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From the *Department of Anesthesia, Kyoto University Hospital, and National Hospital Organization Kyoto Medical Center, Kyoto, Japan.
Address correspondence and reprint requests to Dr. Sahoko Koyanagi, Department of Anesthesia, Kyoto University Hospital, 54 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan. Address e-mail to sahoko{at}kuhp.kyoto-u.ac.jp.
Abstract
BACKGROUND: The mechanism of the antinociceptive effects of nitrous oxide (N2O) has not been completely elucidated. On the other hand, numerous studies have indicated that mesolimbic dopaminergic neurons, which are thought to be involved in rewarding and reinforcement processes, play important roles in the supraspinal pain-suppression system. We hypothesized that the mesolimbic dopaminergic system is involved in the antinociceptive effect of N2O.
METHODS: Adult male Fischer rats were used in this study. To examine whether the dopaminergic system is activated by N2O, frozen sections of the ventral tegmental area of rats exposed to 75% N2O were double-stained for c-Fos and tyrosine hydroxylase. To clarify whether the dopaminergic system is involved in the antinociceptive action of N2O, saline or raclopride, a dopamine D2-like receptor antagonist, was injected into the nucleus accumbens (NAc) shell region. After exposure to 25% oxygen–75% nitrogen or 25% oxygen–75% N2O for 30 min, rats were subjected to formalin test, and the spinal cord was examined immunohistochemically.
RESULTS: Exposure to 75% N2O increased c-Fos expression in tyrosine hydroxylase-positive cells in the ventral tegmental area. Raclopride, injected into the NAc shell region, attenuated the antinociceptive effect of N2O in the formalin test, and blocked the suppressive effect of N2O on the formalin-induced c-Fos expression in the dorsal horn of the spinal cord by N2O.
CONCLUSION: It is possible that inhalation of N2O activates mesolimbic dopaminergic neurons, and that the antinociceptive effect of N2O is at least partially mediated by dopamine D2-like receptors in the NAc shell region.
Nitrous oxide (N2O) has been used widely in clinical anesthesia for more than 160 years. N2O exhibits a variety of pharmacological effects, including antinociception, sedation, euphoric effects, and addiction, but its mechanism of action has not been completely understood. It has been suggested that opioid receptors in the brainstem and descending inhibitory pathways in the spinal cord are activated by N2O, resulting in attenuation of pain sensation.1–4 However, a number of points remain to be discovered with respect to the mechanism of the antinociceptive effects of N2O. Although N2O affects benzodiazepine5,6 and N-methyl-d-aspartate receptors,7 there have been no reports that suggest the involvement of these receptors in the antinociceptive effects of N2O.
Although it has been previously suggested that dopaminergic neurons in the central nervous system are affected by inhalation of N2O, the implication of this phenomenon in the pharmacological action of N2O has not been clarified. Dopaminergic neurons originating in the ventral tegmental area (VTA) and projecting to the nucleus accumbens (NAc) are considered to form not only a rewarding mechanism but also a part of the supraspinal pain-suppression system. Several studies have shown that the mesolimbic dopaminergic system plays important roles in the suppression of tonic pain or persistent pain and these studies have provided direct evidence that the NAc plays a major role in the pain-suppression system.8–14 Therefore, it might be possible that activation of the dopaminergic system by N2O is involved in its antinociceptive effect.
In this study, we hypothesized that the mesolimbic dopaminergic system is involved in the antinociceptive effect of N2O. To test this hypothesis, we examined N2O-induced activation of dopaminergic neurons in the rat VTA by the immunohistochemical method. We further tested the effect of a dopamine receptor antagonist infused into the rat NAc on the behavioral responses and c-Fos expression in the spinal cord induced by formalin injection into the paw.
METHODS
Animals
Adult male Fischer rats weighing between 250 and 300 g were used throughout the study (Shimizu Laboratory CO Ltd., Kyoto, Japan). Rats were provided with access to food and water ad libitum and were kept in a 12-h light and dark cycle (lights on at 06:00 h and off at 18:00 h). All experiments were performed between 12:00 and 18:00. The study protocol was approved by the Animal Research Committee, Graduate School of Medicine, Kyoto University.
