Cryo-EM structures reveal native GABAA receptor assemblies and pharmacology.

Type A γ-aminobutyric acid receptors (GABAARs) are the principal inhibitory receptors in the brain and the target of a wide range of clinical agents, including anaesthetics, sedatives, hypnotics and antidepressants1-3. However, our understanding of GABAAR pharmacology has been hindered by the vast number of pentameric assemblies that can be derived from 19 different subunits4 and the lack of structural knowledge of clinically relevant receptors. Here, we isolate native murine GABAAR assemblies containing the widely expressed α1 subunit and elucidate their structures in complex with drugs used to treat insomnia (zolpidem (ZOL) and flurazepam) and postpartum depression (the neurosteroid allopregnanolone (APG)). Using cryo-electron microscopy (cryo-EM) analysis and single-molecule photobleaching experiments, we uncover three major structural populations in the brain: the canonical α1ß2γ2 receptor containing two α1 subunits, and two assemblies containing one α1 and either an α2 or α3 subunit, in which the single α1-containing receptors feature a more compact arrangement between the transmembrane and extracellular domains. Interestingly, APG is bound at the transmembrane α/ß subunit interface, even when not added to the sample, revealing an important role for endogenous neurosteroids in modulating native GABAARs. Together with structurally engaged lipids, neurosteroids produce global conformational changes throughout the receptor that modify the ion channel pore and the binding sites for GABA and insomnia medications. Our data reveal the major α1-containing GABAAR assemblies, bound with endogenous neurosteroid, thus defining a structural landscape from which subtype-specific drugs can be developed.

Quetiapine Add-On Therapy May Improve Persistent Sleep Disturbances in Patients with PTSD on Stabile Combined SSRI and Benzodiazepine Combination: A One-Group Pretest-Posttest Study.

BACKGROUND: To assess potential benefits of quetiapine for persistent sleep disturbances in patients with posttraumatic stress disorder (PTSD) on stable combined SSRI and benzodiazepine therapy, who previously failed to respond to various benzodiazepine and non-benzodiazepine hypnotic adjuvant treatment as well as to first-generation antipsychotic add-on treatment. SUBJECTS AND METHODS: Fifty-two male PTSD outpatients on stable combination treatment with SSRI and benzodiazepines, with persistent sleep disturbances not responding to prescription of zolpidem, flurazepam, nitrazepam, promazine, and levopromazine, were assessed for sleep disturbances improvements after prescription of quetiapine in the evening. Each patient met both ICD-10 and DSM-IV criteria for PTSD. Psychiatric comorbidity and premorbidity were excluded using the Mini-International Neuropsychiatric Interview (MINI). Improvement on the CAPS recurrent distressing dream item, reduction in the amount of time needed to fall asleep, prolongation of sleep duration, and reduction in average number of arousals per night in the last 7 days before the assessment period were used as efficacy measures. RESULTS: All sleep-related parameters improved significantly at the end of a five-week follow-up: sleep duration increased by one hour (p<0.001), sleep latency decreased by 52.5 minutes (p<0.001), median number of arousals per night decreased from two to one (p<0.001), CAPS recurrent distressing dream item median decreased from five to four (p<0.001), and the number of patients dissatisfied with their sleep quality and quantity decreased from 45 to two (p<0.001). CONCLUSION: Quetiapine prescribed in the evening may be successful therapy for persistent sleep disturbances in patients with PTSD and generally good response to an SSRI and benzodiazepine combination, who previously failed to respond to some of the usual hypnotic medication or addition of first-generation antipsychotics: zolpidem, flurazepam, nitrazepam, promazine, and levopromazine.

Interventions to reduce bruxism in children and adolescents: a systematic scoping review and critical reflection.

