Volatile Anesthetics Depress Glutamate Transmission Via Presynaptic Actions

Background: Recent evidence for a presynaptic depression of glutamate release produced by volatile anesthetics prompted the current study of isoflurane and halothane effects on glutamate-mediated transmission in the mammalian central nervous system.

Methods: Electrophysiologic recordings from CA1 neurons in rat hippocampal brain slices were used to measure anesthetic effects on glutamate-mediated excitatory postsynaptic potential (EPSP) amplitudes and paired pulse facilitation. Paired pulse facilitation is known to be altered when the calcium-dependent release of glutamate is depressed, but not when EPSP amplitudes are depressed by postsynaptic mechanisms.

Results: Isoflurane depressed EPSP amplitudes over a concentration range of 0.35-2.8 vol %, with a 50% depression (EC50) occurring at 1.0 vol % (0.71 rat minimum alveolar concentration). This depression was accompanied by an increase in paired-pulse facilitation of approximately 30% at 1.7 vol %, using interpulse intervals of 120 ms. Halothane depressed EPSP amplitudes in a concentration-dependent manner (0.3-2.4 vol %, EC50 = 1.1 minimum alveolar concentration; 1.3 vol %) and also increased facilitation by approximately 20% at 1.2 vol %. These effects persisted in the presence of 10 micro Meter bicuculline, indicating that enhanced gamma-aminobutyric acid-mediated inhibition was not involved. The anesthetic-induced increase in facilitation and EPSP depression was mimicked by lowering extracellular calcium, which is known to depress glutamate release at these synapses. The postsynaptic glutamate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione depressed EPSP amplitudes with no change in facilitation.     

Volatile Anesthetics Increase Intracellular Calcium in Cerebrocortical and Hippocampal Neurons

Background: An increase in intracellular calcium concentration ([Ca2+]i) in neurons has been proposed as an important effect of volatile anesthetics, because they alter signaling pathways that influence neurotransmission. However, the existing data for anesthetic-induced increases in [Ca2+]i conflict.

Methods: Changes in [Ca2+]i were measured using fura-2 fluorescence spectroscopy in rat cortical brain slices at 90, 185, 370, and 705 [micro sign]M isoflurane. To define the causes of an increase in [Ca2+]i, slices were studied in Ca2+-free medium, in the presence of Ca2+-channel blockers, and in the presence of the Ca2+-release inhibitor azumolene. The authors compared the effect of the volatile anesthetic with that of the nonanesthetic compound 1,2-dichlorohexafluorocyclobutane. Single-dose experiments in CA1 neurons in hippocampal slices with halothane (360 [micro sign]M) and in acutely dissociated CA1 neurons with halothane (360 [micro sign]M) and isoflurane (445 [micro sign]M) also were performed.

Results: Isoflurane at 0.5, 1, and 2 minimum alveolar concentrations increased basal [Ca2+]i in cortical slices in a dose-dependent manner (P < 0.05). This increase was not altered by Ca2+-channel blockers or Ca2+-free medium but was reduced 85% by azumolene. The nonanesthetic 1,2-dichlorohexafluorocyclobutane did not increase [Ca2+] (i). In dissociated CA1 neurons, isoflurane reversibly increased basal [Ca (2+)]i by 15 nM (P < 0.05). Halothane increased [Ca2+]i in dissociated CA1 neurons and CA1 neurons in hippocampal slices by approximately 30 nM (P < 0.05).     

Effects of Small Concentrations of Volatile Anesthetics on Action Potential Firing of Neocortical Neurons In Vitro

Background: Volatile general anesthetics depress neuronal activity in the mammalian central nervous system and enhance inhibitory Cl- currents flowing across the [Greek small letter gamma]-aminobutyric acidA (GABA (A)) receptor - ion channel complex. The extent to which an increase in GABAA-mediated synaptic inhibition contributes to the decrease in neuronal firing must be determined, because many further effects of these agents have been reported on the molecular level.

