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Anesth Analg 2007;104:1422-1429
© 2007 International Anesthesia Research Society
doi: 10.1213/01.ane.0000261519.04083.3e

ANESTHETIC PHARMACOLOGY

Increased {gamma}-Aminobutyric Acid Levels in Mouse Brain Induce Loss of Righting Reflex, but Not Immobility, in Response to Noxious Stimulation

Sohtaro Katayama, DDS, PhD*, Masahiro Irifune, DDS, PhD*, Nobuhito Kikuchi, DDS, PhD*, Tohru Takarada, DDS, PhD*, Yoshitaka Shimizu, DDS*, Chie Endo, DDS*, Takashi Takata, DDS, PhD{dagger}, Toshihiro Dohi, PhD{ddagger}, Tomoaki Sato, DDS, PhD§, and Michio Kawahara, MD, PhD*

From the *Department of Dental Anesthesiology, Division of Clinical Medical Science, Programs for Applied Biomedicine, {dagger}Department of Oral Maxillofacial Pathobiology, Division of Frontier Medical Science, and {ddagger}Department of Dental Pharmacology, Division of Integrated Medical Science, Programs for Biomedical Research, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan; and §Department of Applied Pharmacology, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan.

Address correspondence and reprint requests to Masahiro Irifune, DDS, PhD, Department of Dental Anesthesiology, Division of Clinical Medical Science, Programs for Applied Biomedicine, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan. Address e-mail to mirifun{at}hiroshima-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
BACKGROUND: The general anesthetic state comprises behavioral and perceptual components, including amnesia, unconsciousness, and immobility. {gamma}-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.


   Introduction
Top
 Abstract
 Introduction
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The general anesthetic state comprises behavioral and perceptual components, including amnesia, sedation, unconsciousness or hypnosis, analgesia, immobility in response to noxious stimulation, and attenuation of autonomic responses to noxious stimulation (1,2). The sedative and analgesic properties of nitrous oxide, a gaseous general anesthetic, may be modulated at different, independent sites (3). Hence, it is imperative that each component be explored separately (4).

In vitro, electrophysiological studies have revealed that ligand-gated ion channels are important targets for anesthetic action at the cellular level (1,5). Chloride channels gated by the inhibitory neurotransmitter, {gamma}-aminobutyric acid (GABA) are sensitive to clinical concentrations of a wide variety of anesthetics, including volatile inhaled anesthetics and many IV anesthetics (6). At clinical concentrations, general anesthetics increase the sensitivity of the GABAA receptor to GABA, thus enhancing inhibitory neurotransmission and depressing nervous system activity. In vivo, however, the functional relevance of enhancing GABAergic neurotransmission in mediating essential components of the general anesthetic state, such as unconsciousness and immobility, is unknown.

The action of synaptically released GABA is terminated by uptake into neurons and glial cells via high-affinity Na+-dependent transporters. NO-711 potently inhibits GABA uptake and readily penetrates the blood–brain barrier (7). Released GABA is degraded by mitochondrial GABA-transaminase (GABA-T). Gabaculine is a potent, specific GABA-T inhibitor (8,9). These inhibitors increase endogenous GABA in the mammalian central nervous system (CNS) via different mechanisms. Subsequently, the increased GABA levels in the brain selectively stimulate GABA neurons. Thus, in this study, we examined, behaviorally, whether increased GABA levels could produce components of the general anesthetic state using two end points. 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). In addition, the action of these GABA mimetics on the righting reflex and response to noxious stimulation was compared to the effects of volatile anesthetics.


   METHODS
Top
 Abstract
 Introduction
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
The study was approved by the Committee of Research Facilities for Laboratory Animal Science, Graduate School of Biomedical Sciences, Hiroshima University. Adult male ddY mice (Japan SLC, Shizuoka, Japan) weighing 33–56 g were used in this study. Animals were housed five per cage in an air-conditioned room maintained at 25°C ± 1°C with 50% relative humidity on a 12-h light/dark cycle (lights on at 8:00 am). Food and water were available ad libitum. Animals were used only once in all experiments. All behavioral experiments were performed between 10:00 am and 6:00 pm.

3-Amino-2,3-dihydrobenzoic acid (gabaculine) hydrochloride and 1-[2-([(diphenylmethylene)imino]oxy)ethyl]- 1,2,5,6-tetrahydro-3-pyridinecarboxylic acid (NO-711) hydrochloride were purchased from Sigma-Aldrich (St. Louis, MO). Halothane was obtained from Takeda Chemical Industries (Osaka, Japan). Enflurane and isoflurane were from Abbot Laboratories (North Chicago, IL). Sevoflurane was from Maruishi Pharmaceutical Co. (Osaka, Japan). Chloral hydrate was from Nacalai Tesque (Kyoto, Japan).

