Neurosteroids Mediate Neuroprotection in an In Vitro Model of Hypoxic/Hypoglycaemic Excitotoxicity via δ-GABAA Receptors without Affecting Synaptic Plasticity.

Neurosteroids and benzodiazepines are modulators of the GABAA receptors, thereby causing anxiolysis. Furthermore, benzodiazepines such as midazolam are known to cause adverse side-effects on cognition upon administration. We previously found that midazolam at nanomolar concentrations (10 nM) blocked long-term potentiation (LTP). Here, we aim to study the effect of neurosteroids and their synthesis using XBD173, which is a synthetic compound that promotes neurosteroidogenesis by binding to the translocator protein 18 kDa (TSPO), since they might provide anxiolytic activity with a favourable side-effect profile. By means of electrophysiological measurements and the use of mice with targeted genetic mutations, we revealed that XBD173, a selective ligand of the translocator protein 18 kDa (TSPO), induced neurosteroidogenesis. In addition, the exogenous application of potentially synthesised neurosteroids (THDOC and allopregnanolone) did not depress hippocampal CA1-LTP, the cellular correlate of learning and memory. This phenomenon was observed at the same concentrations that neurosteroids conferred neuroprotection in a model of ischaemia-induced hippocampal excitotoxicity. In conclusion, our results indicate that TSPO ligands are promising candidates for post-ischaemic recovery exerting neuroprotection, in contrast to midazolam, without detrimental effects on synaptic plasticity.

Midazolam at Low Nanomolar Concentrations Affects Long-term Potentiation and Synaptic Transmission Predominantly via the α1-γ-Aminobutyric Acid Type A Receptor Subunit in Mice.

BACKGROUND: Midazolam amplifies synaptic inhibition via different γ-aminobutyric acid type A (GABAA) receptor subtypes defined by the presence of α1-, α2-, α3-, or α5-subunits in the channel complex. Midazolam blocks long-term potentiation and produces postoperative amnesia. The aims of this study were to identify the GABAA receptor subtypes targeted by midazolam responsible for affecting CA1 long-term potentiation and synaptic inhibition in neocortical neurons. METHODS: The effects of midazolam on hippocampal CA1 long-term potentiation were studied in acutely prepared brain slices of male and female mice. Positive allosteric modulation on GABAA receptor-mediated miniature inhibitory postsynaptic currents was investigated in organotypic slice cultures of the mouse neocortex. In both experiments, wild-type mice and GABAA receptor knock-in mouse lines were compared in which α1-, α5-, α1/2/3-, α1/3/5- and α2/3/5-GABAA receptor subtypes had been rendered benzodiazepine-insensitive. RESULTS: Midazolam (10 nM) completely blocked long-term potentiation (mean ± SD, midazolam, 98 ± 11%, n = 14/8 slices/mice vs. control 156 ± 19%, n = 20/12; P < 0.001). Experiments in slices of α1-, α5-, α1/2/3-, α1/3/5-, and α2/3/5-knock-in mice revealed a dominant role for the α1-GABAA receptor subtype in the long-term potentiation suppressing effect. In slices from wild-type mice, midazolam increased (mean ± SD) charge transfer of miniature synaptic events concentration-dependently (50 nM: 172 ± 71% [n = 10/6] vs. 500 nM: 236 ± 54% [n = 6/6]; P = 0.041). In α2/3/5-knock-in mice, charge transfer of miniature synaptic events did not further enhance when applying 500 nM midazolam (50 nM: 171 ± 62% [n = 8/6] vs. 500 nM: 175 ± 62% [n = 6/6]; P = 0.454), indicating two different binding affinities for midazolam to α2/3/5- and α1-subunits. CONCLUSIONS: These results demonstrate a predominant role of α1-GABAA receptors in the actions of midazolam at low nanomolar concentrations. At higher concentrations, midazolam also enhances other GABAA receptor subtypes. α1-GABAA receptors may already contribute at sedative doses to the phenomenon of postoperative amnesia that has been reported after midazolam administration.

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.

Effects of Diazepam on Low-Frequency and High-Frequency Electrocortical γ-Power Mediated by α1- and α2-GABAA Receptors.

Patterns of spontaneous electric activity in the cerebral cortex change upon administration of benzodiazepines. Here we are testing the hypothesis that the prototypical benzodiazepine, diazepam, affects spectral power density in the low (20-50 Hz) and high (50-90 Hz) γ-band by targeting GABAA receptors harboring α1- and α2-subunits. Local field potentials (LFPs) and action potentials were recorded in the barrel cortex of wild type mice and two mutant strains in which the drug exclusively acted via GABAA receptors containing either α1- (DZα1-mice) or α2-subunits (DZα2-mice). In wild type mice, diazepam enhanced low γ-power. This effect was also evident in DZα2-mice, while diazepam decreased low γ-power in DZα1-mice. Diazepam increased correlated local LFP-activity in wild type animals and DZα2- but not in DZα1-mice. In all genotypes, spectral power density in the high γ-range and multi-unit action potential activity declined upon diazepam administration. We conclude that diazepam modifies low γ-power in opposing ways via α1- and α2-GABAA receptors. The drug's boosting effect involves α2-receptors and an increase in local intra-cortical synchrony. Furthermore, it is important to make a distinction between high- and low γ-power when evaluating the effects of drugs that target GABAA receptors.

