123 research outputs found
Propofol suppresses synaptic responsiveness of somatosensory relay neurons to excitatory input by potentiating GABA(A )receptor chloride channels
Propofol is a widely used intravenous general anesthetic. Propofol-induced unconsciousness in humans is associated with inhibition of thalamic activity evoked by somatosensory stimuli. However, the cellular mechanisms underlying the effects of propofol in thalamic circuits are largely unknown. We investigated the influence of propofol on synaptic responsiveness of thalamocortical relay neurons in the ventrobasal complex (VB) to excitatory input in mouse brain slices, using both current- and voltage-clamp recording techniques. Excitatory responses including EPSP temporal summation and action potential firing were evoked in VB neurons by electrical stimulation of corticothalamic fibers or pharmacological activation of glutamate receptors. Propofol (0.6 – 3 μM) suppressed temporal summation and spike firing in a concentration-dependent manner. The thalamocortical suppression was accompanied by a marked decrease in both EPSP amplitude and input resistance, indicating that a shunting mechanism was involved. The propofol-mediated thalamocortical suppression could be blocked by a GABA(A )receptor antagonist or chloride channel blocker, suggesting that postsynaptic GABA(A )receptors in VB neurons were involved in the shunting inhibition. GABA(A )receptor-mediated inhibitory postsynaptic currents (IPSCs) were evoked in VB neurons by electrical stimulation of the reticular thalamic nucleus. Propofol markedly increased amplitude, decay time, and charge transfer of GABA(A )IPSCs. The results demonstrated that shunting inhibition of thalamic somatosensory relay neurons by propofol at clinically relevant concentrations is primarily mediated through the potentiation of the GABA(A )receptor chloride channel-mediated conductance, and such inhibition may contribute to the impaired thalamic responses to sensory stimuli seen during propofol-induced anesthesia
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Polypharmacological in Silico Bioactivity Profiling and Experimental Validation Uncovers Sedative-Hypnotic Effects of Approved and Experimental Drugs in Rat
In this work, we describe the computational ("in silico") mode-of-action analysis of CNS-active drugs, which is taking both multiple simultaneous hypotheses as well as sets of protein targets for each mode-of-action into account, and which was followed by successful prospective in vitro and in vivo validation. Using sleep-related phenotypic readouts describing both efficacy and side effects for 491 compounds tested in rat, we defined an "optimal" (desirable) sleeping pattern. Compounds were subjected to in silico target prediction (which was experimentally confirmed for 21 out of 28 cases), followed by the utilization of decision trees for deriving polypharmacological bioactivity profiles. We demonstrated that predicted bioactivities improved classification performance compared to using only structural information. Moreover, DrugBank molecules were processed via the same pipeline, and compounds in many cases not annotated as sedative-hypnotic (alcaftadine, benzatropine, palonosetron, ecopipam, cyproheptadine, sertindole, and clopenthixol) were prospectively validated in vivo. Alcaftadine, ecopipam cyproheptadine, and clopenthixol were found to promote sleep as predicted, benzatropine showed only a small increase in NREM sleep, whereas sertindole promoted wakefulness. To our knowledge, the sedative-hypnotic effects of alcaftadine and ecopipam have not been previously discussed in the literature. The method described extends previous single-target, single-mode-of-action models and is applicable across disease areas.This research was supported by Unilever (A.B.), the EPSRC (G.D.), and Eli Lilly (G.D.). A.B. thanks the ERC for an ERC Starting Grant
Withdrawal-induced delirium associated with a benzodiazepine switch: a case report
<p>Abstract</p> <p>Introduction</p> <p>Introduced in the early 1960s, diazepam remains among the most frequently prescribed benzodiazepine-type sedatives and hypnotics. Patients with chronic use of short-acting benzodiazepines are frequently switched to diazepam because the accumulating, long-acting metabolite, N-desmethyl-diazepam, prevents benzodiazepine-associated withdrawal symptoms, which can occur during trough plasma levels of short-acting benzodiazepines. Although mild to moderate withdrawal symptoms are frequently observed during benzodiazepine switching to diazepam, severe medical complications associated with this treatment approach have thus far not been reported.</p> <p>Case presentation</p> <p>A 64-year-old female Caucasian with major depression, alcohol dependence and benzodiazepine dependence was successfully treated for depression and, after lorazepam-assisted alcohol detoxification, was switched from lorazepam to diazepam to facilitate benzodiazepine discontinuation. Subsequent to the benzodiazepine switch, our patient unexpectedly developed an acute delirious state, which quickly remitted after re-administration of lorazepam. A newly diagnosed early form of mixed dementia, combining both vascular and Alzheimer-type lesions, was found as a likely contributing factor for the observed vulnerability to benzodiazepine-induced withdrawal symptoms.</p> <p>Conclusion</p> <p>Chronic use of benzodiazepines is common in the elderly and a switch to diazepam often precedes benzodiazepine discontinuation trials. However, contrary to common clinical practice, benzodiazepine switching to diazepam may require cross-titration with slow tapering of the first benzodiazepine to allow for the build-up of N-desmethyl-diazepam, in order to safely prevent severe withdrawal symptoms. Alternatively, long-term treatment with low doses of benzodiazepines may be considered, especially in elderly patients with chronic use of benzodiazepines and proven vulnerability to benzodiazepine-associated withdrawal symptoms.</p