Gabaculine, NO-711, and chloral hydrate were dissolved in 0.9% saline solution. Each drug was freshly prepared on the day of the experiment. All drugs, except for volatile anesthetics, were administered intraperitoneally (IP) in a volume of 5 mL/kg. The volatile anesthetics halothane, enflurane, isoflurane, and sevoflurane were administered with 33% oxygen at a total flow rate of 6 L/min (1 L/min of oxygen plus 5 L/min of fresh air). The mixed gas entered one end of an airtight clear Plexiglas chamber (30 cm wide x 30 cm deep x 25 cm high) that was vented at the other end. Each volatile anesthetic was delivered from a dedicated, calibrated vaporizer, and the anesthetic concentration in the chamber was continuously monitored. A gas sample was continuously drawn from the chamber, and the concentration of anesthetic was measured with an infrared analyzer (Capnox; Colin, Aichi, Japan).

Behavioral Study
Anesthetic states were evaluated using two end-points: loss of rolling response (as a measure of unconsciousness) and loss of response to noxious stimulation (as a measure of immobility). In this study, righting reflex was used to indicate the rolling response, and clamping the mouse's tail was the noxious stimulation.

The mice were examined individually in a circular glass beaker (13.5 cm diameter x 19 cm high). To examine the righting reflex, we tilted the beaker by hand to an angle of approximately 45° from a horizontal plane. The beaker was tilted three times at each recording time after IP administration of gabaculine or NO-711. Righting reflex was assessed and recorded every 2 min for the first 1 h and every 10 min for the following 6 h after administration of NO-711, and every 1 h for 24 h after administration of gabaculine, by a blinded observer. Righting reflex scores were evaluated according to the rating scale of Irifune et al. (10): a score of 0 indicated a normal righting reflex; +1 indicated that the mouse righted itself within 2 s on all three trials (slightly impaired righting reflex); +2 indicated that the latency to righting was >2 s, but <10 s at the best response in three trials (moderately or severely impaired righting reflex); +3 corresponded to absence of righting reflex (no righting within 10 s on all three trials).

To determine immobility, a tail clamp was applied with arterial forceps close to the base of the tail for 1 min or until the animal moved at the time when each drug produced a peak effect on the righting reflex. Purposeful movement of head and/or legs after tail-clamp stimulation was considered a response. Purposeless movement, such as coughing or hyperventilation, was excluded.

In the behavioral study of volatile anesthetics (halothane, enflurane, isoflurane, and sevoflurane), mice were placed in the chambers after the target concentrations were achieved. Anesthetic partial pressures were continuously monitored with the infrared analyzer. Animals were equilibrated with anesthetic partial pressure (a target concentration) for 20 min of spontaneous breathing. The target concentration in the chamber was maintained by adjusting vaporizers throughout the study. After 20 min exposure, the righting reflex and tail-clamp response were evaluated as described above.

Five to eight mice were used per dose or concentration for each drug. First, doses or concentrations of the drug large enough to eliminate and small enough not to affect the righting reflex or tail-clamp response for all animals were determined. Then, 3–4 doses or concentrations were administered between these maximum and minimum doses or concentrations. The anesthetized animals were kept warm with an overhead heat lamp.

Because the response is all-or-none, the number of animals losing the righting reflex (scored +3) or the tail-clamp response of the total that received a specific treatment (5–8 mice were used per dose) was used to calculate the percentage loss of response. The 50% effective dose (ED50) for LORR (righting-reflex ED50) and for loss of movement in response to tail-clamp stimulation (tail-clamp ED50) with 95% confidence limits, and the parallelism between the dose-response curves were determined according to the method of Litchfield and Wilcoxon (11). The ratio of tail-clamp ED50 to righting-reflex ED50 was calculated for each anesthetic.

Microdialysis Study
Microdialysis is a powerful technique for monitoring events occurring in the extracellular fluid, including neurotransmission and cell metabolism, as well as application of drugs directly into the brain.

Mice were anesthetized with chloral hydrate (400 mg/kg, IP) and placed in a stereotaxic frame (KOPF model 900; David Kopf Instruments, CA). A microdialysis guide cannula (CUP/7; CMA/microdialysis AB, Solna, Sweden) was implanted into the hippocampus (AP: –2.6 mm, L: +2.0 mm, V: –1.2 mm from bregma and top of the skull) according to the atlas of Franklin and Paxinos (12). The guide cannula was secured to the skull using a bone anchor screw and dental acrylic cement. The animals were allowed to recover from the surgery for at least 7 days before perfusion experiments.

