Incompatibility of the short-acting benzodiazepine remimazolam with common perioperative medication.

BACKGROUND: Remimazolam is a relatively new benzodiazepine with growing use in procedural sedation and general anaesthesia. Initiated by case reports, the physical incompatibility of remimazolam with ringer's acetated and ringer's lactated solution has been reported. More recently, remifentanil, fentanyl, rocuronium, vecuronium, dexmedetomidine, and midazolam, have been investigated and suggested safe for coadministration with remimazolam. Apart from case reports, incompatibility for other frequently used drugs remains unknown. METHODS: Sixty-five drugs and intravenous fluids were tested for possible precipitation with remimazolam in a simulated y-site administration. Equal volumes of the test drug were injected into the remimazolam solution, examined and photo documented at 1, 15, 30 and 60 min after mixture. Examination was taken by two independent investigators. pH was measured before, and 60 min after mixing the drugs. RESULTS: Seventeen (26.15%) drugs or fluids showed precipitation, 47 (72.31%) did not show any sign of interaction. Propofol could not be assessed, because of the turbidity of the substance itself. Precipitation occurred immediately and remained stable in all timestamps. The incompatible drug-remimazolam-mixtures had a median pH of 7.15 (6.67, 8.01), the non-precipitating mixtures a median pH of 4.75 (3.8, 5.6). The pH-values of both groups were significantly different (Mann-Whitney-U-test; p < .00001). There is an increasing risk for precipitation with more basic baseline pH-levels of the tested drug. No interaction was seen in baseline pH below 5. CONCLUSIONS: Remimazolam (Byfavo®) is incompatible with ampicillin/ sulbactam, calcium gluconate, clindamycin, dexamethasone, dimenhydrinate, an 148mval/l electrolyte - glucose 1% solution (E148G1®), furosemide, a 4% gelatine volume expander (gelafundin®), heparin sodium, insulin, meropenem, sodium bicarbonate 8.4%, prednisolone, the crystalloid infusions jonosteril® and sterofundin®, thiopental and tranexamic acid. The results strongly affirm remimazolam's safety requirements: A separate line for remimazolam and an approved compatible baseline infusion is mandatory and an alternative way to administer bolus medication is required.

Allopregnanolone Enhances GABAergic Inhibition in Spinal Motor Networks.

The neurosteroid allopregnanolone (ALLO) causes unconsciousness by allosteric modulation of γ-aminobutyric acid type A (GABAA) receptors, but its actions on the spinal motor networks are unknown. We are therefore testing the hypothesis that ALLO attenuates the action potential firing of spinal interneurons and motoneurons predominantly via enhancing tonic, but not synaptic GABAergic inhibition. We used video microscopy to assess motoneuron-evoked muscle activity in organotypic slice cultures prepared from the spinal cord and muscle tissue. Furthermore, we monitored GABAA receptor-mediated currents by performing whole-cell voltage-clamp recordings. We found that ALLO (100 nM) reduced the action potential firing of spinal interneurons by 27% and that of α-motoneurons by 33%. The inhibitory effects of the combination of propofol (1 µM) and ALLO on motoneuron-induced muscle contractions were additive. Moreover, ALLO evoked a tonic, GABAA receptor-mediated current (amplitude: 41 pA), without increasing phasic GABAergic transmission. Since we previously showed that at a clinically relevant concentration of 1 µM propofol enhanced phasic, but not tonic GABAergic inhibition, we conclude that ALLO and propofol target distinct subpopulations of GABAA receptors. These findings provide first evidence that the combined application of ALLO and propofol may help to reduce intraoperative movements and undesired side effects that are frequently observed under total intravenous anesthesia.

Propofol Affects Cortico-Hippocampal Interactions via ß3 Subunit-Containing GABAA Receptors.

