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From the Department of Anesthesiology, College of Physicians and Surgeons, Columbia University, New York, New York.
Address correspondence and reprint requests to Robert A. Whittington, MD, Columbia University, College of Physicians and Surgeons, Department of Anesthesiology, 622 West 168th Street PH 5, New York, NY 10032. Address e-mail to raw9{at}columbia.edu.
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Abstract |
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 Top Abstract IntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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Introduction |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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Furthermore, it has been demonstrated that inhaled anesthetics can have significant effects at specific 5-HT receptor subtypes. For example, halothane and isoflurane have been shown in cloned human 5-HT3 receptors expressed in Xenopus oocytes to enhance the function of this specific receptor subtype (5). More recently, Stevens et al. (6) demonstrated that volatile anesthetics, at clinical concentrations, can modulate heteromeric human 5-HT3AB receptors, expressed in Xenopus oocytes, suggesting that this receptor subtype may indeed be a target of volatile anesthetics. The potential significance of 5-HT3AB modulation by these anesthetics is further highlighted by the fact that this receptor subtype has been also linked with alterations in the release of other neurotransmitters such as gamma-aminobutyric acid (GABA), glutamate, acetylcholine, glycine, all of which have been more closely linked with volatile anesthetic action (6–9). In addition, several inhaled anesthetics appear to antagonize serotonergic neurotransmission at the 5-HT2A receptor, a 5-HT receptor subtype thought to mediate anesthetic-induced immobility to noxious stimulation (10). The importance of serotonergic receptors appears to be not solely limited to volatile anesthetics, as it has been observed in rats that the administration of serotonergic receptor antagonists enhanced the anesthetic response to ketamine, pentothal, and chloral hydrate (11).
The hippocampus is an area of the brain linked to cognition (12,13) and depressed mood states (14,15), as well as stress and anxiety (16,17). Furthermore, serotonergic neurotransmission in this brain region has been specifically linked to neurobehavioral states such as anxiety and depression (15,18), as well as to cognitive deficits (19). There is a paucity of information regarding the in vivo effects of isoflurane on extracellular 5-HT in the central nervous system, and even less is known as to how the volatile anesthetics affect hippocampal 5-HT. The purpose of this study was to determine, in vivo, the effects of clinically relevant concentrations of isoflurane on extracellular hippocampal levels of 5-HT.
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METHODS |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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Isoflurane (Abbott Laboratories, Chicago, Il) was administered via an Isotec 4 vaporizer (Datex-Ohmeda, Louisville, CO). Perchloric acid, ortho-phosphoric acid, 1-decanesulfonic acid, EDTA, citric acid monohydrate, and NaOH were purchased from Fluka Chemie AG (Milwaukee, WI). Methanol was purchased from Fisher Scientific (Fair Lawn, NJ). The artificial cerebrospinal fluid solution used to perfuse the microdialysis probes was purchased from Harvard Apparatus (Holliston, MA) and contained sterile water and electrolytes in the following concentration: Na+ 150 mM, K+ 3.0 mM, Ca2+ 1.4 mM, Mg2+ 0.8 mM, HPO42– 1.0 mM, Cl– 155.4 mM.
Two days before the study, the mice were anesthetized with ketamine (35 mg/kg IP) and pentobarbital (35 mg/kg IP) and had a CMA/7 microdialysis guide cannula (CMA Microdialysis, North Chelmsford, MA) placed in the ventral hippocampus (VHC). Each cannula was implanted stereotaxically in the VHC using the following coordinates, according to the atlas of Franklin and Paxinos (20): anterior-posterior: –2.8 mm from the bregma, lateral: –3.0 mm from the midline, dorsal-ventral: –2.0 mm from the top of the skull.
