Michio Kawahara

Evidence for GABAA receptor agonistic properties of ketamine: Convulsive and anesthetic behavioral models in mice

We examined the potentiation by ketamine of the &ggr;-aminobutyric acidA (GABAA) receptor function using convulsive and anesthetic behavioral models in adult male ddY mice. General anesthetic potencies were evaluated by a rating scale, which provided the data for anesthetic scores, loss of righting reflex, duration, and recovery time. All drugs were administered intraperitoneally. Small subanesthetic doses of ketamine did inhibit tonic seizures induced by a large dose of the GABAA receptor antagonist bicuculline (8 mg/kg). The 50% effective dose value was 15 (95% confidence limits 10–22) mg/kg. Even large anesthetic doses (100–150 mg/kg) did not suppress clonic seizures in 50% of the animals. The GABAA receptor agonist, muscimol (0.32–1.12 mg/kg), potentiated ketamine-induced anesthesia in a dose-dependent fashion (P < 0.05). Similarly, the benzodiazepine receptor agonist, diazepam (1–3 mg/kg), augmented ketamine anesthesia in a dose-dependent manner (P < 0.05). Bicuculline (2–5 mg/kg) dose-dependently antagonized ketamine-induced anesthesia (P < 0.05). Neither the benzodiazepine receptor antagonist, flumazenil (2–20 mg/kg), nor the GABA synthesis inhibitor, l-allylglycine (200 mg/kg), affected the anesthetic action of ketamine. These results suggest that ketamine has GABAA receptor agonistic properties and that ketamine-induced anesthesia is mediated, at least in part, by GABAA receptors. Implications We examined the potentiation by ketamine of the &ggr;-aminobutyric acidA receptor function using convulsive and anesthetic behavioral models in mice. Subanesthetic doses of ketamine-inhibited tonic convulsions induced by the &ggr;-aminobutyric acidA receptor antagonist bicuculline. The &ggr;-aminobutyric acidA receptor agonist, muscimol, potentiated ketamine-induced anesthesia. Bicuculline antagonized ketamine anesthesia, but the benzodiazepine receptor antagonist, flumazenil, and the &ggr;-aminobutyric acid synthesis inhibitor, l-allyglycine, did not. The effects of ketamine on the &ggr;-aminobutyric acidA receptors appear to correlate with its anesthetic actions.

Propofol-induced Anesthesia in Mice Is Mediated by γ-aminobutyric Acid-a and Excitatory Amino Acid Receptors

To elucidate the role of &ggr;-aminobutyric acid (GABA)A receptor complex and excitatory amino acid receptors (N-methyl-d-aspartate [NMDA] and non-NMDA receptors) in propofol-induced anesthesia, we examined behaviorally the effects of GABAergic and glutamatergic drugs on propofol anesthesia in mice. All drugs were administered intraperitoneally. General anesthetic potencies were evaluated using a righting reflex assay. The GABAA receptor agonist muscimol potentiated propofol (140 mg/kg; 50% effective dose for loss of righting reflex) induced anesthesia. Similarly, the benzodiazepine receptor agonist diazepam and the NMDA receptor antagonist MK-801 augmented propofol anesthesia, but the non-NMDA receptor antagonist CNQX did not. In contrast, the GABAA receptor antagonist bicuculline antagonized propofol (200 mg/kg; 95% effective dose for loss of righting reflex) induced anesthesia. However, neither the benzodiazepine receptor antagonist flumazenil, the GABA synthesis inhibitor l-allylglycine, nor the NMDA receptor agonist NMDA reversed propofol anesthesia. Conversely, the non-NMDA receptor agonist kainate enhanced propofol anesthesia. These results suggest that propofol-induced anesthesia is mediated, at least in part, by both GABAA and excitatory amino acid receptors.

