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 Ca
3.1 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 Ca
3.1 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 Ca
3.1 isoform of T-channels in the effects of various classes of general anesthetics warrants consideration.
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.
•Some ion channels that regulate VB neuronal excitability undergo lasting plasticity after neonatal general anesthesia (GA).•Evoked AMPA and GABA currents increased in female but not male young rats after a single isoflurane/nitrous oxide exposure.•T-type calcium channel and NMDA currents, as well as tonic/rebound burst firing activity were minimally affected by GA.•AMPA receptors may be a target for therapies aimed at treating cognitive impairment induced by GA during development.
Abstract
We previously documented that the Ca
V
3.3 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 Ca
V
3.3 channels in anaesthetic-induced hypnosis and underlying neuronal oscillations. To address this issue, we used Ca
V
3.3 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 Ca
V
3.3 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 Ca
V
3.3 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.
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•Effects of LPS applied in late embryogenesis were tested at postnatal days 40 and 60.•Male rats exhibited spatial learning and memory impairments only in early adulthood.•Female rats ...displayed decreased reactivity to amphetamine only in early adulthood.•Gender differences may mimic cognitive dysfunction and deficit symptoms, respectively.
Numerous basic and epidemiological studies have connected prenatal maternal immune activation with the occurrence of schizophrenia and/or autism. Depending on subtle differences in protocols of the used animal model, a variety of behavioral abnormalities has been reported. This study investigated behavioral differences in Wistar rat offspring of both genders, exposed to the 100μg/kg per day dose of lipopolysaccharide (LPS) in late embryogenesis (embryonic days 15 and 16), while tested at their adolescent and young adult age (postnatal days 40 and 60, respectively). Immune activation was confirmed by detecting high levels of TNF-α and IL-6 in dam blood withdrawn 2h after the first dose of LPS. The animals were assessed in three consecutive trials of locomotor activity (novelty exploration, response to i.p. saline injection and challenge with 0.5mg/kg amphetamine), Morris water maze and social interaction tests. Overt behavioral dysfunction was perceived in adult rats only, and these changes were gender-distinctive. When compared with control rats, LPS females displayed baseline hypolocomotion and a decreased reactivity to amphetamine, while LPS males exhibited spatial learning (acquisition trials) and memory (probe trial) impairments. Prenatal treatment did not affect the time spent in social interaction. As maternal exposure to LPS in late gestation resulted in behavioral changes in offspring in early adulthood, it may model schizophrenia-like, but not autism-like endophenotypes. However, lack of a potentiated response to amphetamine testified that this model could not mimic positive symptoms, but rather certain traits of cognitive dysfunction and deficit symptoms, in males and females, respectively.
Many neuroactive steroids induce sedation/hypnosis by potentiating γ-aminobutyric acid (GABAA) currents. However, we previously demonstrated that an endogenous neuroactive steroid epipregnanolone ...(3β,5β)-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).
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.
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.
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.
•This study seeks to investigate hypnotic effects of epipregnanolone (EpiP) and possible role of T-type calcium channels.•EpiP induced hypnotic state in wild type (WT) mice and oscillations in slower electroencephalogram (EEG) frequencies.•Only CaV3.1, but not CaV3.2 and CaV3.3 KO mice showed decreased duration of EpiP-induced hypnosis 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 when compared to WT mice.•EpiP may have an important role as a novel sedative/hypnotic agent with distinct effects across all three T-channel isoforms.
The ventral tegmental area (VTA) is a midbrain region highly involved in motivation and reward. A large body of work has investigated synaptic plasticity and ion channel excitability in this area, ...which has strong implication in drug abuse. We recently provided electrophysiological and pharmacological evidence that the Ca
3.1 isoform of T-type voltage-gated calcium channels contributes to the excitability of VTA dopamine (DA) neurons. However, the role of T-channels in excitability of VTA gamma-amino-butyric acid (GABA) neurons remained unaddressed. Here, with a population study of rat VTA GABA neurons, we provide evidence that T-channels contribute to rebound spiking activity in two phenotypically distinct subpopulations of GABAergic neurons, each with differing electrophysiological characteristics. Additionally, we provide the first study to investigate the effect of α-lipoic acid (ALA) on ion channels in mesolimbic reward circuitry. Taken together, our population study and pharmacology experiments implicate T-channels as a target for therapies aimed at tempering VTA and mesolimbic circuit excitability.
► PWZ-029 is an α5 GABAA selective inverse agonist. ► We tested PWZ-029, on its own and with scopolamine, in two memory paradigms. ► It improved object recognition in the novel object recognition ...test. ► PWZ-029 was ineffective in the Morris water maze test. ► Testing of putative procognitive compounds needs use of a variety of models.
Inverse agonism at the benzodiazepine site of α5 subunit-containing GABAA receptors is an attractive approach for the development of putative cognition-enhancing compounds, which are still far from clinical application. Several ligands with binding and/or functional selectivity for α5 GABAA receptors have been synthesized and tested in a few animal models. PWZ-029 is an α5 GABAA selective inverse agonist whose memory enhancing effects were demonstrated in the passive avoidance task in rats and in Pavlovian fear conditioning in mice. In the present study we investigated the effects of PWZ-029 administration in novel object recognition test and Morris water maze, in normal and scopolamine-treated rats. All the three doses of PWZ-029 (2, 5 and 10mg/kg) improved object recognition after the 24-h delay period, as shown by significant differences between the exploration times of the novel and old object, and the respective discrimination indices. PWZ-029 (2mg/kg) also successfully reversed the 0.3mg/kg scopolamine-induced deficit in recognition memory after the 1-h delay. In the Morris water maze test, PWZ-029 (5, 10 and 15mg/kg) did not significantly influence swim patterns, either during five acquisition days or during the treatment-free probe trial. PWZ-029 (2, 5 and 10mg/kg) also proved to be ineffective in the reversal of the 1mg/kg scopolamine-induced memory impairment in the water maze. The present mixed results encourage use of a variety of tests and experimental conditions in order to increase the predictability of preclinical testing of selective α5 GABAA inverse agonists.
