Action of isoflurane on the substantia gelatinosa neurons of the adult rat spinal cord

BACKGROUND: Although isoflurane, a volatile anesthetic, can block the motor response to noxious stimulation (immobility and analgesia) and suppress autonomic responsiveness, how it exerts these effects at the neuronal level in the spinal cord is not fully understood. METHODS: The effects of a clinically relevant concentration (1 rat minimum alveolar concentration [MAC]) of isoflurane on electrically evoked and spontaneous excitatory/inhibitory transmission and on the response to exogenous administration of the gamma-aminobutyric acid type A receptor agonist muscimol were examined in lamina II neurons of adult rat spinal cord slices using the whole cell patch clamp technique. The effect of isoflurane on the action potential-generating membrane property was also examined. RESULTS: Bath-applied isoflurane (1.5%, 1 rat MAC) diminished dorsal root-evoked polysynaptic but not monosynaptic excitatory postsynaptic currents. Glutamatergic miniature excitatory postsynaptic currents were also unaffected by isoflurane. In contrast, isoflurane prolonged the decay phase of evoked and miniature gamma-aminobutyric acid type A receptor-mediated inhibitory postsynaptic currents and increased the amplitude of the muscimol-induced current. Isoflurane had little effect on action potential discharge activity. CONCLUSIONS: Isoflurane augments gamma-aminobutyric acid-mediated inhibitory transmission, leading to a decrease in the excitability of spinal dorsal horn neurons. This may be a possible mechanism for the antinociceptive effect of isoflurane in the spinal cord.     

Actions of Midazolam on Excitatory Transmission in Dorsal Horn Neurons of Adult Rat Spinal Cord

Background: Although intrathecal administration of midazolam, a water-soluble imidazobenzodiazepine derivative, has been found to produce analgesia, how it exerts this effect at the neuronal level in the spinal cord is not fully understood.

Methods: The effects of midazolam on electrically evoked and spontaneous excitatory transmission were examined in lamina II neurons of adult rat spinal cord slices using the whole cell patch clamp technique.

Results: Bath-applied midazolam (1 [mu]m) diminished A[delta]- and C-fiber evoked polysynaptic excitatory postsynaptic currents in both amplitude and integrated area. However, it affected neither A[delta]- and C-fiber evoked monosynaptic excitatory postsynaptic currents in amplitude nor miniature excitatory postsynaptic currents in amplitude, frequency, and decay time constant. In the presence of a benzodiazepine receptor antagonist, flumazenil (5 [mu]m), midazolam (1 [mu]m) did not diminish A[delta]-fiber evoked polysynaptic excitatory postsynaptic currents, suggesting that midazolam modulate the [gamma]-aminobutyric acid interneurons in the dorsal horn.     

Actions of Midazolam on GABAergic Transmission in Substantia Gelatinosa Neurons of Adult Rat Spinal Cord Slices

Background: Although intrathecal administration of midazolam has been found to produce analgesia, how midazolam exerts this effect is not understood fully at the neuronal level in the spinal cord.

Methods: The effects of midazolam on either electrically evoked or spontaneous inhibitory transmission and on a response to exogenous [gamma]-aminobutyric acid (GABA), a GABAA-receptor agonist, muscimol, or glycine were evaluated in substantia gelatinosa neurons of adult rat spinal cord slices by using the whole-cell patch-clamp technique.

Results: Bath-applied midazolam (1 [mu]M) prolonged the decay phase of evoked and miniature inhibitory postsynaptic currents (IPSCs), mediated by GABAA receptors, without a change in amplitudes, while not affecting glycine receptor-mediated miniature inhibitory postsynaptic currents in both the decay phase and the amplitude. Either GABA- or muscimol-induced currents were enhanced in amplitude by midazolam (0.1 [mu]M) in a manner sensitive to a benzodiazepine receptor antagonist, flumazenil (1 [mu]M); glycine currents were, however, unaltered by midazolam.     

