M. Bruce MacIver

Carbachol-induced EEG ‘theta’ activity in hippocampal brain slices

Application of the cholinergic agonist carbachol (50 microM) produced theta-like rhythmical waveforms, recorded in the stratum moleculare of the dentate gyrus. Atropine sulfate (50 microM) antagonized the carbachol-induced theta-like activity, consistent with this action of atropine in vivo. These results provide the first direct evidence that hippocampal neurons are capable of producing synchronized slow-wave activity when isolated from pulsed rhythmic inputs of the medial septum and other brain regions.

Halothane enhances tonic neuronal inhibition of elevating intracellular calcium

Whether the major action of anesthetics is to depress the central nervous system (CNS) by reducing excitation or enhancing inhibition remains unknown. Using whole cell patch-clamp recording in hippocampal slices, halothane and pentobarbital were found to prolong the decay time constant (TAU(D)) of GABAA-mediated spontaneous inhibitory postsynaptic currents (sIPSCs). Intracellular administration of the Ca2+ chelator BAPTA or the Ca2+ release inhibitor dantrolene significantly (ANOVA, P less than 0.005) reduced halothanes effect; in contrast, the pentobarbital effect was unchanged. Halothane induced depression of population spike amplitude was blocked by the GABAA antagonist bicuculline. Together, these findings suggest that a major depressant effect of halothane involves enhancement of GABAA-mediated inhibition through release of intraneuronally stored Ca2+.

Excitatory Synaptic Transmission Mediated by NMDA Receptors Is More Sensitive to Isoflurane than Are Non-NMDA Receptor-mediated Responses

Background Effects of volatile anesthetic agents on N-methyl-D-aspartate (NMDA) receptor–mediated excitatory synaptic transmission have not been well characterized. The authors compared effects produced by halothane and isoflurane on electrophysiologic properties of NMDA and non-NMDA receptor–mediated synaptic responses in slices from the rat hippocampus. Methods Field excitatory postsynaptic potentials (fEPSPs) in the CA1 area were recorded with extracellular electrodes after electrical stimulation of Schaffer-collateral-commissural fiber inputs. NMDA or non-NMDA receptor–mediated fEPSPs were pharmacologically isolated using selective antagonists. Clinically relevant concentrations of halothane or isoflurane were applied to slices in an artificial cerebrospinal fluid perfusate. Paired pulse facilitation was used as a measure of presynaptic effects of the anesthetic agents. Results Clinically relevant concentrations of halothane (1.2 vol% ≈ 0.35 mM) depressed fEPSP amplitudes mediated by NMDA receptors and non-NMDA receptors to a similar degree (mean ± SD: 63.3 ± 14.0% of control, n = 5; 60.2 ± 7.3% of control, n = 7, respectively). In contrast, isoflurane (1.4 vol% ≈ 0.50 mM) preferentially depressed fEPSP amplitudes mediated by NMDA receptors (44.0 ± 7.4% of control, n = 6, P < 0.001) compared with those for non-NMDA receptors (68.7 ± 5.4% of control, n = 6), indicating a selective, additional postsynaptic effect. Paired pulse facilitation of fEPSPs was increased significantly by both anesthetic agents from 1.37 ± 0.13 to 1.91 ± 0.25 (n = 5, P < 0.05 for halothane) and from 1.44 ± 0.04 to 1.64 ± 0.08 (n = 5, P < 0.01 for isoflurane), suggesting that presynaptic mechanisms are also involved in fEPSP depression produced by the anesthetic agents. Neither rise times nor decay times of fEPSPs were changed in the presence of the anesthetic agents. Conclusions These results indicate that fEPSPs mediated by postsynaptic NMDA receptors are more sensitive to clinically relevant concentrations of isoflurane than are non-NMDA receptor–mediated responses, but this selective effect was not observed for halothane. Both agents also appeared to depress release of glutamate from nerve terminals via presynaptic actions.

Agent-selective effects of volatile anesthetics on GABAA receptor-mediated synaptic inhibition in hippocampal interneurons.