Methods: The actions of halothane, isoflurane, and enflurane on the firing patterns of single neurons were investigated by extracellular recordings in organotypic slice cultures derived from the rat neocortex.

Results: Volatile anesthetics depressed spontaneous action potential firing of neocortical neurons in a concentration-dependent manner. The estimated median effective concentration (EC50) values were about one half the EC50 values for general anesthesia. In the presence of the GABA (A) antagonist bicuculline (20 [micro sign]M), the effectiveness of halothane, isoflurane, and enflurane in reducing the discharge rates were diminished by 48 - 65%, indicating that these drugs act via the GABAA receptor.     

Propofol and Sevoflurane Depress Spinal Neurons In Vitro via Different Molecular Targets

Background: The capacity of general anesthetics to produce immobility is primarily spinally mediated. Recently, compelling evidence has been provided that the spinal actions of propofol involve [gamma]-aminobutyric acid type A (GABAA) receptors, whereas the contribution of glycine receptors remains uncertain. The relevant molecular targets of the commonly used volatile anesthetic sevoflurane in the spinal cord are largely unknown, but indirect evidence suggests a mechanism of action distinct from propofol.

Methods: The effects of sevoflurane and propofol on spontaneous action potential firing were investigated by extracellular voltage recordings from ventral horn interneurons in cultured spinal cord tissue slices obtained from embryonic rats (embryonic days 14-15).

Results: Propofol and sevoflurane reduced spontaneous action potential firing of neurons. Concentrations causing half-maximal effects (0.11 [mu]m propofol, 0.11 mm sevoflurane) were lower than the median effective concentration immobility (1-1.5 [mu]m propofol, 0.35 mm sevoflurane). At higher concentrations, complete inhibition of action potential activity was observed with sevoflurane but not with propofol. Effects of sevoflurane were mediated predominantly by glycine receptors (45%) and GABAA receptors (38%), whereas propofol acted almost exclusively via GABAA receptors (96%).     

吸入麻醉药的心肌保护机制

日渐增多的证据表明,吸入麻醉药可保护心肌免受可复性或不可复性缺血性损伤。探讨吸入麻醉药介导抗缺血性效应的机制近来备受关注,由于吸入麻醉药可明显影响心血管功能,如降低动脉及冠脉灌注压、剂量依赖性抑制心肌收缩、舒张冠脉、影响电生理、不同程度调节自主神经系统活性等,使得机制的探讨较为困难。因此心肌氧供需关系的适度     

Volatile General Anesthetics Produce Hyperpolarization of Aplysia Neurons by Activation of a Discrete Population of Baseline Potassium Channels

Background: The mechanism by which volatile anesthetics act on neuronal tissue to produce reversible depression is unknown. Previous studies have identified a potassium current in invertebrate neurons that is activated by volatile anesthetics. The molecular components generating this current are characterized here in greater detail.

Methods: The cellular and biophysical effects of halothane and isoflurane on neurons of Aplysia californica were studied. Isolated abdominal ganglia were perfused with anesthetic-containing solutions while membrane voltage changes were recorded. These effects were also studied at the single-channel level by patch clamping cultured neurons from the abdominal and pleural ganglia.

Results: Clinically relevant concentrations of halothane and isoflurane produced a slow hyperpolarization in abdominal ganglion neurons that was sufficient to block spontaneous spike firings. Single-channel studies revealed specific activation by volatile anesthetics of a previously described potassium channel. In pleural sensory neurons, halothane and isoflurane increased the open probability of the outwardly rectifying serotonin-sensitive channel (S channel). Halothane also inhibited a smaller noninactivating channel with a linear slope conductance of approximately 40 pS. S channels were activated by halothane with a median effective concentration of approximately 500 micro Meter (0.013 atm), which increased channel activity about four times. The mechanism of channel activation involved shortening the closed-time durations between bursts and apparent recruitment of previously silent channels.     

