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.

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.

Hyperexcitability of rat thalamocortical networks after exposure to general anesthesia during brain development.

Prevailing literature supports the idea that common general anesthetics (GAs) cause long-term cognitive changes and neurodegeneration in the developing mammalian brain, especially in the thalamus. However, the possible role of GAs in modifying ion channels that control neuronal excitability has not been taken into consideration. Here we show that rats exposed to GAs at postnatal day 7 display a lasting reduction in inhibitory synaptic transmission, an increase in excitatory synaptic transmission, and concomitant increase in the amplitude of T-type calcium currents (T-currents) in neurons of the nucleus reticularis thalami (nRT). Collectively, this plasticity of ionic currents leads to increased action potential firing in vitro and increased strength of pharmacologically induced spike and wave discharges in vivo. Selective blockade of T-currents reversed neuronal hyperexcitability in vitro and in vivo. We conclude that drugs that regulate thalamic excitability may improve the safety of GAs used during early brain development.

Inhibition of T-type calcium current in rat thalamocortical neurons by isoflurane.

Thalamocortical (TC) neurons provide the major sensory input to the mammalian somatosensory cortex. Decreased activity of these cells may be pivotal in the ability of general anesthetics to induce loss of consciousness and promote sleep (hypnosis). T-type voltage-gated calcium currents (T-currents) have a key function regulating the cellular excitability of TC neurons and previous studies have indicated that volatile general anesthetics may alter the excitability of these neurons. Using a patch-clamp technique, we investigated the mechanisms whereby isoflurane, a common volatile anesthetic, modulates isolated T-currents and T-current-dependent excitability of native TC neurons in acute brain slices of the rat. In voltage-clamp experiments, we found that isoflurane strongly inhibited peak amplitude of T-current, yielding an IC(50) of 1.1 vol-% at physiological membrane potentials. Ensuing biophysical studies demonstrated that inhibition was more prominent at depolarized membrane potentials as evidenced by hyperpolarizing shifts in channel availability curves. In current-clamp experiments we found that isoflurane decreased the rate of depolarization of low-threshold-calcium spikes (LTCSs) and consequently increased the latency of rebound spike firing at the same concentrations that inhibited isolated T-currents. This effect was mimicked by a novel selective T-channel blocker 3,5-dichloro-N-[1-(2,2-dimethyl-tetrahydro-pyran-4-ylmethyl)-4-fluoro-piperidin-4-ylmethyl]-benzamide (TTA-P2). In contrast, isoflurane and TTA-P2 had minimal effect on resting membrane potential and cell input resistance. We propose that the clinical properties of isoflurane may at least partly be provided by depression of thalamic T-currents.

T-type voltage-gated calcium channels as targets for the development of novel pain therapies.

It is well recognized that voltage-gated calcium (Ca(2+)) channels modulate the function of peripheral and central pain pathways by influencing fast synaptic transmission and neuronal excitability. In the past, attention focused on the modulation of different subtypes of high-voltage-activated-type Ca(2+) channels; more recently, the function of low-voltage-activated or transient (T)-type Ca(2+) channels (T-channels) in nociception has been well documented. Currently, available pain therapies remain insufficient for certain forms of pain associated with chronic disorders (e.g. neuropathic pain) and often have serious side effects. Hence, the identification of selective and potent inhibitors and modulators of neuronal T-channels may help greatly in the development of safer, more effective pain therapies. Here, we summarize the available information implicating peripheral and central T-channels in nociception. We also discuss possible future developments aimed at selective modulation of function of these channels, which are highly expressed in nociceptors.

Mechanisms of inhibition of CaV3.1 T-type calcium current by aliphatic alcohols.

Many aliphatic alcohols modulate activity of various ion channels involved in sensory processing and also exhibit anesthetic capacity in vivo. Although the interaction of one such compound, 1-octanol (octanol) with different T-type calcium channels (T-channels) has been described, the mechanisms of current modulation and its functional significance are not well studied. Using patch-clamp technique, we investigated the mechanisms of inhibition of T-currents by a series of aliphatic alcohols in recombinant human Ca(V)3.1 (alpha1G) T-channel isoform expressed in human embryonic kidney (HEK) 293 cells and thalamocortical (TC) relay neurons in brain slices of young rats. Octanol, 1-heptanol (heptanol) and 1-hexanol (hexanol) inhibited the recombinant Ca(V)3.1 currents in concentration-dependent manner yielding IC(50) values of 362 microM, 1063 microM and 3167 microM, respectively. Octanol similarly inhibited native thalamic Ca(V)3.1 T-currents with an IC(50) of 287 microM and diminished burst firing without significant effect on passive membrane properties of these neurons. Inhibitory effect of octanol on T-currents in both native and recombinant cells was accompanied with accelerated macroscopic inactivation kinetics and hyperpolarizing shift in the steady-state inactivation curve. Additionally, octanol induced a depolarizing shift in steady-state activation curves of T-current in TC neurons. Surprisingly, the recovery from fast inactivation at hyperpolarized membrane potentials was accelerated by octanol up 3-fold in native but not recombinant channels. Given the importance of thalamocortical pathways in providing sleep, arousal, and anesthetic states, modulation of thalamic T-currents may at least contribute to the pharmacological effects of aliphatic alcohols.