Isoflurane Differentially Modulates Inhibitory and Excitatory Synaptic Transmission to the Solitary Tract Nucleus

Background: Isoflurane anesthesia produces cardiovascular and respiratory depression, although the specific mechanisms are not fully understood. Cranial visceral afferents, which innervate the heart and lungs, synapse centrally onto neurons within the medial portion of the nucleus tractus solitarius (NTS). Isoflurane modulation of afferent to NTS synaptic communication may underlie compromised cardiorespiratory reflex function.

Methods: Adult rat hindbrain slice preparations containing the solitary tract (ST) and NTS were used. Shocks to ST afferents evoked excitatory postsynaptic currents with low-variability (SEM <200 [mu]s) latencies identifying neurons as second order. ST-evoked and miniature excitatory postsynaptic currents as well as miniature inhibitory postsynaptic currents were measured during isoflurane exposure. Perfusion bath samples were taken in each experiment to measure isoflurane concentrations by gas chromatography-mass spectrometry.

Results: Isoflurane dose-dependently increased the decay-time constant of miniature inhibitory postsynaptic currents. At greater than 300 [mu]m isoflurane, the amplitude of miniature inhibitory postsynaptic currents was decreased, but the frequency of events remained unaffected, whereas at equivalent isoflurane concentrations, the frequency of miniature excitatory postsynaptic currents was decreased. ST-evoked excitatory postsynaptic current amplitudes decreased without altering event kinetics. Isoflurane at greater than 300 [mu]m increased the latency to onset and rate of synaptic failures of ST-evoked excitatory postsynaptic currents.     

Effect of Isoflurane and Other Potent Inhaled Anesthetics on Minimum Alveolar Concentration, Learning, and the Righting Reflex in Mice Engineered to Express α1 γ-Aminobutyric Acid Type A Receptors Unresponsive to Isoflurane

Background: Enhancement of the function of [gamma]-aminobutyric acid type A receptors containing the [alpha]1 subunit may underlie a portion of inhaled anesthetic action. To test this, the authors created gene knock-in mice harboring mutations that render the receptors insensitive to isoflurane while preserving sensitivity to halothane.

Methods: The authors recorded miniature inhibitory synaptic currents in hippocampal neurons from hippocampal slices from knock-in and wild-type mice. They also determined the minimum alveolar concentration (MAC), and the concentration at which 50% of animals lost their righting reflexes and which suppressed pavlovian fear conditioning to tone and context in both genotypes.

Results: Miniature inhibitory postsynaptic currents decayed more rapidly in interneurons and CA1 pyramidal cells from the knock-in mice compared with wild-type animals. Isoflurane (0.5-1 MAC) prolonged the decay phase of miniature inhibitory postsynaptic currents in neurons of the wild-type mice, but this effect was significantly reduced in neurons from knock-in mice. Halothane (1 MAC) slowed the decay of miniature inhibitory postsynaptic current in both genotypes. The homozygous knock-in mice were more resistant than wild-type controls to loss of righting reflexes induced by isoflurane and enflurane, but not to halothane. The MAC for isoflurane, desflurane, and halothane did not differ between knock-in and wild-type mice. The knock-in mice and wild-type mice did not differ in their sensitivity to isoflurane for fear conditioning.     

Norepinephrine Facilitates Inhibitory Transmission in Substantia Gelatinosa of Adult Rat Spinal Cord (Part 1): Effects on Axon Terminals of GABAergic and Glycinergic Neurons

Background: The activation of descending norepinephrine-containing fibers from the brain stem inhibits nociceptive transmission at the spinal level. How these descending noradrenergic pathways exert the analgesic effect is not understood fully. Membrane hyperpolarization of substantia gelatinosa (Rexed lamina II) neurons by the activation of [alpha]2 receptors may account for depression of pain transmission. In addition, it is possible that norepinephrine affects transmitter release in the substantia gelatinosa.

Methods: Adult male Sprague-Dawley rats (9-10 weeks of age, 250-300 g) were used in this study. Transverse spinal cord slices were cut from the isolated lumbar cord. The blind whole-cell patch-clamp technique was used to record from neurons. The effects of norepinephrine on the frequency and amplitude of miniature excitatory and inhibitory postsynaptic currents were evaluated.

