Bestimmte Hirnareale und neuronale Schaltkreise wurden als Entstehungsort von Teilwirkungen der Allgemeinanästhesie eingegrenzt, z.B. thalamocorticale Schleifen für die Ausschaltung des Bewusstseins [1]. In der vorliegenden Studie untersuchten wir die Wirkungen von Isofluran auf die synaptische Transmission im in vitro Präparat der Amygdala. Trotz der anerkannten Bedeutung dieses paläokortikalen Kerngebiets für Schmerzverarbeitung und aversive Gedächtnisinhalte wurde der Einfluss volatiler Anästhetika auf die synaptische Transmission in der Amygdala bislang nicht beschrieben.

Attempts have been made to attribute the particular features of general anaesthesia such as hypnosis, analgesia, amnesia and autonomic stability to certain brain regions. In the present study, we examined the effects of the commonplace volatile anaesthetic isoflurane on synaptic transmission in an in vitro slice preparation of the murine amygdala. Despite the established role of this limbic structure in the formation of aversive memories, conditioned fear and anxiety, as well as pain processing and regulation of sympathetic tone, the influence of volatile anaesthetics on synaptic signalling has not yet been investigated in this region of the brain. Evoked postsynaptic currents were monitored from principal neurons in the basolateral nucleus of the amygdala by means of patch‐clamp recording. The mixed postsynaptic currents were mediated by non‐NMDA, NMDA, GABA A and GABA B receptors. Isoflurane added to the perfusion medium reduced the strength of synaptic signalling following the activation of non‐NMDA, NMDA, and GABA B receptors, whereas the GABA A receptor‐mediated responses were enhanced. The overall reduction of neuronal excitability was also reflected in a reduction of field potential amplitudes. Isoflurane neither changed the membrane resting potential nor the input resistance of principal neurons in the amygdala. The present results may contribute to the understanding of how stress reactions and long‐lasting neuroplastic processes are suppressed under general anaesthesia.

Flupirtine is a centrally acting nonopioid analgesic with muscle-relaxant properties. Flupirtine has been found to activate inwardly rectifying potassium conductances and hence to indirectly inhibit the activation of NMDA receptors. NMDA receptor activation is crucial for the induction of long-term potentiation (LTP) of synaptic transmission, which is considered as cellular correlate of learning and memory and of central sensitization in chronic pain states. Although flupirtine has been widely used for the management of pain, its effects on synaptic plasticity have not yet been investigated. We, therefore, performed extracellular and whole-cell patch-clamp recordings in hippocampal slices of mice to examine the effects of flupirtine on synaptic plasticity and neuronal membrane properties. Excitatory postsynaptic potentials (EPSPs) in the CA1 region were evoked alternately by stimulating two independent Schaffer collateral-commissural inputs. LTP and long-term depression (LTD) were induced by different stimulation paradigms (100 Hz, 10 Hz, 5 Hz, and 1 Hz). Flupirtine (30 μM) diminished the degree of LTP and enhanced LTD. This effect is most likely due to the hyperpolarization of CA1 pyramidal neurons and the reduction of their input resistance found after application of flupirtine. The observed effects on synaptic strength could underly the beneficial effects of flupirtine on different types of chronic pain.

UNLABELLED:The minimum alveolar concentration (MAC) of a volatile anesthetic defines anesthetic potency in terms of a suppressed motor response to a noxious stimulus. Therefore, the MAC of an anesthetic might in part reflect depression of motor neuron excitability. In the present study we evaluated the effect of isoflurane (ISO) on neurons in the substantia gelatinosa driven synaptically by putative nociceptive inputs in an in vitro spinal cord preparation of the rat. Whole-cell patch-clamp recordings were performed in neurons with their soma in the substantia gelatinosa of transverse rat spinal cord slices. We investigated the effect of ISO on excitatory postsynaptic currents (EPSC) evoked by dorsal root stimulation (eEPSC), spontaneous (sEPSC), and miniature (mEPSC) EPSC. ISO reversibly reduced the amplitude of eEPSC to 39% +/- 22% versus control. ISO decreased the frequency of sEPSC and mEPSC to 39% +/- 26% and 63% +/- 7%. Neither the amplitudes nor the kinetics of mEPSC and sEPSC were altered by ISO. We conclude that ISO depresses glutamatergic synaptic transmission of putative nociceptive primary-afferent inputs, presumably by reducing the release of the excitatory transmitter. This effect may contribute to an antinociceptive action of volatile anesthetics at the spinal cord level. IMPLICATIONS:The present electrophysiological in vitro experiments provide evidence that the volatile anesthetic isoflurane reduces excitatory transmitter release at the first site of synaptic integration of nociceptive inputs, the spinal cord superficial dorsal horn. This effect may contribute to the anesthetic action of volatile anesthetics at the spinal cord level.

