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.

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.

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.

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.

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.

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.

[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.

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.

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.

Spinal cord--skeletal muscle cocultures detect muscle-relaxant action of botulinum neurotoxin A.

The mouse LD50 assay is routinely used for potency testing of botulinum toxins. Unfortunately, this test is associated with severe pain and distress in animals and requires large quantities of mice. Here we used cocultures of spinal cord and muscle tissue as an alternative for probing botulinum toxins. Cocultures were prepared from mouse embryonic tissue (C57/BL6J) and cultured for 24-27 days. In these cultures spontaneous muscle activity was quantified in sham- and botulinum toxin-treated cultures for up to 3 days by video microscopy. At a concentration of 58 fmol/L or higher, incobotulinumtoxin A significantly reduced the frequency of muscle contractions within 24 hours after incubation. Hence, nerve-muscle-cultures are similar sensitive as the mouse LD50 assay. The limit of detection, as observed in our study, is close to the most sensitive cell-based bioassays, capable to detect concentrations of botulinum neurotoxin A between 30 and 50 fmol/L. However, spontaneous muscle activity of individual cultures displayed considerable fluctuations when evaluated on a day-to-day basis. Generally, the authors would like to emphasize, that in its present form, this in vitro assay might be too laborious for botulinum toxin potency testing. Thus, methodical improvements to decrease data variability are the next milestone to be passed towards developing this model into an assay that can be utilized for reducing animal experimentation.

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.

Effects of succinylcholine in an organotypic spinal cord-skeletal muscle coculture of embryonic mice.

Intoxication with organophosphorus compounds is an important clinical problem worldwide. Although the core treatments - atropine, oximes and diazepam - are defined, high case fatalities were reported for intoxication with organophosphorus insecticides. In particular the role of oximes is not completely understood since they might benefit only patients poisoned by specific pesticides or patients with moderate poisoning and few randomised trials of such poisoning have been performed. This justifies the need for new in vitro test-systems like cocultures of spinal cord and muscle tissue, which have been recently introduced. However this test-system is not yet fully characterized. In order to estimate the applicability of cocultures of spinal cord and muscle tissue their sensitivity to succinylcholine (di-acetylcholine), a depolarizing muscle relaxant in clinical use, was tested. The test-system evaluated in the current study showed sensitivity to succinylcholine with an EC50 as low as 1µM thereby being close to the EC50 value in adult human patients (2.6µM). Furthermore, action potential activity of spinal ventral horn neurons was not altered by succinylcholine. The latter observations strongly suggest that our preparation well predicts the qualitative and quantitative actions of novel drugs targeting the neuromuscular system in vivo. In summary, cocultures of spinal cord and muscle tissue seem to be a valid test-system for the development and investigation of new oximes. Moreover, practical aspects like transport over long distances to further laboratories, the opportunity to conduct long-term studies and the reduction of animal usage display further advantages of its use.

Sevoflurane-induced loss of consciousness is paralleled by a prominent modification of neural activity during cortical down-states.

Networks of neocortical neurons display a bistable activity pattern characterised by phases of high frequency action potential firing, so called up-states, and episodes of low discharge activity (down-states). We hypothesised that during down-states neocortical neurons are vulnerable to anaesthetic agents. To tackle this issue, it is necessary to identify analytical methods, which are sufficiently sensitive for resolving anaesthetic effects during phases of scarce neuronal activity. The local field potential was recorded in organotypic cultures (OTC) from rat neocortex under control conditions and in the presence of increasing concentrations of sevoflurane by extracellular electrodes. Epochs from down-states were cut from the local field potential and analysed using power spectrum density as well as non-linear parameters approximate entropy (ApEn) and order recurrence rate (ORR). ApEn and ORR proved to be suitable tools for analysing the actions of volatile anaesthetics on cortical down-states. During these phases of low neuronal activity, sevoflurane caused prominent changes in the local field potential. Time series analysis using ApEn showed a reduction of signal predictability in the presence of sevoflurane. Furthermore, the ORR displayed an abrupt decrease at sevoflurane concentrations corresponding to loss of consciousness in vivo, indicating a drug-induced decrease in the signal to noise ratio. The actions of volatile anaesthetics on cortical down-states have been neglected so far, perhaps due to the lack of suitable analysis tools. In the current in vitro study the non-linear parameters ApEn and ORR are introduced to characterise volatile anaesthetics actions. Sevoflurane alters cortical down-states as indicated by non-linear parameter analysis of local field potential recording from cultured neuronal networks. ORR even displays an abrupt change, i.e., a step-like behaviour indicating an increased signal complexity at concentrations of sevoflurane corresponding to loss of consciousness in humans.

