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ANESTHETIC PHARMACOLOGY

Activity of the Serotonergic System During Isoflurane Anesthesia

From the Department of Anesthesia, Kyoto University Hospital, Kyoto, Japan.

Address correspondence and reprint requests to Kumiko Mukaida, MD, Department of Anesthesia, Kyoto University Hospital, 54 Shogoin-Kawaharacho, Sakyo-ku, Kyoto 606-8507, Japan. Address e-mail to kumicom{at}kuhp.kyoto-u.ac.jp.

	Abstract

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BACKGROUND: Microdialysis studies have demonstrated that the release of serotonin (5-hydroxytryptamine, 5-HT) in the serotonergic projection areas increases during waking and decreases during sleep in rat and cat, suggesting that 5-HT plays an important role in modulation of sleep. Although it might be expected that 5-HT release is also decreased during general anesthesia, the functional contribution of serotonergic neurons in pharmacological effects of volatile anesthetics has not been fully investigated.

METHODS: Using an in vivo microdialysis technique, we measured extracellular 5-HT in rat frontal cortex during waking, slow-wave sleep, and isoflurane anesthesia. To assess the involvement of the serotonergic system in the hypnotic action of isoflurane, the concentration of isoflurane required for loss of righting reflex was determined with or without pretreatment of fluoxetine hydrochloride, a selective 5-HT reuptake inhibitor.

RESULTS: During slow-wave sleep and isoflurane anesthesia (0.1–1.5 MAC), 5-HT release decreased to 21%–44% of that during the waking state. Loss of righting reflex occurred at significantly higher isoflurane concentrations in fluoxetine-treated rats (0.76% ± 0.03% [n = 8]) than in control rats (0.60% ± 0.01% [n = 8]).

CONCLUSIONS: It is suggested that a change in the activity of the serotonergic system in the brain is involved in the hypnotic action of isoflurane.

	Introduction

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Serotonin (5-hydroxytryptamine, 5-HT) has been known for many years to play an important role in modulation of the sleep/wake cycle (1,2). Microdialysis studies have demonstrated that 5-HT release in the serotonergic projection areas increases during waking and decreases during sleep in the rat (3) and cat (4). Neuronal 5-HT is produced and released by the neurons of the midbrain raphe nuclei, where the neurons are most densely grouped in the dorsal raphe nucleus (DRN). Serotonergic ascending projections from the DRN spread throughout the midbrain and forebrain, which include the ascending reticular formation (5). The brainstem ascending reticular formation has been the target of research attempting to elucidate the physiological basis of consciousness, waking, sleeping, or attentional switching (6–8).

Sleep and anesthesia both produce loss of responsiveness to environmental stimuli, although sleep is reversible with external stimuli. Tung et al. (9) have demonstrated that sleep deprivation enhances the onset and prolongs the duration of the loss of righting reflex induced by propofol and isoflurane in rats, suggesting that neuronal networks active in sleep are also involved in the anesthetized state. Thus, functional changes in the serotonergic system may also contribute to general anesthesia. In fact, several reports have suggested the involvement of 5-HT in general anesthesia (10–12). Therefore, we hypothesized that volatile anesthetics decrease serotonergic activity and the effect takes part in the hypnotic action of volatile anesthetics. In this study, we aimed to assess the activity of serotonergic neurons during isoflurane anesthesia in comparison to slow-wave sleep, using in vivo microdialysis technique. Furthermore, to analyze the effect of 5-HT on the hypnotic action of isoflurane, we tested the effect of the selective serotonin reuptake inhibitor, fluoxetine on isoflurane-induced loss of righting reflex.

	METHODS

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The study was approved by the Animal Research Committee of Kyoto University Faculty of Medicine. Male Wistar rats, weighing 230–300 g, were used. They were housed, one per cage, under a 12-h light–dark cycle (lights on from 07:00 to 19:00) with free access to food and water. All experiments except cannula implantation were started between 10:00 and 11:00 am.

In Vivo Microdialysis
Male Wistar rats were anesthetized with pentobarbital 40 mg/kg, IP, and placed on a stereotaxic frame (NARISHIGE, Tokyo, Japan). The skull was exposed through a midline incision in the scalp, and a burr hole of 3 mm diameter was drilled. A cannula was implanted at 2.0 mm in depth in the frontal cortex at A 3 mm and L 2 mm from the bregma, according to Paxinos and Watson's stereotaxic map (13), and a stainless steel screw was placed on the opposite side of the skull for electroencephalogram (EEG) monitoring. They were fixed in place with dental cement. The rats were allowed to recover for at least one day.