GABA(A) receptor-targeted drug development -New perspectives in perioperative anesthesia.

Introduction: Perioperative anesthesia delivers pre-, intra-, and postoperative care to meet the needs of patients undergoing diagnostic and surgical procedures. Major challenges are patients at the extremes of age and individuals with a pre-existing disease burden. Frequent problems are the development of chronic pain and cognitive dysfunction upon surgery. Current perioperative pharmacotherapy utilizes a number of drugs acting at GABAA receptors. Area covered: This review evaluates novel formulations and newly designed GABAergic drugs, offering future improvements in perioperative anesthesia, especially for reducing mortality and avoiding cognitive dysfunction and chronic pain as an outcome of surgery. Expert opinion: There are multiple reasons for mounting efforts in the development of novel GABAergic medications. First, requirements in perioperative anesthesia care have substantially changed during the last two decades. In this respect, the dramatic increase in life expectancy is the most important factor. Moreover, research has considerably expanded our knowledge of how drugs in current clinical use act on the molecular level. Almost all ongoing developmental programs choose chemical structures of well-tried agents as a starting point for exploring the properties of structural analogs. This strategy aims to maintain the clinically desired actions of mother compounds while attempting to extinguish adverse side effects.

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.

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.

New insights in the systemic and molecular underpinnings of general anesthetic actions mediated by γ-aminobutyric acid A receptors.

PURPOSE OF REVIEW: The review highlights novel insights into the role of γ-aminobutyric acid A (GABAA) receptors in mediating clinically relevant actions of anesthetic agents. RECENT FINDINGS: GABAA receptors in the hippocampus are located on glutamatergic pyramidal cells and GABAergic interneurons. Etomidate-induced inhibition of a synaptic correlate of learning and memory is caused by receptors on nonpyramidal neurons, likely on interneurons that incorporate α5 subunits. Selective enhancement of α2 subunit containing GABAA receptors in the spinal cord provides antihyperalgesia against inflammatory and neuropathic pain without causing sedation, motor impairment, and tolerance development. Inflammation, traumatic brain injury, and exposure to anesthetic agents modify the expression patterns of GABAA receptors in a subtype-specific manner. These modifications may persist for weeks. The neuroactive steroid alphaxalone causes fast-onset and short-duration anesthesia in humans. Cardiovascular and respiratory side-effects are less severe than with propofol. SUMMARY: Identification of the molecular and cellular substrates involved in anesthesia and insights into disease and drug-induced alterations in the expression patterns of GABAA receptors in the central nervous system are emphasizing the need for individualized anesthesia care. Introducing neuroactive steroids into clinical anesthesia is expected to reduce cardiovascular and respiratory side-effects.

Anesthetic actions of thiopental remain largely unaffected during cholinergic overstimulation in cultured cortical networks.

In case of military or terrorist use of organophosphorus (OP) compounds victims are likely to suffer from not only intoxication but physical trauma as well. Appropriate emergency care may therefore include general anesthesia to allow life-saving surgical intervention. Since there is evidence that drug potency and efficacy of several anesthetics are attenuated by high concentrations of acetylcholine in the CNS, this study was designed to evaluate the anesthetic actions of thiopental during cholinergic overstimulation. Making use of organotypic slice cultures derived from the mouse neocortex, drug effects were assessed by extracellular voltage recordings of network activity at basal cholinergic tone and during simulated cholinergic crisis (high cholinergic tone). The latter was achieved by inhibition of acetylcholinesterases via soman and an ambient acetylcholine concentration of 10µM. The induction of cholinergic crisis in vitro increased the network activity of cortical neurons significantly. Surprisingly, differences in network activity between basal and high cholinergic tone became less pronounced with rising concentrations of thiopental and drug potency and efficacy were almost equivalent. These results clearly distinguish thiopental from previously tested general anesthetics and make it a promising candidate for in vivo studies to identify suitable anesthetics for victims of OP intoxication.

Self-regeneration of neuromuscular function following soman and VX poisoning in spinal cord-skeletal muscle cocultures.

Aside from nerve agents, various highly toxic pesticides belong to the group of organophosphorus (OP) compounds, thereby causing a large number of intoxications every year. Unfortunately, there are still shortcomings in the current treatment for OP poisoning and research on novel therapeutic options is restricted in several aspects. In this study we investigated the suitability of organotypic cocultures for pharmacological in vitro studies involving OP compounds. These slice cultures are derived from murine spinal cord and muscle tissue forming functional neuromuscular synapses, which trigger spontaneous contractions of muscle fibers. Using video microscopy to quantify muscle activity, we assessed the viability of cocultures after exposure to soman and VX, and the associated loss and recovery of neuromuscular function. Antidotal treatment was not provided. The application of nerve agents led to an almost complete loss of muscle activity. However, cell cultures regained equivalent muscular function to the control situation three and seven days after intoxication. In summary, the tested in vitro system could be a promising tool for the investigation of long term effects and therapeutic options for OP poisoning.