Results: In the majority of substantia gelatinosa neurons tested, norepinephrine (10-100 [mu]M) dose-dependently increased the frequency of [gamma]-aminobutyric acid (GABA)-ergic and glycinergic miniature inhibitory postsynaptic currents; miniature excitatory postsynaptic currents were unaffected. This augmentation was mimicked by an [alpha]1-receptor agonist, phenylephrine (10-60 [mu]M), and inhibited by [alpha]1-receptor antagonists prazosin (0.5 [mu]M) and 2-(2,6-dimethoxyphenoxyethyl) aminomethyl-1,4-benzodioxane (0.5 [mu]M). Neither postsynaptic responsiveness to exogenously applied GABA and glycine nor the kinetics of GABAergic and glycinergic inhibitory postsynaptic currents were affected by norepinephrine.     

α2 Adrenoceptor-mediated Presynaptic Inhibition of Primary Afferent Glutamatergic Transmission in Rat Substantia Gelatinosa Neurons

Background: Although intrathecal administration of norepinephrine is known to produce analgesia, cellular mechanisms for this action have not yet been fully understood.

Methods: The actions of norepinephrine (50 [mu]m) on glutamatergic transmission were examined by using the whole cell patch clamp technique in substantia gelatinosa neurons of an adult rat spinal cord slice with an attached dorsal root.

Results: Norepinephrine inhibited the amplitude of monosynaptically evoked A[delta]-fiber and C-fiber excitatory postsynaptic currents in a reversible manner. When compared in magnitude between the A[delta]-fiber and C-fiber excitatory postsynaptic currents, the former inhibition (50 +/- 4%, n = 20) was significantly larger than the latter one (28 +/- 4%, n = 8). Both actions of norepinephrine were mimicked by an [alpha]2 adrenoceptor agonist, clonidine (10 [mu]m), and an [alpha]2A agonist, oxymetazoline (10 [mu]m), but not by an [alpha]1 agonist, phenylephrine (10 [mu]m), and a [beta] agonist, isoproterenol (40 [mu]m). The inhibitory actions were antagonized by an [alpha]2 antagonist, yohimbine (1 [mu]m), all of the results of which indicate an involvement of [alpha]2 adrenoceptors. Norepinephrine did not affect the amplitude of miniature excitatory postsynaptic current and of a response of substantia gelatinosa neurons to AMPA, indicating that its action on evoked excitatory postsynaptic currents is presynaptic in origin.     

Contrasting Synaptic Actions of the Inhalational General Anesthetics Isoflurane and Xenon

Background: The mechanisms by which the inhalational general anesthetics isoflurane and xenon exert their effects are unknown. Moreover, there have been surprisingly few quantitative studies of the effects of these agents on central synapses, with virtually no information available regarding the actions of xenon.

Methods: The actions of isoflurane and xenon on [gamma]-aminobutyric acid-mediated (GABAergic) and glutamatergic synapses were investigated using voltage-clamp techniques on autaptic cultures of rat hippocampal neurons, a preparation that avoids the confounding effects of complex neuronal networks.

Results: Isoflurane exerts its greatest effects on GABAergic synapses, causing a marked increase in total charge transfer (by approximately 70% at minimum alveolar concentration) through the inhibitory postsynaptic current. This effect is entirely mediated by an increase in the slow component of the inhibitory postsynaptic current. At glutamatergic synapses, isoflurane has smaller effects, but it nonetheless significantly reduces the total charge transfer (by approximately 30% at minimum alveolar concentration) through the excitatory postsynaptic current, with the N-methyl-D-aspartate (NMDA) and [alpha]-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptor-mediated components being roughly equally sensitive. Xenon has no measurable effect on GABAergic inhibitory postsynaptic currents or on currents evoked by exogenous application of GABA, but it substantially inhibits total charge transfer (by approximately 60% at minimum alveolar concentration) through the excitatory postsynaptic current. Xenon selectively inhibits the NMDA receptor-mediated component of the current but has little effect on the AMPA/kainate receptor-mediated component.     

