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NEUROPHARMACOLOGY
Departments of Emergency Medicine (D.E.R., M.V.Z.) and Pharmacology and Toxicology (D.E.R., D.V.Z., M.V.Z., J.A.D.), Indiana University School of Medicine, Indianapolis, Indiana
Received May 24, 2007; accepted August 15, 2007.
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Abstract |
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). Although hyperthermia remains the best studied physiologic effect related to MDMA usage, hypothermia can also occur, a phenomenon that is often ignored or, when acknowledged, is attributed to low ambient temperatures (Malberg and Seiden, 1998). MDMA, however, can evoke decreases in body temperature at normal or even elevated ambient temperatures (Malberg et al., 1996; Bexis and Docherty, 2006). Even in studies in which MDMA causes hyperthermia, a transient initial period of hypothermia or a delay in the development of hyperthermia is often seen (Gordon et al., 1991; Malberg et al., 1996). To date few studies have addressed the mechanisms responsible for MDMA-induced hypothermia.
The principal pharmacologic action of MDMA is facilitation of transmission at monoaminergic nerve terminals, causing the release of monoamines in the central nervous system and more specifically elevating extracellular levels of serotonin, dopamine, and norepinephrine (Rothman et al., 2001; Gough et al., 2002; Sprague et al., 2007), all of which have been implicated in hyperthermia induced by MDMA. The mechanisms behind hypothermia induced by MDMA are, however, largely unknown. Serotonin can elicit opposite effects on body temperature, depending on whether it binds to serotonergic 1A (5-HT1A) or 2A (5-HT2A) receptors. The divergent effects of serotonin on body temperature present an attractive hypothesis for changes in temperature induced by MDMA. To date, the role of serotonin in body temperature changes evoked by MDMA has focused on 5-HT2A receptors with 5-HT2A antagonists reducing hyperthermia and neurotoxicity elicited by MDMA (Schmidt et al., 1990). 5-HT2A receptors are involved in hyperthermia evoked by MDMA by impairing heat dissipation through the constriction of cutaneous vascular beds (Blessing et al., 2003; Ootsuka et al., 2004) and by increasing heat generation through interscapular brown adipose tissue (IBAT) (Blessing et al., 2006). Whereas 5-HT2A receptors contribute to MDMA-mediated hyperthermia, the role of 5-HT1A receptors in temperature effects elicited by MDMA is largely unknown. As serotonin has a >1000-fold affinity for 5-HT1A receptors over that of 5-HT2A receptors (Peroutka et al., 1981), its release by MDMA probably activates 5-HT1A receptors. Activation of 5-HT1A receptors by agonists such as 8-hydroxy-2-di-n-propylamino-tetralin (DPAT) causes, similar to MDMA, the serotonin behavioral syndrome (Goodwin et al., 1987) and in a manner opposite to 5-HT2A receptor activation, hypothermia (Gudelsky et al., 1986; Ootsuka and Blessing, 2003). Along with their individual contributions, 5-HT1A and 5-HT2A receptors have complex interactions affecting body temperature. Hyperthermia elicited by stimulating 5-HT2A receptors with (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride is potentiated by the 5-HT1A antagonist WAY 100635 and prevented by the 5-HT1A agonist DPAT (Salmi and Ahlenius, 1998). Similarly, WAY 100635 augments the hyperthermia induced by MDMA in a warm environment (Saadat et al., 2005). Together these data suggest that the overall effect of MDMA on body temperature may reflect the net sum of the combined activities of serotonin on 5-HT1A and 5-HT2A receptors.
Muscle activity represents a key component of thermoregulation in the rat in the form of shivering and work-related motor activity. A direct relationship exists between work intensity and core body temperature (Gordon, 1990). MDMA increases locomotion in rats, a factor that probably contributes to the development of hyperthermia (Spanos and Yamamoto, 1989). Locomotion by itself however, is insufficient to explain the effects of MDMA on body temperature as the degree of locomotion induced by a dose of MDMA is similar regardless of whether the animal develops hypo- or hyperthermia, depending on the ambient temperature in which it is given (Dafters, 1994). The role 5-HT1A receptors play in the locomotor effects induced by MDMA is unclear as 5-HT1A antagonists have been reported to both cause reductions and have no effect on MDMA-evoked locomotion (Callaway et al., 1992; McCreary et al., 1999).
