Anesth Analg 2003;96:1027-1031
© 2003 International Anesthesia Research Society
ANESTHETIC PHARMACOLOGY
5-HT3 Receptors Partially Mediate Halothane Depression of Spinal Dorsal Horn Sensory Neurons
Masayuki Koshizaki, MD,
Mikito Kawamata, MD PhD,
Steven G. Shimada, PhD,
Yoji Saito, MD PhD, and
J. G. Collins, PhD
Department of Anesthesiology, Yale University School of Medicine, New Haven, Connecticut
Address correspondence and reprint requests to J. G. Collins, PhD, Department of Anesthesiology, Yale University School of Medicine, 333 Cedar St., PO Box 208051, New Haven, CT 06520-8051. Address e-mail to j.collins{at}yale.edu
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Abstract
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We recently reported that 
-aminobutyric acid type A- and strychnine-sensitive glycine receptor systems partially mediate halothane depression of spinal dorsal horn low-threshold neurons. Serotonin subtype 3 (5-HT3) receptors belong to the same ligand-activated ion-channel family as -aminobutyric acid type A- and strychnine-sensitive glycine receptors, so we examined the possible involvement of 5-HT receptor systems in halothane depression of spinal sensory neurons. Extracellular recordings of spinal low-threshold neurons were obtained in decerebrate, spinally transected rats. Receptive field size and brush-induced activity were recorded in the presence or absence of 5-HT antagonists and in the presence or absence of 1.1% (1 minimum alveolar anesthetic concentration) halothane. In the absence of halothane, antagonists had no effect on receptive field size or brush-induced activity. In the presence of halothane, methysergide, a nonselective 5-HT antagonist, and tropisetron, a selective 5-HT3 antagonist, significantly reversed the halothane-induced reduction in receptive field size but did not alter halothane depression of brush-induced activity. Methiothepin, a 5-HT1 antagonist, and ketanserin, a 5-HT2 antagonist, did not reverse halothane depression. These results support the hypothesis that 5-HT3 receptors partially mediate some inhibitory effects of halothane on spinal dorsal horn neurons.
IMPLICATIONS: The results of this study support the hypothesis that halothane depression of spinal sensory neuronal responses to low-intensity stimuli is mediated, to a minor extent, by serotonin subtype 3 neurotransmitter systems.
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Introduction
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Spinal dorsal horn sensory neurons receive input from primary afferents and serve as cells of origin for important fiber tracts within the spinal cord (1–3) . It is, therefore, reasonable to hypothesize that anesthetic depression of those neurons may, by reducing the level of afferent input that reaches the supraspinal regions of the central nervous system, contribute to the complex of behavioral changes associated with the state of general anesthesia. We recently reported that halothane depression of spinal low-threshold (LT) dorsal horn neurons is dependent, in part, on -aminobutyric acid type A (GABAA)- and strychnine-sensitive glycine inhibitory neurotransmitter systems (4). Of particular relevance to this study, we observed that although GABAA and glycine were involved, they were not the sole mediators of the observed anesthetic depression, something that we also observed for spinal wide dynamic range neurons (5). Zhang et al. (6) also reported that halothane depression of behavior was not totally dependent on spinal GABAA and glycine. Although there are many other inhibitory neurotransmitter systems in the spinal cord, the fact that the serotonin subtype 3 (5-HT3) system is a member of the same superfamily of fast-acting ligand-gated ion channels as GABAA and glycine (7) makes it a likely candidate for an additional system that could mediate halothane depression of spinal dorsal horn LT neurons.
The possibility that 5-HT receptors are involved in anesthetic modulation of spinal LT neurons is strengthened by reports that 5-HT can inhibit spinal sensory processing (8–10) . In addition, we previously reported that systemic administration of the nonspecific 5-HT antagonist methysergide increased the size of peripheral receptive fields of spinal dorsal horn neurons in physiologically intact, awake, drug-free cats, suggesting tonic 5-HT inhibition of those neurons (11). On the basis of those reports and observations, we hypothesized that 5-HT receptor systems (most likely 5-HT3) help to mediate halothane depressive effects on spinal dorsal horn neurons. This study was conducted both to test that hypothesis and to define the 5-HT receptor subtypes that may be involved in those anesthetic-induced modulations.