All dialysis experiments were performed on conscious, freely moving mice. On the day of experiment, the inlet of the microdialysis probe (CUP/7; 0.24 mm membrane diameter x 2.0 mm membrane length; CMA/Microdialysis AB) was connected to a microinfusion pump (CMA/102; CMA/Microdialysis AB), and its outlet was connected to a fraction collector (CMA/170; CMA/Microdialysis AB). Then, the probe was filled with an artificial cerebrospinal fluid (ACSF). The ACSF consisted of 120 mM NaCl, 5 mM KCl, 1.2 mM MgCl2, 1.8 mM CaCl2, 1.2 mM KH2PO4, and 1.6 mM Na2HPO4 (pH 7.4). After preparation of the probe, the mouse was lightly anesthetized with chloral hydrate (200 mg/kg, IP). The guide obturator was removed and the probe was introduced. Then, the mouse hippocampus was perfused with the ACSF. Thereafter, the mouse was placed in a large hemispherical bowl (40 cm diameter). A 3 h equilibration period was allowed before sampling for stabilization of GABA concentrations after probe implantation. Dialysate samples were then collected and stored at 4°C in plastic vials containing 6 µL of 0.1 N HCl to conserve the intrinsic acidic amino acid. In the gabaculine-treated group, the flow rate of the perfusion medium (ACSF) was maintained at 0.5 µL/min using the microperfusion pump, and samples were collected every 60 min. This slow flow rate permits the avoidance of amino-acid depletion by gabaculine in the mouse hippocampus. Gabaculine was administered IP 6 h after the start of sampling, and the alteration of GABA was measured for 27 h. In the NO-711-treated group, the flow rate was 1.0 µL/min, and samples were collected every 30 min. NO-711 was injected IP 3 h after the start of sampling, and samples were collected for 10 h. The microdialysis samples were stored at –20°C until a GABA assay was performed. The microdialysis probes were evaluated in vitro to determine the mean percentage of relative recovery of GABA at 37°C. The means were 23.9% ± 2.9% (mean ± sem, n = 6) at the flow rate of 0.5 µL/min and 15.0% ± 0.7% (n = 6) at the flow rate of 1.0 µL/min, respectively.

At the end of each microdialysis experiment, the brains of the mice were removed after decapitation under deep chloral hydrate anesthesia. The brains were fixed in 10% formalin, cut on a cryomicrotome, and examined by microscope. The location of the microdialysis probe was determined histologically on serial coronal sections. Because all the implanted probes were situated at the proper positions in the hippocampus of animals studied, all the data obtained were used.

GABA in the dialysate was measured by reverse-phase high-performance liquid chromatography with fluorescence detection after precolumn derivatization with orthophthaldialdehyde reagent. The derivatization was performed using a refrigerated autoinjector (SIL-10AD VP; Shimadzu, Kyoto, Japan) with computer (SCL-10A VP; Shimadzu). A 20-µL dialysate sample was mixed with 20-µL of the orthophthaldialdehyde regent. After 150 s of reaction time, a 20-µL aliquot of the mixture was injected into the high-performance liquid chromatography apparatus, which consisted of a delivery pump (LC-100; BAS Japan, Tokyo, Japan), a degasser (LC-27A; BAS Japan), a reverse-phase column (CMA/GABA analysis column, 1.0 mm internal diameter x 100 mm length; CMA/ Microdialysis), a fluorescence detector (CMA/280; CMA/Microdialysis) with "amplify" set to 100 and "rise time" to 1, and a computing integrator-printer (C-R6A Chromatopac; Shimadzu). The analytical column temperature was controlled at 40°C (CTO-10AS VP; Shimadzu). The mobile phase consisted of 0.1 M acetate buffer and 20% acetonitrile adjusted to pH 5.4 with concentrated phosphoric acid. It was pumped through the column at a rate of 60 µL/min. The basal level of GABA in each animal was calculated as the mean of the last three consecutive samples immediately before drug treatment. Subsequent results were expressed as a percentage of the basal GABA level in each individual animal. The data are expressed as the mean ± sem.

The data were analyzed by one-way analysis of variance (ANOVA) followed by Student t-test with Bonferroni correction for multiple comparisons. The results were considered statistically significant when P < 0.05.


   RESULTS
Top
 Abstract
 Introduction
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The IP administration of gabaculine (35–200 mg/kg) in mice increased righting reflex scores in a dose-dependent fashion. The peak effect of gabaculine occurred at approximately 17 h postinjection, followed by a plateau. Gabaculine increased the percentage of LORR in a dose-dependent manner with an ED50 value of 100 (75–134; 95% confidence limits) mg/kg. However, even larger doses of gabaculine (285–400 mg/kg) produced no loss of movement in response to tail-clamp stimulation (Fig. 1).


Figure 118
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  		Figure 1. Effects of gabaculine on righting reflex (open circles) and tail-clamp response (closed circles) in mice. Gabaculine was administered intraperitoneally. Righting reflex was assessed every 1 h for 24 h after administration, and tail-clamp response was evaluated when the drug produced its peak effect on the righting reflex (5–8 animals per dose, 5–6 doses per dose-response curve). The doses against percent effect of animals that lost the righting reflex or tail-clamp response are plotted on the logarithmic-probability (X–Y axis) scale graph. Each point represents a percent effect of 5–8 animals per dose of gabaculine (see Methods in the text for ED50 values).

 

NO-711 (50–100 mg/kg, IP) induced neither LORR nor any immobility in response to tail-clamp. Larger doses of NO-711 (70–100 mg/kg) produced myoclonic- like seizures. NO-711 at 50 mg/kg produced an ataxic gait, with peak effect occurring at 6 min postinjection. This behavior lasted for more than 60 min.