The cardiovascular system plays a critical role in normal thermoregulation. Activation of the sympathetic nervous system in response to a cold environment results in the constriction of cutaneous vessels, along with an increase in heart rate and cardiac output (Blessing, 2004; Deussen, 2007). The net result of these effects is to conserve heat by shunting blood away from a cool skin surface while simultaneously supporting heat generation by diverting blood to heat-producing central organs and brown adipose tissue. The reverse of these effects occurs in a warm environment, facilitating heat release (Ootsuka and Blessing, 2006b). Whereas the effects of MDMA on cutaneous blood flow have been well studied (Pedersen and Blessing, 2001), its effects on other components of the cardiovascular system are less well known. Although it causes tachycardia in humans (Lester et al., 2000), the cardiovascular effects of MDMA in rats have not been clearly delineated, as both an increase (Gordon et al., 1991) and no effect on heart rate have been reported (Irvine et al., 2001; Bexis and Docherty, 2006).
The rostral raphe pallidus (rRP) is a serotonin-rich region of the medulla containing sympathetic thermoregulatory premotor neurons that control the sympathetic innervation of cutaneous blood vessels and IBAT in the rat (DiMicco et al., 2006). Neurons in the rRP are known to express 5-HT1A receptors (Helke et al., 1997), and studies to date have demonstrated that microinjections of DPAT into the rRP block thermogenesis induced by leptin and cutaneous vasoconstriction induced by cold (Morrison, 2004; Ootsuka and Blessing, 2006a). Likewise microinjections of DPAT into various regions of the brainstem and rRP inhibit the cardioexcitatory responses to leptin and to hypothalamic stimulation (Morrison, 2004; Horiuchi et al., 2005).
The cardiovascular and thermogenic responses to various stressors share similar hypothalamic and brainstem mechanisms (DiMicco et al., 2006). Thus, we sought to investigate the role of 5-HT1A receptors in the region of the rRP of conscious, freely moving rats in 1) the thermoregulatory, cardiovascular, and locomotor effects induced by MDMA and 2) the hypothermia induced by systemic administration of DPAT.
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Materials and Methods |
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Drugs
For systemic administration, WAY 100635 (Sigma-Aldrich, St. Louis, MO) was dissolved to concentration with normal saline and used on the day of the experiment. For microinjections, WAY 100635 was dissolved in 0.1 M phosphate-buffered saline (PBS) and used on the same day or frozen at–80°C and used within 2 weeks. DPAT (Sigma-Aldrich) was dissolved in normal saline and used on the day of the experiment. MDMA was generously provided by the National Institutes of Health and prepared at the time of the experiments in normal saline.
Animals
Male Sprague-Dawley rats (325 ± 25 g; Harlan, Indianapolis, IN) were maintained in a 12-h light/dark cycle beginning at 7 A.M. and fed ad libitum. Animals were housed singly to avoid interference from competing radiotelemetric signals and to avoid potential chewing or damage to central nervous system guide cannulas from other animals.
Surgical Preparation
For studies using MDMA and WAY 100635, rats were anesthetized (80 mg/kg ketamine and 11.5 mg/kg xylazine i.p., supplemented as required) and a telemetric transmitter (model C50-PXT; Data Sciences, St. Paul, MN) was implanted as described previously for the measurement of mean arterial pressure, heart rate, locomotion, and core body temperature (Zaretsky et al., 2003). For studies with DPAT and those to determine the effects of different doses of MDMA on body temperature, a telemetric transmitter measuring only core temperature and locomotion (model TA-F40; Data Sciences) was implanted into the abdominal cavity.
For experiments involving microinjections into the rRP, rats were anesthetized 5 days after transmitter implantation and placed in a stereotaxic apparatus with the incisor bar set 3.3 mm below the interaural line for placement of a guide cannula as described previously (Zaretsky et al., 2003). The target coordinates were anterior-posterior–2.8 mm, left-right 0.0 mm, and height-depth–1.1 mm with the interaural line as a reference point. Dummy wire cannulas were inserted in the guides, and rats were returned to their cages and left undisturbed until they regained their approximate presurgery weights (approximately 5–7 days).