Volatile Anesthetics Bind Rat Synaptic Snare Proteins

Background: Volatile general anesthetics (VAs) have a number of synaptic actions, one of which is to inhibit excitatory neurotransmitter release; however, no presynaptic VA binding proteins have been identified. Genetic data in Caenorhabditis elegans have led to the hypothesis that a protein that interacts with the presynaptic protein syntaxin 1A is a VA target. Motivated by this hypothesis, the authors measured the ability of syntaxin 1A and proteins that interact with syntaxin to bind to halothane and isoflurane.

Methods: Recombinant rat syntaxin 1A, SNAP-25B, VAMP2, and the ternary SNARE complex that they form were tested. Binding of VAs to these proteins was detected by 19F-nuclear magnetic resonance relaxation measurements. Structural alterations in the proteins were examined by circular dichroism and ability to form complexes.

Results: Volatile anesthetics did not bind to VAMP2. At concentrations in the clinical range, VAs did bind to SNAP-25B; however, binding was detected only in preparations containing SNAP-25B homomultimers. VAs also bound at clinical concentrations to both syntaxin and the SNARE complex. Addition of an N-terminal His6 tag to syntaxin abolished its ability to bind VAs despite normal secondary structure and ability to form SNARE complexes; thrombin cleavage of the tag restored VA binding. Thus, the VA binding site(s) has structural requirements and is not simply any [alpha]-helical bundle. VAs at supraclinical concentrations produced an increase in helicity of the SNARE complex; otherwise, VA binding produced no gross alteration in the stability or secondary structure of the SNARE complex.     

Use of Volatile Anesthetics in Extracorporeal Circulation

Background and objectivesThe use of volatile anesthetics in cardiac surgery is not recent. Since the introduction of halothane in clinical practice, several cardiac surgery centers started to use these anesthetics constantly.ContentIn the last years a great number of studies have shown that the volatile anesthetics have a protecting effect against myocardial ischemic dysfunction. Experimental evidences have shown that the halogenated anesthetics have cardioprotective effects that cannot be only explained by coronary flow alterations or by the balance between myocardium available and consumed oxygen. In addition to that, the use of volatile anesthetics during extracorporeal circulation (ECC) in cardiac surgery plays an important role. Recent studies have proven that these agents have cardioprotective properties and produce better results when the volatile anesthetic is used during the whole surgery procedure, including ECC. The use of halogenated anesthetics through calibrated vaporizers adapted to the ECC circuit via oxygenator membranes has become popular. Therefore, the professionals involved such as anesthesiologists and perfusionists should learn specifcs in order to solve possible doubts.     

注射挥发性麻醉药对动物的效应

研究了注射挥发性麻醉药对动物的效应，在小鼠，乙醚的催眠ＥＤ５０是５．３５（ｓｃ）和１．６２（ｉｐ）ｍｌ／ｋｇ；ＬＤ５０是１３．７（ｓｃ）和２．３２（ｉｐ）ｍｌ／ｋｇ。甲氧氟烷的ＥＤ５０为０．７５（ｉｐ）和４．１２（ｉｍ）ｍｌ／ｋｇ；ＬＤ５０为１．２３（ｉｐ）５．８１（ｉｍ）ｍｌ／ｋｇ，ｉｐ氟烷的ＥＤ５０和ＬＤ５０分别是１．２０和１．９４ｍｌ／ｋｇ，ｉｐ安氟醚的ＥＤ５０和ＬＤ５０分别是３．０１和７．     

Differential Inhibition of Lysophosphatidate Signaling by Volatile Anesthetics

Background: Volatile anesthetics have been found to interfere with the functioning of several G protein-coupled receptors, effects that may be relevant to the mechanism of anesthetic action. Lysophosphatidate (1-acyl-2-sn-glycero-3-phosphate; LP) is the simplest natural phospholipid. It has pronounced biological effects and signals through a specific G protein-coupled receptor. Because of its lipophilicity, the LP receptor is a feasible site of anesthetic interaction. Therefore, the authors investigated the effects of halothane and isoflurane on LP signaling using Xenopus oocytes.