BackgroundA relatively small number of inhibitory interneurons can control the excitability and synchronization of large numbers of pyramidal cells in hippocampus and other cortical regions. Thus, anesthetic modulation of interneurons could play an important role for the maintenance of anesthesia. The aim of this study was to compare effects produced by volatile anesthetics on inhibitory postsynaptic currents (IPSCs) of rat hippocampal interneurons. MethodsPharmacologically isolated &ggr;-aminobutyric acid type A (GABAA) receptor–mediated IPSCs were recorded with whole cell patch-clamp techniques in visually identified interneurons of rat hippocampal slices. Neurons located in the stratum radiatum–lacunosum moleculare of the CA1 region were studied. The effects of clinically relevant concentrations (1.0 rat minimum alveolar concentration) of halothane, enflurane, isoflurane, and sevoflurane were compared on kinetics of both stimulus-evoked and spontaneous GABAA receptor–mediated IPSCs in interneurons. ResultsHalothane (1.2 vol% ≈ 0.35 mm), enflurane (2.2 vol% ≈ 0.60 mm), isoflurane (1.4 vol% ≈ 0.50 mm), and sevoflurane (2.7 vol% ≈ 0.40 mm) preferentially depressed evoked IPSC amplitudes to 79.8 ± 9.3% of control (n = 5), 38.2 ± 8.6% (n = 6), 52.4 ± 8.4% (n = 5), and 46.1 ± 16.0% (n = 8), respectively. In addition, all anesthetics differentially prolonged the decay time constant of evoked IPSCs to 290.1 ± 33.2% of control, 423.6 ± 47.1, 277.0 ± 32.2, and 529 ± 48.5%, respectively. The frequencies of spontaneous IPSCs were increased by all anesthetics (twofold to threefold). Thus, the total negative charge transfer mediated by GABAA receptors between synaptically connected interneurons was enhanced by all anesthetics. ConclusionsVolatile anesthetics differentially enhanced GABAA receptor–mediated synaptic inhibition in rat hippocampal interneurons, suggesting that hippocampal interneuron circuits are depressed by these anesthetics in an agent-specific manner.

Membrane and Synaptic Actions of Halothane on Rat Hippocampal Pyramidal Neurons and Inhibitory Interneurons

A relatively small number of inhibitory interneurons can control the excitability and synchronization of large numbers of pyramidal neurons in hippocampus and other cortical regions. Thus, anesthetic modulation of interneurons could play an important role during anesthesia. The aim of this study was to investigate effects of a general anesthetic, halothane, on membrane and synaptic properties of rat hippocampal interneurons. GABA receptor-mediated IPSCs were recorded with whole-cell patch-clamp techniques in visually identified CA1 pyramidal cells and interneurons located at the border of stratum lacunosum-moleculare and stratum radiatum. Halothane (0.35 mm ≅ 1.2 vol%) depressed evoked IPSC amplitudes recorded from both pyramidal cells and inhibitory interneurons. Also, halothane considerably prolonged the decay time constant of evoked IPSCs in pyramidal cells and interneurons. The frequencies of miniature IPSCs were increased by halothane (two- to threefold) in both types of neuron. On the other hand, halothane effects on resting membrane potentials were variable but minimal in both types of neurons. In current-clamp recordings, halothane depressed EPSP amplitudes and increased IPSP amplitudes recorded from both types of neurons. In addition, halothane increased the failure rate of synaptically evoked action potentials. Taken together, these data provide evidence that halothane increases GABAAreceptor-mediated synaptic inhibition between synaptically connected interneurons and depresses excitatory transmission, similar to effects observed in pyramidal neurons.