In contrast to the GABA mimetics, all the tested volatile anesthetics concentration-dependently abolished both righting and tail-clamp response (Fig. 2). Table 1 summarizes the righting-reflex ED50, the tail-clamp ED50, and the ratios of the tail-clamp ED50 to the righting-reflex ED50 for various volatile anesthetics.


Figure 218
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  		Figure 2. Effects of halothane, enflurane, isoflurane, and sevoflurane on righting reflex (open circles) and tail-clamp response (closed circles) in mice. After 20 min exposure to a volatile anesthetic, righting reflex and tail-clamp response were assessed (5–8 animals per concentration, 5–6 concentrations per concentration-response curve). The concentrations against percent effect of animals that lost the righting reflex or tail-clamp response are plotted on the logarithmic-probability (X–Y axis) scale graph. Each point represents a percent effect of 5–8 animals per concentration of an anesthetic (see Methods in the text for ED50 values).

 

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  		Table 1. General Anesthetic Potency of Volatile Anesthetics

 

In a microdialysis study, the basal levels of GABA in the hippocampus were 9.99 ± 0.80 (n = 36) nM at a flow rate of 0.5 µL/min and 4.46 ± 0.85 (n = 33) nM at a flow rate of 1.0 µL/min, respectively. The IP administration of vehicle saline produced no significant change from the basal GABA levels (n = 5, F(15,64) = 1.014, P > 0.05). The time-course effects of gabaculine and NO-711 on extracellular GABA levels and righting reflex scores are shown in Figure 3. Gabaculine (100 mg/kg, IP) produced a steep increase in GABA levels of the hippocampus up to 10 h after the injection; thereafter, the increase was gradual during a 24-h period. Extracellular GABA levels were significantly increased 4 h postinjection and reached 3393.0% ± 447.0% at 17 h postinjection, when the anesthetic scores peaked, as compared to the basal levels (n = 6, F(24,125) = 9.608, P < 0.0001). These changes in GABA level in mouse brain corresponded with righting reflex scores (Fig. 3a). In contrast, a small dose of gabaculine (10 mg/kg), which did not affect the righting reflex, increased GABA to 1103.8% ± 329.7% of baseline (n = 6) (data not shown).


Figure 318
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  		Figure 3. Time-course effects of gabaculine (a) or NO-711 (b) on extracellular GABA levels in the hippocampus and on righting reflex scores in mice. Gabaculine (100 mg/kg) or NO-711 (50 mg/kg) was administered intraperitoneally at 0 h. Righting reflex was assessed every 1 h for 24 h after administration of gabaculine, and every 2 min for the first 1 h and every 10 min for the following 6 h after administration of NO-711. Closed circles represent extracellular GABA levels, expressed as a percentage of basal level calculated from three consecutive samples before injection of gabaculine or NO-711 (means ± sem, n = 6). Hatched bars represent means of righting reflex scores (n = 6). A score of 0 indicates a normal righting reflex; +1 indicates that the mouse rights itself within 2 s on all three trials; +2 indicates that the latency to righting is >2 s, but <10 s at the best response in three trials; +3 indicates no righting within 10 s on all three trials. *P < 0.05, **P < 0.01 compared to basal level, one-way ANOVA followed by Student t-test with Bonferroni correction for multiple comparisons.

 

NO-711 at a dose of 50 mg/kg, which is large enough to induce anticonvulsant effects, significantly increased GABA to 269.7% ± 24.2% of basal levels at 90 min postinjection and returned to baseline at 210 min (n = 6, F(15,80) = 14.345, P < 0.0001), but did not affect the righting reflex (Fig. 3b).


   DISCUSSION
Top
 Abstract
 Introduction
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study showed that IP administration of gabaculine induced LORR in a dose-dependent fashion (Fig. 1). The behavioral and microdialysis studies revealed that the endogenous GABA-induced LORR occurred in a brain concentration-dependent manner (Fig. 3). However, even larger doses of gabaculine produced no immobility in response to noxious stimulation (Fig. 1). In contrast, all the tested volatile anesthetics concentration-dependently produced both LORR and immobility (Fig. 2), supporting the evidence that volatile anesthetics act on a variety of molecular targets (1). These findings indicate that LORR is associated with enhanced GABAergic neurotransmission, but that immobility is not.

Local perfusion of the thalamus in vivo with GABA through an indwelling microdialysis probe produces a significant increase in physiological sleep, and induces long-lasting inhibition of somatosensory event-related potentials in cats (13). However, no studies evaluated the effects of GABA itself on general anesthetic components. The present study was the first to examine the effects of increased endogenous GABA in the brain by gabaculine on LORR and immobility, and no studies have yet investigated whether gabaculine could induce anesthetic effects (amnesia, sedation, and attenuation of autonomic responses to noxious stimulation, etc.) other than analgesia (14). Further studies will be required.