BACKGROUND: General anesthetics depress neuronal activity. The depression and uncoupling of cortico-hippocampal activity may contribute to anesthetic-induced amnesia. However, the molecular targets involved in this process are not fully characterized. GABAA receptors, especially the type with ß3 subunits, represent a main molecular target of propofol. We therefore hypothesized that GABAA receptors with ß3 subunits mediate the propofol-induced disturbance of cortico-hippocampal interactions. METHODS: We used local field potential (LFP) recordings from chronically implanted cortical and hippocampal electrodes in wild-type and ß3(N265M) knock-in mice. In the ß3(N265M) mice, the action of propofol via ß3subunit containing GABAA receptors is strongly attenuated. The analytical approach contained spectral power, phase locking, and mutual information analyses in the 2-16 Hz range to investigate propofol-induced effects on cortico-hippocampal interactions. RESULTS: Propofol caused a significant increase in spectral power between 14 and 16 Hz in the cortex and hippocampus of wild-type mice. This increase was absent in the ß3(N265M) mutant. Propofol strongly decreased phase locking of 6-12 Hz oscillations in wild-type mice. This decrease was attenuated in the ß3(N265M) mutant. Finally, propofol reduced the mutual information between 6-16 Hz in wild-type mice, but only between 6 and 8 Hz in the ß3(N265M) mutant. CONCLUSIONS: GABAA receptors containing ß3 subunits contribute to frequency-specific perturbation of cortico-hippocampal interactions. This likely explains some of the amnestic actions of propofol.

Diazepam and ethanol differently modulate neuronal activity in organotypic cortical cultures.

BACKGROUND: The pharmacodynamic results of diazepam and ethanol administration are similar, in that each can mediate amnestic and sedative-hypnotic effects. Although each of these molecules effectively reduce the activity of central neurons, diazepam does so through modulation of a more specific set of receptor targets (GABAA receptors containing a γ-subunit), while alcohol is less selective in its receptor bioactivity. Our investigation focuses on divergent actions of diazepam and ethanol on the firing patterns of cultured cortical neurons. METHOD: We used electrophysiological recordings from organotypic slice cultures derived from Sprague-Dawley rat neocortex. We exposed these cultures to either diazepam (15 and 30 µM, n = 7) or ethanol (30 and 60 mM, n = 11) and recorded the electrical activity at baseline and experimental conditions. For analysis, we extracted the episodes of spontaneous activity, i.e., cortical up-states. After separation of action potential and local field potential (LFP) activity, we looked at differences in the number of action potentials, in the spectral power of the LFP, as well as in the coupling between action potential and LFP phase. RESULTS: While both substances seem to decrease neocortical action potential firing in a not significantly different (p = 0.659, Mann-Whitney U) fashion, diazepam increases the spectral power of the up-state without significantly impacting the spectral composition, whereas ethanol does not significantly change the spectral power but the oscillatory architecture of the up-state as revealed by the Friedman test with Bonferroni correction (p < 0.05). Further, the action potential to LFP-phase coupling reveals a synchronizing effect of diazepam for a wide frequency range and a narrow-band de-synchronizing effect for ethanol (p < 0.05, Kolmogorov-Smirnov test). CONCLUSION: Diazepam and ethanol, induce specific patterns of network depressant actions. Diazepam induces cortical network inhibition and increased synchronicity via gamma subunit containing GABAA receptors. Ethanol also induces cortical network inhibition, but without an increase in synchronicity via a wider span of molecular targets.

[Development of Anaesthesia-Related Mortality and Impact On Perioperative Outcome]. / Entwicklung der anästhesieassoziierten Letalität und Einfluss auf das Outcome.

Achievements in anaesthesiology form the basis for the tremendous development of surgical therapy. Reliable monitoring technology, which is scrutinized on a regular basis using checklists, is allowing anaesthesia at the borders of physiology. During the last decades, anaesthesia-related mortality has been decreasing considerably. Well-trained anaesthesiology staff, who considers patient- and procedure-specific risks in anaesthetic management, is of major importance. Furthermore, postoperative care in specialised units and intensive care wards is a key factor of improved patient outcome after surgery. In the future, unravelling the interactions of anaesthesia, surgical trauma and postoperative complications will further contribute to improved patient safety.

Synergistic Modulation of γ-Aminobutyric Acid Type A Receptor-Mediated Synaptic Inhibition in Cortical Networks by Allopregnanolone and Propofol.