The aim of the present study was to perform a critical reflection about intervention options for bruxism reduction in children and adolescents. Search was conducted based on the PICO-structured question: "What are the intervention options to reduce bruxism in children/adolescents?". No language, year, or study design restrictions were imposed. Studies reporting interventions to reduce bruxism in children (< 10) and adolescents (10 to 19 years old) were included. Reviews and letters to editors were not included. From 2723 records, 17 papers were included. Included studies were primarily randomized clinical trials performed in Brazil (35.3%) and using different criteria for the diagnosis of bruxism. Reduction in self-reported bruxism and headaches associated with bruxism were observed in studies that used medications (hydroxyzine/trazodone/flurazepam), occlusal splints, orthodontic interventions, and psychological and physical therapy interventions. Reduction in Rhythmic Masticatory Muscle Activity was observed with the use of the occlusal splint and in orthodontic interventions. Alternative treatments (medicinal extracts such as Melissa officinalis-L) have shown inconclusive results.Conclusions: Several intervention options are available to inhibit or reduce bruxism activity. The respective indication, contraindications, and side effects of each treatment option must be assessed individually and carefully, taking into account that bruxism is not considered a disorder in otherwise healthy individuals.What is known• Biological and psychological factors have been strongly correlated to the development of bruxism• Bruxism prevalence ranging from 6 to 50% in childrenWhat is new• Reduction in self-reported bruxism and headaches associated with bruxism were observed in studies that used medication (Hydroxyzine/ Trazodone/ Flurazepam), occlusal splints, orthodontic interventions, psychological, and physical therapy interventions• A reduction in Rhythmic Masticatory Muscle Activity was observed with the use of the occlusal splint and orthodontic interventions. Alternative treatments (medicinal extracts such as Melissa officinalis L) show inconclusive results in respect of the reduction in bruxism.

Synaptic GABAA receptor clustering without the γ2 subunit.

Rapid activation of postsynaptic GABAA receptors (GABAARs) is crucial in many neuronal functions, including the synchronization of neuronal ensembles and controlling the precise timing of action potentials. Although the γ2 subunit is believed to be essential for the postsynaptic clustering of GABAARs, synaptic currents have been detected in neurons obtained from γ2(-/-) mice. To determine the role of the γ2 subunit in synaptic GABAAR enrichment, we performed a spatially and temporally controlled γ2 subunit deletion by injecting Cre-expressing viral vectors into the neocortex of GABAARγ2(77I)lox mice. Whole-cell recordings revealed the presence of miniature IPSCs in Cre(+) layer 2/3 pyramidal cells (PCs) with unchanged amplitudes and rise times, but significantly prolonged decays. Such slowly decaying currents could be evoked in PCs by action potentials in presynaptic fast-spiking interneurons. Freeze-fracture replica immunogold labeling revealed the presence of the α1 and ß3 subunits in perisomatic synapses of cells that lack the γ2 subunit. Miniature IPSCs in Cre(+) PCs were insensitive to low concentrations of flurazepam, providing a pharmacological confirmation of the lack of the γ2 subunit. Receptors assembled from only αß subunits were unlikely because Zn(2+) did not block the synaptic currents. Pharmacological experiments indicated that the αßγ3 receptor, rather than the αßδ, αßε, or αßγ1 receptors, was responsible for the slowly decaying IPSCs. Our data demonstrate the presence of IPSCs and the synaptic enrichment of the α1 and ß3 subunits and suggest that the γ3 subunit is the most likely candidate for clustering GABAARs at synapses in the absence of the γ2 subunit.

Comparison of alternative models for linking drug exposure with adverse effects.

Pharmacoepidemiology investigates associations between time-varying medication use/dose and risk of adverse events. Applied research typically relies on a priori chosen simple conventional models, such as current dose or any use in the past 3 months. However, different models imply different risk predictions, and only one model can be etiologically correct in any specific applications. We first formally defined several candidate models mapping the time vector of past drug doses (X (t), t = 1, … ,u) into the value of a time-varying exposure metric M(u) at current time u. In addition to conventional one-parameter models, we considered two-parameter models accounting for recent dose increase or withdrawal and a flexible spline-based weighted cumulative exposure (WCE) model that defines M(u) as the weighted sum of past doses. In simulations, we generated event times assuming one of the models was correct and then analyzed the data with all candidate models. We demonstrated that the minimum AIC criterion is able to identify the correct model as the best-fitting model or one of the equivalent (within 4 AIC points of the minimum) models in a vast majority of simulated samples, especially with 500 or more events. We also showed how relying on an incorrect a priori chosen model may largely reduce the power to test for an association. Finally, we demonstrated how the flexible WCE estimates may help with model diagnostics even if the correct model is not WCE. We illustrated the practical advantages of AIC-based a posteriori model selection and WCE modeling in a real-life pharmacoepidemiology example.

