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).     

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 alpha2 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 microM) 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 alpha1-receptor agonist, phenylephrine (10-60 microM), and inhibited by alpha1-receptor antagonists prazosin (0.5 microM) and 2-(2,6-dimethoxyphenoxyethyl) amino-methyl-1,4-benzodioxane (0.5 microM). Neither postsynaptic responsiveness to exogenously applied GABA and glycine nor the kinetics of GABAergic and glycinergic inhibitory postsynaptic currents were affected by norepinephrine. CONCLUSION: These results suggest that norepinephrine enhances inhibitory synaptic transmission in the substantia gelatinosa through activation of presynaptic alpha1 receptors, thus providing a mechanism underlying the clinical use of alpha1 agonists with local anesthetics in spinal anesthesia.     

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.     

Alpha 2 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 microm) 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 alpha2 adrenoceptor agonist, clonidine (10 microm), and an alpha 2A agonist, oxymetazoline (10 microm), but not by an alpha1 agonist, phenylephrine (10 microm), and a beta agonist, isoproterenol (40 microm). The inhibitory actions were antagonized by an alpha 2 antagonist, yohimbine (1 microm), 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. CONCLUSIONS: Norepinephrine inhibits A delta-fiber- and C-fiber-mediated sensory transmission to substantia gelatinosa neurons through the activation of the alpha 2 adrenoceptor (possibly alpha2A type, based on the current, published behavioral and anatomical data) existing in primary afferent terminals; this action of norepinephrine is more effective in A delta-fiber than C-fiber transmission. This could contribute to at least a part of inhibitory modulation of pain sensation in the substantia gelatinosa by intrathecally administered 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.     

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.     

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 GABA(A)-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 microM) prolonged the decay phase of evoked and miniature inhibitory postsynaptic currents (IPSCs), mediated by GABA(A) 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 microM) in a manner sensitive to a benzodiazepine receptor antagonist, flumazenil (1 microM); glycine currents were, however, unaltered by midazolam. CONCLUSIONS: Midazolam augmented both the duration of GABA-mediated synaptic current and the amplitude of GABA-induced current by acting on the GABA(A)-benzodiazepine receptor in substantia gelatinosa neurons; this would increase the inhibitory GABAergic transmission. This may be a possible mechanism for antinociception by midazolam.     

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 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.     

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.     

Differential presynaptic effects of opioid agonists on Adelta- and C-afferent glutamatergic transmission to the spinal dorsal horn

BACKGROUND: Although intrathecal administration of opioids produces antinociceptive effects in the spinal cord, it has not been established whether intrathecal opioid application more effectively terminates C fiber-mediated pain than A fiber-mediated pain. Here, the authors focus on the differences in opioid actions on Adelta- and C-afferent responses. METHODS: Using the whole cell patch clamp technique, the authors investigated the presynaptic inhibitory actions of micro-, delta-, and kappa-opioid receptor agonists on primary afferent-evoked excitatory postsynaptic currents (EPSCs) in substantia gelatinosa neurons of adult rat spinal cord slices. RESULTS: The micro agonist DAMGO (0.1, 1 microM) reduced the amplitude of glutamatergic monosynaptic Adelta- or C fiber-evoked EPSCs. C fiber-evoked EPSCs were inhibited to a greater extent than Adelta fiber-evoked EPSCs. The delta agonist DPDPE (1, 10 microM) produced modest inhibition of Adelta- or C fiber-evoked EPSCs. In contrast, the kappa agonist U69593 (1 microM) did not affect the amplitude of either Adelta or C fiber-evoked EPSCs. CONCLUSIONS: These results indicate that opioids suppress excitatory synaptic transmission mainly through activation of micro receptors on primary afferent C fibers. Given that the substantia gelatinosa is the main termination of Adelta and C fibers transmitting nociceptive information, the current findings may partially explain the different potency of opioid agonists.     

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 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.     

Modulation of gamma-aminobutyric acid A receptor function by thiopental in the rat spinal dorsal horn neurons

To assess the actions of thiopental at the spinal dorsal horn level, we examined the effects of thiopental using the whole cell patch-clamp technique on mechanically dissociated rat spinal dorsal horn neurons. Thiopental, at large concentrations, elicited a current (I(Thio)) through activation of chloride conductance, and its threshold concentration was approximately 50 microM. I(Thio) was sensitive to bicuculline, a gamma-aminobutyric acid (GABA)A receptor antagonist, but not to strychnine, a glycine receptor antagonist. At a clinically relevant concentration (30 muM), thiopental markedly enhanced the peak amplitude of a subsaturating GABA-induced current (I(GABA)) but not that of a saturating GABA-induced current. Furthermore, thiopental prolonged the time constants of both desensitization and deactivation of I(GABA). At a large concentration (300 muM), it inhibited the peak amplitude of I(GABA), which may be the result of open-channel blockade. In addition, at 30 microM, thiopental increased the duration and decreased the frequency of GABAergic miniature inhibitory postsynaptic currents. These results indicate that thiopental enhances GABAergic inhibitory transmission and suggest that GABA(A) receptors in the spinal cord are a potential target through which thiopental causes immobility and depresses the response to noxious stimuli.     