Botulinum toxin B increases intrinsic muscle activity in organotypic spinal cord-skeletal muscle co-cultures.

In organotypic spinal cord-skeletal muscle co-cultures, motoneurons are driven by locomotor commands and induce contractions in surrounding muscle fibres. Using these co-cultures, it has been shown that effects of organophosphorus compounds on neuromuscular synapses can be determined in vitro. In the present study we aimed to extend this in vitro tool for pharmacologic testing of botulinum toxin B. This neurotoxin is widely used for the treatment of dystonia. Besides its effects on the neuromuscular junction, botulinum toxins may also act at centrally located synapses. Incubation with botulinum toxin B (Neurobloc(®)) induced a significant increase in muscular activity after 24, 48 and 72h. Application of the NMDA- and AMPA-receptor antagonists AP5 (20µM) and CNQX (15µM) induced a similar augmentation of muscle activity after 48 and 72h, respectively. Administration of the glycine- and GABA(A)-receptor antagonists strychnine (1µM) and bicuculline (100µM) did not alter intrinsic muscle activity. In contrast, application of a non-depolarizing muscle relaxant rocuronium bromide reduced the muscle activity in a dose-dependent manner. Our findings suggest that glutamatergic synapses in the spinal cord are more sensitive to botulinum toxin B than synaptic contacts between spinal motoneurons and muscle fibres.

Closing the gap between the molecular and systemic actions of anesthetic agents.

Genetic approaches have been successfully used to relate the diverse molecular actions of anesthetic agents to their amnestic, sedative, hypnotic, and immobilizing properties. The hypnotic effect of etomidate, quantified as the duration of the loss of righting reflex in mice, is equally mediated by GABAA receptors containing ß2- and ß3-protein subunits. However, only ß3-containing receptors are involved in producing electroencephalogram (EEG)-patterns typical of general anesthesia. The sedative action of diazepam is produced by α1-subunit-containing receptors, but these receptors do not contribute to the drug's characteristic EEG-"fingerprint." Thus, GABAA receptors with α1- and ß2-subunits take a central role in causing benzodiazepine-induced sedation and etomidate-induced hypnosis, but the corresponding EEG-signature is difficult to resolve. Contrastingly, actions of etomidate and benzodiazepines mediated via α2- and ß3-subunits modify rhythmic brain activity in vitro and in vivo at least in part by enhancing neuronal synchrony. The immobilizing action of GABAergic anesthetics predominantly involves ß3-subunit-containing GABAA receptors in the spinal cord. Interestingly, this action is self-limiting as GABA-release is attenuated via the same receptors. Anesthetic-induced amnesia is in part mediated by GABAA receptors harboring α5-subunits that are highly enriched in the hippocampus and, in addition, by α1-containing receptors in the forebrain. Because there is accumulating evidence that in patients the expression pattern of GABAA receptor subtypes varies with age, is altered by the long-term use of drugs, and is affected by pathological conditions like inflammation and sepsis, further research is recommended to adapt the use of anesthetic agents to the specific requirements of individual patients.

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.

Impairment of GABA transporter GAT-1 terminates cortical recurrent network activity via enhanced phasic inhibition.

In the central nervous system, GABA transporters (GATs) very efficiently clear synaptically released GABA from the extracellular space, and thus exert a tight control on GABAergic inhibition. In neocortex, GABAergic inhibition is heavily recruited during recurrent phases of spontaneous action potential activity which alternate with neuronally quiet periods. Therefore, such activity should be quite sensitive to minute alterations of GAT function. Here, we explored the effects of a gradual impairment of GAT-1 and GAT-2/3 on spontaneous recurrent network activity--termed network bursts and silent periods--in organotypic slice cultures of rat neocortex. The GAT-1 specific antagonist NO-711 depressed activity already at nanomolar concentrations (IC50 for depression of spontaneous multiunit firing rate of 42 nM), reaching a level of 80% at 500-1000 nM. By contrast, the GAT-2/3 preferring antagonist SNAP-5114 had weaker and less consistent effects. Several lines of evidence pointed toward an enhancement of phasic GABAergic inhibition as the dominant activity-depressing mechanism: network bursts were drastically shortened, phasic GABAergic currents decayed slower, and neuronal excitability during ongoing activity was diminished. In silent periods, NO-711 had little effect on neuronal excitability or membrane resistance, quite in contrast to the effects of muscimol, a GABA mimetic which activates GABAA receptors tonically. Our results suggest that an enhancement of phasic GABAergic inhibition efficiently curtails cortical recurrent activity and may mediate antiepileptic effects of therapeutically relevant concentrations of GAT-1 antagonists.