Assessing the cumulative effects of exposure to selected benzodiazepines on the risk of fall-related injuries in the elderly.

BACKGROUND: The use of benzodiazepines is associated with increased risk of fall-related injuries in the elderly. However, it is unclear if the risks vary across the products and how they depend on the pattern of use and dosage. Specifically, the possibility of cumulative effects of past benzodiazepine use has not been thoroughly investigated. METHODS: We used the administrative database for a cohort of 23,765 new users of benzodiazepines, aged 65 years and older, in Quebec, Canada, between 1990 and 1994. The associations between the use of seven benzodiazepines and the risk of fall-related injuries were assessed using several statistical models, including a novel weighted cumulative exposure model. That model assigns to each dose taken in the past a weight that represents the importance of that dose in explaining the current risk of fall. RESULTS: For flurazepam, the best-fitting model indicated a cumulative effect of doses taken in the last two weeks. Uninterrupted use of flurazepam in the past months was associated with a highly significant increase in the risk of fall-related injuries (HR = 2.83, 95% CI: 1.45-4.34). The cumulative effect of a 30-day exposure to alprazolam was 1.27 (1.13-1.42). For temazepam, the results suggested a potential withdrawal effect. CONCLUSIONS: Mechanisms affecting the risk of falls differ across benzodiazepines, and may include cumulative effects of use in the previous few weeks. Thus, benzodiazepine-specific analyses that account for individual patterns of use should be preferred over simpler analyses that group different benzodiazepines together and limit exposure to current use or current dose.

Regulation of Ca²âº/calmodulin-dependent protein kinase II signaling within hippocampal glutamatergic postsynapses during flurazepam withdrawal.

Cessation of one-week oral administration of the benzodiazepine flurazepam (FZP) to rats results in withdrawal anxiety after 1 day of withdrawal. FZP withdrawal is correlated with synaptic incorporation of homomeric GluA1-containing α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors (AMPARs) in the proximal stratum radiatum of CA1 neurons. After 2 days of withdrawal, Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) phosphorylates GluA1 subunits at Ser(831), increasing channel conductance. Secondary to AMPAR potentiation, GluN2B-containing N-methyl-D-aspartate receptors (NMDARs), known binding partners of CaMKII, are selectively removed from the postsynaptic density (PSD). While activation of synaptic CaMKII is known to involve translocation to the PSD, CaMKII bound to NMDARs may be removed from the PSD. To distinguish these possibilities, the current studies used postembedding immunogold electron microscopy to investigate alterations in CaMKII signaling at CA1 stratum radiatum synapses after 2 days of FZP withdrawal. These studies revealed decreased total, but not autophosphorylated (Thr(286)) CaMKIIα expression in CA1 PSDs. The removal of CaMKII-GluN2B complexes from the PSD during drug withdrawal may serve as a homeostatic mechanism to limit AMPAR-mediated CA1 neuron hyperexcitability and benzodiazepine withdrawal anxiety.

Effects of discontinuing benzodiazepine-derivative hypnotics on postural sway and cognitive functions in the elderly.