Effect of dorsal-column stimulation on gelatinosa and marginal neurons of cat spinal cord

Single neuronal units with physiological characteristics of superficial dorsal-horn neurons were recorded extracellularly in laminae 1, 2, and 3 of cat spinal cord. When focal electrical stimulation was applied to the ipsilateral dorsal column, most of the units were excited transsynaptically at various latencies consistent with an effect mediated by large myelinated axons. Units recorded in laminae 2 and 3 had earlier latencies of activation than units in lamina 1. Units with cutaneous receptive fields only for noxious stimuli were activated at significantly longer latencies than units responsive to innocuous stimuli. The time course of these effects was consistent with the concept that many cells in laminae 1 to 3 receive direct excitatory synaptic input from collaterals of dorsal-column fibers, and some lamina 1 cells receive excitatory synaptic input from lamina 2 neurons. Previous reports have emphasized the inhibitory action of dorsal-column stimulation on nociceptive responses of cells in laminae 4 and 5 of the dorsal-horn, particularly those of the spinocervical tract in cats and the spinothalamic tract in primates. The present study suggests that some of this inhibition might be sustained by a network of interneurons in or near the substantia gelatinosa and marginal layer. The therapeutic efficiency of dorsal-column stimulation for pain relief in humans may depend in part on the activation of neurons in the superficial layers of the dorsal horn.     

Midazolam presynaptically inhibits excitatory synaptic transmission in the superficial dorsal horn of the rat spinal cord]

Whole-cell voltage-clamp recordings were made from superficial dorsal horn neurons in thin slices of neonatal rat spinal cord. Spontaneously occurring glutaminergic miniature excitatory postsynaptic currents (mEPSCs) were recorded in the presence of tetrodotoxin, strychnine and bicuculline. Bath-application of midazolam (5-60 microM) reduced the frequency of mEPSCs dose-dependently and this depression was antagonized by flumazenil 15 microM. In 8 neurons studied, midazolam 15 microM affected neither the amplitude profiles nor the mean amplitudes of the mEPSCs, suggesting a presynaptic site of action. In a nominally Ca(2+)-free solution, midazolam still reduced the frequency of mEPSCs, but to a lesser extent than in a standard solution. We conclude that midazolam may presynaptically inhibit excitatory synaptic transmission in the superficial dorsal horn by affecting both Ca2+ entry into the nerve terminals and transmitter release mechanisms downstream to Ca2+ entry.     

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.     

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 micrometer) or strychnine (2 micrometer) 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 micrometer) or 6-cyano-7-nitroquinoxaline-2,3-dione disodium (10 micrometer) was applied to block NMDA or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-kainate receptors respectively. CONCLUSIONS: Enflurane exerts direct depressant effects on both alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and NMDA glutamate currents in motor neurons. Enhancement of gamma-aminobutyric acid A and glycine inhibition is not needed for this effect. Direct depression of glutamatergic excitatory transmission by a postsynaptic action on motor neurons thus may contribute to general anesthesia as defined by immobility in response to a noxious stimulus.     

Anatomy,physiology and pharmacology of pain

Nociception is conveyed from the periphery to the brain at three levels: the peripheral nociceptor, the spinal cord, and the supra-spinal (brain) levels. Physiological (first or ‘fast’) pain is produced by stimulation of high threshold thermo/mechanical nociceptors, which transmit via fast conducting myelinated A delta fibres. These enter the dorsal horn of the spinal cord and synapse at laminae I and V. Pathophysiological (second or ‘slow’) pain originates from stimulation of the high threshold polymodal nociceptors (free endings) present in all tissues. The nociceptors respond to mechanical, chemical and thermal stimuli and are transmitted via slow conducting unmyelinated C fibres. These synapse at laminae II and III (substantia gelatinosa) of the dorsal horn. The second order neurons are either nociceptive specific (substantia gelatinosa) or wide dynamic range (WDR) neurons (in laminae V and VI) that respond to a wide range of noxious and non-noxious input. Both pathways ascend up the spinal cord via the spinothalamic tracts to the thalamus, which synapse and project on to the somatosensory cortex. Inhibitory inter-neurons in the substantia gelatinosa prevent activation of the dorsal root ganglia. Interneurons can be activated by A beta and inhibited by A beta and C fibre activity. Pain can be ‘gated-out’ by stimulating the large A beta fibres in the painful area. This is the working mechanism behind transcutaneous electrical nerve stimulation. The descending inhibition pathways originate at the level of the cortex and thalamus, and descend via the brainstem (periaqueductal grey) and the dorsal columns to terminate at the dorsal horn of the spinal cord. Neurotransmitters noradrenaline, serotonin (5-HT) and the endogenous opioids are released to provide antinociception.     

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.