Immunohistochemical Examination of the VTA
Immunohistochemical examination of the VTA was performed in rats exposed to N2 (75%)–O2 (25%) or N2O (75%)–O2 (25%). Each group included six animals.
Gas Exposure
Gas exposure was performed in clear plastic chambers (20 x 17 x 16 cm). A mixture of N2–O2 or N2O–O2 was delivered continuously at 6 L/min into the chamber. Concentrations of N2O, O2, and CO2 in the chamber were measured continuously by infrared gas spectrometry (Capnomac Ultima, Datex-Ohmeda, Helsinki, Finland). After gas exposure for 90 min, under deep anesthesia with pentobarbital (100 mg/kg, i.p.), the rats were perfused with 0.1 M phosphate-buffered saline (PBS), followed by 4% paraformaldehyde in 0.1 M PBS (4°C). The brains were excised and stored in 4% paraformaldehyde overnight, and then in 30% sucrose in 0.1 M PBS for at least 48 h at 4°C. The brains were frozen, and transverse 30-µm sections were cut at –15°C and collected in 0.1 M PBS as floating sections.
Double Staining for c-Fos and Tyrosine Hydroxylase
Brain sections were first incubated for 1 h at room temperature in blocking solution consisting of 0.3% Triton X with 2% skim milk in 0.1 M PBS. Samples were then incubated with primary antibodies, sheep anti-tyrosine hydroxylase (TH) (1:100, Chemicon Inc., AB1542, Temecula, CA), and rabbit anti-c-Fos (1:100, Cat.no.sc-52, Santa Cruz Biotechnology, CA), in 2% skim milk in 0.1 M PBS overnight at 4°C. After three washes in 0.1 M PBS, samples were incubated for 2 h at room temperature with the secondary antibodies, fluorescein isothiocyanate-labeled anti-rabbit immunoglobulin (1:200, Jackson Immunoresearch Laboratories Inc., West Grove, PA) and Cy3-labeled anti-sheep immunoglobulin (1:200, Jackson Immunoresearch Laboratories Inc.). Three well-preserved, undamaged sections were randomly selected from each animal and examined under a fluorescent microscope with a DP70 digital CCD camera (Olympus Optics, CO, Tokyo, Japan). c-Fos-positive neurons and the TH-positive neurons were identified by green staining of the nucleus and red staining of the cytoplasm, respectively. For each animal, the number of cells expressing TH or expressing both c-Fos and TH was counted in three sections and averaged. The investigator was blinded to the origin of the sections. Colocalization of c-Fos and TH was confirmed using a Fluoview FV300 laser confocal microscope (Olympus Optics, CO).
Effects of Microinjection of Raclopride into the NAc on the Antinociceptive Effect of N2O
Four groups of rats were examined: saline–N2–O2 group, raclopride–N2–O2 group, saline–N2O–O2 group, and raclopride–N2O–O2 group. Each group included six animals.
Microinjection of Raclopride into the NAc
Seven to 10 days after arrival, rats were anesthetized with injection of sodium pentobarbital (50 mg/kg i.p.). For the NAc injections, 11-mm-long, 22-gauge guide cannulae (Plastics One, Roanoke, VA) were stereotactically implanted bilaterally 1.0 mm above the NAc shell, anteroposterior: +1.6, mediolateral: +1.0, dorsoventral: –6.5 mm, according to the atlas by Paxinos and Watson.15 After surgery, 28-gauge double-dummy cannulae were inserted into the guide cannulae. A recovery period of 5–7 days was allowed before testing.
Rats were placed in a clear plastic chamber (as described earlier) during the testing. After removal of the double-dummy cannulae, 28-gauge double-internal injection cannulae extending 1.0 mm beyond the tip of the double-guide cannulae were inserted. The injection cannulae were connected via a polyethylene tube to 1-mL Hamilton syringes. Raclopride or saline was infused bilaterally into the NAc shell region of unrestrained rats in a volume of 1.0 µL/side over 60 s using the microsyringe pump (ESP-32, Eicom Corp., Kyoto, Japan). Raclopride (Sigma Aldrich, Japan) was dissolved in saline and infused at doses of 50 µg/side. The doses used in these experiments were based on pilot experiments in which the nociceptive behavior of rats was observed (data not shown) according to a previous study.10
Immediately after drug administration, rats were exposed to N2 (75%)–O2 (25%) or N2O (75%)–O2 (25%) for 30 min. After gas exposure, 50 µL of 5% formalin was injected subcutaneously into the plantar surface of their right hindpaws with a 27-gauge needle. After experiments, the bilateral sites of cannulation in the brain were confirmed by histological examination.