Experiments
Experiments were conducted in the animal's home cage in quiet and isolated rooms between 10:00 A.M. and 2:00 P.M. Animals acclimated to their new environment for a minimum of 2 h before experiments were initiated. Throughout the experiment ambient temperature was maintained at 24 to 25°C, a temperature typically associated with MDMA-induced hyperthermia (Malberg and Seiden, 1998). In all the experiments, animals were randomized to study groups in blocks of eight using a random number generator.
Dose Response. To determine the dose causing hypothermia and the time of maximal effect, rats received an i.p. dose of either saline or one of three doses of MDMA (1.5, 7.5, or 15 mg/kg). We recorded temperature in degrees Celsius and locomotion in units of activity every minute for 120 min after vehicle or MDMA.
Effect of Systemic Treatment with WAY 100635 on MDMA-Induced Hypothermia. Animals were assigned to one of four groups (saline/saline, WAY/saline, saline/MDMA, or WAY/MDMA; n = 8 rats/group). After an acclimation period, each animal received a 0.3-ml i.p. injection of either saline vehicle (0.9%) or WAY 100635 (0.5 mg/kg) followed 5 min later by an equal volume of either saline or MDMA (7.5 mg/kg). Temperature (degrees Celsius), locomotion (units of activity), heart rate in beats per minute, and mean arterial pressure in millimeters of mercury were recorded every minute for 120 min after MDMA.
Effect of Microinjection of WAY 100635 into the rRP on DPAT-Induced Hypothermia. To confirm the dosing of WAY 100635 for microinjection experiments, we investigated the role of 5-HT1A receptors in the region of the rRP in body temperature decreases elicited by systemic administration of the 5-HT1A agonist DPAT. In these experiments, telemetric probes measured only temperature and locomotion. Animals were assigned to one of four groups (PBS/saline, WAY/saline, PBS/DPAT, or WAY/DPAT). The dummy cannula was removed, and a microinjector (33-gauge; Plastics One, Roanoke, VA) connected to a 10-µl Hamilton syringe with Teflon tubing (i.d. 0.12 mm; o.d. 0.65 mm; BAS Bioanalytic Systems, West Lafayette, IN) was placed through the guide cannula and positioned above the region of the rRP. The syringe was mounted in an infusion pump (KD Scientific, Holliston, MA) that was used to deliver 100 nl of a solution of either PBS (0.1 M) vehicle or WAY 100635 (5 mM solution; 0.5 nmol) over 30 s. Five minutes after microinjection, animals received an i.p. injection of a solution (0.3 ml) of either 0.9% saline vehicle or DPAT (0.2 mg/kg). Preliminary studies demonstrated that 0.2 mg/kg DPAT evoked a decrease in body temperature similar in magnitude to that produced by 7.5 mg/kg MDMA (data not shown). We recorded temperature (degrees Celsius) and locomotion (units of activity) every minute for 120 min after DPAT injection. Two days later, animals receiving PBS/saline were crossed over to receive WAY/saline, and those animals originally receiving WAY/saline were crossed over to PBS/saline. In a similar fashion, animals originally receiving PBS/DPAT were crossed over to receive WAY/DPAT and vice versa. To assess the potential impact of damage caused by the placement of the guide cannula and microinjector into the region of the rRP on the response to DPAT, animals completing the above protocols, received an i.p. injection of DPAT (0.2 mg/kg). We defined a positive response to DPAT as a decrease of greater than 0.5°C within 30 min of injection. Data from four rats that failed to respond to DPAT were excluded from the final analysis. Fluorescent microspheres, 0.04 µm in diameter (Invitrogen, Carlsbad, CA), were included in the microinjection solutions at a concentration of 0.25% to mark the exact site of injection. After the last session, rats were injected with pentobarbital (100 mg/kg), a 14-gauge feeding tube was placed in the left ventricle, and the brains were perfused in situ with 30 ml of cold saline (4°C) followed by 30 ml of 4% paraformaldehyde. The brains were then removed and post-fixed in 4% paraformaldehyde overnight or longer, followed by placement in a solution saturated with 30% sucrose. Brains were cut into 40-µm coronal sections on a freezing microtome and injection sites were determined using a fluorescent microscope according to the atlas of Paxinos and Watson (1998) by an observer blinded to group allocation.