It has been reported that systemic administration of the GABAA receptor agonist, THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol), induces LORR in mice and rats (15). Moreover, direct injection of muscimol, another GABAA receptor agonist, into the tuberomammillary nucleus (a posterior hypothalamic cell group thought to be important in promoting arousal) produces LORR in rats (4). These findings suggest that the production of LORR (unconsciousness) occurs at a supraspinal site of GABA neurons in the mammalian CNS. However, GABA and GABAA receptor ligands, such as THIP and muscimol, each have individual agonist binding sites in GABAA receptors (16). Therefore, endogenous GABA and exogenous GABAA receptor agonists may induce different physiological behaviors. In addition, Meldrum has pointed out that desensitization of GABA receptors does not occur with increased endogenous GABA by GABA-T inhibitors, although GABAA receptor agonists induce such desensitization (17). The current study was the first to demonstrate that endogenous GABA produces LORR in a brain concentration-dependent fashion in mice (Figs. 1 and 3).

In this study, two end-points were used to assess the general anesthetic state: 1) LORR, and 2) loss of response to noxious stimulation. LORR is thought to be a primary end-point for unconsciousness (4,18). The loss of response to noxious stimulation is used to determine a minimum alveolar (anesthetic) concentration (MAC) of an anesthetic that produces immobility in 50% of those subjects exposed to noxious stimulation. However, the methods by which MAC was determined in previous studies were not precisely consistent for type and strength of noxious stimulation. In the present study, we demonstrated that tail-clamp ED50 (MAC) values for halothane, enflurane, isoflurane, and sevoflurane were 1.35, 2.50, 1.35, and 2.40%, respectively (Table 1). These results are consistent with data reported previously (19–22). Thus, the method used in this study to determine the tail-clamp ED50 (MAC) appears appropriate.

Blood/gas partition coefficients for halothane, enflurane, isoflurane, and sevoflurane are 2.5, 1.8, 1.4, and 0.65, respectively. A larger blood/gas partition coefficient produces a greater uptake, hence a lower alveolar anesthetic concentration (FA)/inspired concentration (FI) ratio. Therefore, at the time of testing (after 20 min exposure of the inhaled anesthetics), the FA/FI ratios of these anesthetics might be different. In humans, the ratios for halothane and sevoflurane have been reported to be approximately 0.5 and 0.75, respectively, after 20 min inhalation (23). Thus, longer exposure time might be required especially for halothane.

GABA, on its own, does not readily pass through the blood–brain barrier. For this reason, we used gabaculine which penetrates the barrier easily (8,9), is the most potent GABA-T inhibitor available, and increases brain GABA concentrations; these properties of gabaculine make it possible to administer systemically and stimulate GABAergic neurons selectively. Gabaculine is a catalytic inhibitor of GABA-T, and may also inhibit glutamic acid decarboxylase (GAD), which is a synthetic enzyme responsible for the conversion of l-glutamic acid to GABA. However, gabaculine has a fair degree of specificity because it is about 1000-fold less effective as a GAD inhibitor than as a GABA-T inhibitor. GABA-T is the primary catabolic enzyme for GABA, occurring in both neurons and glial cells. GABA is metabolized largely at extraneuronal intercellular sites or in the postsynaptic neurons (24). In fact, systemic administration of gabaculine increases GABA concentrations in both the intracellular and extracellular compartments of rat brain (25). In this study, gabaculine increased extracellular GABA levels of the hippocampus in mice in a time course-dependent manner (Fig. 3a). This microdialysis result is consistent with the time-course data for GABA content measured from the mouse whole brain tissue (8).

Gabaculine was administered systemically in the present study. Therefore, it may be problematic to interpret the effects of systemically administered drugs mechanistically, unless one excludes action at targets outside the CNS that may affect the measured variables. In addition, the drugs can interact at numerous levels of integration above the direct receptor level within the CNS. GABA is found in large concentrations in the brain and spinal cord but is absent, or present only in trace amounts, in peripheral tissues in mammals (24). Thus, the action of GABA appears insignificant in the peripheral tissues. However, the present study could not clarify the responsible site of gabaculine action to induce LORR in the CNS because of its systemic administration.

The primary mechanism by which the action of synaptically released GABA is terminated is uptake into presynaptic terminals and surrounding glial cells via high-affinity Na+-dependent transporters. The pharmacological inhibition of this uptake appears beneficial in conditions where decreased GABAergic transmission has been implicated, such as epilepsy. Using molecular biological techniques, four subtypes of mouse GABA transporter (GAT) have been cloned: GAT-1, GAT-2, GAT-3, and GAT-4 (26). NO-711 is a potent and selective GABA uptake inhibitor that exhibits the highest affinity at human GAT-1 (7). Distributions of GAT-1 mRNA are observed over many regions of the brain, including the retina, olfactory bulb, neocortex, ventral pallidum, hippocampus, and cerebellum (27). Therefore, NO-711 has been found to exhibit potent anticonvulsant effects in many rodent seizure models (7). In this behavioral study, however, although gabaculine induced LORR in a dose-dependent manner, even large doses of NO-711 did not. This discrepancy may be due to the different brain GABA concentrations they induce. To explore this possibility, GABA brain levels were measured after administration of gabaculine or NO-711 using an in vivo microdialysis technique.