Methods: Mature oocytes were harvested from Xenopus frogs, isolated, and defolliculated manually. Lysophosphatidate receptors are endogenously present in these cells. Angiotensin receptors were expressed recombinantly to study anesthetic effects on intracellular signaling. Oocytes were studied individually with a two-electrode voltage clamp at room temperature. Integrated Ca2+ -activated Cl sup - currents (ICl(Ca)) were used to evaluate the effects of anesthetics on changes in intracellular Ca2+ concentration in response to receptor agonists (10 sup -7 M LP or 10 sup -7 M angiotensin II) or intracellular inositoltrisphosphate (IP3) injection.

Results: Halothane depressed LP signaling in a concentration-dependent manner, with half-maximal inhibition at 0.23 mM and virtually complete inhibition at 0.34 mM. Responses could be recovered after an anesthetic-free wash. Oocyte injection with heparin, an IP3 receptor antagonist, completely blocked LP and angiotensin signaling, indicating similar IP3 -dependent pathways. However, ICl(Ca) induced by angiotensin receptor activation or intracellular IP3 injection were not inhibited by halothane. Isoflurane, at comparable concentrations, did not depress LP responses in oocytes significantly.     

Behavioral Effects of Volatile Anesthetics in Caenorhabditis elegans

Background: The nematode Caenorhabditis elegans offers many advantages as a model organism for studying volatile anesthetic action: It has a simple, well-understood nervous system; it allows the researcher to do forward genetics; and its genome will soon be completely sequenced. C. elegans is immobilized by volatile anesthetics only at high concentrations and with an unusually slow time course. Here other behavioral dysfunctions are considered as anesthetic endpoints in C. elegans.

Methods: The potency of halothane for disrupting eight different behaviors was determined by logistic regression of concentration and response data. Other volatile anesthetics were also tested for some behaviors. Established protocols were used for behavioral endpoints that, except for pharyngeal pumping, were set as complete disruption of the behavior. Time courses were measured for rapid behaviors. Recovery from exposure to 1 or 4 vol% halothane was determined for mating, chemotaxis, and gross movement. All experiments were performed at 20 to 22 degrees Celsius.

Results: The median effective concentration values for halothane inhibition of mating (0.30 vol% - 0.21 mM), chemotaxis (0.34 vol% - 0.24 mM), and coordinated movement (0.32 vol% - 0.23 mM) were similar to the human minimum alveolar concentration (MAC; 0.21 mM). In contrast, halothane produced immobility with a median effective concentration of 3.65 vol% (2.6 mM). Other behaviors had intermediate sensitivities. Halothane's effects reached steady-state in 10 min for all behaviors tested except immobility, which required 2 h. Recovery was complete after exposure to 1 vol% halothane but was significantly reduced after exposure to immobilizing concentrations.     

GDNF对脊髓前角运动神经元的保护作用

目的：证实胶质源性神经营养因子（glial cell line-derived neurotrophic factor,GDNF)对脊髓前角运动神经元有保护作用。方法：切断SD大鼠一侧坐骨神经建立脊髓前角运动神经元损伤模型。借助单盲端硅胶管系统在损伤神经局部给予GDNF，术后不同时间分别取L5脊髓切片，利用酶组织化学染色方法显示胆碱酯酶（CHE）和酸性磷酸酶（ACP）活性并进行图像分析。结果：坐骨神经切断可导致腰段脊髓前角运动神经元明显损害，表现为CHE活性降低。ACP活性升高；应用GDNF可显著改善上述酶学变化。结论：GDNF对损伤的脊髓前角运动神经元有保护作用。