Effects of ketamine on dopamine metabolism during anesthesia in discrete brain regions in mice: comparison with the effects during the recovery and subanesthetic phases

The effects of ketamine on the levels of dopamine (DA), norepinephrine (NE), 5-hydroxytryptamine (5-HT, serotonin) and their metabolites were examined in discrete brain regions in mice. A high dose of ketamine (150 mg/kg, i.p.) did not change DA metabolism in the frontal cortex, nucleus accumbens, striatum and hippocampus, but did decrease it in the brainstem during anesthesia. In contrast, during recovery from the ketamine anesthesia, the high dose increased the level of homovanillic acid (HVA) in all brain regions. A low subanesthetic dose of ketamine (30 mg/kg, i.p.) increased the concentrations of both 3,4-dihydroxyphenylacetic acid (DOPAC) and HVA only in the nucleus accumbens. The DA level was not affected by any ketamine treatment. During ketamine anesthesia, the content of 3-methoxy-4-hydroxy-phenylglycol (MHPG) was decreased in the brainstem, whereas during recovery from anesthesia, the MHPG level was increased in the frontal cortex, nucleus accumbens and brainstem. The NE content was not altered in any region by ketamine treatment. The concentration of 5-hydroxyindoleacetic acid (5-HIAA) was reduced in the frontal cortex, striatum, hippocampus and brainstem during ketamine anesthesia. The 5-HT level was unaltered in all regions except the brainstem where it was reduced. In contrast, after anesthesia, the concentrations of both 5-HT and 5-HIAA were increased in the striatum. During the subanesthetic phase, however, the levels of NE, 5-HT and their metabolites were unchanged. These neurochemical results are consistent with the electrophysiological findings that a high dose of ketamine does not change the basal firing rates of nigrostriatal DA neurons during anesthesia, while low subanesthetic doses significantly increase those of ventral tegmental DA neurons.

Suppression of Noxiously Evoked WDR Dorsal Horn Neuronal Activity by Spinally Administered Morphine

The present study was carried out in order to examine the ability of spinally administered morphine to suppress noxiously evoked activity of wide-dynamic-range (WDR) neurons in the dorsal horn of the spinal cord in decerebrate, spinal cord-transected cats. All cells (n = 25) responded maximally to high-intensity noxious heat stimulation (51°C) and were classified as wide dynamic range neurons. The spinal administration of 0.1 mg of morphine caused a significant reduction of noxiously evoked activity but did not significantly change spontaneous activity. The 0.25-mg dose caused a significant reduction of both types of activity. Thirty minutes after spinal administration, 0.1 mg of morphine caused a 27% reduction of spontaneous activity and a 43% reduction of noxiously evoked activity. The 0.25-mg dose reduced spontaneous activity by 44% and the evoked activity by 70%. Naloxone partially reversed the morphine-induced neuronal suppression. In addition, in the four neurons in which it was tried, spinally administered epinephrine was found to further suppress the remaining neuronal activity following the spinal morphine effect. These results demonstrate for the first time that spinally administered morphine is capable of suppressing noxiously evoked activity of wide-dynamic-range neurons in the dorsal horn of the spinal cord. They also demonstrate the dosedependent nature of this effect and the potential importance of the interaction between morphine and adrenergic agonists in blocking information about noxious events. This information provides a probable mechanism of action for spinal opiate analgesia.

Effects of volatile and intravenous anesthetics on the uptake of GABA, glutamate and dopamine by their transporters heterologously expressed in COS cells and in rat brain synaptosomes

Although the neurotransmitter uptake system is considered a possible target for the presynaptic action of anesthetic agents, observations are inconsistent concerning effects on the transporter and their clinical relevance. The present study examined the effects of volatile and intravenous anesthetics on the uptake of GABA, glutamate and dopamine in COS cells heterologously expressing the transporters for these neurotransmitters and in the rat brain synaptosomes. Halothane and isoflurane, but not thiamylal or thiopental, significantly inhibited uptake by COS cell systems of GABA, dopamine and glutamic acid in a concentration-dependent manner within clinically relevant ranges for anesthesia induced by these agents. Similarly, in synaptosomes halothane and isoflurane but not thiopental significantly suppressed the uptake of GABA and glutamic acid, respectively. These results do not support the hypothesis that volatile and intravenous anesthetics exert their action via specific inhibition of GABA uptake to enhance inhibitory GABAergic neuronal activity. Rather, they suggest that presynaptic uptake systems for various neurotransmitters including GABA may be the molecular targets for volatile anesthetic agents.