Mechanisms of inhibition of T-type calcium current in the reticular thalamic neurons by 1-octanol: implication of the protein kinase C pathway.

Recent studies indicate that T-type calcium channels (T-channels) in the thalamus are cellular targets for general anesthetics. Here, we recorded T-currents and underlying low-threshold calcium spikes from neurons of nucleus reticularis thalami (nRT) in brain slices from young rats and investigated the mechanisms of their modulation by an anesthetic alcohol, 1-octanol. We found that 1-octanol inhibited native T-currents at subanesthetic concentrations with an IC(50) of approximately 4 muM. In contrast, 1-octanol was up to 30-fold less potent in inhibiting recombinant Ca(V)3.3 T-channels heterologously expressed in human embryonic kidney cells. Inhibition of both native and recombinant T-currents was accompanied by a hyperpolarizing shift in steady-state inactivation, indicating that 1-octanol stabilized inactive states of the channel. To explore the mechanisms underlying higher 1-octanol potency in inhibiting native nRT T-currents, we tested the effect of the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) and PKC inhibitors. We found that PMA caused a modest increase of T-current, whereas the inactive PMA analog 4alpha-PMA failed to affect T-current in nRT neurons. In contrast, 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole (Go 6976), an inhibitor of calcium-dependent PKC, decreased baseline T-current amplitude in nRT cells and abolished the effects of subsequently applied 1-octanol. The effects of 1-octanol were also abolished by chelation of intracellular calcium ions with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Taken together, these results suggest that inhibition of calcium-dependent PKC signaling is a possible molecular substrate for modulation of T-channels in nRT neurons by 1-octanol.

Are neuroactive steroids promising therapeutic agents in the management of acute and chronic pain?

Neuroactive steroids with potentiating effects on GABA(A) channels and inhibitory effects on T-type Ca2+ channels which are located in peripheral sensory neurons are potent modulators of pain perception. The focus of this review is on peripheral anti-nociceptive properties of 5alpha- and 5beta-reduced neuroactive steroids with either selective or combined modulatory action on GABA(A) and T-type Ca2+ channel-mediated neurotransmission. We report that these neuroactive steroids are very effective in alleviating peripheral nociception in both acute and chronic pain conditions in animal models of pain. We believe that promising animal data warrant the exploration of their usefulness in clinical settings especially considering the fact that chronic pain sufferers are often young and otherwise healthy people.

Isoflurane-sensitive presynaptic R-type calcium channels contribute to inhibitory synaptic transmission in the rat thalamus.

Because inhibitory synaptic transmission is a major mechanism of general anesthesia, we examined the effects of isoflurane on properties of GABAergic inhibitory currents in the reticular thalamic nucleus (nRT) in brain slices. The evoked IPSCs (eIPSCs) and spontaneous miniature synaptic currents (mIPSCs) of visualized nRT cells in young and adult rats were recorded. Consistent with postsynaptic effects on GABA(A) receptors, isoflurane prolonged the decay-time constants of both eIPSCs and mIPCSs. Surprisingly, isoflurane completely inhibited the amplitude of eIPSCs at clinically relevant concentrations (IC(50) of 240+/-20 microm), increased the paired-pulse ratio, and decreased the frequency of mIPSCs, indicating that presynaptic mechanisms may also contribute to the effects of isoflurane on IPSCs. The overall effect of isoflurane on eIPSCs in nRT cells was a decrease of net charge-transfer across the postsynaptic membrane. The application of 100 microm nickel (Ni(2+)) and the more specific R-type Ca(2+) channel blocker SNX-482 (0.5 microm) decreased eIPSC amplitudes, increased the paired-pulse ratio, and attenuated isoflurane-induced inhibition of eIPSCs. In addition, isoflurane potently blocked currents in recombinant human Ca(V)2.3 (alpha1E) channels with an IC(50) of 206 +/- 22 mum. Importantly, in vivo electroencephalographic (EEG) recordings in adult Ca(V)2.3 knock-out mice demonstrated alterations in isoflurane-induced burst-suppression activity. Because the thalamus has a key function in processing sensory information, sleep, and cognition, modulation of its GABAergic tone by presynaptic R-type Ca(2+) channels may contribute to the clinical effects of general anesthesia.

Functional regulation of T-type calcium channels by s-nitrosothiols in the rat thalamus.