Propofol Modulates γ-Aminobutyric Acid-mediated Inhibitory Neurotransmission to Cardiac Vagal Neurons in the Nucleus Ambiguus

Background: Although it is well recognized that anesthetics modulate the central control of cardiorespiratory homeostasis, the cellular mechanisms by which anesthetics alter cardiac parasympathetic activity are poorly understood. One common site of action of anesthetics is inhibitory neurotransmission. This study investigates the effect of propofol on [gamma]-aminobutyric acid-mediated (GABAergic) and glycinergic neurotransmission to cardiac parasympathetic neurons.

Methods: Cardiac parasympathetic neurons were identified in vitro by the presence of a retrograde fluorescent tracer, and spontaneous GABAergic and glycinergic synaptic currents were examined using whole cell patch clamp techniques.

Results: Propofol at concentrations of 1.0 [mu]m and greater significantly (P < 0.05) increased the duration and decay time of spontaneous GABAergic inhibitory postsynaptic currents. To determine whether the action of propofol was at presynaptic or postsynaptic sites, tetrodotoxin was applied to isolate miniature inhibitory postsynaptic currents. Propofol at concentrations of 1.0 [mu]m and greater significantly (P < 0.05) prolonged the decay time and duration of miniature inhibitory postsynaptic currents, indicating that propofol directly alters GABAergic neurotransmission at a postsynaptic site. Propofol at high concentrations (>=50 [mu]m) also inhibited the frequency of both GABAergic inhibitory postsynaptic currents and miniature inhibitory postsynaptic currents. Propofol at concentrations up to 50 [mu]m had no effect on glycinergic neurotransmission.     

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. 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.     

Enflurane Actions on Spinal Cords from Mice That Lack the β3 Subunit of the GABAA Receptor

Background: [gamma]-Aminobutyric acid type A (GABAA) receptors are considered important in mediating anesthetic actions. Mice lacking the [beta]3 subunit of this receptor ([beta]3-/-) have a higher enflurane minimum alveolar concentration (MAC) than wild types (+/+). MAC is predominantly determined in spinal cord.

Methods: The authors measured three population-evoked responses in whole spinal cords, namely, the excitatory postsynaptic potential (pEPSP), the slow ventral root potential (sVRP), and the dorsal root potential. Synaptic and glutamate-evoked currents from motor neurons in spinal cord slices were also measured.

Results: Sensitivity of evoked responses to enflurane did not differ between +/+ and -/- cords. The GABAA receptor antagonist bicuculline significantly (P < 0.05) attenuated the depressant effects of enflurane on pEPSP, sVRP and glutamate-evoked currents in +/+ but not -/- cords. The glycine antagonist strychnine elevated the pEPSP to a significantly greater extent in -/- than in +/+ cords, but the interactions between strychnine and enflurane did not differ between -/- and +/+ cords.     

Enflurane Directly Depresses Glutamate AMPA and NMDA Currents in Mouse Spinal Cord Motor Neurons Independent of Actions on GABAA or Glycine Receptors

Background: The spinal cord is an important anatomic site at which volatile agents act to prevent movement in response to a noxious stimulus. This study was designed to test the hypothesis that enflurane acts directly on motor neurons to inhibit excitatory synaptic transmission at glutamate receptors.

Methods: Whole-cell recordings were made in visually identified motor neurons in spinal cord slices from 1- to 4-day-old mice. Excitatory postsynaptic currents (EPSCs) or potentials (EPSPs) were evoked by electrical stimulation of the dorsal root entry area or dorsal horn. The EPSCs were isolated pharmacologically into glutamate N-methyl-d-aspartate (NMDA) receptor- and non-NMDA receptor-mediated components by using selective antagonists. Currents also were evoked by brief pulse pressure ejection of glutamate under various conditions of pharmacologic blockade. Enflurane was made up as a saturated stock solution and diluted in the superfusate; concentrations were measured using gas chromatography.