BACKGROUND: The neuroactive steroid allopregnanolone (ALLO) is an endogenous allosteric modulator of γ-aminobutyric acid type A (GABAA) receptors. There is evidence that ALLO, at physiologically relevant concentrations, modulates GABAA receptor function in the cerebral cortex. The widely used anesthetic agent propofol and ALLO share a similar mode of molecular action. Here, we ask how GABAA receptor-mediated synaptic inhibition and action potential firing of neurons in cultured cortical slices are altered by either ALLO or propofol or by coapplying both agents. METHODS: We explored the effects of ALLO and propofol on spontaneous action potential activity of neocortical neurons in organotypic slices cultured from C57BL6 mice by performing extracellular multiunit recordings. Furthermore, we carried out whole-cell voltage-clamp experiments to quantify the drug effects on GABAA receptor-mediated tonic and phasic currents. RESULTS: We found that ALLO (100 nM) decreased multiunit action potential firing of neocortical neurons by approximately 21%. Moreover, the duration of GABAA receptor-mediated inhibitory postsynaptic currents (IPSCs) was prolonged (mean Δdecay time prolongation: 12.9 ± 2.2 milliseconds; n = 23), and a bicuculline-sensitive tonic current was induced (mean Δbaseline shift: -24.6 ± 13.6 pA; P = .002; n = 6). A subanesthetic concentration of propofol (250 nM) decreased the discharge rates of cortical neurons to a similar degree as ALLO (100 nM). ALLO and propofol administered in combination acted in an additive manner to reduce action potential firing. However, during ALLO administration, propofol was significantly more effective in enhancing GABAergic synaptic transmission. Propofol (250 nM) prolonged the inhibitory postsynaptic currents decay times by 10.4 ± 6.1 milliseconds (n = 9) with ALLO added to the bathing solution; in the absence of ALLO, however, propofol prolonged the decay time by only 3.8 ± 2 milliseconds (n = 13). CONCLUSIONS: In cortical neurons, GABAA receptor-mediated synaptic transmission is potentiated by ALLO and propofol in a synergistic manner, whereas the effects on spontaneous action potential activity appear additive. A coapplication of neurosteroids and propofol in general anesthesia and intensive care medicine may open new ways to reduce anesthetic dose requirements and, thus, avoid undesired anesthetic-induced side effects.

Brain electrical activity obeys Benford's law.

BACKGROUND: Monitoring and automated online analysis of brain electrical activity are frequently used for verifying brain diseases and for estimating anesthetic depth in subjects undergoing surgery. However, false diagnosis with potentially catastrophic consequences for patients such as intraoperative awareness may result from unnoticed irregularities in the process of signal analysis. Here we ask whether Benford's Law can be applied to detect accidental or intended modulation of neurophysiologic signals. This law states that the first digits of many datasets such as atomic weights or river lengths are distributed logarithmically and not equally. In particular, we tested whether data obtained from electrophysiological recordings of human patients representing global activity and organotypic slice cultures representing pure cortical activity follow the predictions of Benford's Law in the absence and in the presence of an anesthetic drug. METHODS: Electroencephalographic (EEG) recordings from human subjects and local field potential recordings from cultured cortical brain slices were obtained before and after administration of sevoflurane. The first digit distribution of the datasets was compared with the Benford distribution. RESULTS: All datasets showed a Benford-like distribution. Nevertheless, distributions belonging to different anesthetic levels could be distinguished in vitro and in human EEGs. With sevoflurane, the first digit distribution of the in vitro data becomes steeper, while it flattens for EEG data. In the presence of high frequency noise, the Benford distribution falls apart. CONCLUSIONS: In vitro and EEG data show a Benford-like distribution which is altered by sevoflurane or destroyed by noise used to simulate artefacts. These findings suggest that algorithms based on Benford's Law can be successfully used to detect sevoflurane-induced signal modulations in electrophysiological recordings.

Preoperative Midazolam and Patient-Centered Outcomes of Older Patients: The I-PROMOTE Randomized Clinical Trial.