On the day of the microdialysis study, the stylet was removed from the cannula and a microdialysis probe was inserted. The microdialysis probe (A-I-8, inner diameter 0.28 mm; EICOM, Kyoto, Japan) had a diameter of 500 µm and a tip length of 2 mm. Rats were placed in clear plastic boxes (20 x 15 x 15 cm) which were gassed continuously with 6 L/min mixture of 25% oxygen and 75% nitrogen from the port. The dialysis probe was connected to the perfusion pump (EP-50; EICOM) using polyethylene tubing, and perfused continuously with artificial cerebrospinal fluid solution (147 mM Na⁺, 2.3 mM Ca²⁺, 4 mM K⁺, 156 mM Cl^–, pH 7.4) at a rate of 2.0 µL/min. Perfusate samples collected during the first 2 h after implantation of the probe were discarded. The location of the probe was confirmed by visual examination of the brain at the end of each experiment.

Using EEG monitoring, the status of the rat before inhaling isoflurane was divided into either wakefulness, slow-wave sleep, or rapid eye movement (REM) sleep, according to the criteria of Ursin and Larsen (14). For each rat, several 20-µL perfusate fractions were manually collected during wakefulness and slow-wave sleep. Perfusates during REM sleep were not collected, because the duration of REM sleep was too short to collect enough samples for 5-HT measurement.

After sampling during wakefulness and slow-wave sleep, 0.1 MAC (0.15%), 0.3 MAC (0.45%), 0.5 MAC (0.75%), 1.0 MAC (1.5%), or 1.5 MAC (2.25%) isoflurane was added to the nitrogen–oxygen mixture gas for 2 h. The MAC value for isoflurane (1.52%) was reported previously (15). Each animal was randomized to receive only one of the isoflurane concentrations. Isoflurane, oxygen, nitrogen, and carbon dioxide concentrations in the box were monitored by an anesthetic gas monitor (Type 1304; Brel and Kjær, Denmark). The isoflurane concentration in the box reached expected concentrations within 10 min. During isoflurane administration, perfusate was collected every 20 min by the fraction collector (EF-80; EICOM). All perfusates were cooled to approximately 6°C with the cooler (cool pump CP-80; TAITEC, Japan) to prevent degradation of 5-HT.

Perfusate samples obtained by in vivo microdialysis were manually applied to the high-performance liquid chromatography system for quantification of 5-HT. The 5-HT in the perfusates was separated by high-performance liquid chromatography using Eicompack PP-ODS column (particle size 2 mm, 4.6 x 30 mm; EICOM) maintained at 25°C. The mobile phase, which contains 0.1 M sodium phosphate buffer (pH 6.0), 450 mg/L sodium 1-decansulfonate, and 50 mg/L EDTA acid, was delivered by a pump (model 203, EICOM) at 0.5 mL/min to the column. Substances in the mobile phase were quantified by electrochemical detection using a glassy carbon working electrode set at 400 mV against a silver-silver chloride reference electrode (WE-3G; EICOM).

Assessment of Righting Reflex
Rats were injected IP with 20 mg/kg fluoxetine hydrochloride (Nacalai Tesque, Kyoto, Japan) dissolved in 0.5 mL of normal saline, or vehicle (0.5 mL of normal saline), and individually placed in clear airtight plastic boxes (30 x 30 x 35 cm). Thirty minutes after IP injection, isoflurane at initial concentrations of 0.4%, 0.5%, 0.6%, or 0.7% was administered in 6 L/min mixture of 25% oxygen and 75% nitrogen from the port of the boxes. Isoflurane, oxygen, nitrogen, and carbon dioxide concentrations in the box were monitored by an anesthetic gas monitor (Type 1304; Brel and Kjær). The concentration of isoflurane was maintained for at least 15 min, and then repeatedly increased or decreased in 0.05% steps and allowed to re-equilibrate at each concentration. To assess righting reflex at each isoflurane concentration, each unrestrained rat was placed in a wire-mesh cage that could be rotated at 20 rpm in the box. The minimum isoflurane concentration required for loss of righting reflex was obtained. The observer was blinded to the concentrations of isoflurane. Each experiment was completed within 90 min.