The serotonin type 3 (5-HT3) receptor is the only ligand-gated ion channel receptor for serotonin (5-HT). 5-HT3 receptors play an important role in modulating the inhibitory action of dopamine in mesocorticolimbic brain regions. Neuroleptic drugs are commonly thought to exert their psychopharmacological action mainly through dopamine and serotonin type 2 (5-HT2) receptors. Except for clozapine, a direct pharmacological interaction of neuroleptics with 5-HT3 receptors has not yet been described. Using the concentration-clamp technique, we investigated the effects of flupentixol, various phenothiazines, haloperidol, clozapine and risperidone on Na þ-inward currents through 5-HT3 receptors stably expressed in human embryonic kidney 293 cells, and through endogenous 5-HT3 receptors of murine N1E-115 neuroblastoma cells. In addition, we studied their effects on Ca2 þ influx, measured as a change in intracellular Ca2 þ concentrations ((Ca2 þ)i). All neuroleptic drugs, but not risperidone, antagonized Na þ- and Ca2 þ-inward currents evoked by 5-HT (10 lM for 2 s and 1 lM, respectively) in a voltage-independent manner. Only clozapine was a competitive antagonist, while all other compounds turned out to be noncompetitive. Fluphenazine and haloperidol affected membrane anisotropy at concentrations below their IC50 values, indicating that a change in membrane anisotropy might contribute to their antagonistic effect at the 5-HT3 receptor. Only structure analogues of flupentixol and fluphenazine with a lipophilic side chain were potent antagonists against 5-HT-evoked Na þ and Ca2 þ currents. Since 5-HT3 receptors modulate mesolimbic and mesocortical dopaminergic activity, the functional antagonism of neuroleptics at 5-HT3 receptors may contribute to their antipsychotic efficacy and may constitute a not yet recognized pharmacological principle of these drugs. Molecular Psychiatry (2004) 9, 846-858. doi:10.1038/sj.mp.4001490 Published online 16 March 2004

The serotonin type 3 (5-HT 3 ) receptor is the only ligand-gated ion channel receptor for serotonin (5-HT). 5-HT 3 receptors play an important role in modulating the inhibitory action of dopamine in mesocorticolimbic brain regions. Neuroleptic drugs are commonly thought to exert their psychopharmacological action mainly through dopamine and serotonin type 2 (5-HT 2 ) receptors. Except for clozapine, a direct pharmacological interaction of neuroleptics with 5-HT 3 receptors has not yet been described. Using the concentration-clamp technique, we investigated the effects of flupentixol, various phenothiazines, haloperidol, clozapine and risperidone on Na + -inward currents through 5-HT 3 receptors stably expressed in human embryonic kidney 293 cells, and through endogenous 5-HT 3 receptors of murine N1E-115 neuroblastoma cells. In addition, we studied their effects on Ca 2+ influx, measured as a change in intracellular Ca 2+ concentrations ([Ca 2+ ] i ). All neuroleptic drugs, but not risperidone, antagonized Na + - and Ca 2+ -inward currents evoked by 5-HT (10 μ M for 2 s and 1 μ M, respectively) in a voltage-independent manner. Only clozapine was a competitive antagonist, while all other compounds turned out to be noncompetitive. Fluphenazine and haloperidol affected membrane anisotropy at concentrations below their IC 50 values, indicating that a change in membrane anisotropy might contribute to their antagonistic effect at the 5-HT 3 receptor. Only structure analogues of flupentixol and fluphenazine with a lipophilic side chain were potent antagonists against 5-HT-evoked Na + and Ca 2+ currents. Since 5-HT 3 receptors modulate mesolimbic and mesocortical dopaminergic activity, the functional antagonism of neuroleptics at 5-HT 3 receptors may contribute to their antipsychotic efficacy and may constitute a not yet recognized pharmacological principle of these drugs.