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Methods
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This protocol was approved by the Yale University Institutional Animal Care and Use Committee. Electrophysiological experiments were performed with Sprague-Dawley rats (250–400 g).
Anesthesia was induced by placing animals in a chamber that contained 3% halothane in 100% oxygen. After loss of the righting reflex, anesthesia was maintained with 2% halothane in 100% oxygen delivered through a tightly fitting mask. Body temperature was monitored with a rectal probe and maintained between 36°C and 38°C throughout the experiment. A carotid artery and a jugular vein were cannulated to allow for blood pressure monitoring and for fluid and drug administration, respectively. A tracheostomy was performed, and the trachea was intubated. The animals were paralyzed with a continuous infusion of the neuromuscular blocking drug pancuronium bromide (0.15 mg · kg-1 · h-1) and mechanically ventilated for the remainder of the experiment. End-tidal oxygen, CO2, and halothane concentrations were monitored throughout the experiment. Mean arterial blood pressure was maintained at greater than 60 mm Hg by continuous administration of lactated Ringer’s solution to avoid the hypotensive effect of halothane on receptive field size (12). All animals were spinally transected at the third thoracic level and were rendered decerebrate by aspiration of cranial contents rostral to the mesencephalon. Spinal cord transection allowed us to study drug effects at the level of the spinal cord without interference from supraspinal drug actions. Decerebration rendered the animals permanently unconscious and allowed us to obtain baseline data in the absence of an anesthetic. A laminectomy (T12-13) was performed to expose the lumbar enlargement where the recordings were made. Halothane was discontinued after surgical preparations.
After at least 30 min of recovery from halothane anesthesia, tungsten microelectrodes (impedance, 10 M
; FHC Inc., Bowdinham, ME) were advanced in the spinal dorsal horn to isolate the activity of a single neuron. When the extracellular activity of a single dorsal horn neuron was isolated, its response profile to peripheral receptive field stimulation was examined. Only LT neurons were included in this study. LT neurons were identified as those neurons that were excited only by nonnoxious tactile stimulation. High-intensity mechanical stimuli can also activate these neurons, but the firing frequency is not greater than that seen with low-intensity stimuli.
The animals were divided into two groups. Group 1 animals received a 5-HT receptor antagonist in the absence of halothane and provided control data for antagonist effects alone. Group 2 animals received a 5-HT receptor antagonist in the presence of halothane.
After isolation of a single LT neuron, by using a von Frey filament (0.3556 mm in diameter, 4.08 g), the LT receptive field within which the stimulus always elicited a response was carefully determined and marked on the shaved skin (baseline). At the perimeter of this marked area, stimuli elicited a response on approximately 50% of trials. After the receptive field area was mapped, the most sensitive portion of it was stimulated 10 times by brushing with an artist’s paintbrush. Unit activity was fed to a computer through a data acquisition system (CED1401) for storage and analyzed by Spike 2 software (Cambridge Electronic Design, Cambridge, UK). Group 1 rats then received 2 IV injections (cumulative dosing) of a 5-HT receptor antagonist at 15-min intervals. Five minutes after each injection, the receptive field area and brush-induced activity were reevaluated. Maximally effective antagonist doses were determined by information from pilot studies and from literature references. In Group 2, after baseline determinations, 1.1% (1 minimum alveolar anesthetic concentration [MAC]) halothane was administered, and the receptive field area and brush-induced activity were reevaluated. Then, with continuing halothane anesthesia, rats received a 5-HT receptor antagonist in a dose-dependent manner, and the receptive field area and brush-induced activity were again reevaluated.
Only one neuron was studied per rat, and each animal received only one 5-HT receptor antagonist. At the end of the experiment, the outline of the receptive field area marked on the skin was traced, digitized, and used to measure the receptive field size.