Statistical Analysis
Data are shown as mean ± sem (standard error of mean). Release of 5-HT during slow-wave sleep and during isoflurane inhalation was expressed as percentage of the mean value of 5-HT release during wakefulness. The differences in 5-HT release among tested groups were assessed for statistical significance by analysis of variance (repeated-measures ANOVA), and when significant F values were encountered, the Fisher's protected least significant differences test was used to confirm significant differences among treatments. A probability level of P < 0.05 was considered statistically significant.

The isoflurane concentration required for loss of righting reflex was compared between fluoxetine-treated and control groups using the unpaired Student's t-test with significance at P < 0.05.

	RESULTS

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Effect of Isoflurane on 5-HT Release in the Rat Frontal Cortex
The amplitude of EEG activity did not change at 0.1 or 0.3 MAC compared with slow-wave sleep, and then increased at 0.5 MAC. The frequency of EEG activity decreased at 0.5 MAC. At 1.0 MAC, spike burst and suppression patterns appeared, and at 1.5 MAC the suppression periods became dominant and the EEG became almost silent. All rats were quiet and immobile at every isoflurane concentration during the experiments.

Release of 5-HT in the frontal cortex was stable during control examination for 2 h without isoflurane anesthesia. The 5-HT release during wakefulness was 101.9 ± 14.3 fmol/20 µL fraction (n = 33). The 5-HT content in the perfusate during slow-wave sleep and that during isoflurane inhalation (0.1, 0.3, 0.5, 1.0, and 1.5 MAC) were significantly smaller than that during wakefulness (100%) (P < 0.05) (Fig. 1). There was no significant difference between 5-HT release during slow-wave sleep and isoflurane inhalation (0.1–1.5 MAC).

View larger version (35K): [in this window] [in a new window]   Figure 1. Release of 5-HT in the rat frontal cortex measured by in vivo microdialysis during wakefulness (W), slow-wave sleep (SWS), 0.1, 0.3, 0.5, 1.0, and 1.5 MAC isoflurane. Values are expressed as percentage of the mean value of 5-HT content in 3–5 samples obtained during wakefulness. Each group consisted of 6–7 rats. *P < 0.05 versus wakefulness. There were no significant differences in 5-HT release among slow-wave sleep and all of the isoflurane concentrations tested.

Effect of Fluoxetine on the Isoflurane Concentration Required for Loss of Righting Reflex
Apparently fluoxetine alone did not affect the behavior of rats, although 5-HT release was increased almost twice by administration of fluoxetine (data not shown). The isoflurane concentration required for loss of righting reflex in control and fluoxetine-pretreated rats was 0.60% ± 0.01% (n = 8) and 0.76% ± 0.03% (n = 8), respectively. The difference was statistically significant (P < 0.05) (Fig. 2).

View larger version (60K): [in this window] [in a new window]   Figure 2. Effect of fluoxetine on the isoflurane concentration required for loss of righting reflex. After intraperitoneal injection of saline (control) or fluoxetine, isoflurane concentration required for loss of righting reflex was measured. Data were obtained from eight rats for each group. *P < 0.05 versus control group.

	DISCUSSION

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In this study, we demonstrated that inhalation of isoflurane, as well as slow-wave sleep, caused a significant decrease in 5-HT release in rat frontal cortex when compared with the release during wakefulness. Release of 5-HT during isoflurane inhalation was not dependent on isoflurane concentration (0.1–1.5 MAC), but was similar to that during slow-wave sleep.

Tung et al. (9) demonstrated that sleep-deprived rats require less anesthetic agents than control rats to achieve loss of righting reflex, which may suggest that neuronal networks active in sleep are also involved in the anesthetized state. Accordingly, it might be possible that changes in neurotransmitter release similar to sleep-induced changes are observed during general anesthesia. It has been shown that the extracellular level of 5-HT in the frontal cortex in the rat (3) and cat (4) is significantly lower during slow-wave sleep than wakefulness. In accordance with these reports, Adell et al. (11) showed that extracellular content of 5-HT in rat raphe nucleus is reduced by 60% when anesthetized with pentobarbital or chloral hydrate compared with wakefulness. Some reports suggested the possible involvement of 5-HT in modulation of consciousness by inhaled anesthetics. Roizen et al. (10) demonstrated that lesions in DRN, where serotonergic neurons distribute in high density, decreased halothane and cyclopropane MAC in rats. The administration of ketanserin, 5-HT_2A receptor antagonist, decreased the MAC of isoflurane (12). In contrast, Rampil et al. (16) showed that 5-HT₃ receptor antagonist did not alter the isoflurane MAC in rats. Although involvement of the 5-HT system in general anesthesia has been suggested, changes in activity of the serotonergic system induced by inhaled anesthetics have not been reported.