We recently reported that a neurosteroid analogue with T-channel-blocking properties (3β,5β,17β)-3-hydroxyandrostane-17-carbonitrile (3β-OH), induced hypnosis in rat pups without triggering neuronal ...apoptosis. Furthermore, we found that the inhibition of the Ca
3.1 isoform of T-channels contributes to the hypnotic properties of 3β-OH in adult mice. However, the specific mechanisms underlying the role of other subtypes of voltage-gated calcium channels in thalamocortical excitability and oscillations
during 3β-OH-induced hypnosis are largely unknown. Here, we used patch-clamp recordings from acute brain slices,
electroencephalogram (EEG) recordings, and mouse genetics with wild-type (WT) and Ca
2.3 knock-out (KO) mice to further investigate the molecular mechanisms of neurosteroid-induced hypnosis. Our voltage-clamp recordings showed that 3β-OH inhibited recombinant Ca
2.3 currents. In subsequent current-clamp recordings in thalamic slices
, we found that selective Ca
2.3 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 3β-OH inhibited spike-firing more profoundly in WT than in mutant mice. Furthermore, 3β-OH reduced bursting frequencies in WT but not mutant animals. In ensuing
experiments, we found that intra-peritoneal injections of 3β-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, β, and low γ EEG power was more profound in WT than in Ca
2.3 KO females over time, while at 60 min after injections of 3β-OH, the increase in relative β power was higher in mutant females. In addition, 3β-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 Ca
2.3 subtype of voltage-gated calcium channels in thalamocortical excitability and the oscillations that underlie neurosteroid-induced hypnosis.
► Benzodiazepines cause anterograde rather than retrograde amnesia. ► We studied the effects of midazolam on the water maze behavior in rats. ► It impaired acquisition and retention, but not ...consolidation of spatial learning. ► Midazolam administered before the probe test impaired retrieval of reference memory. ► There is a possibility of retrograde amnesia when midazolam is clinically used.
Amnesia is one of the most discussed properties of the benzodiazepine class of drugs. The effects of benzodiazepines on human memory are usually anterograde, while changes in retrograde memory functions were seldom reported. Such inconsistent findings have prompted numerous animal studies investigating the influences of these positive modulators of inhibitory neurotransmission on different stages of memory. Among the benzodiazepines, memory effects of midazolam are of special interest due to its many and varied clinical applications. The present Morris water maze study in adult male Wistar rats was performed in three experiments in which midazolam was administered at doses of 0.5, 1 and 2mg/kg intraperitoneally, before or immediately after each of five daily learning sessions, with two trials in a session, as well as before the probe test. Midazolam impaired acquisition and subsequent retention of spatial learning of the position of the hidden platform even at a pre-training dose of 0.5mg/kg. This low dose was not associated with impairment of the procedural component of learning, manifested by increased time spent in the periphery of the pool. The lack of midazolam effect on consolidation has not been confounded by the observed below-chance performance of the control group since our additional experiment using diazepam also administered immediately after each of five learning sessions has revealed a similar pattern of results. Finally, midazolam administered before the probe test impaired retrieval of reference memory at all tested doses. Hence, induction of retrograde, besides anterograde amnesia should be kept in mind as a possibility when midazolam is used in clinical settings.
Rationale
Synthesis of ligands inactive or with low activity at α
1
GABA
A
receptors has become the key concept for development of novel, more tolerable benzodiazepine (BZ)-like drugs. WYS8, a ...remarkably (105 times) α
1
-subtype selective partial positive modulator, may serve as a pharmacological tool for refining the role of α
1
GABA
A
receptors in mediation of BZs’ effects.
Objectives
Here, the effects of WYS8 on GABA-induced currents and on diazepam-induced potentiation of recombinant BZ-sensitive GABA
A
receptors were studied in more detail. In addition, the behavioral profile of WYS8 (0.2, 1, and 10 mg/kg i.p.), on its own and in combination with diazepam, was tested in the spontaneous locomotor activity, elevated plus maze, grip strength, rotarod, and pentylenetetrazole tests.
Results
WYS8, applied at an in vivo attainable concentration of 100 nM, reduced the stimulation of GABA currents by 1 μM diazepam by 57 % at α
1
β
3
γ
2
, but not at α
2
β
3
γ
2
, α
3
β
3
γ
2
, or α
5
β
3
γ
2
GABA
A
receptors. The administration of WYS8 alone induced negligible behavioral consequences. When combined with diazepam, WYS8 caused a reduction in sedation, muscle relaxation, and anticonvulsant activity, as compared with this BZ alone, whereas ataxia was preserved, and the anxiolytic effect of 2 mg/kg diazepam was unmasked.
Conclusions
Hence, a partial instead of full activation at α
1
GABA
A
receptors did not necessarily result in the attenuation of the effects assumed to be mediated by activation of these receptors, or in the full preservation of the effects mediated by activation of other GABA
A
receptors. Thus, the role of α
1
GABA
A
receptors appears more complex than that proposed by genetic studies.
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