M. Bruce MacIver - US grants

The development of safer and more efficacious general anesthetics has been limited by a lack of knowledge about their neuronal sites and mechanisms of action. Synaptic transmission in the central nervous system (CNS) is regarded as a primary target of anesthetic action, however, only a few studies have investigated possible presynaptic actions. Preliminary results from our laboratory have demonstrated that clinically effective concentrations of volatile anesthetics (e.g. halothane at 0.5 to 1.5 vol %) enhance the release of gamma-aminobutyric acid (GABA) from presynaptic terminals, evidenced by an increased frequency of GABA-mediated miniature inhibitory postsynaptic currents (IPSCs). This presynaptic action would add to the well established post synaptic prolongation of IPSCs produced by halothane, resulting in a marked increase in synaptic inhibition. Increased synaptic inhibition is thought to play a significant role in the CNS depression associated with general anesthesia. The long-term goal of the proposed research is to define the synaptic sites of action which contribute to anesthetic-induced CNS depression. The actions of six clinically used anesthetics (halothane, isoflurane, sevoflurane, pentobarbital, propofol and midazolam) will be compared on five independent measures of presynaptic function. Electrophysiological recording in rat hippocampal cortex slices will be combined with the use of selective pharmacological probes to isolate and identify possible presynaptic sites of anesthetic action. The specific aims are to compare anesthetic effects on: 1) the excitability of afferent fibers, 2) the frequency-dependant facilitation at excitatory synapses, 3) the excitability of GABAergic interneurons, 4) the frequency of miniature GABA-mediated IPSCs, 5) the frequency-dependant depression of inhibitory (GABA) synapses. The proposed study will provide detailed information about anesthetic-induced alterations in presynaptic function and will contribute to an understanding of anesthetic mechanisms of action; prerequisite to the development of better and safer therapeutic agents for general anesthesia.

DESCRIPTION: (Adapted from the applicant's abstract) Evidence exists for anesthetic actions at central nervous system (CNS) synapses. Some anesthetics may enhance GABA-mediated inhibitory transmission while others appear to produce CNS depression by reducing glutamate-mediated excitation. Preliminary results from these investigators suggests that several anesthetics act at both types of synapses and may summate to reduce synaptically-evoked discharge of neurons. The investigator suggests that it may be possible to develop selectively targeted anesthetics based on an understanding of actions at glutamate and GABA synapses. To do this a determination of common synaptic actions of different chemical/pharmacological classes of anesthetics is proposed. The Specific Aims are to determine: 1) the extent to which anesthetic-induced depression of CA 1 neuron discharge results from enhanced GABA-mediated transmission. 2) whether anesthetic-induced depression of glutamate-mediated synaptic responses involves enhanced GABA inhibition. 3) the cellular and molecular mechanisms of anesthetic effects at glutamate and GABA synapses. The proposed research will use the well-characterized CA 1 neuron circuit in rat hippocampus, in which GABA and glutamate mediate fast monosynaptic transmission. Evoked responses will be completely blocked by selective GABA and glutamate receptor antagonists. Electrophysiological recordings will be combined with selective NMDA and AMPA receptor anatagonists to investigate anesthetic-induced depression of glutamate-mediated excitatory postsynaptic potentials. Parallel studies will determine the extent to which an anesthetic-induced depression of CA 1 neuron discharge can be reversed by blocking GABA receptor/Cl- channels.

DESCRIPTION: (Adapted from the applicant's abstract) The goal of the investigators is to understand mechanisms of general anesthetic action. Attenuated neuronal excitability during anesthesia results in part from enhanced GABA-mediated synaptic inhibition. The major direction of this proposal is to investigate anesthetic actions on inhibitory GABAergic interneurons. Interneurons mediate synaptic inhibition by releasing GABA. Whole-cell recordings and infrared imaging make it now possible to study anesthetic actions on previous difficult to access interneurons. Unexpectedly, because general anesthetics usually depress transmitter release, is the preliminary finding that general anesthetics increase the spontaneous release of GABA from inhibitory interneurons. Enhanced GABA release can combine with anesthetic-postsynaptic GABA receptor actions to augment inhibition. The proposed study will identify the effects of anesthetics on inhibitory GABAergic interneurons, characterize the mechanisms underlying these actions, and determine the consequences of these effects on neuronal excitability. The investigators believe that these studies on inhibitory GABAergic interneurons will provide information regarding mechanisms of anesthetic action.