Behavioral Study
The testing was performed in clear chambers, which were placed on a clear acrylic plastic board. A digital video camera (DCR-HC90, Sony, Japan) was positioned approximately 55 cm beneath the chambers to record animal behavior. Behavior was monitored continuously for 60 min from the moment when the rats were injected with formalin. The intensity of pain was rated according to four behavioral categories, using a scale of 0 to 3: a score of 0—-no pain, if the rat walked or sat normally with weight placed equally on both hindpaws; 1—-favoring, if the rat favored the injected paw (e.g., if it limped); 2—-lifting, if the rat held the injected paw off the floor; and 3—-licking, if the rat chewed or licked the injected paw. The investigator was blinded to the pretreatment. The nociceptive score was calculated as follows, based on a previous report.16
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T1, T2, and T3 are the duration(s) of nociceptive behavior of scores 1, 2, and 3, respectively, during each 5-min period.
Immunohistochemical Detection of c-Fos
Ninety minutes after formalin injection, rats were injected with sodium pentobarbital (100 mg/kg, i.p.) and perfused as described earlier. The spinal cord was excised and stored as described earlier. A portion of the spinal cord at the lumbar enlargement (approximately 5 mm in length) was frozen, and transverse 30-µm sections were cut at –15°C and collected in 0.1 M PBS as floating sections.
Spinal cord sections were incubated for 1 h at room temperature in blocking solution as described earlier, incubated overnight at 4°C with rabbit anti-c-Fos antibody (1:10,000, Cat. no. sc-52, Santa Cruz Biotechnology, CA) in blocking solution, rinsed with 0.1 M PBS, incubated for 1 h at room temperature with biotinylated goat anti-rabbit immunoglobulin (1:200, Vector Laboratories, Burlingame, CA), rinsed with 0.1 M PBS and, finally, incubated for 1 h at room temperature in avidin–biotin–peroxidase complex (Vector Laboratories) in 0.1 M PBS. Visualization of the immunohistochemical reaction was achieved by incubation in DAB solution containing nickel-ammonium sulfate to which hydrogen peroxidase was added (DAB kit, Vector Laboratories). Three randomly selected well-preserved sections from each animal were examined using a bright-field microscope (Olympus Model BX60, Olympus Optics) and, according to the method by Presley et al.,17 the number of c-Fos-positive cells was counted for each lamina in the right side of the spinal cord; laminae I–II (superficial area), laminae III–IV (nucleus proprius area), laminae V–VI (neck area), and laminae VII–X (ventral area). The effect of formalin injection was examined on five different sections taken from each rat, and the mean number of c-Fos-positive cells per section was calculated. The number of c-Fos-positive cells was expressed as the mean ± sd for each group. The investigator was blinded to the treatment group.
Statistical Analysis
Statistical analyses and calculation of the area under the curve were performed with Prism 3 software (GraphPad Software, Inc., San Diego, CA). Results from double staining for c-Fos and TH were analyzed using unpaired two-tailed t-test. Differences in the area under the nociceptive score curves and differences in numbers of c-Fos-positive cells in each lamina in the spinal cord among groups were analyzed by one-way analysis of variance, and Bonferroni correction was used as a post hoc test. P values <0.05 were considered to be statistically significant.
RESULTS
Induction of c-Fos Expression by N2O in TH-Positive Cells
The number of TH-positive cells in the bilateral VTA was 117 ± 5 (mean ± sd, n = 6) in the control rats exposed to N2–O2. In the rats exposed to N2O–O2, the number of TH-positive cells was 110% ± 2% of the control rats and was not statistically different. The double staining experiment demonstrated that c-Fos staining was colocalized with the TH staining. Confocal microscopic examination of double staining for c-Fos and TH (Fig. 1A) showed that c-Fos was expressed in TH-positive cells. In the rats exposed to N2–O2, 10.1% ± 2.3% (n = 6) of the TH-positive cells in the bilateral VTA expressed c-Fos, and the ratio increased significantly by inhalation of N2O–O2 to 23.6% ± 3.5% (n = 6) (Fig. 1B). These results suggest that N2O induces activation of the dopaminergic neurons in the VTA.