On the day of the study, a CMA/7 cerebral microdialysis probe (2 mm membrane length, 0.24 mm O.D.) was placed in the VHC of the awake mouse. These microdialysis probes were found to have an in vitro relative recovery for 5-HT of 27% ± 4%. Relative recovery is an in vitro measurement of how much of the analyte (5-HT) passes through the probe membrane at a given perfusion rate. The probe was then perfused with artificial cerebrospinal fluid at a rate of 0.5 µL/min, using a gastight syringe pump (Beestinger®, BAS Inc., West Lafayette, IN). The awake mouse was placed in a transparent study cage (BeeKeeper®, Bioanalytical Systems Inc., West Lafayette, IN) equipped with a rotating swivel, and the cage was mounted on a rotating platform (Raturn®, BAS Inc., West Lafayette, IN), which allowed the mouse to move about freely before the administration of isoflurane. This cage system was fitted with a specially designed lid, which allowed cerebral microdialysis to continue during the administration of isoflurane without animal manipulation (Fig. 1). Isoflurane and O2 concentrations in the microdialysis study cage were determined using a respiratory gas monitor (Ohmeda 5250 RGM; Datex-Ohmeda, Louisville, CO). Spontaneous ventilation was maintained throughout the entire study.
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Two hours after probe insertion, hippocampal dialysate samples were collected throughout the study at 20-min intervals in vials pretreated with 2 µL of 0.33 M sodium dihydrogen phosphate buffer with 0.5 µM EDTA (pH 6) to minimize 5-HT degradation. The dialysates were immediately analyzed for 5-HT, and once a stable baseline was obtained (3 consecutive samples with <10% variation in peak heights between samples), isoflurane in a 40% O2 in air mixture or a 40% O2 in air mixture alone (control) was administered.
The analysis of 5-HT in brain microdialysates was performed by high performance liquid chromatography with electrochemical detection, using a system consisting of a GBC LC1150 pump (GBC Scientific, Dandenong, Australia), a Rheodyne 9725i manual injector (Rohnert Park, CA), and an Antec Intro electrochemical detector with a VT-03 flow cell (Antec Leyden, The Netherlands). The mobile phase contained 150 mM sodium dihydrogen phosphate, 4.76 mM citric acid, 3 mM 1-decanesulfonic acid (sodium salt), 100 µM EDTA, 6% methanol and 9% acetonitrile, and was adjusted to pH 5.6. Chromatographic separation was achieved on a microbore (1 x 100 mm) C-18 column with a 3-µm particle size, at a flow rate of 50 µL/min and at 30°C. Detection was performed at a potential of +500 mV. The assay was characterized by excellent linearity in the calibration range of 1 to 100 fmol (in 10 µL injection volume), and a limit of detection of 0.3 fmol (30 pM).
Isoflurane was administered in 3 different studies, designed to further detail the effects of the volatile anesthetic on hippocampal 5-HT. In study 1, once stable basal extracellular 5-HT levels were achieved, the 129/SvEv mice received one of the following treatments: 40% O2 in air (control, n = 8), isoflurane 1 minimum alveolar concentration (MAC) (1.3%, n = 6) or isoflurane 1.5 MAC (1.9%, n = 4) both in 40% O2 in air for a period of 80 min.
Study 2 was designed to determine whether changes in extracellular hippocampal 5-HT occur in the absence of normal serotonin transporter (SERT) function. In these studies, wild-type (SERT +/+, n = 7) or homozygous SERT knockout (SERT –/–, n = 6) mice, from a C57BL/6 genetic background, received 1 MAC (1.3%) isoflurane in 40% O2 in air for a period of 80 min.
Study 3 was designed to determine the effects of 1.5 MAC isoflurane on extracellular hippocampal 5-HT both during and after isoflurane exposure. During these studies, once the 129/SvEv mice used in study 1 received 1.5 MAC isoflurane (n = 4) in 40% O2 in air for a period of 80 min, the isoflurane was discontinued and the mice were returned to room air. Hippocampal dialysates for 5-HT were collected for an additional 80 min immediately after the termination of isoflurane exposure. A final dialysate sample for 5-HT was obtained the next morning, approximately 18–20 h after exposure to isoflurane (24 h after probe insertion). As a control group for this study, another group of 129/SvEv mice (n = 5) was exposed to 40% O2 in air for 80 min. After 80 min of 40% O2 in air exposure, they were returned to room air and extracellular hippocampal microdialysates for 5-HT were similarly collected for an additional 80 min as well as the next am.
