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 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.     

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.     

Differential Presynaptic Effects of Opioid Agonists on Aδ- 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 A[delta]- and C-afferent responses.

Methods: Using the whole cell patch clamp technique, the authors investigated the presynaptic inhibitory actions of [mu]-, [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 [mu] agonist DAMGO (0.1, 1 [mu]m) reduced the amplitude of glutamatergic monosynaptic A[delta]- or C fiber-evoked EPSCs. C fiber-evoked EPSCs were inhibited to a greater extent than A[delta] fiber-evoked EPSCs. The [delta] agonist DPDPE (1, 10 [mu]m) produced modest inhibition of A[delta]- or C fiber-evoked EPSCs. In contrast, the [kappa] agonist U69593 (1 [mu]m) did not affect the amplitude of either A[delta] or C fiber-evoked EPSCs.     

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).     

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.     

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.     

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.     

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.     

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.     

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.     

Activation of μ- and δ-Opioid Receptors Causes Presynaptic Inhibition of Glutamatergic Excitation in Neocortical Neurons

Background: The mechanism underlying the depressant effect of opioids on neuronal activity within the neocortex is still not clear. Three modes of action have been suggested: (1) inhibition by activation of postsynaptic potassium channels, (2) interaction with postsynaptic glutamate receptors, and (3) presynaptic inhibition of glutamate release. To address this issue, the authors investigated the effects of [mu]- and [delta]-receptor agonists on excitatory postsynaptic currents (EPSCs) and on membrane properties of neocortical neurons.

Methods: Intracellular recordings were performed in rat brain slices. Stimulus-evoked EPSCs mediated by different glutamate receptor subtypes were pharmacologically isolated, and opioids were applied by addition to the bathing medium. Possible postsynaptic interactions between glutamate and opioid receptors were investigated using microiontophoretic application of glutamate on neurons functionally isolated from presynaptic input.

Results: [delta]-Receptor activation by d-Ala2-d-Leu5-enkephalin (DADLE) reduced the amplitudes of EPSCs by maximum 60% in a naltrindole-reversible manner (EC50: 6-15 nm). In 30-40% of the neurons investigated, higher concentrations (0.1-1 [mu]m) of DADLE activated small outward currents. The [mu]-receptor selective agonist d-Ala2-N-MePhe5-Gly5-ol-enkephalin (0.1-1 [mu]m) depressed the amplitudes of EPSCs by maximum 30% without changes in postsynaptic membrane properties. In the absence of synaptic transmission, inward currents induced by microiontophoretic application of glutamate were not affected by DADLE.     

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.     

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.     

Modulation of γ-Aminobutyric Acid Type A Receptor-mediated Spontaneous Inhibitory Postsynaptic Currents in Auditory Cortex by Midazolam and Isoflurane

Background: Anesthetic agents that target [gamma]-aminobutyric acid type A (GABAA) receptors modulate cortical auditory evoked responses in vivo, but the cellular targets involved are unidentified. Also, for agents with multiple protein targets, the relative contribution of modulation of GABAA receptors to effects on cortical physiology is unclear. The authors compared effects of the GABAA receptor-specific drug midazolam with the volatile anesthetic isoflurane on spontaneous inhibitory postsynaptic currents (sIPSCs) in pyramidal cells of auditory cortex.

Methods: Whole cell recordings were obtained in murine brain slices at 34[degrees]C. GABAA sIPSCs were isolated by blocking ionotropic glutamate receptors. Effects of midazolam and isoflurane on time course, amplitude, and frequency of sIPSCs were measured.

Results: The authors detected no effect of midazolam at 0.01 [mu]m on sIPSCs, whereas midazolam at 0.1 and 1 [mu]m prolonged the decay of sIPSCs by approximately 25 and 70%, respectively. Isoflurane at 0.1, 0.25, and 0.5 mm prolonged sIPSCs by approximately 45, 150, and 240%, respectively. No drug-specific effects were observed on rise time or frequency of sIPSCs. Isoflurane at 0.5 mm caused a significant decrease in sIPSC amplitude.     

Ketamine Inhibits Presynaptic and Postsynaptic Nicotinic Excitation of Identified Cardiac Parasympathetic Neurons in Nucleus Ambiguus

Background: Ketamine increases both blood pressure and heart rate, effects commonly thought of as sympathoexcitatory. The authors investigated possible central nervous system actions of ketamine to inhibit cardiac parasympathetic neurons in the brainstem by inhibiting multiple nicotinic excitatory mechanisms.

Methods: The authors used a novel in vitro approach to study the effect of ketamine on identified cardiac parasympathetic preganglionic neurons in rat brainstem slices. The cardiac parasympathetic neurons in the nucleus ambiguus were retrogradely prelabeled with the fluorescent tracer by placing rhodamine into the pericardial sac. Dye-labeled neurons were visually identified for patch clamp recording. The effects of ketamine were tested on nicotine-evoked ligand-gated currents and spontaneous glutamatergic miniature synaptic currents (mini) in cardiac parasympathetic preganglionic neurons.

Results: Ketamine (10 [mu]m) inhibited (1) the nicotine (1 [mu]m)-evoked presynaptic facilitation of glutamate release (mini frequency, 18 +/- 7% of control; n = 9), and (2) the direct postsynaptic ligand-gated current (27 +/- 8% of control; n = 9), but ketamine did not alter the amplitude of postsynaptic miniature non-N-methyl-d-aspartate currents. [alpha] Bungarotoxin, an antagonist of [alpha]7 containing nicotinic presynaptic receptors, blocked ketamine actions on mini frequency (n = 10) but not mini amplitude.     