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Effect of Microinjection of Raclopride into the NAc on Nociceptive Behavior
Figure 2A shows the time-dependent changes in the nociceptive scores after formalin injection into the paw. Before formalin injection, the rats that inhaled N2–O2 were awake and active, whereas those that inhaled N2O–O2 were excited for the first 5–10 min of exposure, followed by a relatively calm state. No difference in behavior was observed between the saline-injected and the raclopride-injected rats. Behaviors induced by formalin injection in rodents can be divided into two phases that reflect different physiological processes.18,19 The first and second phases were defined as the first 5 min and 30–50 min after formalin injection, respectively. Also in this study, biphasic time-dependent changes in behaviors were observed after formalin injection. In the first phase, the nociceptive score was lower in rats that inhaled N2O–O2 compared with those that inhaled N2–O2. Fifteen minutes after formalin injection, none of the animals exhibited any nociceptive behavior. Thereafter, the rats that inhaled N2–O2 exhibited high-intensity nociceptive behavior, which persisted during the entire second phase. Analysis of the areas under the curve for each group is shown in Figure 2B. There was a statistically significant difference in area under the curve among the four groups. Comparison between the saline–N2–O2 group and the saline–N2O–O2 group demonstrated that N2O almost completely suppressed the nociceptive behavior induced by formalin injection. The raclopride–N2O–O2 group exhibited a 51.6% reduction in nociceptive scores compared with the saline–N2–O2 group, and the reduction rate was significantly smaller when compared with the reduction rate between the saline–N2–O2 group and the saline–N2O–O2 group (93.7%). In the absence of N2O, raclopride did not significantly affect the nociceptive score.
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Formalin injection into the paw induced c-Fos expression in the laminae I–II and V–VI in the dorsal horn of the spinal cord. The number of c-Fos-positive cells in each of the laminae was compared among the four study groups. (Fig. 3) The number of c-Fos-positive cells in laminae I–II and V–VI in the saline–N2O–O2 group was significantly smaller than that in the saline–N2–O2 group. In the raclopride–N2O–O2 group, c-Fos expression was significantly higher compared with the saline–N2O–O2 group. In the absence of N2O, raclopride did not significantly affect the number of c-Fos-positive cells in each lamina.
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DISCUSSION
In this study, we demonstrated that exposure to N2O increased expression of c-Fos in the TH-positive cells in the VTA, indicating that N2O activates dopaminergic neurons. We further showed that the injection of raclopride into the NAc shell region partially blocked the antinociceptive effect of N2O, suggesting the importance of dopamine receptors in the antinociceptive effect of N2O.
It has previously been suggested that dopaminergic neurons in the central nervous system are affected by inhalation of N2O. Neurochemical studies have demonstrated that N2O induced significant increases of dopamine metabolites in the brain20,21 and changes in dopamine concentration or turnover in the brain.22 Furthermore, Sakamoto et al. showed in a microdialysis study that N2O, but not xenon, increased the dopamine levels in the NAc.23 However, it has not been directly demonstrated whether N2O activates dopaminergic neurons. Our findings that N2O increased the expression of c-Fos in the TH-positive cells in the VTA are consistent with previous reports, and to our knowledge, the present study is the first to demonstrate that dopaminergic neurons were activated by N2O.
To examine the involvement of mesolimbic dopaminergic neurons in the antinociceptive effect of N2O, we tested the effect of injection of raclopride, a dopamine D2-like receptor antagonist, into the NAc shell region. Our results showed that N2O suppressed the formalin-induced nociceptive behavior, which is consistent with previous reports,16,24 and raclopride partially suppressed the antinociceptive effect of N2O; raclopride alone did not significantly affect the nociceptive behaviors. The effect of raclopride on the antinociceptive effect of N2O was apparently different between the two phases of behaviors induced by formalin injection. One possibility is that the dose of raclopride was not high enough to block the antinociceptive effect of N2O in the first phase of the formalin test. Alternatively, it might be possible that the involvement of dopaminergic neurons in the antinociceptive effect of N2O is different between the two phases of the formalin-induced behaviors, in which different physiological mechanisms are involved.