Results: Excitatory postsynaptic currents and EPSPs recorded from motor neurons by stimulation in the dorsal horn were mediated by glutamate receptors of both non-NMDA and NMDA subtypes. Enflurane at a general anesthetic concentration (one minimum alveolar anesthetic concentration) reversibly depressed EPSCs and EPSPs. Enflurane also depressed glutamate-evoked currents in the presence of tetrodotoxin (300 nm), showing that its actions are postsynaptic. Block of inhibitory [gamma]-aminobutyric acid A and glycine receptors by bicuculline (20 [mu]m) or strychnine (2 [mu]m) or both did not significantly reduce the effects of enflurane on glutamate-evoked currents. Enflurane also depressed glutamate-evoked currents if the inhibitory receptors were blocked and if either D,L-2-amino-5-phosphonopentanoic acid (50 [mu]m) or 6-cyano-7-nitroquinoxaline-2,3-dione disodium (10 [mu]m) was applied to block NMDA or [alpha]-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-kainate receptors respectively.     

Dual Actions of Enflurane on Postsynaptic Currents Abolished by the γ-Aminobutyric Acid Type A Receptor β3(N265M) Point Mutation

Background: At concentrations close to 1 minimum alveolar concentration (MAC)-immobility, volatile anesthetics display blocking and prolonging effects on [gamma]-aminobutyric acid type A receptor-mediated postsynaptic currents. It has been proposed that distinct molecular mechanisms underlie these dual actions. The authors investigated whether the blocking or the prolonging effect of enflurane is altered by a point mutation (N265M) in the [beta]3 subunit of the [gamma]-aminobutyric acid type A receptor. Furthermore, the role of the [beta]3 subunit in producing the depressant actions of enflurane on neocortical neurons was elucidated.

Methods: Spontaneous inhibitory postsynaptic currents were sampled from neocortical neurons in cultured slices derived from wild-type and [beta]3(N265M) mutant mice. The effects of 0.3 and 0.6 mm enflurane on decay kinetics, peak amplitude, and charge transfer were quantified. Furthermore, the impact of enflurane-induced changes in spontaneous action potential firing was evaluated by extracellular recordings in slices from wild-type and mutant mice.

Results: In slices derived from wild-type mice, enflurane prolonged inhibitory postsynaptic current decays and decreased peak amplitudes. Both effects were almost absent in slices from [beta]3(N265M) mutant mice. At clinically relevant concentrations between MAC-awake and MAC-immobility, the anesthetic was less effective in depressing spontaneous action potential firing in slices from [beta]3(N265M) mutant mice compared with wild-type mice.     

Actions of midazolam on excitatory transmission in dorsal horn neurons of adult rat spinal cord

BACKGROUND: Although intrathecal administration of midazolam, a water-soluble imidazobenzodiazepine derivative, has been found to produce analgesia, how it exerts this effect at the neuronal level in the spinal cord is not fully understood. METHODS: The effects of midazolam on electrically evoked and spontaneous excitatory transmission were examined in lamina II neurons of adult rat spinal cord slices using the whole cell patch clamp technique. RESULTS: Bath-applied midazolam (1 microm) diminished Adelta- and C-fiber evoked polysynaptic excitatory postsynaptic currents in both amplitude and integrated area. However, it affected neither Adelta- and C-fiber evoked monosynaptic excitatory postsynaptic currents in amplitude nor miniature excitatory postsynaptic currents in amplitude, frequency, and decay time constant. In the presence of a benzodiazepine receptor antagonist, flumazenil (5 microm), midazolam (1 microm) did not diminish Adelta-fiber evoked polysynaptic excitatory postsynaptic currents, suggesting that midazolam modulate the gamma-aminobutyric acid interneurons in the dorsal horn. CONCLUSIONS: Midazolam reduced excitatory synaptic transmission by acting on the gamma-aminobutyric acid type A/benzodiazepine receptor in interneurons, leading to a decrease in the excitability of spinal dorsal horn neurons. This may be a possible mechanism for the antinociception by midazolam in the spinal cord.     

