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.

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.

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.

The T-type calcium channel isoform Cav3.1 is a target for the hypnotic effect of the anaesthetic neurosteroid (3ß,5ß,17ß)-3-hydroxyandrostane-17-carbonitrile.

BACKGROUND: The mechanisms underlying the role of T-type calcium channels (T-channels) in thalamocortical excitability and oscillations in vivo during neurosteroid-induced hypnosis are largely unknown. METHODS: We used patch-clamp electrophysiological recordings from acute brain slices ex vivo, recordings of local field potentials (LFPs) from the central medial thalamic nucleus in vivo, and wild-type (WT) and Cav3.1 knock-out mice to investigate the molecular mechanisms of hypnosis induced by the neurosteroid analogue (3ß,5ß,17ß)-3-hydroxyandrostane-17-carbonitrile (3ß-OH). RESULTS: Patch-clamp recordings showed that 3ß-OH inhibited isolated T-currents but had no effect on phasic or tonic γ-aminobutyric acid A currents. Also in acute brain slices, 3ß-OH inhibited the spike firing mode more profoundly in WT than in Cav3.1 knockout mice. Furthermore, 3ß-OH significantly hyperpolarised neurones, reduced the amplitudes of low threshold spikes, and diminished rebound burst firing only in WT mice. We found that 80 mg kg-1 i.p. injections of 3ß-OH induced hypnosis in >60% of WT mice but failed to induce hypnosis in the majority of mutant mice. A subhypnotic dose of 3ß-OH (20 mg kg-1 i.p.) accelerated induction of hypnosis by isoflurane only in WT mice, but had similar effects on the maintenance of isoflurane-induced hypnosis in both WT and Cav3.1 knockout mice. In vivo recordings of LFPs showed that a hypnotic dose of 3ß-OH increased δ, θ, α, and ß oscillations in WT mice in comparison with Cav3.1 knock-out mice. CONCLUSIONS: The Cav3.1 T-channel isoform is critical for diminished thalamocortical excitability and oscillations that underlie neurosteroid-induced hypnosis.

Global genetic deletion of CaV3.3 channels facilitates anaesthetic induction and enhances isoflurane-sparing effects of T-type calcium channel blockers.

We previously documented that the CaV3.3 isoform of T-type calcium channels (T-channels) is inhibited by clinically relevant concentrations of volatile anaesthetics, including isoflurane. However, little is understood about the functional role of CaV3.3 channels in anaesthetic-induced hypnosis and underlying neuronal oscillations. To address this issue, we used CaV3.3 knock-out (KO) mice and a panselective T-channel blocker 3,5-dichloro-N-[1-(2,2-dimethyltetrahydro-pyran-4-ylmethyl)-4-fluoro-piperidin-4-ylmethyl]-benzamide (TTA-P2). We found that mutant mice injected with the vehicle showed faster induction of hypnosis than wild-type (WT) mice, while the percent isoflurane at which hypnosis and immobility occurred was not different between two genotypes. Furthermore, we found that TTA-P2 facilitated isoflurane induction of hypnosis in the CaV3.3 KO mice more robustly than in the WT mice. Isoflurane-induced hypnosis following injections of TTA-P2 was accompanied with more prominent delta and theta EEG oscillations in the mutant mice, and reached burst-suppression pattern earlier when compared to the WT mice. Our findings point to a relatively specific value of CaV3.3 channels in anaesthetic induced hypnosis. Furthermore, we propose that T-channel blockers may be further explored as a valuable adjunct to reducing the usage of potent volatile anaesthetics, thereby improving their safety.

Neonatal general anesthesia causes lasting alterations in excitatory and inhibitory synaptic transmission in the ventrobasal thalamus of adolescent female rats.

Ample evidence has surfaced documenting the neurotoxic effects of various general anesthetic (GA) agents in the mammalian brain when administered at critical periods of synaptogenesis. However, little is known about how this neurotoxic insult affects persisting neuronal excitability after the initial exposure. Here we investigated synaptic activity and intrinsic excitability of the ventrobasal nucleus (VB) of the thalamus caused by neonatal GA administration. We used patch-clamp recordings from acute thalamic slices in young rats up to two weeks after neurotoxic GA exposure of isoflurane and nitrous oxide for 6 h at postnatal age of 7 (P7) days. We found that GA exposure at P7 increases evoked excitatory postsynaptic currents (eEPSCs) two fold by means through AMPA mediated mechanisms, while NMDA component was spared. In addition, miniature EPSCs showed a faster decay rate in neurons from GA treated animals when compared to sham controls. Likewise, we discovered that the amplitudes of evoked inhibitory postsynaptic currents (eIPSCs) were increased in VB neurons from GA animals about two-fold. Interestingly, these results were observed in female but not male rats. In contrast, intrinsic excitability and properties of T-type voltage gated calcium currents were minimally affected by GA exposure. Together, these data further the idea that GAs cause lasting alterations in synaptic transmission and neuronal excitability depending upon the placing and connectivity of neurons in the thalamus. Given that function of thalamocortical circuits critically depends on the delicate balance between excitation and inhibition, future development of therapies aimed at addressing consequences of altered excitability in the developing brain by GAs may be an attractive possibility.