Importance: The effect of oral midazolam premedication on patient satisfaction in older patients undergoing surgery is unclear, despite its widespread use. Objective: To determine the differences in global perioperative satisfaction in patients with preoperative administration of oral midazolam compared with placebo. Design, Setting, and Participants: This double-blind, parallel-group, placebo-controlled randomized clinical trial was conducted in 9 German hospitals between October 2017 and May 2019 (last follow-up, June 24, 2019). Eligible patients aged 65 to 80 years who were scheduled for elective inpatient surgery for at least 30 minutes under general anesthesia and with planned extubation were enrolled. Data were analyzed from November 2019 to December 2020. Interventions: Patients were randomized to receive oral midazolam, 3.75 mg (n = 309), or placebo (n = 307) 30 to 45 minutes prior to anesthesia induction. Main Outcomes and Measures: The primary outcome was global patient satisfaction evaluated using the self-reported Evaluation du Vécu de l'Anesthésie Generale (EVAN-G) questionnaire on the first postoperative day. Key secondary outcomes included sensitivity and subgroup analyses of the primary outcome, perioperative patient vital data, adverse events, serious complications, and cognitive and functional recovery up to 30 days postoperatively. Results: Among 616 randomized patients, 607 were included in the primary analysis. Of these, 377 (62.1%) were male, and the mean (SD) age was 71.9 (4.4) years. The mean (SD) global index of patient satisfaction did not differ between the midazolam and placebo groups (69.5 [10.7] vs 69.6 [10.8], respectively; mean difference, -0.2; 95% CI, -1.9 to 1.6; P = .85). Sensitivity (per-protocol population, multiple imputation) and subgroup analyses (anxiety, frailty, sex, and previous surgical experience) did not alter the primary results. Secondary outcomes did not differ, except for a higher proportion of patients with hypertension (systolic blood pressure ≥160 mm Hg) at anesthesia induction in the placebo group. Conclusion and Relevance: A single low dose of oral midazolam premedication did not alter the global perioperative patient satisfaction of older patients undergoing surgery or that of patients with anxiety. These results may be affected by the low dose of oral midazolam. Further trials-including a wider population with commonplace low-dose intravenous midazolam and plasma level measurements-are needed. Trial Registration: ClinicalTrials.gov Identifier: NCT03052660.

Occurrence and Severity of Venous Air Embolism During Neurosurgical Procedures: Semisitting Versus Supine Position.

BACKGROUND: At our institution, patients undergoing neurosurgical procedures in the posterior cranial fossa are placed either in the semisitting or in the supine position. The major risk of the semisitting positioning is a venous air embolism (VAE), which may, however, also occur in the supine position. METHODS: In a prospective single-center study with 137 patients, we evaluated the occurrence of VAEs in patients in the supine and in the semisitting position during the period from January 2014 until April 2015. All patients were monitored for VAE by the use of a transesophageal echocardiography (TEE). RESULTS: In total, 50% of the patients experienced a VAE (56% of these patients underwent surgery in the semisitting and 11% in the supine position). In total, 86% of the VAEs were detected by the use of a TEE and did not lead to any changes in the end-expiratory CO2. We only observed VAEs with a decrease in end-expiratory CO2 in the semisitting position. However, none of the patients had any hemodynamic changes due to the VAE. CONCLUSIONS: The semisitting position with TEE monitoring and a standardized protocol is a safe and advantageous technique, taking account of a significant rate of VAEs. VAEs also occur in the supine position, but less frequently.

Identification and characterization of anesthetic targets by mouse molecular genetics approaches.

PURPOSE: It is now generally accepted that proteins are the primary targets of general anesthetics. However, the demonstration that the activity of a protein is altered by general anesthetics at clinically relevant concentrations in vitro does not provide direct evidence that this target mediates pharmacological actions of general anesthetics. Here we report on advances that have been made in identifying the contribution of individual ligand-gated ion channels to defined anesthetic endpoints using molecular mouse genetics. PRINCIPAL FINDINGS: Gamma-aminobutyric acid (GABA)(A) receptor subtypes defined by the presence of the α1, α4, α5, ß2, and ß3 subunits and two-pore domain potassium channels (TASK-1, TASK-3, and TREK) have been discovered to mediate, at least in part, the hypnotic, immobilizing or amnestic actions of intravenous and volatile general anesthetics. Moreover, using tissues from genetically modified mice, specific functions of GABA(A) receptor subtypes in cortical and spinal neuronal networks were identified. CONCLUSION: Genetically modified mice have been very useful for research on mechanisms of anesthesia and have contributed to the functional identification of general anesthetic targets and of the role of these targets in neuronal networks.

Outcomes in Critically Ill Patients Sedated with Intravenous Lormetazepam or Midazolam: A Retrospective Cohort Study.