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 [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.     

Sevoflurane Enhances γ-Aminobutyric Acid Type A Receptor Function and Overall Inhibition of Inspiratory Premotor Neurons in a Decerebrate Dog Model

Background: Inspiratory premotor neurons in the caudal ventral medulla relay excitatory drive to phrenic and inspiratory intercostal motoneurons in the spinal cord. These neurons are subject to tonic [gamma]-aminobutyric acid type A (GABAA)ergic inhibition. In a previous study, 1 minimum alveolar concentration (MAC) sevoflurane depressed overall glutamatergic excitatory drive and enhanced overall GABAAergic inhibitory drive to the neurons. This study investigated in further detail the effects of sevoflurane on GABAAergic inhibition by examining postsynaptic GABAA receptor activity in these neurons.

Methods: Studies were performed in decerebrate, vagotomized, paralyzed, and mechanically ventilated dogs during hypercapnic hyperoxia. The effect of 1 MAC sevoflurane on extracellularly recorded neuronal activity was measured during localized picoejection of the GABAA receptor antagonist bicuculline and the GABAA agonist muscimol. Complete blockade of GABAAergic inhibition by bicuculline allowed estimation of the prevailing overall inhibition of the neuron. The neuronal response to muscimol was used to assess the anesthetic effect on the postsynaptic GABAA receptor function.

Results: One MAC sevoflurane depressed the spontaneous activity of 21 inspiratory premotor neurons by (mean +/- SD) 32.6 +/- 20.5% (P < 0.001). Overall excitatory drive was depressed 17.9 +/- 19.8% (P < 0.01). Overall GABAAergic inhibition was enhanced by 18.5 +/- 18.2% (P < 0.001), and the postsynaptic GABAA receptor function was increased by 184.4 +/- 121.8% (n = 20; P < 0.001).     

Excitatory and Inhibitory Actions of Isoflurane on the Cholinergic Ascending Arousal System of the Rat

Background: The cholinergic arousal systems are known to critically regulate the state of consciousness. The aim of this study was to determine the effect of isoflurane on the inhibitory or excitatory neurotransmitters efflux in important nuclei within the cholinergic arousal system using in vivo intracerebral microdialysis.

Methods: The efflux of glutamate, [gamma]-aminobutyric acid (GABA), or acetylcholine in the posterior hypothalamus (PH), the basal forebrain (BF), and the somatosensory cortex (S1BF) of rats was detected using intracerebral microdialysis under an awake condition and at 0.5-2.0 minimum alveolar concentration (MAC) isoflurane anesthesia. The intrabasalis perfusion of [alpha]-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and N-methyl-d-aspartate on the cortical acetylcholine effluxes was also examined under both conditions.

Results: Isoflurane had no influence on the glutamate and GABA efflux in the PH, whereas in the BF, it dose-dependently increased glutamate efflux and decreased GABA efflux. A transient increase in glutamate efflux at 1.0 MAC and a decrease in GABA at 0.5-1.5 MAC were observed in the S1BF. Isoflurane dose-dependently decreased acetylcholine efflux in the S1BF. Perfusion of the BF with AMPA increased acetylcholine efflux in the S1BF with electroencephalographic activation during 0.75 MAC isoflurane anesthesia, suggesting an inhibitory action of isoflurane on AMPA receptors in the BF. However, N-methyl-d-aspartate had no effect on these parameters.     

Halothane Enhances γ-Aminobutyric Acid Receptor Type A Function but Does Not Change Overall Inhibition in Inspiratory Premotor Neurons in a Decerebrate Dog Model

Background: Inspiratory premotor neurons in the caudal ventral medulla relay excitatory drive to phrenic and inspiratory intercostal motoneurons in the spinal cord. These neurons are subject to tonic [gamma]-aminobutyric acid type A (GABAA)-mediated (GABAAergic) inhibition. In a previous study, 1 minimum alveolar concentration (MAC) halothane depressed overall glutamatergic excitatory drive but did not change overall inhibitory drive to the neurons. This study investigated in further detail the effects of halothane on GABAAergic inhibition by examining postsynaptic GABAA receptor activity in these neurons.