OBJECTIVE: Benzodiazepines (BZDs) have been reported to cause negative impacts on body stability and cognitive functions, which in turn could result in lethal incidents, including falls, especially in the elderly. This fact notwithstanding, no systematic trial has evaluated the feasibility and benefits of discontinuing BZD-derivative hypnotics in this population, which was addressed in this study. METHODS: In this 8-week open-label study, subjects aged ≥ 60 living in a nursing home who received BZD as a hypnotic were recruited. The BZD dose was tapered off over 3 weeks. The following assessments were performed 12 h post-dose at baseline and at endpoint: the Clinical Stabilometric Platform (CSP), the Critical Flicker Fusion Test (CFF), the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS), and the Leeds Sleep Evaluation Questionnaire (LSEQ). RESULTS: Thirty subjects were enrolled (mean ± SD age = 79.1 ± 8.9 years, mean ± SD flurazepam equivalent BZD dose = 19.5 ± 10.9 mg/day). Psychiatric diagnoses (DSM-IV) of subjects were as follows: schizophrenia (n = 12), primary insomnia (n = 9), dementia (n = 7), and bipolar disorder (n = 2). In 26 completers, significant changes were found in a total length and a range of trunk motion with eyes closed. Significant improvements were also observed in the CFF and RBANS immediate memory, language, and attention index scores. Subjective worsening in sleep was not reported in those completers, assessed with the LSEQ. CONCLUSIONS: Our results suggest that discontinuation of BZD hypnotics is feasible in a majority of elderly persons and leads to an improvement in the stability of body and a recovery in cognitive functions during the daytime.

Mice lacking Gad2 show altered behavioral effects of ethanol, flurazepam and gabaxadol.

Gamma-aminobutyric acid (GABA) is synthesized in brain by two isoforms of glutamic acid decarboxylase (Gad), Gad1 and Gad2. Gad1 provides most of the GABA in brain, but Gad2 can be rapidly activated in times of high GABA demand. Mice lacking Gad2 are viable whereas deletion of Gad1 is lethal. We produced null mutant mice for Gad2 on three different genetic backgrounds: predominantly C57BL/6J and one or two generations of backcrossing to 129S1/SvimJ (129N1, 129N2). We used these mice to determine if actions of alcohol are regulated by synthesis of GABA from this isoform. We also studied behavioral responses to a benzodiazepine (flurazepam) and a GABAA receptor agonist (gabaxadol). Deletion of Gad2 increased ethanol palatability and intake and slightly reduced the severity of ethanol-induced withdrawal, but these effects depended strongly on genetic background. Mutant mice on the 129N2 background showed the above three ethanol behavioral phenotypes, but the C57BL/6J inbred background did not show any of these phenotypes. Effects on ethanol consumption also depended on the test as the mutation did not alter consumption in limited access models. Deletion of Gad2 reduced the effect of flurazepam on motor incoordination and increased the effect of extrasynaptic GABAA receptor agonist gabaxadol without changing the duration of loss of righting reflex produced by these drugs. These results are consistent with earlier proposals that deletion of Gad2 (on 129N2 background) reduces synaptic GABA but also suggest changes in extrasynaptic receptor function.

[Drugs and the mechanism for reversing the tolerance of flurazepan in rats].

OBJECTIVE: Benzodiazepines (BDZ) have many effects on various kinds of epilepsies, but long-term treatment with BDZ often leads to drug tolerance. This study aimed to seek drugs which can reverse the tolerance of flurazepam (FZP), and to explore the role of neuropeptide Y (NPY) in the reversal effect. METHODS: A rat model of anticonvulsant tolerance to FZP was prepared. The rats with FZP tolerance were randomly assigned to seven groups: FZP-tolerance, and nifedipine, levetiracetam, topiramate, flumazenil, L-NAME and pyridoxamine treatment groups. The tolerance to FZP was evaluated through pentylenetetrazol (PTZ) infusion into a tail vein. The latency to onset of clonic seizure and the PTZ threshold were recorded. The mRNA of NPY receptor Y2 in the hippocampus was determined by RT-PCR, and the distribution of NPY in the hippocampus was examined by immunohistochemistry. RESULTS: In comparison with the blank control group, the average latency to the onset of clonic seizure was shortened, the average PTZ threshold decreased and the expression of NYT and NPY receptor Y2 mRNA decreased significantly in the FZP-tolerance group (p<0.01). In comparison with the FZP-tolerance group, the average latency to onset of clonic seizure was prolonged by 2 times and the average PTZ threshold doubled in the topiramate treatment group. The average latency to onset of clonic seizure was prolonged by 1 time and the average PTZ threshold increased 1 time in the nifedipine, the levetiracetam and the flumazenil treatment groups. The mRNA expression of NPY receptor Y2 increased by 1 or 2 times in the flumazenil, the nifedipine and the topiramate treatment groups when compared with the FZP-tolerance group. CONCLUSIONS: Nifedipine, levetiracetam, topiramate and flumazenil can reverse the anticonvulsant tolerance to flurazepam. NPY may play a role in mediating the reversal effect.