Microdialysis is the only technique that can collect virtually any substance from distinct brain regions with minimal tissue trauma. The major advantage of the in vivo microdialysis method is that it can be used in the freely moving animal, allowing one to make correlations between changes in extracellular neurotransmitter levels and behavior (28). It has been reported that in awake, freely moving rats, even at 3 h postimplantation of the microdialysis probe, both the basal and high K+-evoked release of GABA are decreased in a Ca2+-dependent manner (29). In addition, it has recently been shown that the blockade of GABA release by tetrodotoxin, or Ca2+-free high Mg2+ buffer perfusion, is proportional to the release of this neurotransmitter, indicating that the corresponding increase or decrease of GABA in the brain reflects neuronal activity. In a microdialysis study, approximately 40%– 60% of GABA in the dialysate is believed to be of neuronal origin, although the remainder may be from glial metabolism or reversal of uptake sites (30).

The primary function of the hippocampus is disputed, but it is generally associated with the coding of visual and auditory inputs, arousal and attention, voluntary movements, exploratory behavior and, especially, formation of memory and retention of learned behavior. A particularly important hippocampal function with respect to anesthesia may be the association of this structure with memory formation. The general anesthetic state comprises many components, including loss of sensation, absence of awareness of surroundings, and unconsciousness. These manifestations may result from the loss of short-term memory traces. The major inhibitory neurotransmitter in the hippocampus is GABA. Therefore, the link between amnesia and anesthesia suggests that alterations in hippocampal function via GABAergic neurons may contribute to the state of anesthesia. In fact, an electrophysiological study has revealed that volatile anesthetics prolong GABA-induced inhibition in the hippocampus (31). In a behavioral study, direct injection of muscimol into the hippocampus decreased the dose of a general anesthetic needed to induce LORR and loss of response to noxious stimulation (32). In addition, GABA-T and GAT-1 are abundant in the hippocampus. Thus, we selected the hippocampus as the region in which to measure extracellular levels of GABA using an in vivo microdialysis method.

NO-711 induced much less increase in extracellular GABA levels than did gabaculine. The precise mechanism producing this difference is uncertain. However, NO-711 is a potent and selective GABA uptake inhibitor, exhibiting specific affinity at GAT-1. Therefore, it may be that extracellular GABA could still be taken up into presynaptic terminals or surrounding glial cells through other types of GAT (GAT-2, GAT-3, and GAT-4), even after administration of NO-711. Moreover, synaptically released GABA is also metabolized by GABA-T at extraneuronal intercellular sites. The brain ratio of GABA-T/GAD activity is always more than 1 (24). In fact, anticonvulsant doses of tiagabine, another selective GABA uptake inhibitor with high affinity at GAT-1, increased extracellular GABA levels to almost 3.5 times higher than baseline in a microdialysis study, but even large doses produced no LORR (33,34).

Larger doses of NO-711 induced myoclonic-like seizures. Similarly, some direct-acting GABAA receptor agonists, like muscmol and THIP, have been reported to produce epileptogenic action (35). These findings suggest that some adverse effects may occur with some GABA mimetics, although the precise mechanism producing the paradoxical convulsions is uncertain.

Gabaculine at doses four times larger than the ED50 for LORR induced no loss of movement to tail-clamp stimulation (immobility) (Fig. 1). In contrast, all the volatile anesthetics used in this study produced both LORR and immobility in response to noxious stimulation in a concentration-dependent manner (Fig. 2 and Table 1). The inhaled anesthetics (halothane, enflurane, isoflurane, and sevoflurane) enhance the actions of GABA at clinically relevant concentrations (6,36). Therefore, many clinical and laboratory anesthetics share the ability to regulate GABAA receptor function. The present and previous behavioral findings suggest that GABA mimetics themselves can produce LORR (unconsciousness) through GABA neurons at a supraspinal site (Figs. 1 and 3) (4,13). Thus, GABAA receptors may play an important role in mediating the effects of general anesthetics related to consciousness.

Closely related to the GABAA receptors are other ligand-gated ion channels, including glycine and neuronal nicotinic acetylcholine receptors. Clinical concentrations of the inhaled anesthetics enhance the ability of glycine to activate glycine-gated Cl– channels that significantly affect inhibitory neurotransmission in the spinal cord and brainstem. Therefore, glycine receptors may play a role in mediating inhibition by anesthetics of response to noxious stimulation. Subanesthetic concentrations of inhaled anesthetics inhibit some classes of neuronal nicotinic acetylcholine receptors, suggesting that the receptors may have a part in mediating the analgesic effects of the anesthetics (1). Rampil et al. (37), Rampil (38), and Antognini and Schwartz (39) have demonstrated that immobility in response to a surgical incision (the end-point used in determining MAC) results from inhaled anesthetic action in the spinal cord. Furthermore, in vivo, behavioral and neurophysiological studies have shown that immobility induced by inhaled anesthetics is mediated by glycine and N-methyl-d-aspartate receptors and Na+ channels in the spinal cord, but not by GABAA, nicotinic acetylcholine, serotonin, opioid and {alpha}2-adrenergic receptors and K+ channels [see Sonner et al. (40) for review].