In vivo microdialysis is a bioanalytical sampling technique that allows on-line monitoring of biochemical events occurring in the extracellular space of living tissue. The presence of the small probe does not disturb the animals, which are free to move, eat and sleep at will. This feature of microdialysis makes it suited for in vivo analysis of changes in neurotransmitter release induced by inhaled anesthetics. Therefore, we attempted to assess isoflurane-induced modulation of serotonergic system activity by assessing 5-HT release by in vivo microdialysis. To our knowledge, this is the first report on the change in serotonergic activity induced by inhaled anesthetics.

We demonstrated that the decrease of 5-HT release is not dependent on the isoflurane concentration in the concentration range of 0.1–1.5 MAC, which might mean that the decrease is not a direct effect of isoflurane. In contrast, EEG activity was similar to slow-wave sleep at an isoflurane concentration 0.3 MAC, and it then changed in a concentration-dependent manner at higher than 0.5 MAC. These results suggest that a decrease in 5-HT release in the frontal cortex during isoflurane inhalation does not directly cause suppression of EEG activity. However, it might be possible that a decrease in 5-HT release is at least partially involved in the isoflurane-induced behavioral change, because the rats were quiet at isoflurane concentrations larger than 0.1 MAC, which was apparently indistinguishable from slow-wave sleep. To assess the relationship between the change in 5-HT release and the hypnotic effect of isoflurane, the effect of pretreatment with fluoxetine on the requirement of isoflurane for loss of righting reflex was examined. Fluoxetine is an antidepressant drug that increases the availability of 5-HT by inhibition of 5-HT reuptake at the synapses in the brain. The duration of extracellular 5-HT increase induced by IP administration of fluoxetine is from 20 to 120 min after drug injection, as shown by our pilot study (data not shown) and a previous study (17). Our results indicated that increases of 5-HT availability partially antagonize the isoflurane-induced loss of righting reflex, suggesting that modulation of serotonergic neuron activity is at least partially involved in the hypnotic action of isoflurane. Serotonergic projection from raphe nucleus in the medulla and pons is involved in the brainstem reticular formation (18). Because the brainstem reticular formation plays an important role in the generation of consciousness and arousal, anatomical distribution of serotonergic neurons is consistent with our results. However, fluoxetine can produce pharmacological effects other than 5-HT receptor-mediated effects, including modulation of potassium channels (19,20). We cannot completely exclude the possibility that fluoxetine affects the hypnotic action of isoflurane independently of 5-HT.

The role of 5-HT in behavioral processes has been confounded by the lack of selectivity of the drugs available. Most of the work on the functional role of 5-HT in the sleep–waking cycle has been done on 5-HT_1A (21) and 5-HT₂ receptors. The 5-HT₂ receptor antagonist ritanserin promotes sleep in both humans (22) and rats (23), suggesting that the 5-HT₂ receptor mediates promotion of waking activity. The 5-HT_1A agonist 8-OH-DPAT perfused into DRN increased REM sleep, but had no effect on other sleep stages (24,25). In contrast, systemic administration of 5-HT_1A agonists increased waking (23). The differential effects of 5-HT_1A agonists, depending on the route of administration, might be explained by the presence of the 5-HT_1A autoreceptors in DRN. Thus, further investigation is necessary to identify the 5-HT receptor subtype involved in the sleep–waking cycle and anesthesia.

In conclusion, isoflurane inhalation, similar to slow-wave sleep, caused a decrease in 5-HT release in the frontal cortex, compared with that during wakefulness. In addition, inhibition of 5-HT reuptake increased the requirement of isoflurane for loss of righting reflex. These results may suggest that modification of serotonergic system activity is involved in the hypnotic action of isoflurane.

	Footnotes

 
Accepted for publication November 22, 2006.

Supported by Ministry of Education, Culture, Sports, Science, and Technology of Japan.

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