Hyperlocomotion during Recovery from Isoflurane Anesthesia Is Associated with Increased Dopamine Turnover in the Nucleus Accumbens and Striatum in Mice

Background It was recently reported that isoflurane increases dopamine release in the striatum in rats both in vivo and in vitro, and that isoflurane inhibits uptake of dopamine in the rat brain synaptosomes. However, the functional role of these effects of isoflurane on dopamine neurons is uncertain. Dopaminergic mechanisms within the nucleus accumbens and striatum play an important role in the control of locomotor activity, and a change in dopamine turnover depends essentially on a change in impulse flow in the dopamine neurons. In this study, the effects of isoflurane on locomotor activity and on dopamine turnover were investigated in discrete brain regions in mice. Methods Mice were placed in individual airtight clear plastic chambers and spontaneously breathed isoflurane in 25% oxygen and 75% nitrogen (fresh gas flow, 4 l/min). Locomotor activity was measured with an Animex activity meter. Animals were decapitated after treatments with or without isoflurane, and the concentrations of monoamines and their metabolites in different brain areas were measured by high‐performance liquid chromatography. Results During the 10 min after the cessation of the 20‐min exposure to isoflurane, there was a significant increase in locomotor activity in animals breathing 1.5% isoflurane but not 0.7% isoflurane. This increase in locomotor activity produced by 1.5% isoflurane was abolished by a low dose of haloperidol (0.1 mg/kg), a dopamine receptor antagonist. Regional brain monoamine assays revealed that 1.5% isoflurane significantly increased the 3,4‐dihydroxyphenylacetic acid:dopamine ratio (one indicator of transmitter turnover) in the nucleus accumbens and striatum, but a concentration of 0.7% did not. This significant increase in dopamine turnover in these regions continued during 20 min after the cessation of the administration of 1.5% isoflurane. Conclusions These results suggest that isoflurane‐induced hyperlocomotion during emergence may be associated with increased dopamine turnover in the nucleus accumbens and striatum.

Riluzole, a glutamate release inhibitor, induces loss of righting reflex, antinociception, and immobility in response to noxious stimulation in mice

BACKGROUND:The general anesthetic state comprises behavioral and perceptual components, including amnesia, unconsciousness, analgesia, and immobility. In vitro, glutamatergic excitatory neurons are important targets for anesthetic action at the cellular and microcircuits levels. Riluzole (2-amino-6-[trifluoromethoxy]benzothiazole) is a neuroprotective drug that inhibits glutamate release from nerve terminals in the central nervous system. Here, we examined in vivo the ability of riluzole to produce components of the general anesthetic state through a selective blockade of glutamatergic neurotransmission. METHODS:Riluzole was administered intraperitoneally in adult male ddY mice. To assess the general anesthetic components, three end-points were used: 1) loss of righting reflex (LORR; as a measure of unconsciousness), 2) loss of movement in response to noxious stimulation (as a measure of immobility), and 3) loss of nociceptive response (as a measure of analgesia). RESULTS:The intraperitoneal administration of riluzole induced LORR in a dose-dependent fashion with a 50% effective dose value of 27.4 (23.3–32.2; 95% confidence limits) mg/kg. The behavioral and microdialysis studies revealed that time-course changes in impairment and LORR induced by riluzole corresponded with decreased glutamate levels in the mouse brain. This suggests that riluzole-induced LORR (unconsciousness) could result, at least in part, from its ability to decrease brain glutamate concentrations. Riluzole dose-dependently produced not only LORR, but also loss of movement in response to painful stimulation (immobility), and loss of nociceptive response (analgesia) with 50% effective dose values of 43.0 (37.1–49.9), and 10.0 (7.4–13.5) mg/kg, respectively. These three dose–response curves were parallel, suggesting that the behavioral effects of riluzole may be mediated through a common site of action. CONCLUSIONS:These findings suggest that riluzole-induced LORR, immobility, and antinociception appear to be associated with its ability to inhibit glutamatergic neurotransmission in the central nervous system.