Treatment of a 12-hour shift of sleep schedule with benzodiazepines.

Normal sleepers underwent sleep recordings and daytime tests of sleep tendency, performance, and mood while being shifted 180 degrees in their sleep-wake schedule. After two baseline 24-hour periods, subjects postponed sleep until noon. For the next three 24-hour periods, they were in bed from 1200 to 2000 and received triazolam, flurazepam, or placebo at bedtime in parallel groups. Placebo subjects showed significant sleep loss after the shift. Active medication reversed this sleep loss. Despite good sleep, flurazepam subjects appeared most impaired of the three groups on objective assessments of waking function; triazolam subjects were least impaired.

A benzodiazepine receptor antagonist decreases sleep and reverses the hypnotic actions of flurazepam.

The benzodiazepine receptor antagonist 3-hydroxymethyl-beta-carboline, which blocks several of the pharmacological actions of benzodiazepines, induces a dose-dependent increase in sleep latency in the rat. Furthermore, at a low dose that by itself does not affect sleep, 3-hydroxymethyl-beta-carboline blocks sleep induction by a large dose of flurazepam. The benzodiazepine receptor may play a role in both the physiological regulation and pharmacological induction of sleep.

Inosine may be an endogenous ligand for benzodiazepine receptors on cultured spinal neurons.

Mouse spinal neurons grown in tissue culture were used to study the membrane effects of the benzodiazepine flurazepam and the naturally occurring purine nucleoside inosine, which competes for benzodiazepine receptor sites in the central nervous system. Application of inosine elicited two types of transmitter-like membrane effects: a rapidly desensitizing excitatory response and a nondesensitizing inhibitory response. Flurazepam produced a similar excitatory response which showed cross-desensitization with the purine excitation. Flurazepam also blocked the inhibitory inosine response. The results provide electrophysiological evidence that an endogenous purine can activate two different conductances on spinal neurons and that flurazepam can activate one of the conductances and antagonize the other.

Early morning insomnia with rapidly eliminated benzodiazepines.

Early morning insomnia, a significant increase in wakefulness during the final hours of drug nights, occurred after 1 or 2 weeks of nightly administration of benzodiazepine hypnotics with short elimination half-lives, when tolerance had begun to develop. Early morning insomnia may be a variant of rebound insomnia and therefore specific to benzodiazepines, or it may occur with any rapidly eliminated sedative-hypnotic agent.

Flurazepam effects on slow-wave sleep: stage 4 suppressed but number of delta waves constant.

Repeated administration of flurazepam reduced stage 4 sleep (high delta-wave concentration) but produced a greater increase in stage 2 duration so that total sleep time was increased. Computer analysis revealed that the increased amount of stage 2 (low delta-wave concentration) sleep provided a number and duration of delta waves sufficient to offset the loss of delta activity in stage 4. However, the amplitude of the average delta wave was reduced. These results demonstrate the value of direct quantification of delta-wave activity, the variable that underlies visual classification of slow-wave sleep into stages 2 to 4. They also give rise to new hypotheses regarding the relative absence of side effects in spite of profound stage 4 suppression by flurazepam and the mechanisms by which total sleep time is increased by this drug.

Fyn-kinase as a determinant of ethanol sensitivity: relation to NMDA-receptor function.

Animals vary in their sensitivity to ethanol, a trait at least partly determined by genetic factors. In order to identify possible responsible genes, mice lacking Fyn, a non-receptor type tyrosine kinase, were investigated. These mice were hypersensitive to the hypnotic effect of ethanol. The administration of ethanol enhanced tyrosine phosphorylation of the N-methyl-D-aspartate receptor (NMDAR) in the hippocampus of control mice but not in Fyn-deficient mice. An acute tolerance to ethanol inhibition of NMDAR-mediated excitatory postsynaptic potentials in hippocampal slices developed in control mice but not in Fyn-deficient mice. These results indicate that Fyn affects behavioral, biochemical, and physiological responses to ethanol.