Opposing actions of sevoflurane on GABAergic and glycinergic synaptic inhibition in the spinal ventral horn.

BACKGROUND: The ventral horn is a major substrate in mediating the immobilizing properties of the volatile anesthetic sevoflurane in the spinal cord. In this neuronal network, action potential firing is controlled by GABA(A) and glycine receptors. Both types of ion channels are sensitive to volatile anesthetics, but their role in mediating anesthetic-induced inhibition of spinal locomotor networks is not fully understood. METHODOLOGY/PRINCIPAL FINDINGS: To compare the effects of sevoflurane on GABAergic and glycinergic inhibitory postsynaptic currents (IPSCs) whole-cell voltage-clamp recordings from ventral horn interneurons were carried out in organotypic spinal cultures. At concentrations close to MAC (minimum alveolar concentration), decay times of both types of IPSCs were significantly prolonged. However, at 1.5 MAC equivalents, GABAergic IPSCs were decreased in amplitude and reduced in frequency. These effects counteracted the prolongation of the decay time, thereby decreasing the time-averaged GABAergic inhibition. In contrast, amplitudes and frequency of glycinergic IPSCs were not significantly altered by sevoflurane. Furthermore, selective GABA(A) and glycine receptor antagonists were tested for their potency to reverse sevoflurane-induced inhibition of spontaneous action potential firing in the ventral horn. These experiments confirmed a weak impact of GABA(A) receptors and a prominent role of glycine receptors at a high sevoflurane concentration. CONCLUSIONS: At high concentrations, sevoflurane mediates neuronal inhibition in the spinal ventral horn primarily via glycine receptors, and less via GABA(A) receptors. Our results support the hypothesis that the impact of GABA(A) receptors in mediating the immobilizing properties of volatile anesthetics is less essential in comparison to glycine receptors.

Enhancing the function of alpha5-subunit-containing GABAA receptors promotes action potential firing of neocortical neurons during up-states.

Neocortical neurons mediate the sedative and anticonvulsant properties of benzodiazepines. These agents enhance synaptic inhibition via positive modulation of γ-aminobutyric acid (GABAA) receptors harboring α1-, α2-, α3- or α5-protein subunits. Benzodiazepine-sensitive GABAA receptors containing the α5-subunit are abundant in the neocortex, but their impact in controlling neuronal firing patterns is unknown. Here we studied how the discharge rates of cortical neurons are modified by a positive (SH-053-2'F-R-CH3) and a negative (L 655,708) α5-subunit-preferring allosteric modulator in comparison to diazepam, the classical non-selective benzodiazepine. Drug actions were characterized in slice cultures from wild-type and α5(H105R) knock-in mice by performing extracellular multi-unit-recordings. In knock-in mice, receptors containing the α5 subunit are insensitive to benzodiazepines. The non-selective positive allosteric modulator diazepam decreased the discharge rates of neocortical neurons during episodes of ongoing neuronal activity (up states). In contrast to diazepam, the α5-preferring positive modulator SH-053-2'F-R-CH3 accelerated action potential firing during up states. This promoting action was absent in slices from α5(H105R) mice, confirming that it is mediated by the α5-subunit. Consistent with these observations, the negative α5-selective modulator L 655,708 inhibited up state action potential activity in slices from wild-type mice. The opposing actions of diazepam and SH-053-2'F-R-CH3, which both enhance GABAA receptor function but differ in subtype-selectivity, uncovers contrasting roles of GABAA receptor subtypes in controlling the firing rates of cortical neurons. These findings may have important implications for the design of novel anaesthetic and anticonvulsant benzodiazepines displaying an improved efficacy and fewer side effects.