MK-801 enhances gabaculine-induced loss of the righting reflex in mice, but not immobility.

Purpose: γ-Aminobutyric acid (GABA) and N-methyl-D-aspartate (NMDA) receptors are important targets for anesthetic action at thein vitro cellular level. Gabaculine is a GABA-transaminase inhibitor that increases endogenous GABA in the brain, and enhances GABA activity. We have recently shown that unconsciousness is associated with the enhanced GABA activity due to gabaculine, but that immobility is not. MK-801 is a selective NMDA channel blocker. In this study, we examined behaviourally whether gabaculine in combination with MK-801 could produce these components of the general anesthetic state. We further compared the effect of MK-801 with ketamine, another NMDA channel blocker.Methods: All drugs were administered intraperitoneally to adult male ddY mice. To assess the general anesthetic components, two endpoints were used. One was loss of the righting reflex (LORR; as a measure of unconsciousness) and the other was loss of movement in response to tail-clamp stimulation (as a measure of immobility).Results: Large doses of MK-801 alone (10–50 mg·kg−1) induced neither LORR nor immobility in response to noxious stimulation. However, even a small dose (0.2 mg·kg−1) significantly enhanced gabaculine-induced LORR (P<0.05), although gabaculine in combination with MK-801 (0.2–10 mg·kg−1) produced no immobility. However, gabaculine plus a subanesthetic dose of ketamine (30 mg·kg−1), which acts on NMDA, opioid and nicotinic acetylcholine receptors and neuronal Na+ channels, suppressed the pain response, but did not achieve a full effect. Ketamine alone dose-dependently produced both LORR and immobility.Conclusion: These findings suggest that gabaculine-induced LORR is modulated by blocking NMDA receptors, but that immobility is not mediated through GABA or NMDA receptors.RésuméObjectif: Les récepteurs GABA (acide gamma-aminobutyrique) et NMDA (N-méthyl-D-aspartate) constituent d’importantes cibles pour l’action des anesthésiques au niveau cellulaire in vitro. La ga-baculine est un inhibiteur des GABA-transaminases qui augmente le GABA endogène dans le cerveau, et stimule l’activité GABA. Nous avons récemment démontré que la perte de conscience est associée à l’activité GABA stimulée par la gabaculine, mais que l’immobilité ne l’est pas. Le MK-801 est un bloqueur sélectif du canal NMDA. Dans cette étude, nous avons examiné si la gabaculine combinée à du MK-801 pouvait produire ces composantes de l’état d’anesthésie générale au niveau comportemental. Nous avons également comparé l’effet du MK-801 à celui de la kétamine, un autre bloqueur du canal NMDA.Méthode: Tous les médicaments ont été administrés à des souris mâles adultes ddY par voie intrapéritonéale. Deux paramètres ont été utilisés afin d’évaluer les composantes de l’anesthésie générale. L’un était la perte du réflexe de redressement (LORR — loss of righting reflex ; pour mesurer la perte de conscience), et l’autre l’absence de mouvement en réaction à la stimulation d’une pince à la queue (pour mesurer l’immobilité).Résultats: D’importantes doses de MK-801 seul (10–50 mg·kg−1) n’ont provoqué ni LORR ni l’immobilité en réaction à une stimulation nociceptive. Toutefois, une dose même faible (0.2 mg·kg−1) a significativement accentué le LORR provoqué par la gabaculine (P<0,05), bien que la gabaculine associée à du MK-801 (0,2–10 mg·kg−1)n’ait pas provoqué d’immobilité. Cependant, la gabaculine additionnée d’une dose sous-anesthésique de kétamine (30 mg·kg−1),laquelle agit sur les récepteurs NMDA, opiacés et cholinergiques nicotiniques ainsi que sur les canaux Na+, a supprimé la réaction douloureuse, mais n’a pas eu un effet complet. La kétamine seule a provoqué LORR et immobilité, de façon dose-dépendante.Conclusion: Ces résultats suggèrent que le LORR provoqué par la gabaculine est modulé en bloquant les récepteurs NMDA, mais que l’immobilité n’est pas médiée par les récepteurs GABA ou NMDA.

