Painful diabetic neuropathy occurs in approximately 20% of diabetic patients with underlying pathomechanisms not fully understood. We evaluated the contribution of the CaV3.2 isoform of T-type calcium channel to hyperglycemia-induced changes in cutaneous sensory C-fiber functions and neuropeptide release employing the streptozotocin (STZ) diabetes model in congenic mouse strains including global knockouts (KOs). Hyperglycemia established for 3-5 weeks in male C57BL/6J mice led to major reorganizations in peripheral C-fiber functions. Unbiased electrophysiological screening of mechanosensitive single-fibers in isolated hairy hindpaw skin revealed a relative loss of (polymodal) heat sensing in favor of cold sensing. In healthy CaV3.2 KO mice both heat and cold sensitivity among the C-fibers seemed underrepresented in favor of exclusive mechanosensitivity, low-threshold in particular, which deficit became significant in the diabetic KOs. Diabetes also led to a marked increase in the incidence of spontaneous discharge activity among the C-fibers of wildtype mice, which was reduced by the specific CaV3.2 blocker TTA-P2 and largely absent in the KOs. Evaluation restricted to the peptidergic class of nerve fibers - measuring KCl-stimulated CGRP release - revealed a marked reduction in the sciatic nerve by TTA-P2 in healthy but not diabetic wildtypes, the latter showing CGRP release that was as much reduced as in healthy and, to the same extent, in diabetic CaV3.2 KOs. These data suggest that diabetes abrogates all CaV3.2 functionality in the peripheral nerve axons. In striking contrast, diabetes markedly increased the KCl-stimulated CGRP release from isolated hairy skin of wildtypes but not KO mice, and TTA-P2 reversed this increase, strongly suggesting a de novo expression of CaV3.2 in peptidergic cutaneous nerve endings which may contribute to the enhanced spontaneous activity. De-glycosylation by neuraminidase showed clear desensitizing effects, both in regard to spontaneous activity and stimulated CGRP release, but included actions independent of CaV3.2. However, as diabetes-enhanced glycosylation is decisive for intra-axonal trafficking, it may account for the substantial reorganizations of the CaV3.2 distribution. The results may strengthen the validation of CaV3.2 channel as a therapeutic target of treating painful diabetic neuropathy.

Different roles of T-type calcium channel isoforms in hypnosis induced by an endogenous neurosteroid epipregnanolone.

BACKGROUND: Many neuroactive steroids induce sedation/hypnosis by potentiating γ-aminobutyric acid (GABAA) currents. However, we previously demonstrated that an endogenous neuroactive steroid epipregnanolone [(3ß,5ß)-3-hydroxypregnan-20-one] (EpiP) exerts potent peripheral analgesia and blocks T-type calcium currents while sparing GABAA currents in rat sensory neurons. This study seeks to investigate the behavioral effects elicited by systemic administration of EpiP and to characterize its use as an adjuvant agent to commonly used general anesthetics (GAs). METHODS: Here, we utilized electroencephalographic (EEG) recordings to characterize thalamocortical oscillations, as well as behavioral assessment and mouse genetics with wild-type (WT) and different knockout (KO) models of T-channel isoforms to investigate potential sedative/hypnotic and immobilizing properties of EpiP. RESULTS: Consistent with increased oscillations in slower EEG frequencies, EpiP induced an hypnotic state in WT mice when injected alone intra-peritoneally (i.p.) and effectively facilitated anesthetic effects of isoflurane (ISO) and sevoflurane (SEVO). The CaV3.1 (Cacna1g) KO mice demonstrated decreased sensitivity to EpiP-induced hypnosis when compared to WT mice, whereas no significant difference was noted between CaV3.2 (Cacna1h), CaV3.3 (Cacna1i) and WT mice. Finally, when compared to WT mice, onset of EpiP-induced hypnosis was delayed in CaV3.2 KO mice but not in CaV3.1 and CaV3.3 KO mice. CONCLUSION: We posit that EpiP may have an important role as novel hypnotic and/or adjuvant to volatile anesthetic agents. We speculate that distinct hypnotic effects of EpiP across all three T-channel isoforms is due to their differential expression in thalamocortical circuitry.

Differential effects of the novel neurosteroid hypnotic (3ß,5ß,17ß)-3-hydroxyandrostane-17-carbonitrile on electroencephalogram activity in male and female rats.

BACKGROUND: We recently showed that a neurosteroid analogue, (3ß,5ß,17ß)-3-hydroxyandrostane-17-carbonitrile (3ß-OH), induced hypnosis in rats. The aim of the present study was to evaluate the hypnotic and anaesthetic potential of 3ß-OH further using electroencephalography. METHODS: We used behavioural assessment and cortical electroencephalogram (EEG) spectral power analysis to examine hypnotic and anaesthetic effects of 3ß-OH (30 and 60 mg kg-1) administered intraperitoneally or intravenously to young adult male and female rats. RESULTS: We found dose-dependent sex differences in 3ß-OH-induced hypnosis and EEG changes. Both male and female rats responded similarly to i.p. 3ß-OH 30 mg kg-1. However, at the higher dose (60 mg kg-1, i.p.), female rats had two-fold longer duration of spontaneous immobility than male rats (203.4 [61.6] min vs 101.3 [32.1] min), and their EEG was suppressed in the low-frequency range (2-6 Hz), in contrast to male rats. Although a sex-dependent hypnotic effect was not confirmed after 30 mg kg-1 i.v., female rats appeared more sensitive to 3ß-OH with relatively small changes within delta (1-4 Hz) and alpha (8-13 Hz) bands. Finally, 3ß-OH had a rapid onset of action and potent hypnotic/anaesthetic effect after 60 mg kg-1 i.v. in rats of both sexes; however, all female rats and only half of the male rats reached burst suppression, an EEG pattern usually associated with profound inhibition of thalamocortical networks. CONCLUSIONS: Based on its behavioural effects and EEG signature, 3ß-OH is a potent hypnotic in rats, with female rats being more sensitive than male rats.

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.