Antidepressants are commonly supposed to enhance serotonergic and/or noradrenergic neurotransmission by inhibition of neurotransmitter reuptake through binding to the respective neurotransmitter transporters or through inhibition of the monoamine oxidase. Using the concentration-clamp technique and measurements of intracellular Ca2+, we demonstrate that different classes of antidepressants act as functional antagonists at the human 5-HT3A receptor stably expressed in HEK 293 cells and at endogenous 5-HT3 receptors of rat hippocampal neurons and N1E-115 neuroblastoma cells. The tricyclic antidepressants desipramine, imipramine, and trimipramine, the serotonin reuptake inhibitor fluoxetine, the norepinephrine reuptake inhibitor reboxetine, and the noradrenergic and specific serotonergic antidepressant mirtazapine effectively reduced the serotonin-induced Na+- and Ca2+-currents in a dose-dependent fashion. This effect was voltage-independent and, with the exception of mirtazapine, noncompetitive. Desipramine, imipramine, trimipramine, and fluoxetine also accelerated receptor desensitization. Moclobemide and carbamazepine had no effect on the serotonin-induced cation current. By analyzing analogues of desipramine and carbamazepine, we found that a basic propylamine side chain increases the antagonistic potency of tricyclic compounds, whereas it is abolished by an uncharged carboxamide group. The antagonistic effects of antidepressants at the 5-HT3 receptor did not correlate with their effects on membrane fluidity. In conclusion, structurally different types of antidepressants modulate the function of this ligand-gated ion channel. This may represent a yet unrecognized pharmacological principle of antidepressants.

Homomeric complexes of 5-HT3A receptor subunits form a ligand-gated ion channel. This assembly does not fully reproduce the biophysical and pharmacological properties of native 5-HT3 receptors which might contain the recently cloned 5-HT3B receptor subunit. In the present study, heteromeric assemblies containing human 5-HT3A and 5-HT3B subunits were expressed in HEK 293 cells to detail the functional diversity of 5-HT3 receptors. We designed patch-clamp experiments with homomeric (5-HT3A) and heteromeric (5-HT3AB) receptors to emphasize the kinetics of channel activation and desensitization. Co-expression of the 5-HT3B receptor subunit reduced the sensitivity for 5-HT (5-HT3A receptor: EC50 3 μM, Hill coefficient 1.8; 5-HT3AB receptor: EC50 25 μM, Hill coefficient 0.9) and markedly altered receptor desensitization. Kinetic modeling suggested that homomeric receptors, but not heteromeric receptors, desensitize via an agonist-induced open-channel block. Furthermore, heteromeric 5-HT3AB receptor assemblies recovered much faster from desensitization than homomeric 5-HT3A receptor assemblies. Unexpectedly, the specific 5-HT3 receptor agonist mCPBG induced an open-channel block at both homomeric and heteromeric receptors. Because receptor desensitization and resensitization massively affect amplitude, duration, and frequency of synaptic signaling, these findings are evidence in favor of a pivotal role of subunit composition of 5-HT3 receptors in serotonergic transmission.

Activation of adrenoreceptors modulates synaptic transmission in the basolateral amygdala. Here, we investigated the effects of alpha(2) -adrenoreceptor activation on long-term depression and long-term potentiation in an in vitro slice preparation of the mouse basolateral amygdala. Field potentials and excitatory postsynaptic currents were evoked in the basolateral amygdala by stimulating the lateral amygdala. Norepinephrine (20 mum) reduced synaptic transmission and completely blocked the induction of long-term potentiation and long-term depression. The alpha(2) -adrenoreceptor antagonist yohimbine (2 mum) reversed this effect. The alpha(2) -adrenoreceptor agonist clonidine (10 mum) mimicked the effects of norepinephrine. The G(i/o) -protein inhibitor pertussis toxin (5 mug/mL) reversed the effect of clonidine. Long-term depression was blocked in the presence of omega-conotoxin GVIA, but not omega-agatoxin IVA. Clonidine inhibited voltage-activated Ca2+ currents mediated via N- or P/Q-type Ca2+ -channels. The inhibitory action of clonidine on long-term depression was reversed when inwardly rectifying K+ -channels were blocked by Ba2+ (300 mum). The present data suggest that alpha(2) -adrenoreceptor activation impairs the induction of long-term depression in the basolateral amygdala by a G(i/o) -protein-mediated inhibition of presynaptic N-type Ca2+ -channels and activation of inwardly-rectifying K+ -channels.