At the end of each study, all mice were killed with an overdose of pentobarbital administered by IP injection. The brain was immediately harvested and preserved in a 10% formalin and 15% sucrose solution. Coronal sections were obtained (50 µm) and the probe placement was verified using the atlas of Franklin and Paxinos (20). Only data obtained from animals with proper probe placement were analyzed.
Because there can normally be significant variability in basal 5-HT levels among mice, changes in extracellular 5-HT over time were expressed as a percentage of each group's respective average baseline level. In study 1 and when comparing basal 5-HT concentrations (Table 1), between-group comparisons of 5-HT levels were performed using analysis of variance with Bonferroni post hoc test applied when appropriate. In studies 2 and 3, between-group changes in extracellular 5-HT as a function of time were performed using an unpaired Student's t-test. In all studies, within-group treatment-induced changes in extracellular 5-HT were compared with each group's respective baseline and were analyzed using analysis of variance followed by Dunnett's post hoc test when appropriate. Statistical calculations were performed using InStat® statistical analysis software (GraphPad Software, Inc., San Diego, CA), and all data are reported as mean ± sd, with a value of P < 0.05 considered statistically significant.
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RESULTS |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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In study 2, which examined the role of SERT in mediating isoflurane-induced decreases in hippocampal 5-HT, when compared with each group's respective baseline, within 20–40 min of exposure, 1 MAC isoflurane significantly decreased hippocampal 5-HT in both SERT +/+ and SERT –/– groups, and this decrease lasted throughout the study (Fig. 3). Interestingly, the decrease in 5-HT induced by isoflurane was significantly greater in SERT –/– when compared to SERT +/+. The peak decrease in hippocampal 5-HT was to 50.2% ± 17.4% and 22.4% ± 8.5% of the baseline 5-HT levels in SERT +/+ and SERT –/–, respectively. No significant strain-related differences in isoflurane-induced decreases in 5-HT were observed between the wild-type (SERT +/+) C57BL/6 and 129/SvEv mice (Fig. 4). By the end of the study, hippocampal 5-HT in both strains of wild-type mice had decreased to a similar degree: 50.2% ± 17.4% and 45.5% ± 11.6%, respectively.
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Study 3 examined extracellular hippocampal 5-HT during and after exposure to 1.5 MAC isoflurane in 129/SvEv mice. Within 20–40 min of administration, extracellular hippocampal 5-HT levels significantly decreased to 51.3% ± 24.6% of baseline, and this decrease persisted throughout the entire sampling period (Fig. 5). The effect of isoflurane on hippocampal 5-HT reached its nadir, 30.7% ± 10.0% of baseline, 20–40 min following the end of isoflurane exposure (100–120 min from the start of anesthesia). Interestingly, towards the end of the study (beginning 120 min post-isoflurane), an increasing trend in extracellular hippocampal 5-HT towards baseline levels was observed; however, when compared with its respective baseline, extracellular 5-HT remained significantly decreased, 58.7% ± 26.0% of baseline, at the end of the study, i.e., 18–20 h after exposure to isoflurane.
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DISCUSSION |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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Although the serotonergic system may be affected by, and also linked to, volatile anesthetic action (4,6,10), the present study is the first to examine extracellular brain levels of 5-HT, in vivo, after the administration of isoflurane. Our findings support the notion that volatile anesthetics can have significant serotonergic effects in the hippocampus. Furthermore, the finding that the isoflurane-induced decrease in extracellular 5-HT was significantly larger in the absence of SERT, the major transport protein involved in the synaptic reuptake of 5-HT, is an important observation in terms of elucidating the mechanism underlying this change in synaptic 5-HT. This finding specifically suggests that SERT is probably not involved in mediating the isoflurane-induced decrease in extracellular 5-HT. Of note, the larger isoflurane-induced decrease in extracellular 5-HT observed in SERT –/–, was probably because basal 5-HT levels in SERT –/– were approximately 10-fold higher than in the SERT +/+ (Table 1). Thus, these higher basal 5-HT levels in SERT –/– would allow for a greater decrement in extracellular 5-HT before reaching the maximum isoflurane-induced decrease; whereas, the lower basal levels in SERT +/+ were probably already closer to the nadir of the isoflurane-induced response.