The benzodiazepine, midazolam, is one of the most frequently used sedatives in intensive care medicine, but it has an unfavorable pharmacokinetic profile when continuously applied. As a consequence, patients are frequently prolonged and more deeply sedated than intended. Due to its distinct pharmacological features, including a cytochrome P450-independent metabolization, intravenous lormetazepam might be clinically advantageous compared to midazolam. In this retrospective cohort study, we compared patients who received either intravenous lormetazepam or midazolam with respect to their survival and sedation characteristics. The cohort included 3314 mechanically ventilated, critically ill patients that received one of the two drugs in a tertiary medical center in Germany between 2006 and 2018. A Cox proportional hazards model with mortality as outcome and APACHE II, age, gender, and admission mode as covariates revealed a hazard ratio of 1.75 [95% CI 1.46-2.09; p < 0.001] for in-hospital mortality associated with the use of midazolam. After additionally adjusting for sedation intensity, the HR became 1.04 [95% CI 0.83-1.31; p = 0.97]. Thus, we concluded that excessive sedation occurs more frequently in critically ill patients treated with midazolam than in patients treated with lormetazepam. These findings require further investigation in prospective trials to assess if lormetazepam, due to its ability to maintain light sedation, might be favorable over other benzodiazepines for sedation in the ICU.

Diazepam decreases action potential firing of neocortical neurons via two distinct mechanisms.

BACKGROUND: Benzodiazepines are widely used in clinical anesthesia as premedication, but also to induce general anesthesia. Recent in vitro studies suggest that γ-aminobutyric acid type A receptors, harboring a classical high-affinity benzodiazepine binding site, possess another "nonclassical" binding site for benzodiazepines. At present, it is unclear if, and to what extent, this novel nonclassical binding site is of relevance for the actions of benzodiazepines in the central nervous system. METHODS: Because neocortex is involved in mediating the sedative and hypnotic properties of general anesthetics, we quantified the actions of diazepam over a wide range of concentrations (from 10 nM up to 100 µM) in organotypic slice cultures using extracellular multiunit recordings of spontaneous action potential activity. RESULTS: Up to a concentration of 6.25 µM, diazepam reduced the activity of neocortical neurons, approaching a maximum of approximately 20%. This action was nullified by the benzodiazepine antagonist flumazenil. At concentrations >12.5 µM, diazepam evoked a second concentration-dependent dampening of network activity. Unlike the low concentration effect, this high concentration component was resistant to flumazenil. CONCLUSIONS: Diazepam induced a biphasic attenuation of spontaneous action potential firing of neocortical neurons. Low to moderate concentrations caused a monotonic, mild depression that is mediated via the classical binding site as it is antagonized by flumazenil. However, the effects of diazepam observed at high concentrations were not affected by flumazenil. Hence, these findings support the concept of at least 2 different binding sites for benzodiazepines on γ-aminobutyric acid type A receptors. Furthermore, our results are consistent with the hypothesis that the classical high-affinity binding site mediates low-dose diazepam actions, such as amnesia, anxiolysis, and sedation, while a second, nonclassical and independent site contributes to the anesthetic effects of diazepam, such as hypnosis and immobility.

Impact of soman and acetylcholine on the effects of propofol in cultured cortical networks.

Patients intoxicated with organophosphorous compounds may need general anaesthesia to enable mechanical ventilation or for control of epileptiform seizures. It is well known that cholinergic overstimulation attenuates the efficacy of general anaesthetics to reduce spontaneous network activity in the cortex. However, it is not clear how propofol, the most frequently used intravenous anaesthetic today, is affected. Here, we investigated the effects of cholinergic overstimulation induced by soman and acetylcholine on the ability of propofol to depress spontaneous action potential activity in organotypic cortical slices measured by extracellular voltage recordings. Cholinergic overstimulation by co-application of soman and acetylcholine (10 µM each) did not reduce the relative inhibition of propofol (1.0 µM; mean normalized action potential firing rate 0.49 ± 0.06 of control condition, p < 0.001, Wilcoxon signed rank test) but clearly reduced its efficacy. Co-application of atropine (10 nM) did not improve the efficacy. Propofol preserved its relative inhibitory potential but did not produce a degree of neuronal depression which can be expected to assure hypnosis in humans. Since a combination with atropine did not improve its efficacy, an increase in dosage will probably be necessary when propofol is used in victims suffering from organophosphorous intoxication.

Impact of PReOperative Midazolam on OuTcome of Elderly patients (I-PROMOTE): study protocol for a multicentre randomised controlled trial.