Supraspinal Anesthesia: Behavioral and Electroencephalographic Effects of Intracerebroventricularly Infused Pentobarbital, Propofol, Fentanyl, and Midazolam

Background: Anesthetic endpoints of unconsciousness and immobility result from agent effects on both brain and spinal cord that are difficult to separate during systemic administration. To investigate cerebral mechanism of anesthetic-induced unconsciousness, the authors studied behavioral and electrophysiologic effects of four anesthetics infused intracerebroventricularly to conscious rats. The authors aimed to produce progressively increasing anesthetic depths, indicated by electro-encephalographic synchronization and behavioral change.

Methods: During anesthesia, rats were equipped with intracerebroventricular infusion catheters, hind-paw stimulation, and epidural electrodes to record the electroencephalogram from the somatosensory cortex. Silicone bolus was injected into the fourth ventricle to minimize drug distribution to the spinal cord. 60 min later, 50-min infusion of pentobarbital (6.0 mg/h), fentanyl (0.75 [mu]g/h), propofol (3.0 mg/h), or midazolam (0.24 mg/h) was initiated. Vibrissal, olfactory, corneal, and tail-pinch responses were tested every 10 min.

Results: All agents depressed vibrissal, olfactory, and corneal responses; propofol and pentobarbital produced the strongest effect. All agents except propofol depressed tail-pinch response; fentanyl and pentobarbital produced the strongest effect. All agents except midazolam increased [delta] power. Pentobarbital enhanced [theta] power. All agents except fentanyl enhanced [alpha] and [beta] power. Pentobarbital and midazolam slightly increased, whereas fentanyl decreased, [gamma] power. Pentobarbital increased and midazolam decreased somatosensory evoked potential; these changes were small and apparently unrelated to behavior.     

Differential Block of Fast and Slow Inactivating Tetrodotoxin-sensitive Sodium Channels by Droperidol in Spinal Dorsal Horn Neurons

Background: Dorsal horn neurons of the spinal cord participate in neuronal pain transmission. During spinal and epidural anesthesia, dorsal horn neurons are exposed to local anesthetics and opioids. Droperidol is usually given with opioids to avoid nausea and vomiting. A recently developed method of "entire soma isolation" has made it possible to study directly the action of droperidol on different components of Na+ current in dorsal horn neurons.

Methods: Using a combination of the whole-cell patch-clamp recording from spinal cord slices and the entire soma isolation method, we studied the direct action of droperidol on two types of Na+ currents in dorsal horn neurons of young rats.

Results: The tetrodotoxin-sensitive Na+ current in isolated somata consisted of a fast inactivating ([tau]F, 0.5-2 ms; 80-90% of the total amplitude) and a slow inactivating ([tau]S, 6-20 ms; 10-20% of the total amplitude) component. Droperidol, at concentrations relevant for spinal and epidural anesthesia, selectively and reversibly suppressed the fast component with a half-maximum inhibiting concentration (IC50) of 8.3 [mu]m. The slow inactivating component was much less sensitive to droperidol; the estimated IC50 value was 809 [mu]m.     

Opioid Action on Respiratory Neuron Activity of the Isolated Respiratory Network in Newborn Rats

Background: Underlying mechanisms behind opioid-induced respiratory depression are not fully understood. The authors investigated changes in burst rate, intraburst firing frequency, membrane properties, as well as presynaptic and postsynaptic events of respiratory neurons in the isolated brainstem after administration of opioid receptor agonists.

Methods: Newborn rat brainstem-spinal cord preparations were used and superfused with [mu]-, [kappa]-, and [delta]-opioid receptor agonists. Whole cell recordings were performed from three major classes of respiratory neurons (inspiratory, preinspiratory, and expiratory).

Results: Mu- and [kappa]-opioid receptor agonists reduced the spontaneous burst activity of inspiratory neurons and the C4 nerve activity. Forty-two percent of the inspiratory neurons were hyperpolarized and decreased in membrane resistance during opioid-induced respiratory depression. Furthermore, under synaptic block by tetrodotoxin perfusion, similar changes of inspiratory neuronal membrane properties occurred after application of [mu]- and [kappa]-opioid receptor agonists. In contrast, resting membrane potential and membrane resistance of preinspiratory and majority of expiratory neurons were unchanged by opioid receptor agonists, even during tetrodotoxin perfusion. Simultaneous recordings of inspiratory and preinspiratory neuronal activities confirmed the selective inhibition of inspiratory neurons caused by [mu]- and [kappa]-opioid receptor agonists. Application of opioids reduced the slope of rising of excitatory postsynaptic potentials evoked by contralateral medulla stimulation, resulting in a prolongation of the latency of successive first action potential responses.     

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 microM) significantly increased both the frequency and the amplitude of GABAergic inhibitory postsynaptic currents. These increases were blocked by tetrodotoxin (1 microM). The effects of norepinephrine were mimicked by the alpha1-receptor agonist phenylephrine (10-80 microM) and inhibited by the alpha1-receptor-antagonist WB-4101 (0.5 microM). 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 microM). CONCLUSION: The observations suggest that GABAergic interneurons possess somatodendritic alpha1 receptors, and activation of these receptors excites inhibitory interneurons. The alpha1 actions reported herein may contribute to the analgesic action of intrathecally administered phenylephrine.