Interestingly, although published studies examining the effects of isoflurane on extracellular 5-HT are scarce, the findings in the present study are contrary to what would be expected based on the only published study examining the effects of isoflurane on 5-HT uptake. Using a rat brain synaptosome preparation, Martin et al. (4) demonstrated that isoflurane, at clinically relevant concentrations, produced a concentration-dependent decrease in 5-HT uptake, which kinetically was observed to occur in a noncompetitive manner. Based on the findings of Martin et al., an isoflurane-induced decrease in synaptosomal reuptake of 5-HT would have been expected to produce an increase in extracellular 5-HT; however, we observed a decrease in extracellular 5-HT after isoflurane administration. Although we cannot completely account for this observed disparity, we suggest that this may have been attributable to differences in the in vitro and in vivo methodologies used. Consequently, although isoflurane decreased 5-HT synaptosomal reuptake in their in vitro synaptosome preparation, it should be noted that this isolated system may be devoid of other modulatory inputs, such as 5-HT1A and 5-HT1B autoreceptor function, input from nonserotonergic neurotransmitter systems such as GABA and glutamate (21,22), and pharmacodynamic inputs, all which may have significant modulatory effects on extracellular 5-HT and may produce the net effect of a decrease. Moreover, it may be that, although 5-HT reuptake is decreased by isoflurane in vitro, other processes which alter presynaptic release or 5-HT metabolism have a more significant net effect on synaptic 5-HT than reuptake.
Indeed, although it has been suggested that volatile anesthetics might have effects on 5-HT uptake (23), the persistent decrease observed in extracellular 5-HT in the absence of SERT suggests that this transporter protein is not a major target of volatile anesthetics. We do acknowledge that we did not specifically examine other sites of extracellular serotonergic regulation in the present study; however, there is the possibility that extracellular 5-HT could be altered by other sites besides SERT. These include anesthetic-induced presynaptic changes in 5-HT release, changes in somatodendritic 5-HT1A and presynaptic 5-HT1B autoreceptor function, as well as changes in 5-HT release mediated by nonserotonergic neurotransmitter systems.
There is strong evidence to suggest that volatile anesthetic-induced modulation of extracellular neurotransmitters may be mediated by presynaptic mechanisms. Although their precise mechanism of action is still unknown, it has been demonstrated that volatile anesthetics can presynaptically depress excitatory glutamatergic neurotransmission while similarly increasing inhibitory neurotransmission mediated by GABA and glycine (24,25). Regarding 5-HT, it has been suggested that isoflurane-mediated alterations in serotonergic neurotransmission may primarily involve presynaptic mechanisms as opposed to postsynaptic effects (4).
In terms of possible 5-HT receptor targets, 5-HT1A and 5-HT1B receptors are found in somatodendritic and nerve terminal distributions, respectively, and activation of these 5-HT autoreceptors modulates extracellular 5-HT via a negative feedback loop (26,27). Unfortunately, there are no studies that have examined the effects of isoflurane on 5-HT autoreceptor function in vivo. Based on the in vitro findings of Martin et al., the effects of isoflurane on the 5-HT1A receptor are minimal; hence, it is unclear what role, if any, these autoreceptors have in mediating the decrease in hippocampal 5-HT observed in the current study. Nevertheless, further in vivo studies are warranted to define the role of these autoreceptors in producing the observed change in synaptic 5-HT.