Multiple synaptic and membrane sites of anesthetic action in the CA1 region of rat hippocampal slices

BackgroundAnesthesia is produced by a depression of central nervous system function, however, the sites and mechanisms of action underlying this depression remain poorly defined. The present study compared and contrasted effects produced by five general anesthetics on synaptic circuitry in the CA1 region of hippocampal slices.ResultsAt clinically relevant and equi-effective concentrations, presynaptic and postsynaptic anesthetic actions were evident at glutamate-mediated excitatory synapses and at GABA-mediated inhibitory synapses. In addition, depressant effects on membrane excitability were observed for CA1 neuron discharge in response to direct current depolarization. Combined actions at several of these sites contributed to CA1 circuit depression, but the relative degree of effect at each site was different for each anesthetic studied. For example, most of propofols depressant effect (> 70 %) was reversed with a GABA antagonist, but only a minor portion of isofluranes depression was reversed (< 20 %). Differences were also apparent on glutamate synapses-pentobarbital depressed transmission by > 50 %, but thiopental by only < 25 %.ConclusionsThese results, in as much as they may be relevant to anesthesia, indicate that general anesthetics act at several discrete sites, supporting a multi-site, agent specific theory for anesthetic actions. No single effect site (e.g. GABA synapses) or mechanism of action (e.g. depressed membrane excitability) could account for all of the effects produced for any anesthetic studied.

Electrophysiologic recording and thermodynamic modeling demonstrate that helium-neon laser irradiation does not affect peripheral Aδ- or C-fiber nociceptors

&NA; The effect of helium‐neon laser irradiation (632.5 nm) on A&dgr;‐ and C‐fiber sensory afferents was investigated in the rabbit cornea, to determine the physiologic basis for reports that low power (0.1–5 mW) helium‐neon (He‐Ne) lasers produce acute analgesia and alleviate chronic pain. Multiple and single unit extracellular recordings from nociceptive corneal afferent nerves (C‐fiber cold, C‐fiber chemical, A&dgr; mechanical and A&dgr; bimodal) were used to study the effects of He‐Ne laser radiation upon the electrophysiologic responses to mechanical, thermal, chemical and electrical stimulation of the cornea. Action potentials were analyzed for latency, amplitude, rise time, duration and frequency. Exposure of the neural receptive field and/or nerve bundle to a 4‐mm diameter He‐Ne laser (0–5 mW; 0–1800 sec) did not alter spontaneous or evoked neural activity. In addition, single unit action potential parameters were not altered by laser irradiation. Modeling of thermal changes produced by He‐Ne radiation on corneal nerves indicated that effects predicted for receptor and axonal depths in both skin and cornea were minimal (< 0.15°C) and unlikely to alter sensory transduction or transmission.

Riluzole anesthesia : Use-dependent block of presynaptic glutamate fibers

Background Riluzole (RP 54274) is an experimental benzothiazole with anesthetic properties, but little is known about its synaptic or cellular actions. Methods The authors investigated riluzole effects on synaptic response of CA 1 pyramidal neurons in rat hippocampal brain slices. Electrophysiologic recordings of population spikes (PS), excitatory postsynaptic potentials (EPSP), and fiber volleys were studied. Paired pulse stimulation (120 ms interpulse interval) was used to measure effects on gamma-amino butyric acid (GABA)-mediated synaptic inhibition, and stimulus trains (33 Hz) were used to test for use-dependent effects. Results Synaptically evoked PS discharge was blocked in a concentration-dependent manner by riluzole (2.0-20 micro Meter), similar to effects produced by other anesthetics. Paired pulse inhibition was not altered by riluzole. In contrast, 20 micro Meter thiopental produced a marked increase in paired pulse inhibition. Riluzole (5.0 micro Meter) produced a 46.6 plus/minus 19.8% depression of glutamate-mediated EPSPs, which could account for most of the depression of PS discharge (54.2 plus/minus 12.6%) produced by this concentration. Riluzole produced a 36 plus/minus 17% depression of fiver volley amplitudes, which, based on input/output analysis, could completely account for the depression of EPSPs. The depression of fiber volley amplitudes showed a marked use-dependence; the second and subsequent action potentials in a train were progressively depressed by riluzole to a greater extent than the first action potential. Conclusions Riluzole produced a potent block of excitatory synaptic transmission via depression of presynaptic conduction in glutamatergic nerve fibers. The use-dependent depression observed resembled that produced by some local anesthetics on nerve conduction and sodium channels. The presynaptic action, together with a lack of effect on gamma-amino butyric acid-mediated inhibition, provides a unique mechanism of action for a general anesthestic.