Flexible modeling of the cumulative effects of time-dependent exposures on the hazard.

Many epidemiological studies assess the effects of time-dependent exposures, where both the exposure status and its intensity vary over time. One example that attracts public attention concerns pharmacoepidemiological studies of the adverse effects of medications. The analysis of such studies poses challenges for modeling the impact of complex time-dependent drug exposure, especially given the uncertainty about the way effects cumulate over time and about the etiological relevance of doses taken in different time periods. We present a flexible method for modeling cumulative effects of time-varying exposures, weighted by recency, represented by time-dependent covariates in the Cox proportional hazards model. The function that assigns weights to doses taken in the past is estimated using cubic regression splines. We validated the method in simulations and applied it to re-assess the association between exposure to a psychotropic drug and fall-related injuries in the elderly.

Fatalities associated with clozapine-related constipation and bowel obstruction: a literature review and two case reports.

BACKGROUND: Constipation is an exceedingly common side effect of treatment with clozapine. In rare cases, this side effect has resulted in fatal complications. OBJECTIVE: The authors review the literature on fatal complications of clozapine-related constipation and bowel obstruction. METHOD: The authors provide two new case reports of patients who died of similar causes. RESULTS: There were seven reports of deaths from clozapine-related bowel obstruction in the literature, with the most common mechanisms of death being severe impaction leading either to feculent vomiting or bowel necrosis. DISCUSSION: The discussion outlines potential mechanisms and management of clozapine-related constipation.

Genetically inbred Balb/C mice are more sensitive to an effect of flurazepam and more resistant to an effect of stress than a genetically outbred mouse strain.

The inbred Balb/c mouse strain was more sensitive than the outbred NIH Swiss mouse to flurazepam's ability to antagonize electrically precipitated seizures. In prior work, a reduction in flurazepam's antiseizure efficacy was not observed 24h after forcing Balb/c mice to swim for up to 10 min in ambient temperature water. Thus, we wondered if a stress-induced reduction would be observed after forcing mice to swim for up to 10 min in cold (6 degrees C) water, a more severe stress. The current data show that 24 h after exposure to this stress, the ability of flurazepam to raise the threshold voltage for the elicitation of tonic hindlimb extension in the Balb/c mouse strain was reduced. The genetically inbred Balb/c mouse strain is emerging as an interesting animal model in which to study interactions of stress and genetic factors that affect endogenous neurotransmission mediated by l-glutamate and GABA at the NMDA and GABA(A) receptor complexes, respectively.

Next-day residual effects of gaboxadol and flurazepam administered at bedtime: a randomized double-blind study in healthy elderly subjects.

OBJECTIVE: To evaluate the next-day residual effects of the novel hypnotic, gaboxadol, following bedtime dosing in healthy elderly subjects. METHODS: Healthy women (N = 15) and men (N = 10) aged 65-79 years received a single bedtime (22:00 h) dose of gaboxadol 10 mg, flurazepam 30 mg (positive control), and placebo in a randomized, double-blind, crossover study. Measures of information processing and psychomotor performance (choice reaction time, critical flicker fusion, digit symbol substitution, compensatory tracking, body sway), memory (immediate and delayed word recall), and daytime sleepiness (Multiple Sleep Latency Test), as well as subjective ratings (line analog rating scales, Leeds Sleep Evaluation Questionnaire), were obtained starting at 07:00 h the following morning. Adverse events were recorded. RESULTS: Gaboxadol did not show next-day impairments versus placebo on any pharmacodynamic measures whereas the positive control, flurazepam, did show impairments versus placebo on most measures. Gaboxadol showed improvements versus placebo on some measures including subjective rating of next-day alertness/clumsiness on the Leeds Sleep Evaluation Questionnaire. Gaboxadol was generally well-tolerated; there were no serious adverse experiences and no subjects discontinued due to an adverse experience. CONCLUSIONS: A single oral bedtime dose of gaboxadol 10 mg did not have next-day residual effects in healthy elderly subjects, as measured by a range of pharmacodynamic assessments, in contrast to the clear impairments produced by flurazepam 30 mg.