Deletion of the gabra2 gene results in hypersensitivity to the acute effects of ethanol but does not alter ethanol self administration
Human genetic studies have suggested that polymorphisms of the GABRA2 gene encoding the GABA(A) α2-subunit are associated with ethanol dependence. Variations in this gene also convey sensitivity to the subjective effects of ethanol, indicating a role in mediating ethanol-related behaviours. We therefore investigated the consequences of deleting the α2-subunit on the ataxic and rewarding properties of ethanol in mice. Ataxic and sedative effects of ethanol were explored in GABA(A) α2-subunit wildtype (WT) and knockout (KO) mice using a Rotarod apparatus, wire hang and the duration of loss of righting reflex. Following training, KO mice showed shorter latencies to fall than WT littermates under ethanol (2 g/kg i.p.) in both Rotarod and wire hang tests. After administration of ethanol (3.5 g/kg i.p.), KO mice took longer to regain the righting reflex than WT mice. To ensure the acute effects are not due to the gabra2 deletion affecting pharmacokinetics, blood ethanol concentrations were measured at 20 minute intervals after acute administration (2 g/kg i.p.), and did not differ between genotypes. To investigate ethanol's rewarding properties, WT and KO mice were trained to lever press to receive increasing concentrations of ethanol on an FR4 schedule of reinforcement. Both WT and KO mice self-administered ethanol at similar rates, with no differences in the numbers of reinforcers earned. These data indicate a protective role for α2-subunits, against the acute sedative and ataxic effects of ethanol. However, no change was observed in ethanol self administration, suggesting the rewarding effects of ethanol remain unchange
Ionotropic GABA and glycine receptor subunit composition in human pluripotent stem cell-derived excitatory cortical neurones
We have assessed, using whole-cell patch-clamp recording and RNA-sequencing (RNA-seq), the properties and composition of GABA(A) receptors (GABA(A)Rs) and strychnine-sensitive glycine receptors (GlyRs) expressed by excitatory cortical neurons derived from human embryonic stem cells (hECNs). The agonists GABA and muscimol gave EC(50) values of 278 μm and 182 μm, respectively, and the presence of a GABA(A)R population displaying low agonist potencies is supported by strong RNA-seq signals for α2 and α3 subunits. GABA(A)R-mediated currents, evoked by EC(50) concentrations of GABA, were blocked by bicuculline and picrotoxin with IC(50) values of 2.7 and 5.1 μm, respectively. hECN GABA(A)Rs are predominantly γ subunit-containing as assessed by the sensitivity of GABA-evoked currents to diazepam and insensitivity to Zn(2+), together with the weak direct agonist action of gaboxadol; RNA-seq indicated a predominant expression of the γ2 subunit. Potentiation of GABA-evoked currents by propofol and etomidate and the lack of inhibition of currents by salicylidine salycylhydrazide (SCS) indicate expression of the β2 or β3 subunit, with RNA-seq analysis indicating strong expression of β3 in hECN GABA(A)Rs. Taken together our data support the notion that hECN GABA(A)Rs have an α2/3β3γ2 subunit composition – a composition that also predominates in immature rodent cortex. GlyRs expressed by hECNs were activated by glycine with an EC(50) of 167 μm. Glycine-evoked (500 μm) currents were blocked by strychnine (IC(50) = 630 nm) and picrotoxin (IC(50) = 197 μm), where the latter is suggestive of a population of heteromeric receptors. RNA-seq indicates GlyRs are likely to be composed of α2 and β subunits
GABA Receptors and the Pharmacology of Sleep
Current GABAergic sleep-promoting medications were developed pragmatically, without making use of the immense diversity of GABAA receptors. Pharmacogenetic experiments are leading to an understanding of the circuit mechanisms in the hypothalamus by which zolpidem and similar compounds induce sleep at α2βγ2-type GABAA receptors. Drugs acting at more selective receptor types, for example, at receptors containing the α2 and/or α3 subunits expressed in hypothalamic and brain stem areas, could in principle be useful as hypnotics/anxiolytics. A highly promising sleep-promoting drug, gaboxadol, which activates αβδ-type receptors failed in clinical trials. Thus, for the time being, drugs such as zolpidem, which work as positive allosteric modulators at GABAA receptors, continue to be some of the most effective compounds to treat primary insomnia