Abused Inhalants Enhance GABA-Mediated Synaptic Inhibition

Abused inhalants are widely used, especially among school-age children and teenagers, and are ‘gateway’ drugs leading to the abuse of alcohol and other addictive substances. In spite of this widespread use, little is known about the effects produced by inhalants on the central nervous system. The similarity in behavioral effects produced by inhalants and inhaled anesthetics, together with their common chemical features, prompted this study of inhalant actions on a well-characterized anesthetic target, GABA synapses. Whole-cell patch clamp recordings were conducted on CA1 pyramidal neurons in rat hippocampal brain slices to measure effects on resting membrane properties, action potential discharge, and GABA-mediated inhibitory responses. Toluene, 1,1,1-trichloroethane, and trichloroethylene depressed CA1 excitability in a concentration-dependent and reversible manner. This depression appeared to involve enhanced GABA-mediated inhibition, evident in its reversal by a GABA receptor antagonist. Consistent with this, the abused inhalants increased inhibitory postsynaptic potentials produced using minimal stimulation of stratum radiatum inputs to CA1 neurons, in the presence of CNQX and APV to block excitatory synaptic responses and GGP to block GABAB responses. The enhanced inhibition appeared to come about by a presynaptic action on GABA nerve terminals, because spontaneous inhibitory postsynaptic current (IPSC) frequency was increased with no change in the amplitude of postsynaptic currents, both in the presence and absence of tetrodotoxin used to block interneuron action potentials and cadmium used to block calcium influx into nerve terminals. The toluene-induced increase in mIPSC frequency was blocked by dantrolene or ryanodine, indicating that the abused inhalant acted to increase the release of calcium from intracellular nerve terminal stores. This presynaptic action produced by abused inhalants is shared by inhaled anesthetics and would contribute to the altered behavioral effects produced by both classes of drugs, and could be especially important in the context of a disruption of learning and memory by abused inhalants.

Anesthetic properties of 4-iodopropofol: implications for mechanisms of anesthesia.

Background Positive modulation of &ggr;-aminobutyric acid type A (GABAA) receptor function is recognized as an important component of the central nervous system depressant effects of many general anesthetics, including propofol. The role for GABAA receptors as an essential site in the anesthetic actions of propofol was recently challenged by a report that the propofol analog 4-iodopropofol (4-iodo-2,6-diisopropylphenol) potentiated and directly activated GABAA receptors, yet was devoid of sedative–anesthetic effects in rats after intraperitoneal injection. Given the important implications of these findings for theories of anesthesia, the authors compared the effects of 4-iodopropofol with those of propofol using established in vivo and in vitro assays of both GABAA receptor–dependent and –independent anesthetic actions. Methods The effects of propofol and 4-iodopropofol were analyzed on heterologously expressed recombinant human GABAA &agr;1&bgr;2&ggr;2 receptors, evoked population spike amplitudes in rat hippocampal slices, and glutamate release from rat cerebrocortical synaptosomes in vitro. Anesthetic potency was determined by loss of righting reflex in Xenopus laevis tadpoles, in mice after intraperitoneal injection, and in rats after intravenous injection. Results Like propofol, 4-iodopropofol enhanced GABA-induced currents in recombinant GABAA receptors, inhibited synaptic transmission in rat hippocampal slices, and inhibited sodium channel–mediated glutamate release from synaptosomes, but with reduced potency. After intraperitoneal injection, 4-iodopropofol did not produce anesthesia in mice, but it was not detected in serum or brain. However, 4-iodopropofol did produce anesthesia in tadpoles (EC50 = 2.5 ± 0.5 &mgr;m) and in rats after intravenous injection (ED50 = 49 ± 6.2 mg/kg). Conclusions Propofol and 4-iodopropofol produced similar actions on several previously identified cellular and molecular targets of general anesthetic action, and both compounds induced anesthesia in tadpoles and rats. The failure of 4-iodopropofol to induce anesthesia in rodents after intraperitoneal injection is attributed to a pharmacokinetic difference from propofol rather than to major pharmacodynamic differences.