The convulsant effects of alpha-thujone, the psychotropic component of absinthe, were attributed to inhibitory actions at the GABAA receptor. Here, we investigated for the first time the 5-HT3 receptor as a potential site of the psychotropic actions of alpha-thujone. This cation permeable ligand-gated ion channel shows considerable homology to the GABAA receptor. We previously demonstrated that in homomeric assemblies of cloned human 5-HT,A receptor subunits. the endogenous agonist 5-HT induced desensitization via channel blockade. When the 5-HT3 B receptor subunit was co-expressed, the resulting heteromeric assemblies desensitized independent from channel blockade. In the present study, patch-clamp experiments revealed an inhibitory action of alpha-thujone on both homomeric and heteromeric 5-HT3 receptors. This inhibitory action was mediated via channel blockade. However, it was not alpha-thujone itself which blocked the channel. The present experiments suggested that, in homomeric receptors, alpha-thujone enhanced the inherent channel-blocking potency of the natural ligand. 5-HT. In heteromeric receptors, alpha-thujonerecruited an additional channel-blocking component of the agonist. By means of kinetic modeling, we simulated possible mechanisms by which alpha-thuljone decreased the 5-HT-induced responses. It is suggested that alpha-thujone reduced 5-HT3 receptor activity by an effect on mechanisms involved in receptor desensitization, which depend on receptor subunit composition. It remains to be shown if this inhibitory action on serotonergic responses contributes to behavioral effects of alpha-thujone.

The rapid application of solutions containing the volatile anaesthetics isoflurane or sevoflurane induced inward currents in human embryonic kidney (HEK293) cells carrying rat recombinant alpha(1)beta(2)gamma(2L) GABA(A) receptor assemblies. The responses evoked by the anaesthetics applied via a fast delivery system were recorded using the patch-clamp technique in the whole-cell mode. The anaesthetics induced a fast inward current which was followed by a prominent tail current upon the rapid withdrawal of the agent. These currents were simulated using a kinetic scheme embodying two agonist-like binding steps required for receptor activation, and one binding step by which the anaesthetic induces an open-channel block. According to this model of a biphasic receptor modulation, the open-channel block delays the ion flux through the ligand-gated receptors and, thus, prolongs the overall duration of the current response. Open-channel blocks might also be operative in other ligand-gated ion channels to modulate synaptic strength.

In the present study, well-resolved single-channel events of cloned human and murine homomeric 5-hydroxytryptamine type 3 (h5-HT(3A) and m5-HT(3A)) receptors, expressed in human embryonic kidney 293 cells, are reported for the first time. 5-HT (1 microM) applied in Ca(2+)- and Mg(2+)-free external solution to excised outside-out membrane patches induced non-rectifying single-channel inward currents. These currents were not observed in untransfected cells or in the presence of the antagonist ondansetron (1 microM). The mean single-channel conductance of the h5-HT(3A) and m5-HT(3A) receptors was 48+/-8 pS and 46+/-7 pS, the mean reversal potential was 10.3+/-1.8 mV and 4.7+/-5.3 mV, respectively. The analysis of single-channel open-times revealed for both h5-HT(3A) and m5-HT(3A) receptors only one type of open state, but different mean open-times (7.1+/-2.9 ms vs. 4.1+/-0.8 ms) indicating species-dependent gating mechanisms of 5-HT(3) receptors.

Recombinant alpha1beta2gamma2L gamma-aminobutyric acid (A) receptor (GABA(A)R) channels expressed in human embryonic kidney (HEK293) cells were used for patch-clamp experiments. The currents activated by brief pulses of GABA (10(-4) M) applied with a device for fast solution exchange to cells clamped in the whole-cell configuration mimicked GABA(A)R-mediated inhibitory postsynaptic currents. Isoflurane (ISO) at clinically relevant concentrations (0.6 mM) decreased the amplitude and prolonged the decay of the GABA-evoked response. To further detail the mechanism underlying the prolonged decay time, we made simulations based on these measurements. These simulations suggest that ISO slows the rate of GABA unbinding from the receptor. Under these conditions, ISO increases the GABA-induced charge transfer and, thus, could enhance GABAergic inhibition despite the concomitant open-channel block causing the decrease in the current amplitude.