We also performed an immunohistochemical study to assess the effect of N2O on the nociceptive processes. Exposure to N2O suppressed the increase in expression of c-Fos caused by formalin injection into the paw in laminae I–II and V–VI of the spinal cord, in which most of the primary afferent neurons terminate.25 This result is consistent with a previous report.16 In contrast, there have been reports that N2O does not significantly affect c-Fos expression in the spinal cord induced by noxious stimuli. The difference in the effect of N2O might be due to differences in the protocol and rat strain. In the study by Sun et al., N2O was administered to Sprague-Dawley rats 20 min before formalin injection, but was discontinued 5 min after formalin injection, although the noxious stimuli by formalin lasted at least 1 h.26 On the other hand, we used Fischer rats that exhibit the greatest antinociceptive effect by N2O,27 and administered N2O for 90 min until immediately before killing. Raclopride injected into the NAc shell region blocked the effect of N2O on the c-Fos expression, whereas injection of raclopride into the NAc shell region in the absence of N2O did not significantly affect the formalin-induced increase in c-Fos expression induced. Taken together, our findings in the behavior study and the immunohistochemical study provide plausible evidence suggesting that the antinociceptive effect of N2O is at least partially mediated by the dopamine D2-like receptors in the NAc.
The NAc, a rostral telencephalic gray mass, is thought to contribute to reward and addiction. It has a heterogeneous structure and is divided anatomically into two regions, the shell and the core. The NAc shell region is innervated by dopaminergic neurons and is closely related to the mesolimbic system.28 This region is considered to be the primary site of the reinforcing actions of psychostimulants, and to enhance the motivational effects of reinforcement. In contrast, the NAc core is related to the caudate-putamen and striatum and is considered to be involved in the control of goal-directed behavior.29 Dopamine D1 and D2 receptors are present in the NAc,30–32 and D3 receptors are found particularly in the NAc shell region.33
Dopamine receptor subtypes differ with respect to their structure and distribution in rat brain,34 and are classified into two main groups: the D1-like and the D2-like receptors, which include D1 and D5, and D2, D3, and D4, respectively. The D1 receptor is the most widespread-distributed dopamine receptor, whereas the D2 receptor is found mainly in the striatum, olfactory tubercle, and NAc.30,35,36 In contrast to the D1 and D2 receptors that are highly expressed in almost all dopaminergic projection fields, the expression of the D3, D4, and D5 receptors is low, except in some limbic regions.37 In fact, the D3 receptor is selectively expressed in limbic areas including the NAc shell region.33,38 Because we tested only one dopamine receptor antagonist at a single dose in the present study, we cannot certainly conclude which of the dopamine receptor subtypes is mainly involved in the antinociceptive effect of N2O. To understand the roles of each of the dopamine receptor subtypes, effects of the other subtype-selective antagonists at various doses should be tested in future.
Although many studies have suggested that mesolimbic dopaminergic neurons are involved in the pain-suppression system,8–14 the mechanism by which dopamine release in the NAc modulates nociceptive information transmitted from the periphery remains to be elucidated, and the reciprocal relationship between the mesolimbic dopaminergic pain-suppression system and the descending inhibitory system is not known. Thus, further investigation is needed to understand the mechanism of N2O action via the mesolimbic dopaminergic system and the descending inhibitory system.
In conclusion, the present study demonstrates that mesolimbic dopaminergic neurons are activated by N2O. Furthermore, it is suggested that the anitinociceptive effect of N2O is at least partially mediated by dopamine D2-like receptors in the NAc shell region.
Footnotes
Accepted for publication February 4, 2008.
Supported by the Grant-In-Aid for Scientific Research from the Japan Society for the Promotion of Science.
REFERENCES
2 adrenergic receptors in the rat. Anesthesiology 1996;85:846–52[Web of Science][Medline]
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