Acute Cellular Alterations in the Hippocampus After Status Epilepticus
The critical, fundamental mechanisms that determine the emergence of status epilepticus from a single seizure and the prolonged duration of status epilepticus are uncertain. However, several general concepts of the pathophysiology of status epilepticus have emerged: (a) the hippocampus is consistently activated during status epilepticus; (b) loss of GABA-mediated inhibitory synaptic transmission in the hippocampus is critical for emergence of status epilepticus; and, finally (c) glutamatergic excitatory synaptic transmission is important in sustaining status epilepticus. This review focuses on the alteration of GABAergic inhibition in the hippocampus that occurs during the prolonged seizures of status epilepticus. If reduction in GABAergic inhibition leads to development of status epilepticus, enhancement of GABAergic inhibition would be expected to interrupt status epilepticus. Benzodiazepines and barbiturates are both used in the treatment of status epilepticus and both drugs enhance GABA A receptor-mediated inhibition. However, patients often become refractory to benzodiazepines when seizures are prolonged, and barbiturates are often then used for these refractory cases of status epilepticus. Recent evidence suggests the presence of multiple GABA A receptor isoforms in the hippocampus with different sensitivity to benzodiazepines but similar sensitivity to barbiturates, thus explaining why the two drug classes might have different clinical effects. In addition, rapid functional plasticity of GABA A receptors has been demonstrated to occur during status epilepticus in rats. During status epilepticus, there was a substantial reduction of diazepam potency for termination of the seizures. The loss of sensitivity of the animals to diazepam during status epilepticus was accompanied by an alteration in the functional properties of hippocampal dentate granule cell GABA A receptors. Dentate granule cell GABA A receptor currents from rats undergoing status epilepticus had reduced sensitivity to diazepam and zinc but normal sensitivity to GABA and pentobarbital. Therefore, the prolonged seizures of status epilepticus rapidly altered the functional properties of hippocampal dentate granule cell GABA A receptors, possibly explaining why benzodiazepines and barbiturates may not be equally effective during treatment of the prolonged seizures of status epilepticus. A comprehensive understanding of the cellular and molecular events leading to the development, maintenance, and cytotoxicity of status epilepticus should permit development of more effective treatment strategies and reduction in the mortality and morbidity of status epilepticus.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65664/1/j.1528-1157.1999.tb00873.x.pd
Slow GABAA mediated synaptic transmission in rat visual cortex
<p>Abstract</p> <p>Background</p> <p>Previous reports of inhibition in the neocortex suggest that inhibition is mediated predominantly through GABA<sub>A </sub>receptors exhibiting fast kinetics. Within the hippocampus, it has been shown that GABA<sub>A </sub>responses can take the form of either fast or slow response kinetics. Our findings indicate, for the first time, that the neocortex displays synaptic responses with slow GABA<sub>A </sub>receptor mediated inhibitory postsynaptic currents (IPSCs). These IPSCs are kinetically and pharmacologically similar to responses found in the hippocampus, although the anatomical specificity of evoked responses is unique from hippocampus. Spontaneous slow GABA<sub>A </sub>IPSCs were recorded from both pyramidal and inhibitory neurons in rat visual cortex.</p> <p>Results</p> <p>GABA<sub>A </sub>slow IPSCs were significantly different from fast responses with respect to rise times and decay time constants, but not amplitudes. Spontaneously occurring GABA<sub>A </sub>slow IPSCs were nearly 100 times less frequent than fast sIPSCs and both were completely abolished by the chloride channel blocker, picrotoxin. The GABA<sub>A </sub>subunit-specific antagonist, furosemide, depressed spontaneous and evoked GABA<sub>A </sub>fast IPSCs, but not slow GABA<sub>A</sub>-mediated IPSCs. Anatomical specificity was evident using minimal stimulation: IPSCs with slow kinetics were evoked predominantly through stimulation of layer 1/2 apical dendritic zones of layer 4 pyramidal neurons and across their basal dendrites, while GABA<sub>A </sub>fast IPSCs were evoked through stimulation throughout the dendritic arborization. Many evoked IPSCs were also composed of a combination of fast and slow IPSC components.</p> <p>Conclusion</p> <p>GABA<sub>A </sub>slow IPSCs displayed durations that were approximately 4 fold longer than typical GABA<sub>A </sub>fast IPSCs, but shorter than GABA<sub>B</sub>-mediated inhibition. The anatomical and pharmacological specificity of evoked slow IPSCs suggests a unique origin of synaptic input. Incorporating GABA<sub>A </sub>slow IPSCs into computational models of cortical function will help improve our understanding of cortical information processing.</p
F654A and K558Q Mutations in NMDA Receptor 1 Affect Ethanol‐Induced Behaviors in Drosophila
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