BACKGROUND AND OBJECTIVE The gamma-aminobutyric acidA receptor (GABAAR) is a target for anaesthetic agents. We investigated the interactions of sevoflurane with a recombinant GABAAR. Emphasis was on the mechanism of block, as relevant open-channel block by a volatile anaesthetic would possibly explain prolonged GABAergic postsynaptic currents. METHODS The effect of sevoflurane on GABA-induced currents through recombinant alpha1beta2gamma2 GABAAR channels was studied (patch clamp; HEK293 cells). GABA 0.01 mM or 1 mM was applied alone or together with sevoflurane (0.05 mM to 5 mM). RESULTS Currents elicited by GABA 0.01 mM were increased by low sevoflurane concentrations to 183% and decreased by high sevoflurane concentrations (> 1 mM) to 34% (P < 0.05). Ten- to 90%-rise times of the currents were reduced by sevoflurane concentration dependently. At GABA (1 mM), peak currents and 10-90%-rise times decreased with increasing sevoflurane concentrations. A transient current increase was induced by discontinuation of GABA and sevoflurane. Such rebound currents indicate a reversal of an open-channel block by sevoflurane. CONCLUSIONS Sevoflurane (a) increases the apparent affinity of GABA to the GABAAR, as suggested by the decreased current rise times. This explains the enhancement of the currents induced by low GABA concentrations (0.01 mM). Additionally, sevoflurane (b) induces a picrotoxin-like open-channel block at the GABAAR. The reversal of the open-channel block elicits a delayed GABA response. These findings indicate at least two different sites of action of sevoflurane at this receptor that are both important for an enhanced GABAergic synaptic transmission.

Background The volatile anesthetic isoflurane depresses glutamatergic transmission. In this study, the authors investigated the effects of isoflurane on the induction of long-term potentiation (LTP) and long-term depression (LTD) in slices from the juvenile and adult mouse hippocampus. Both forms of synaptic plasticity involve the activation of glutamate receptors. Methods Field excitatory postsynaptic potentials and excitatory postsynaptic currents from neurons in the CA1 area were evoked by stimulation of the Schaffer collateral-commissural pathway. Two independent synaptic inputs were stimulated. Clinically relevant concentrations (0.2-0.3 mM) of isoflurane were added to the perfusion solution. Results Field excitatory postsynaptic potentials from slices of juvenile and adult mice were depressed to 37.3 +/- 6.1% and 58.3 +/- 7.4%, respectively, and excitatory postsynaptic currents were reduced to 36.7 +/- 5.4% by isoflurane. A brief tetanic stimulation (100 Hz, 1 s) induced stable LTP of field excitatory postsynaptic potentials. In the presence of isoflurane, tetanization failed to induce LTP. The effect of isoflurane on LTP induction was reversible and could be prevented by antagonizing gamma-aminobutyric acid type A receptors (GABAA). Low-frequency stimulation (1 Hz/900 pulses) induced LTD. In the presence of isoflurane, low-frequency stimulation failed to induce LTD. Conclusions The prevention of the isoflurane-induced depression of LTP by the GABAA antagonist picrotoxin suggests an involvement of GABAA receptors. An enhancement of the efficacy of GABA-mediated inhibitory synaptic transmission prevents the depolarization of the postsynaptic membrane during tetanus, necessary for the induction of use-dependent alteration of synaptic strength. An impairment of these processes may be a cause for the transient loss of recall and cognitive impairment after anesthesia in juvenile and adult brains.

Clinically relevant concentrations of isoflurane (ISO) and nitrous oxide (N2O) enhance chloride currents induced by activating gamma-aminobutyric acid(A) receptors (GABA(A)R). Channel blocking by ISO overcomes the enhancing effect at higher concentrations. In this study, the effect of coadministered ISO and N2O on responses evoked by GABA in transfected human embryonic kidney 293 cells carrying alpha1beta2gamma2L GABA(A)R was investigated. Patch-clamp recordings from these cells were performed in the whole cell mode. A piezo-driven "liquid filament" drug application system was used to apply solutions of GABA, ISO, and N2O. Increasing the concentration of ISO in steps from 0.15 to 1.2 mM resulted in a bell-shaped concentration-response curve for GABA-induced currents. The maximum increase in current (1.51 +/- 0.14-fold) was seen at 0.45 mM ISO (about 1 minimum alveolar concentration, EC50). N2O (29.2 mM) increased GABA-evoked currents 1.54 +/- 0.10-fold. The enhancing effects of ISO and N2O on the GABAergic response were not additive. However, a transient current, associated with the rapid withdrawal of ISO from the receptor, was markedly increased by N2O. Such rebound currents probably reflect the transition from a "channel-blocked" to a "reopened" state. An open-channel block at ligand-gated receptors can prolong postsynaptic currents. Thus, we conclude that coadministered N2O could increase the enhancing effect of ISO on the GABAergic transmission by an increase in open-channel block at the GABA(A)R.