All drugs were administered in a cumulative fashion. The antagonists and their doses were as follows: methysergide, a nonspecific 5-HT receptor antagonist (Research Biomedical International, Natic, MA), 1.0, 2.0 mg/kg; methiothepin, a 5-HT1 receptor antagonist (Research Biomedical International), 1.0, 2.0 mg/kg; ketanserin, a 5-HT2 receptor antagonist (Research Biomedical International), 1.0, 2.0 mg/kg; and tropisetron, a 5-HT3 receptor antagonist (Research Biomedical International), 1.0, 2.0 mg/kg.
Data are expressed as mean ± SD and were analyzed with repeated-measures analysis of variance. If analysis of variance resulted in a probability value of <0.05, Bonferroni’s post hoc test was performed. P values of <0.05 were considered significant.
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Results
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The effects of methysergide on halothane-induced reduction in the receptive field size of a single LT neuron are shown in Figure 1. In a decerebrate, spinal cord-transected animal, halothane caused a reduction in LT receptive field size that was partially reversed by the IV administration of methysergide. Figure 2 presents a summary of the effects of methysergide on the receptive field size and brush-induced activity in both the presence and absence of halothane. Methysergide produced a partial but statistically significant reversal of the halothane-induced reduction in receptive field size but not in brush-induced activity. As shown in the bottom portion of Figure 2, methysergide was without an effect in the absence of halothane. Because methysergide is a nonspecific antagonist, we next examined antagonists for identified 5-HT receptor systems in the rat spinal cord.
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Figure 1. Example of receptive field changes with the administration of halothane and methysergide. Methysergide produced a partial, but significant, reversal of halothane-induced reduction in the low-threshold receptive field. MAC = minimum alveolar anesthetic concentration.
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Figure 2. Effects of the nonspecific serotonin receptor antagonist methysergide on spinal dorsal horn neurons with (top, n = 8) and without (bottom, n = 6) halothane. In decerebrate, spinally transected control animals (bottom), methysergide failed to cause any change in the low-threshold receptive field size or the brush-induced response of the spinal dorsal horn neurons. Halothane (top) caused statistically significant reductions of the receptive field size and brush-induced response. Methysergide produced a partial, but statistically significant, reversal of halothane-induced reduction of receptive field size. Data are presented as mean percentage (±SD) of the baseline. *P < 0.05 compared with baseline; #P < 0.05 compared with halothane 1 MAC plus 0 mg/kg of methysergide. MAC = minimum alveolar anesthetic concentration.
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As shown in Figure 3, the 5-HT3 receptor antagonist tropisetron produced a small and partial, but statistically significant, reversal of the halothane-induced reduction in receptive field size. As with methysergide, there was no significant effect on the halothane-induced reduction in the brush-induced activity. Tropisetron, in the absence of halothane, produced no change in either the receptive field size or brush induced activity. As seen in Figures 4 and 5, neither ketanserin (5-HT2 antagonist) nor methiothepin (5-HT1 antagonist) had any effect on the receptive field size or brush-induced activity in the presence or absence of halothane.
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Figure 3. Effects of the serotonin subtype 3 receptor antagonist tropisetron on spinal dorsal horn neurons with (top, n = 12) and without (bottom, n = 6) halothane. Tropisetron failed to cause any change in the low-threshold receptive field size or the brush-induced response of the spinal dorsal horn neurons without halothane (bottom). Tropisetron produced a partial, but statistically significant, reversal of the halothane-induced reduction of receptive field size (top). *P < 0.05 compared with baseline; #P < 0.05 compared with halothane 1 MAC plus 0 mg/kg of tropisetron. MAC = minimum alveolar anesthetic concentration.
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Figure 4. Methiothepin, a serotonin subtype 1 receptor antagonist, failed, both in the absence (bottom, n = 4) and presence (top, n = 7) of halothane, to alter the low-threshold receptive field size and brush-induced activity of any of the neurons on which it was tested. Data are presented as mean percentage (±SD) of the baseline. *P < 0.05 compared with baseline. MAC = minimum alveolar anesthetic concentration.