INTRODUCTION: Premedication of surgical patients with benzodiazepines has become questionable regarding risk-benefit ratio and lack of evidence. Though preoperative benzodiazepines might alleviate preoperative anxiety, a higher risk for adverse events is described, particularly for elderly patients (≥ 65 years). Several German hospitals already withhold benzodiazepine premedication from elderly patients, though evidence for this approach is lacking. The patient-centred outcome known as global postoperative patient satisfaction is recognised as a substantial quality indicator of anaesthesia care incorporated by the American Society of Anesthesiologists. Therefore, we aim to assess whether the postoperative patient satisfaction after premedication with placebo compared to the preoperative administration of 3.75 mg midazolam in elderly patients differs. METHODS: This study is a multicentre, randomised, placebo-controlled, double-blinded, two-arm parallel, interventional trial, conducted in nine German hospitals. In total 614 patients (≥ 65-80 years of age) undergoing elective surgery with general anaesthesia will be randomised to receive either 3.75 mg midazolam or placebo. The primary outcome (global patient satisfaction) will be assessed with the validated EVAN-G questionnaire on the first postoperative day. Secondary outcomes will be assessed until the first postoperative day and then 30 days after surgery. They comprise among other things: functional and cognitive recovery, postoperative delirium, health-related quality of life assessment, and mortality or new onset of serious cardiac or pulmonary complications, acute stroke, or acute kidney injury. Analysis will adhere to the intention-to-treat principle. The primary outcome will be analysed with the use of mixed linear models including treatment effect and study centre as factors and random effects for blocks. Exploratory adjusted and subgroup analyses of the primary and secondary outcomes with regard to gender effects, frailty, pre-operative anxiety level, patient demographics, and surgery experience will also be performed. DISCUSSION: This is, to the best of our knowledge, the first study analysing patient satisfaction after premedication with midazolam in elderly patients. In conclusion, this study will provide high-quality data for the decision-making process regarding premedication in elderly surgical patients. TRIAL REGISTRATION: ClinicalTrials.gov, NCT03052660 . Registered on 14 February 2017. EudraCT 2016-004555-79 .

Midazolam is effective to reduce cortical network activity in organotypic cultures during severe cholinergic overstimulation with soman.

Intoxication with organophosphorus compounds can result in life-threatening organ dysfunction and refractory seizures. Sedation or hypnosis is essential to facilitate mechanical ventilation and control seizure activity. The range of indications for midazolam includes both hypnosis and seizure control. Since benzodiazepines cause sedation and hypnosis by dampening neuronal activity of the cerebral cortex, we investigated the drug's effect on action potential firing of cortical neurons in brain slices. Extensive cholinergic overstimulation was induced by increasing acetylcholine levels and simultaneously treating the slices with soman to block acetylcholinesterase activity. At control conditions midazolam reduced discharge rates (median/95% confidence interval) from 8.8 (7.0-10.5) Hz (in the absence of midazolam) to 2.2 (1.4-2.9) Hz (10 µM midazolam) and 1.6 (0.9-2.2) Hz (20 µM midazolam). Without midazolam, cholinergic overstimulation significantly enhanced neuronal activity to 13.1 (11.0-15.2) Hz. Midazolam attenuated firing rates during cholinergic overstimulation to 6.5 (4.8-8.2) Hz (10 µM midazolam) and 4.1 (3.3-6.0) Hz (20 µM midazolam), respectively. Thus, high cholinergic tone attenuated the drug's efficacy only moderately. These results suggest that midazolam is worth being tested as a promising drug to induce sedation and hypnosis in patients suffering from severe organophosphorous intoxication.

Zolpidem Activation of Alpha 1-Containing GABAA Receptors Selectively Inhibits High Frequency Action Potential Firing of Cortical Neurons.

Introduction: High frequency neuronal activity in the cerebral cortex can be induced by noxious stimulation during surgery, brain injury or poisoning. In this scenario, it is essential to block cortical hyperactivity to protect the brain against damage, e.g., by using drugs that act as positive allosteric modulators at GABAA receptors. Yet, cortical neurons express multiple, functionally distinct GABAA receptor subtypes. Currently there is a lack of knowledge which GABAA receptor subtypes would be a good pharmacological target to reduce extensive cortical activity. Methods: Spontaneous action potential activity was monitored by performing extracellular recordings from organotypic neocortical slice cultures of wild type and GABAAR-α1(H101R) mutant mice. Phases of high neuronal activity were characterized using peri-event time histograms. Drug effects on within-up state firing rates were quantified via Hedges' g. Results: We quantified the effects of zolpidem, a positive modulator of GABAA receptors harboring α1-subunits, and the experimental benzodiazepine SH-053-2'F-S-CH3, which preferably acts at α2/3/5- but spares α1-subunits. Both agents decreased spontaneous action potential activity but altered the firing patterns in different ways. Zolpidem reduced action potential firing during highly active network states. This action was abolished by flumazenil, suggesting that it was mediated by benzodiazepine-sensitive GABAA receptors. SH-053-2'F-S-CH3 also attenuated neuronal activity, but unlike zolpidem, failed to reduce high frequency firing. To confirm that zolpidem actions were indeed mediated via α1-dependent actions, it was evaluated in slices from wild type and α(H101R) knock-in mice. Inhibition of high frequency action potential firing was observed in slices from wild type but not mutant mice. Conclusion: Our results suggest that during episodes of scarce and high neuronal activity action potential firing of cortical neurons is controlled by different GABAA receptor subtypes. Exaggerated firing of cortical neurons is reduced by positive modulation of α1-, but not α2/3/5-subunit containing GABAA receptors.