Inhaled solvent abuse is a growing problem among U.S. teenagers and may lead to the abuse of other drugs. The long-term goal of our research is to gain a better understanding of the mechanisms of action of these drugs, in order to provide rational strategies for the treatment and prevention of inhaled solvent abuse. Evidence exists for inhaled solvent actions at central nervous system (CMS) synapses, but the importance of effects at glutamate and gamma-aminobutyric acid (GABA) synapses remains unknown. Some solvents (e.g. toluene) appear to enhance GABA- mediated inhibitory transmission while others appear to disrupt CMSfunction by altering glutamate- mediated excitation. Preliminary results from our laboratory indicate that toluene can act at both types of synapses and that combined effects disprupt the normal circuit function of neurons. The proposed research will use the well-characterized CA 1 neuron circuit in rat hippocampal brain slices. The CA 1 circuit uses GABA and glutamate for fast monosynaptic transmission and evoked responses can be completely blocked by a combination of GABA and glutamate receptor antagonists. The specific aims are as follows: 1) To determine whether inhaled solvents alter GABA- mediated transmission. 2) To determine whether solvents alter glutamate-mediated synaptic responses. 3) To investigate the cellular and molecular mechanisms of solvent effects at amino acid synapses. Electrophysiological recordings will be combined with selective NMDA and AMPA receptor antagonists to investigate solvent-induced effects on glutamate-mediated excitatory postsynaptic potentials. Parallel studies will determine the extent to which solvent-induced effects on CA 1 neuron discharge come about via effects on GABA receptor/CI- channels. Inhaled solvents will likely produce effects similar to inhalational anesthetics, which we have studied tor over fifteen years in our laboratory. Taken together, the results will allow a quantitative comparison of drug effects on glutamate and GABA-mediated transmission for commonly abused solvents including: toluene, 1,1,1-trichloroethane andtrichloroethylene.

DESCRIPTION (provided by applicant): The long term goal of our research is to define the role played in anesthesia by various forms of GABA-mediated inhibition. There is great controversy at present regarding anesthetic-enhanced GABA inhibition for various anesthetic end-points. Good evidence points to considerable involvement, for several end-points, including loss of recall, loss of consciousness and even immobility, but other experiments have suggested little or no involvement. Nor is it clear whether GABA inhibition mediated by synaptic receptors or extrasynaptic tonic receptors play the more important role in depressing circuit level signaling. Our recent discovery of important effects on GABAB, in addition to well documented effects on GABAA systems, have added to the confusion about relevant effects for a growing array of new anesthetic target sites (e.g TREK and TASK potassium channels, calcium channels, other transmitter receptors, presynaptic SNARE proteins, and sodium channels). We will focus this study on GABAA and GABAB mediated inhibition, because these appear to contribute most to depression (over 60% when combined) from our preliminary studies, but our new circuit level analysis has already provided evidence for other key sites of action as well. Three specific aims will be addressed: 1 - to assess the role that fast and slow synaptic as well as tonic GABAA inhibition plays in CA1 circuit integration, 2 - to assess the roles these forms of inhibition play for anesthetic- induced circuit depression, and 3 - to assess the role GABAB mediated inhibition plays for anesthetics. Information from these aims is essential for designing safer and more effective anesthetics that selectively target the most relevant GABA receptors. In the immediate-term, we will contribute quantitative assessments to resolve current controversies, and assess new GABAB contributions for three classes of general anesthetics. Our results will provide the best quantitative data available upon which to model anesthetic effects using computational approaches - current models use estimates of the involvement of GABA systems that range from ~ 20 to 90 %, and there is little evidence upon which to base these estimates. As the GABA receptor subtypes underlying various forms of inhibition become known, our results will help link desired and unwanted anesthetic effects to these molecular targets.