Alterations in Oscillatory Behavior of Central Medial Thalamic Neurons Demonstrate a Key Role of CaV3.1 Isoform of T-Channels During Isoflurane-Induced Anesthesia.

Although the central medial nucleus (CeM) of the thalamus is an essential part of the arousal system for sleep and anesthesia initiation, the precise mechanisms that regulate its activity are not well studied. We examined the role of CaV3.1 isoform of T-type calcium channels (T-channels) in the excitability and rhythmic activity of CeM neurons during isoflurane (ISO)-induced anesthesia by using mouse genetics and selective pharmacology. Patch-clamp recordings taken from acute brain slices revealed that CaV3.1 channels in CeM are inhibited by prototypical volatile anesthetic ISO (250 and 500 µM) and selective T-channels blocker 3,5-dichloro-N-[1-(2,2-dimethyl-tetrahydro-pyran-4-ylmethyl)-4-fluoro-piperidin-4-ylmethyl]-benzamide (TTA-P2). Both TTA-P2 and ISO attenuated tonic and burst firing modes, and hyperpolarized CeM neurons from wild type (WT) mice. These effects were greatly diminished or abolished in CaV3.1 null mice. Our ensuing in vivo local field potential (LFP) recordings from CeM indicated that the ability of TTA-P2 and anesthetic concentrations of ISO to promote δ oscillation was substantially weakened in CaV3.1 null mice. Furthermore, escalating ISO concentrations induced stronger burst-suppression LFP pattern in mutant than in WT mice. Our results demonstrate for the first time the importance of CaV3.1 channels in thalamocortical oscillations from the non-specific thalamic nuclei that underlie clinically important effects of ISO.

Parenteral nanoemulsions as promising carriers for brain delivery of risperidone: Design, characterization and in vivo pharmacokinetic evaluation.

This paper describes design and evaluation of parenteral lecithin-based nanoemulsions intended for brain delivery of risperidone, a poorly water-soluble psychopharmacological drug. The nanoemulsions were prepared through cold/hot high pressure homogenization and characterized regarding droplet size, polydispersity, surface charge, morphology, drug-vehicle interactions, and physical stability. To estimate the simultaneous influence of nanoemulsion formulation and preparation parameters--co-emulsifier type, aqueous phase type, homogenization temperature--on the critical quality attributes of developed nanoemulsions, a general factorial experimental design was applied. From the established design space and stability data, promising risperidone-loaded nanoemulsions (mean size about 160 nm, size distribution <0.15, zeta potential around -50 mV), containing sodium oleate in the aqueous phase and polysorbate 80, poloxamer 188 or Solutol(®) HS15 as co-emulsifier, were produced by hot homogenization and their ability to improve risperidone delivery to the brain was assessed in rats. Pharmacokinetic study demonstrated erratic brain profiles of risperidone following intraperitoneal administration in selected nanoemulsions, most probably due to their different droplet surface properties (different composition of the stabilizing layer). Namely, polysorbate 80-costabilized nanoemulsion showed increased (1.4-7.4-fold higher) risperidone brain availability compared to other nanoemulsions and drug solution, suggesting this nanoemulsion as a promising carrier worth exploring further for brain targeting.

Negative modulation of α5 GABAA receptors in rats may partially prevent memory impairment induced by MK-801, but not amphetamine- or MK-801-elicited hyperlocomotion.

Reportedly, negative modulation of α5 GABAA receptors may improve cognition in normal and pharmacologically-impaired animals, and such modulation has been proposed as an avenue for treatment of cognitive symptoms in schizophrenia. This study assessed the actions of PWZ-029, administered at doses (2, 5, and 10 mg/kg) at which it reached micromolar concentrations in brain tissue with estimated free concentrations adequate for selective modulation of α5 GABAA receptors, in three cognitive tasks in male Wistar rats acutely treated with the noncompetitive N-methyl-d-aspartate receptor antagonist, MK-801 (0.1 mg/kg), as well in tests of locomotor activity potentiated by MK-801 (0.2 mg/kg) or amphetamine (0.5 mg/kg). In a hormetic-like manner, only 5 mg/kg PWZ-029 reversed MK-801-induced deficits in novel object recognition test (visual recognition memory), whereas in the Morris water maze, the 2 mg/kg dose of PWZ-029 exerted partial beneficial effects on spatial learning impairment. PWZ-029 did not affect recognition memory deficits in social novelty discrimination procedure. Motor hyperactivity induced with MK-801 or amphetamine was not preventable by PWZ-029. Our results show that certain MK-801-induced memory deficits can be ameliorated by negative modulation of α5 GABAA receptors, and point to the need for further elucidation of their translational relevance to cognitive deterioration in schizophrenia.