In conclusion, our findings indicate that endogenous GABA-induced LORR occurs in a brain concentration- dependent fashion. However, the increased GABA levels produced no loss of movement in response to tail-clamp stimulation. In contrast, all the tested volatile anesthetics concentration-dependently abolished both the righting and tail-clamp response, supporting the evidence that volatile anesthetics work on a variety of molecular targets, consistent with their status as complete anesthetics producing all components (1). These findings indicate that LORR is associated with enhancing 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.


   ACKNOWLEDGMENTS
 
We thank Mr. Steven L. Leeper, Transnet, for reviewing English language.


   Footnotes
 
Accepted for publication February 13, 2007.

This work was supported, in part, by Grant-in-Aid Nos. 10671740 and 13672097 for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.


   REFERENCES
Top
 Abstract
 Introduction
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Evers AS, Crowder CM. General anesthetics. In: Hardman JG, Limbird LE, Gilman AG, eds. Goodman & Gilman's the pharmacological basis of therapeutics. 10th ed. New York: McGraw-Hill, 2001:337–65.
  2. Antognini JF, Carstens E. In vivo characterization of clinical anaesthesia and its components. Br J Anaesth 2002;89:156–66.[Abstract/Free Full Text]
  3. Sawamura S, Kingery WS, Davies MF, et al. Antinociceptive action of nitrous oxide is mediated by stimulation of noradrenergic neurons in the brainstem and activation of {alpha}2B adrenoceptors. J Neurosci 2000;20:9242–51.[Abstract/Free Full Text]
  4. Nelson LE, Guo TZ, Lu J, et al. The sedative component of anesthesia is mediated by GABAA receptors in an endogenous sleep pathway. Nat Neurosci 2002;5:979–84.[Web of Science][Medline]
  5. Franks NP, Lieb WR. Molecular and cellular mechanisms of general anaesthesia. Nature 1994;367:607–14.[Medline]
  6. Krasowski MD, Harrison NL. General anaesthetic actions on ligand-gated ion channels. Cell Mol Life Sci 1999;55:1278–303.[Web of Science][Medline]
  7. Kubová H. NNC-711: an inhibitor of GABA uptake with selective affinity to GAT-1. CNS Drug Rev 1999;5:317–30.
  8. Matsui Y, Deguchi T. Effects of gabaculine, a new potent inhibitor of gamma-aminobutyrate transaminase, on the brain gamma-aminobutyrate content and convulsions in mice. Life Sci 1977;20:1291–6.[Web of Science][Medline]
  9. Krantis A. Cerebral endothelial GABA-T activity: effects of in vivo GABA-T inhibition. Neurosci Lett 1986;67:48–52.[Web of Science][Medline]
  10. Irifune M, Takarada T, Shimizu Y, et al. Propofol-induced anesthesia in mice is mediated by {gamma}-aminobutyric acid-A and excitatory amino acid receptors. Anesth Analg 2003;97:424–9.[Abstract/Free Full Text]
  11. Litchfield JT Jr, Wilcoxon F. A simplified method of evaluating dose-effect experiments. J Pharmacol Exp Ther 1949;96:99–113.[Abstract/Free Full Text]
  12. Franklin KBJ, Paxinos G. The mouse brain in stereotaxic coordinates. San Diego: Academic Press, 1997.
  13. Juhász G, Emri Z, Kékesi K, Pungor K. Local perfusion of the thalamus with GABA increases sleep and induces long-lasting inhibition of somatosensory event-related potentials in cats. Neurosci Lett 1989;103:229–33.[Web of Science][Medline]
  14. Sivam SP, Ho IK. GABAergic drugs, morphine and morphine tolerance: a study in relation to nociception and gastrointestinal transit in mice. Neuropharmacology 1983;22:767–74.[Web of Science][Medline]
  15. Cheng SC, Brunner EA. Inducing anesthesia with a GABA analog, THIP. Anesthesiology 1985;63:147–51.[Web of Science][Medline]
  16. Baumann SW, Baur R, Sigel E. Individual properties of the two functional agonist sites in GABAA receptors. J Neurosci 2003; 23:11158–66.[Abstract/Free Full Text]
  17. Meldrum BS. GABAergic mechanisms in the pathogenesis and treatment of epilepsy. Br J Clin Pharmacol 1989;27:S3–S11.[Web of Science]
  18. Jurd R, Arras M, Lambert S, et al. General anesthetic actions in vivo strongly attenuated by a point mutation in the GABAA receptor β3 subunit. FASEB J 2003;17:250–2.[Abstract/Free Full Text]
  19. Deady JE, Koblin DD, Eger EI II, et al. Anesthetic potencies and the unitary theory of narcosis. Anesth Analg 1981;60:380–4.[Abstract/Free Full Text]