Effects of cholinergic overstimulation on isoflurane potency and efficacy in cortical and spinal networks.

In scenarios of terrorist attacks with organophosphorus compounds it appears likely that medical aid is required by victims not only suffering from the intoxication but also from physical trauma. These subjects may have to undergo surgical interventions, raising the need for anaesthesia. This prompts the question of how anaesthetic agents work in intoxicated patients. Organophosphates block acetylcholinesterase activity, thereby inducing excessive cholinergic overstimulation in the central nervous system. As the neocortex and spinal cord are important substrates for general anaesthetics, we investigated to what extent cholinergic overstimulation affects the potency and efficacy of the commonly used volatile anaesthetic isoflurane in depressing action potential activity of cortical and spinal neurons. We first quantified the effects of isoflurane in the absence of acetylcholine by performing extracellular voltage recordings in cultured tissue slices. Isoflurane induced a concentration-dependent decrease of neuronal activity in neocortical (EC(50)=0.43+/-0.08 MAC) and spinal slices (EC(50)=0.41+/-0.03 MAC). At concentrations above 1.5 MAC, the anaesthetic almost completely depressed action potential firing in both preparations. Next, we studied the effects of acetylcholine (10microM) in the absence of isoflurane. Acetylcholine approximately doubled spontaneous activity in neocortical and spinal slices. When applying isoflurane together with acetylcholine, different interactions between these agents were observed in neocortical and spinal networks. Acetylcholine significantly reduced both the potency and efficacy of the anaesthetic in neocortical (efficacy 83%; EC(50)=1.16+/-0.02 MAC) but not in spinal (efficacy 100%; EC(50)=0.41+/-0.04 MAC) slices. Our results indicate that cholinergic overstimulation increases the requirement for anaesthetic agents in patients suffering from organophosphorus poisoning via enhancing neuronal background activity of neocortical and spinal neurons and in addition via decreasing drug potency and efficacy in the cortex. Raising anaesthetic concentrations into a high-dose range may not be an appropriate alternative to compensate the increased excitability, since high concentrations of anaesthetics may worsen cardiac abnormalities and hemodynamic instability frequently observed in these patients.

Differential depression of neuronal network activity by midazolam and its main metabolite 1-hydroxymidazolam in cultured neocortical slices.

The benzodiazepine midazolam is widely used in critical care medicine. Midazolam has a clinically active metabolite, 1-hydroxymidazolam. The contribution of 1-hydroxymidazolam to the effects of midazolam is controversial. The aim of the current study was to compare the actions of midazolam and 1-hydroxymidazolam on network activity of cortical neurons. Midazolam depressed neuronal activity at a low concentration of 5 nM. When midazolam concentration was increased, it depressed neuronal discharge rates in a biphasic manner. In comparison, 1-hydroxymidazolam did not depress the cortical network activity at low nanomolar concentrations. Higher concentrations of 1-hydroxymidazolam consistently inhibited neuronal activity. Moreover, midazolam shortened cortical up states at low, but not at high concentrations, while the opposite effect was observed with 1-hydroxymidazolam. The network depressant action of midazolam at low concentrations was absent in slices from GABAA receptor α1(H101R)mutant mice. The α1(H101R)mutation renders α1-subunit containing GABAA receptors insensitive towards benzodiazepines. This GABAA receptor subtype is thought to mediate sedation. As midazolam is more potent than its metabolite 1-hydroxymidazolam, the major clinical effects are thus likely caused by midazolam itself. However, 1-hydroxymidazolam could add to the effects of midazolam, especially after the application of high doses of midazolam, and in case of impaired drug metabolism.