Increased γ-aminobutyric acid levels in mouse brain induce loss of righting reflex, but not immobility, in response to noxious stimulation

BACKGROUND:The general anesthetic state comprises behavioral and perceptual components, including amnesia, unconsciousness, and immobility. &ggr;-Aminobutyric acidergic (GABAergic) inhibitory neurotransmission is an important target for anesthetic action at the in vitro cellular level. In vivo, however, the functional relevance of enhancing GABAergic neurotransmission in mediating essential components of the general anesthetic state is unknown. Gabaculine is a GABA-transaminase inhibitor that inhibits degradation of released GABA, and consequently increases endogenous GABA in the central nervous system. Here, we examined, behaviorally, the ability of increased GABA levels to produce components of the general anesthetic state. METHODS:All drugs were administered systemically in adult male ddY mice. To assess the general anesthetic components, two end-points were used. One was loss of righting reflex (LORR; as a measure of unconsciousness); the other was loss of movement in response to tail-clamp stimulation (as a measure of immobility). RESULTS:Gabaculine induced LORR in a dose-dependent fashion with a 50% effective dose of 100 (75–134; 95% confidence limits) mg/kg. The behavioral and microdialysis studies revealed that the endogenous GABA-induced LORR occurred in a brain concentration-dependent manner. However, even larger doses of gabaculine (285–400 mg/kg) produced no loss of tail-clamp response. In contrast, all the tested volatile anesthetics concentration-dependently abolished both righting and tail-clamp response, supporting the evidence that volatile anesthetics act on a variety of molecular targets. CONCLUSIONS:These findings indicate that LORR is associated with enhanced GABAergic neurotransmission, but that immobility in response to noxious stimulation is not, suggesting that LORR and immobility are mediated through different neuronal pathways and/or regions in the central nervous system.

Inhibition of serotonin transporters by cocaine and meprylcaine through 5-TH2C receptor stimulation facilitates their seizure activities.

The present study examined whether the inhibition of serotonin transporters (SERT) contributes to cocaine- and other local anesthetics-induced convulsions, and which subtypes of 5-HT receptor are involved in the convulsions. For this purpose, cocaine, meprylcaine and lidocaine, all of which have different effects on SERT, were used as convulsants and the effects of serotonin reuptake inhibitors (SSRIs), specific agonists and antagonists for 5-HT receptor subtypes were evaluated in mice. Administration of SSRI, zimelidine, citalopram and fluoxetine, 5-HT(2A,2C) receptor agonist, R(-)-DOI and the 5-HT2C receptor agonists, mCPP, and MK212 resulted in a marked increase in incidence of convulsions and a reduction in the threshold of lidocaine-induced convulsions, while the 5-HT2B receptor agonist, BW723C86, had little influence. On the other hand, SSRI did not affect the measured parameters in meprylcaine- and cocaine-induced convulsions. R(-)-DOI, mCPP, and MK212 reduced the threshold of meprylcaine or cocaine with less extent than the reduction of lidocaine threshold. Incidence of cocaine- and meprylcaine-induced convulsions was significantly reduced by 5-HT(2A,2B,2C) antagonist, LY-53857, and 5-HT2C antagonist, RS 102221. The threshold of cocaine and meprylcaine was significantly increased by both antagonists. 5-HT2A antagonists MDL 11,939 and ketanserin, and 5-HT2B antagonist SB 204741 except at high doses had little effect on cocaine- and meprylcaine-induced convulsions. None of these antagonists altered the parameters of lidocaine-induced convulsions. Pretreatment with fluoxetine but not citalopram increased the plasma concentration of lidocaine. These results suggest that the increase of serotonergic neuronal activity through 5-HT2C receptor stimulation was responsible for increased activity of local anesthetics-induced convulsions and support the involvement of this mechanism in cocaine- and meprylcaine- but not in lidocaine-induced convulsions through their direct inhibitory action on central SERT.