Methods: Studies were performed in decerebrate, vagotomized, paralyzed, and mechanically ventilated dogs during hypercapnic hyperoxia. The effect of 1 MAC halothane on extracellularly recorded neuronal activity was measured during localized picoejection of the GABAA receptor antagonist bicuculline and the GABAA agonist muscimol. Complete blockade of GABAergic inhibition by bicuculline allowed estimation of the prevailing overall inhibition of the neuron. The neuronal response to muscimol was used to assess the anesthetic effect on the postsynaptic GABAA receptor function.

Results: One minimum alveolar concentration halothane depressed the spontaneous activity of 19 inspiratory premotor neurons by 22.9 +/- 29.1% (mean +/- SD; P < 0.01). Overall excitatory drive was depressed 23.6 +/- 16.9% (P < 0.001). Overall GABAergic inhibition was not changed (+8.7 +/- 27.5%; P = 0.295), but the postsynaptic GABAA receptor function was increased by 110.3 +/- 97.5% (P < 0.001).     

Actions of norepinephrine and isoflurane on inhibitory synaptic transmission in adult rat spinal cord substantia gelatinosa neurons

Volatile inhaled anesthetics and nitrous oxide (N2O) are often used together in clinical practice to produce analgesia. Because the analgesic effect of N2O is, at least in part, mediated by norepinephrine (NE) release in the spinal cord, we examined the interaction between isoflurane (ISO) and NE in the adult rat spinal cord with respect to central nociceptive information processing. The effects of clinically relevant concentrations of ISO (1 MAC) and NE (20 microM) on spontaneous inhibitory transmission in substantia gelatinosa (SG) neurons were examined using the blind whole-cell patch-clamp method. ISO prolonged the decay time and increased the total charge transfer of spontaneous inhibitory postsynaptic currents. NE increased the frequency and mean amplitude of inhibitory postsynaptic currents and the charge transfer as well. Coapplication of both drugs led to an additive increase of the charge transfer and frequent temporal summation of inhibitory postsynaptic currents. We conclude that both ISO and NE enhance the inhibitory synaptic transmission in the rat SG neurons and their interaction is additive, suggesting that ISO may add to the analgesic action of N2O at the spinal cord dorsal horn level.     

Norepinephrine Facilitates Inhibitory Transmission in Substantia Gelatinosa of Adult Rat Spinal Cord (Part 2): Effects on Somatodendritic Sites of GABAergic Neurons

Background: It has been reported previously that norepinephrine, when applied to the spinal cord dorsal horn, excites a subpopulation of dorsal horn neurons, presumably inhibitory interneurons. In the current study, the authors tested whether norepinephrine could activate inhibitory interneurons, specifically those that are "GABAergic."

Methods: A transverse slice was obtained from a segment of the lumbar spinal cord isolated from adult male Sprague-Dawley rats. Whole-cell patch-clamp recordings were made from substantia gelatinosa neurons using the blind patch-clamp technique. The effects of norepinephrine on spontaneous GABAergic inhibitory postsynaptic currents were studied.

Results: In the majority of substantia gelatinosa neurons tested, norepinephrine (10-60 [mu]M) significantly increased both the frequency and the amplitude of GABAergic inhibitory postsynaptic currents. These increases were blocked by tetrodotoxin (1 [mu]M). The effects of norepinephrine were mimicked by the [alpha]1-receptor agonist phenylephrine (10-80 [mu]M) and inhibited by the [alpha]1-receptor antagonist WB-4101 (0.5 [mu]M). Primary-afferent-evoked polysynaptic excitatory postsynaptic potentials or excitatory postsynaptic currents in wide-dynamic-range neurons of the deep dorsal horn were also attenuated by phenylephrine (40 [mu]M).