Gerhard Hapfelmeier

Isoflurane blocks synaptic plasticity in the mouse hippocampus

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 &ggr;-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.

α-Thujone reduces 5-HT3 receptor activity by an effect on the agonist-induced desensitization

Abstract The convulsant effects of α-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 α-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-HT3A receptor subunits, the endogenous agonist 5-HT induced desensitization via channel blockade. When the 5-HT3B 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 α-thujone on both homomeric and heteromeric 5-HT3 receptors. This inhibitory action was mediated via channel blockade. However, it was not α-thujone itself which blocked the channel. The present experiments suggested that, in homomeric receptors, α-thujone enhanced the inherent channel-blocking potency of the natural ligand, 5-HT. In heteromeric receptors, α-thujone recruited an additional channel-blocking component of the agonist. By means of kinetic modeling, we simulated possible mechanisms by which α-thujone decreased the 5-HT-induced responses. It is suggested that α-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 α-thujone.

Isoflurane modulates glutamatergic and GABAergic neurotransmission in the amygdala

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, GABAA and GABAB receptors. Isoflurane added to the perfusion medium reduced the strength of synaptic signalling following the activation of non‐NMDA, NMDA, and GABAB receptors, whereas the GABAA 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.

Isoflurane reduces glutamatergic transmission in neurons in the spinal cord superficial dorsal horn: Evidence for a presynaptic site of an analgesic action

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.

Nitrous oxide and xenon increase the efficacy of GABA at recombinant mammalian GABAA receptors.

We investigated the interactions between recombinant gamma-aminobutyric acid receptor complex (GABAAR) and nitrous oxide (N2O) or xenon (Xe). Human embryonic kidney cells (HEK 293) were transfected with rat cDNA for &agr;1&bgr;2&ggr;2L or for &agr;1&bgr;2 recombinant GABAAR subunits. Patch clamp techniques were used in the whole-cell mode to evaluate the effect of N2O and Xe on GABA-induced currents. A piezo-driven “liquid filament switch” was used for fast application. Both N2O (100%, 29.2 mM) and Xe (100%, 3.9 mM) reversibly increased GABA-induced currents through the &agr;1&bgr;2&ggr;2L and the &agr;1&bgr;2 GABAAR channels. The potentiating effect of N2O or Xe on peak currents was prominent at small GABA concentrations (10−7 to 10−5 M). The addition of N2O or Xe increased the efficacy of GABA (10−7 to 10−3 M). Both N2O and Xe significantly decreased the risetime(10%–90%) of the currents elicited by small GABA concentrations. At the concentrations used, neither N2O nor Xe had an intrinsic effect. We conclude that, similar to other anesthetics, both N2O and Xe increase the efficacy of GABA at the GABAAR and enhance inhibitory GABAergic synaptic transmission. {abs} Implications The anesthetic gases nitrous oxide (laughing gas) and xenon increase the activity of a mammalian GABAA receptor. This receptor is held responsible for the inhibition of important actions of the human brain, e.g., maintenance of consciousness and awareness.

Dual action of isoflurane on the γ-aminobutyric acid (GABA)-mediated currents through recombinant α1β2γ2L-GABAA-Receptor channels

Isoflurane (ISO) increased the agonist-induced chloride flux through the &ggr;-aminobutyric acid A receptor (GABAAR). This may reflect an anesthetic-induced increase in the apparent agonist affinity. A dual effect of anesthetics was postulated for both the nicotinic acetylcholine receptor (nAChR) and the GABAAR. We tested the hypothesis that, in addition to a blocking effect, ISO increases &ggr;-aminobutyric acid (GABA)-gated currents through recombinant GABAAR channels. HEK293 cells were transfected with rat cDNA for &agr;1,&bgr;2,&ggr;2L subunits. Currents elicited by 1 mM or 0.01 mM GABA, respectively, alone, or with increasing concentrations of ISO, were recorded by using standard patch clamp techniques. ISO reduced the peak current elicited by 1 mM GABA. Currents induced by 0.01 mM GABA were potentiated by small ISO (twofold at 0.5 mM ISO) and inhibited by larger concentrations. Withdrawal of ISO and GABA induced rebound currents, suggesting an open-channel block by ISO. These currents increased with increasing concentrations of ISO. At large concentrations of ISO, the inhibitory effect predominated and was caused by, at least partly, an open-channel block. At small concentrations of ISO, potentiation of the GABA-gated currents was more prominent. This dual action of ISO indicates different binding sites at the GABAAR. The balance between potentiation and block depends on the concentrations of both ISO and GABA. Implications Isoflurane (ISO) interacts with the inhibitory &ggr;-aminobutyric acid (GABA) receptor. This patch clamp study demonstrates that it may block or potentiate the type A of GABA receptor studied, depending on the concentrations of ISO and of GABA used. At clinically relevant concentrations, ISO considerably potentiates this receptor. This may partly explain its clinical effect.

GABAA receptor activation and open-channel block by volatile anaesthetics: a new principle of receptor modulation? ☆

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.

The potassium channel modulator flupirtine shifts the frequency-response function of hippocampal synapses to favour LTD in mice.

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 microM) 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.

Cloned human and murine serotonin3A receptors expressed in human embryonic kidney 293 cells display different single-channel kinetics

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.