Although T-type Ca(2+) channels in the reticular thalamic nucleus (nRT) have a central function in tuning neuronal excitability and are implicated in sensory processing, sleep, and epilepsy, the mechanisms involved in their regulation are poorly understood. Here we recorded T-type Ca(2+) currents from intact nRT neurons in brain slices from young rats and investigated the mechanisms of T-type channel modulation by S-nitrosothiols (SNOs). We found that extracellular application of S-nitrosoglutathione (GSNO), S-nitrosocysteine (CSNO) and S-nitroso-N-acetyl-penicillamin (SNAP) rapidly and reversibly reduced T-type currents. The effects of SNOs are strongly stereoselective at physiological concentrations: (L)-CSNO was fourfold more effective in inhibiting T-type current than was (D)-CSNO. The effects of GSNO were abolished if cells had been treated with free hemoglobin or N-ethylmaleimide, an irreversible alkylating agent but not by 8-bromoguanosine-3',5'-cyclomonophosphate sodium salt, a membrane-permeant cGMP analogue or 1H-(1,2,4) oxadiazolo (4,3-a) quinoxalin-1-one, a specific soluble guanylyl cyclase inhibitor. In addition, bath applications of GSNO inhibited T-type currents in nucleated outside-out patches and whole cell recordings to a similar extent, with minimal effect on cell-attached recordings, suggesting a direct effect of GSNO on putative extracellular thiol residues on T-type channels. Biophysical studies indicate that GSNO decreased the availability of T-type channels at physiological potentials by modifying gating and stabilizing inactive states of the channels. In current-clamp experiments, GSNO diminished the amplitude of low-threshold calcium spikes and frequency of spike firing with minimal effects on the passive membrane properties. Collectively, the results indicate that SNOs may be a class of endogenous agents that control the functional states of the thalamus.

CaV3.2 is the major molecular substrate for redox regulation of T-type Ca2+ channels in the rat and mouse thalamus.

Although T-type Ca(2+) channels in the thalamus play a crucial role in determining neuronal excitability and are involved in sensory processing and pathophysiology of epilepsy, little is known about the molecular mechanisms involved in their regulation. Here, we report that reducing agents, including endogenous sulfur-containing amino acid l-cysteine, selectively enhance native T-type currents in reticular thalamic (nRT) neurons and recombinant Ca(V)3.2 (alpha1H) currents, but not native and recombinant Ca(V)3.1 (alpha1G)- and Ca(V)3.3 (alpha1I)-based currents. Consistent with this data, T-type currents of nRT neurons from transgenic mice lacking Ca(V)3.2 channel expression were not modulated by reducing agents. In contrast, oxidizing agents inhibited all native and recombinant T-type currents non-selectively. Thus, our findings directly demonstrate that Ca(V)3.2 channels are the main molecular substrate for redox regulation of neuronal T-type channels. In addition, because thalamic T-type channels generate low-threshold Ca(2+) spikes that directly correlate with burst firing in these neurons, differential redox regulation of these channels may have an important function in controlling cellular excitability in physiological and pathological conditions and fine-tuning of the flow of sensory information into the central nervous system.

Contrasting anesthetic sensitivities of T-type Ca2+ channels of reticular thalamic neurons and recombinant Ca(v)3.3 channels.

Reticular thalamocortical neurons express a slowly inactivating T-type Ca(2+) current that is quite similar to that recorded from recombinant Ca(v)3.3b (alpha1Ib) channels. These neurons also express abundant Ca(v)3.3 mRNA, suggesting that it underlies the native current. Here, we test this hypothesis by comparing the anesthetic sensitivities of recombinant Ca(v)3.3b channels stably expressed in HEK 293 cells to native T channels in reticular thalamic neurons (nRT) from brain slices of young rats. Barbiturates completely blocked both Ca(v)3.3 and nRT currents, with pentobarbital being about twice more potent in blocking Ca(v)3.3 currents. Isoflurane had about the same potency in blocking Ca(v)3.3 and nRT currents, but enflurane, etomidate, propofol, and ethanol exhibited 2-4 fold higher potency in blocking nRT vs Ca(v)3.3 currents. Nitrous oxide (N(2)O; laughing gas) blocked completely nRT currents with IC(50) of 20%, but did not significantly affect Ca(v)3.3 currents at four-fold higher concentrations. In addition, we observed that in lower concentration, N(2)O reversibly increased nRT but not Ca(v)3.3 currents. In conclusion, contrasting anesthetic sensitivities of Ca(v)3.3 and nRT T-type Ca(2+) channels strongly suggest that different molecular structures of Ca(2+) channels give rise to slowly inactivating T-type Ca(2+) currents. Furthermore, effects of volatile anesthetics and ethanol on slowly inactivating T-type Ca(2+) channel variants may contribute to the clinical effects of these agents.