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Figure 5. Ketanserin, a serotonin subtype 2 receptor antagonist, failed, both in the absence (bottom, n = 4) and presence (top, n = 7) of halothane, to alter the low-threshold receptive field size and brush-induced activity of any of the neurons on which it was tested. Data are presented as mean percentage (±SD) of the baseline. *P < 0.05 compared with baseline.
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Discussion
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5-HT is one of the neurotransmitters that has been shown to be capable of inhibiting sensory processing in the spinal dorsal horn (8–10) . Previous work in our laboratory demonstrated that systemic administration of methysergide increased the receptive field size of spinal dorsal horn neurons in physiologically intact, awake, drug-free cats, suggesting a continuing level of serotonergic modulation of neuronal activity (11). Glaum et al. (13,14) reported that spinal intrathecal administration of 5-HT produced an antinociceptive effect that was inhibited by a 5-HT3 receptor antagonist. Of particular importance to this study, it has been reported that halothane enhanced 5-HT3 receptor function when homomeric receptors were expressed in oocytes (15). We therefore hypothesized that halothane-induced reductions in spinal dorsal horn neuronal responses to receptive field stimulation were mediated, in part, by spinal serotonergic systems. The results of this study support that hypothesis. Although the effects of methysergide and tropisetron were limited to the receptive field size and were relatively small in absolute terms, they were statistically significant.
The reversal of inhibition by methysergide or tropisetron was approximately 10%–15% of the control value. Although that is a relatively small amount, it is in keeping with expectations based on our recent study of the combined reversal effects of bicuculline (GABAA antagonist) and strychnine (glycine antagonist) (4). When maximum doses of those two antagonists were administered in the presence of 1.1% halothane, a significant but incomplete reversal of the receptive field size to approximately 78% of the control (preantagonist value) occurred. Although it is not possible to compare absolute amounts of reversal, a 10% to 15% effect of a 5-HT antagonist fits well with the remaining 22% depression when GABAA and glycine antagonism are accounted for.
An obvious difference between this study and our recently reported study of GABAA and glycine is the absence of an antagonist effect on the response of LT neurons to receptive field brushing. The sensory input from stimuli used to map a receptive field is different than that generated by moving a brush across a receptive field. The latter, because it is a dynamic stimulus with a large surface area, is likely to activate more and different types of primary afferents.
We are not aware of any studies that define differences in 5-HT receptor distribution among primary afferents. However, we have observed differential effects of anesthetics on responses to the two types of stimuli used in this study. As we previously reported (16), enflurane depressed the receptive field size at the same time that it enhanced the response to receptive field brushing.
These differential effects emphasize the complexity of anesthetic actions and provide a cautionary note about assuming that such actions are the same on all elements of the nervous system. That cautionary note must also be applied to other sensory neurons within the spinal dorsal horn. On the basis of our previous work (11) with neurons that respond to both LT and high-threshold stimuli (wide dynamic range neurons), we would hypothesize that 5-HT3 receptors are more likely to be involved in a greater degree of anesthetic-induced modulation of noxiously evoked activity within the spinal dorsal horn. However, recent work by Rampil et al. (17) indicated that the administration of ondansetron, a 5-HT3 antagonist, did not change the MAC value for isoflurane in rats. It therefore appears that although 5-HT is capable of profoundly depressing spinal sensory neuronal responses to noxious peripheral receptive stimulation, that action is not important to the immobilizing effects of isoflurane in rats.
In summary, 5-HT3 receptor systems help to mediate halothane depression of LT spinal dorsal horn receptive field size. The results of this study support the hypothesis that anesthetics modulate sensory transmission by influencing the action of multiple neurotransmitter systems.
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Acknowledgments
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Supported in part by National Institutes of Health Grant GM 44954.
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References
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Accepted for publication November 21, 2002.
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Abstract
Introduction