Although G-protein coupled receptors, such as the 5-HT1A and 5-HT1B receptors, are major regulators of extracellular 5-HT and are potentially capable of producing the changes in hippocampal 5-HT observed in these studies, the possibility still remains that the changes in extracellular 5-HT may be mediated by other primary targets of volatile anesthetics, namely the GABAergic, glycinergic, and glutamatergic systems. It has been demonstrated that the discharge rate of serotonergic neurons is influenced by GABA, glycine, and glutamate (21,22). For example, in the dorsal raphe nucleus (DRN), a brainstem region containing 5-HT neuronal cell bodies, GABA and glycine have been demonstrated to suppress serotonergic neuronal activity (21). Moreover, in microdialysis experiments in the DRN, it has been shown that GABAA receptor activation decreases extracellular 5-HT; whereas, antagonism of GABAA receptors with bicuculline significantly increases DRN 5-HT (22). These same investigators also observed that glutamate receptor agonists produce increases in 5-HT, although the major influence overall on 5-HT release appears to be GABAergic in nature. Although we did not specifically examine the influence of these nonserotonergic systems on the changes in hippocampal 5-HT observed in the present study, as these neurotransmitter systems appear to be major targets of isoflurane (9), it is nevertheless conceivable that they may play a role in mediating these serotonergic changes.
Another possible mechanism underlying the observed decrease in hippocampal 5-HT after isoflurane administration may be related to changes in 5-HT metabolism. Although we specifically did not measure levels of 5-hydroxyindole-3-acetic acid (5-HIAA), the major metabolite of 5-HT, there is the possibility that isoflurane may accelerate the metabolism of 5-HT to 5-HIAA. Although increased formation of 5-HIAA has not been described with isoflurane in vivo, there is evidence that volatile anesthetics can alter the metabolism of other monoamines. Indeed, Adachi et al. (28) demonstrated that both sevoflurane and isoflurane increase dopamine metabolite formation in the rat striatum. Despite this evidence that isoflurane can alter the metabolism of dopamine, it is unclear whether isoflurane increases the function of either monoamine oxidase or aldehyde dehydrogenase, the enzymes involved in the metabolism of 5-HT to 5-HIAA.
Another interesting observation in the present study is that 1.5 MAC isoflurane produced a decrease in hippocampal 5-HT, which persisted for several hours after anesthesia. Although the precise mechanism underlying this sustained decrease in extracellular 5-HT is unknown, these persistent changes may not have been related to neuronally mediated 5-HT release, which would be expected to usually recover rapidly after termination of anesthesia exposure but may involve slower processes such as altered 5-HT metabolism or altered 5-HT synthesis. However, as previously mentioned, it is unclear if isoflurane has any direct effects on the enzymes involved in the metabolic breakdown of 5-HT to 5-HIAA. Similarly, it is unknown if isoflurane is involved in altering the function of tryptophan hydroxylase, the enzyme involved in the rate-limiting step during serotonin synthesis.
We acknowledge that it would be premature to extrapolate these preclinical findings to the clinical neurobehavioral level; nevertheless, we speculate that diminished 5-HT levels in the hippocampus after anesthetic exposure could theoretically have significant neurobehavioral and cognitive implications. The basis for this hypothesis is based on the fact that this study specifically demonstrated significant anesthetic-induced serotonergic effects at the level of the VHC, a key brain region involved in regulating these behaviors. Furthermore, our finding that the isoflurane-induced decrease in extracellular 5-HT persisted 18–20 hours after anesthetic exposure suggests that the observed change in hippocampal 5-HT may indeed not be transient. Isoflurane has been demonstrated to affect mood (29). Moreover, there is evidence that even transient anesthetic exposure may have persistent effects on cognitive function (30). Nevertheless, it remains to be seen whether isoflurane is associated with significant neurobehavioral effects in terms of anxiety, depression, or cognition and whether anesthetic-induced changes in hippocampal 5-HT contribute to the altered behavioral and cognitive states described after anesthetic exposure (29,30).
In summary, the present study suggests that extracellular 5-HT is decreased by isoflurane in a process that does not involve changes in SERT function. These data support the notion that the serotonergic system may be significantly altered after inhaled anesthesia. Future studies are warranted to further elucidate the mechanisms underlying this decrease, as well as to determine if there are clinical neurobehavioral implications as a consequence of isoflurane-induced decreases in hippocampal 5-HT.
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Footnotes |
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Supported, in part, by National Institute of General Medical Sciences grant K08 GM00681.
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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P < 0.05 compared with corresponding time of the control group.