  20. Koblin DD, Deady JE, Eger EI II. Potencies of inhaled anesthetics and alcohol in mice selectively bred for resistance and susceptibility to nitrous oxide anesthesia. Anesthesiology 1982;56: 18–24.[Web of Science][Medline]
  21. Sonner JM, Gong D, Li J, et al. Mouse strain modestly influences minimum alveolar anesthetic concentration and convulsivity of inhaled compounds. Anesth Analg 1999;89:1030–4.[Abstract/Free Full Text]
  22. Puig NR, Ferrero P, Bay ML, et al. Effects of sevoflurane general anesthesia: immunological studies in mice. Int Immunopharmacol 2002;2:95–104.[Web of Science][Medline]
  23. Eger EI II. Uptake and distribution. In: Miller RD, ed. Anesthesia. 5th ed. Philadelphia: Churchill Livingstone, 2000:74–95.
  24. Cooper JR, Bloom FE, Roth RH. The biochemical basis of neuropharmacology. 8th ed. New York: Oxford University Press, 2003:105–50.
  25. Piérard C, Pérès M, Satabin P, et al. Effects of GABA-transaminase inhibition on brain metabolism and amino-acid compartmentation: an in vivo study by 2D 1H-NMR spectroscopy coupled with microdialysis. Exp Brain Res 1999;127:321–7.[Web of Science][Medline]
  26. Liu QR, López-Corcuera B, Mandiyan S, et al. Molecular characterization of four pharmacologically distinct {gamma}-aminobutyric acid transporters in mouse brain. J Biol Chem 1993;268:2106–12.[Abstract/Free Full Text]
  27. Durkin MM, Smith KE, Borden LA, et al. Localization of messenger RNAs encoding three GABA transporters in rat brain: an in situ hybridization study. Brain Res Mol Brain Res 1995;33:7–21.[Medline]
  28. Cadogan AK, Marsden CA. Measurement of neurotransmitters and their associated second messengers in vivo. In: Turner AJ, Bachelard HS, eds. Neurochemistry: a practical approach. 2nd ed. Oxford: IRL Press, 1997:175–99.
  29. Banerjee PK, Snead OC III. Presynaptic {gamma}-hydroxybutyric acid (GHB) and {gamma}-aminobutyric acidB (GABAB) receptor-mediated release of GABA and glutamate (GLU) in rat thalamic ventrobasal nucleus (VB): a possible mechanism for the generation of absence-like seizures induced by GHB. J Pharmacol Exp Ther 1995;273:1534–43.[Abstract/Free Full Text]
  30. Wang X, Ai J, Hampson DR, Snead OC III. Altered glutamate and GABA release within thalamocortical circuitry in metabotropic glutamate receptor 4 knockout mice. Neuroscience 2005; 134:1195–203.[Web of Science][Medline]
  31. Pearce RA. Volatile anaesthetic enhancement of paired-pulse depression investigated in the rat hippocampus in vitro. J Physiol 1996;492:823–40.[Abstract/Free Full Text]
  32. Ma J, Shen B, Stewart LS, et al. The eptohippocampal system participates in general anesthesia. J Neurosci 2002;22:RC200.[Abstract/Free Full Text]
  33. Fink-Jensen A, Suzdak PD, Swedberg MDB, et al. The {gamma}-aminobutyric acid (GABA) uptake inhibitor, tiagabine, increases extracellular brain levels of GABA in awake rats. Eur J Pharmacol 1992;220:197–201.[Web of Science][Medline]
  34. Smith SE, Parvez NS, Chapman AG, Meldrum BS. The {gamma}-aminobutyric acid uptake inhibitor, tiagabine, is anticonvulsant in two animal models of reflex epilepsy. Eur J Pharmacol 1995;273:259–65.[Web of Science][Medline]
  35. Ticku MK. Mechanism of GABA agonists and modulators of GABAergic transmission in anticonvulsant activity. In: Tunnicliff G, Raess BU, eds. GABA mechanisms in epilepsy. New York: Wiley-Liss, 1991:149–64.
  36. Krasowski MD, Harrison NL. The actions of ether, alcohol and alkane general anaesthetics on GABAA and glycine receptors and the effects of TM2 and TM3 mutations. Br J Pharmacol 2000;129:731–43.[Web of Science][Medline]
  37. Rampil IJ, Mason P, Singh H. Anesthetic potency (MAC) is independent of forebrain structures in the rat. Anesthesiology 1993;78:707–12.[Web of Science][Medline]
  38. Rampil IJ. Anesthetic potency is not altered after hypothermic spinal cord transaction in rats. Anesthesiology 1994;80:606–10.[Web of Science][Medline]
  39. Antognini JF, Schwartz K. Exaggerated anesthetic requirements in the preferentially anesthetized brain. Anesthesiology 1993; 79:1244–9.[Web of Science][Medline]
  40. Sonner JM, Antognini JF, Dutton RC, et al. Inhaled anesthetics and immobility: mechanisms, mysteries, and minimum alveolar anesthetic concentration. Anesth Analg 2003;97:718–40.[Abstract/Free Full Text]



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