Effect of Microinjection of WAY 100635 into the rRP on MDMA-Induced Hypothermia. After determining microinjection of 0.5 nmol of WAY 100635 in the region of the rRP attenuated hypothermia elicited by systemic administration of DPAT, we sought to determine whether microinjections of WAY 100635 could similarly prevent hypothermia evoked by MDMA. Animals were assigned to one of four groups (PBS/saline, WAY/saline, PBS/MDMA, or WAY/MDMA). As in the DPAT experiment described above, animals receiving i.p. injections of saline received microinjections of either PBS or WAY 100635 in a crossover fashion. To avoid the possibility of MDMA-induced serotonin neurotoxicity confounding the effect on body temperature, animals receiving MDMA were not crossed over. Animals were microinjected into the region of the rRP with 100 nl of PBS (0.1 M) or WAY 100635 (5 mM solution; 0.5 nmol) over 30 s depending on group assignment. Five minutes later animals received an i.p. injection of 0.3 ml of either saline vehicle or MDMA (7.5 mg/kg). We recorded temperature (degrees Celsius), locomotion (units of activity), heart rate (beats per minute), and mean arterial pressure (millimeters of mercury) every minute for 120 min after MDMA injection. Injection sites were verified using fluorescent microspheres as described above.
Data Analysis. Data were captured using a receiver placed under the rat's cage and transmitted to an IBM-compatible computer using the Dataquest A.R.T data acquisition system (Data Sciences). Statistics were run using SPSS 14.0 for Windows (SPSS, Chicago, IL). Graphs were made using Prism 4.0 (GraphPad Software, San Diego, CA) software. Grouped data are reported as 5-min means ± 1 S.E. measurement for the time points shown. To compare changes in temperature and activity from baseline in the dose-response experiment we performed a one-way repeated measures ANOVA with pairwise comparisons done using the LSD procedure. For the remainder of the studies we performed a two-way full factorial repeated measures analysis of variance. We included in the model main effects for study group, time, and their interaction. We performed post hoc comparisons among time points within a study group and between study groups within a time point. Pairwise comparisons were adjusted using a LSD procedure.
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RESULTS |
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Effect of Systemic Treatment with WAY 100635 on MDMA-Induced Changes
There were no differences in baseline values of any measured parameter among any of the treatment groups (Fig. 2).
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Locomotion. Outside of transient effects, secondary to the stress of the i.p. injections, neither the injection of saline/saline nor that of WAY/saline resulted in significant increases in locomotion (Fig. 2B). Regardless of pretreatment, the injection of MDMA resulted in significant increases in locomotor activity at every time point after its injection. Pretreatment with WAY 100635 attenuated the increases in locomotion induced by MDMA at 30 min (9.8 ± 3 versus 21.2 ± 3 au, p = 0.019), but by 60 min these effects were no longer evident (p = 0.2).
Heart Rate. In all the groups, regardless of the pretreatment, the first injection (represented by 0 min) resulted in an increase in heart rate (Fig. 2C). In the saline/saline group no further increases were seen. In those animals injected with WAY/saline, however, a significant increase in heart rate compared with baseline was still evident at 30 min (+51 ± 18 beats/min, p = 0.009). The injection of saline followed by MDMA caused a steady increase in heart rate beginning at 30 min and becoming significantly increased compared with baseline by 90 min (+79 ± 18 beats/min, p < 0.001). In those rats pretreated with WAY 100635, increases in heart rate after the injection of MDMA were immediate and sustained throughout the experiment. At both 30 and 60 min, values for heart rate in the WAY/MDMA group were greater then those of any of the other groups.
Mean Arterial Pressure. Similar to heart rate, mean arterial pressure transiently increased after pretreatment injections (represented by 0 min) in each group (Fig. 2D). In only those animals injected with WAY 100635 followed by MDMA, however, were increases in MAP significant compared with baseline at subsequent time points.
Identification of Microinjection Sites
Microinjection sites (Fig. 3) were determined using different colored fluorescent microspheres with the corresponding centers of the visualized microspheres plotted on representative drawings adapted from Paxinos and Watson (1998) and shown in Fig. 3D. Shown in Figs. 3, A to C are examples of fluorescent micrographs with the matching counterstained section. We considered positive injections as those in which the center of the fluorescence was contiguous with or within the area of the raphe pallidus.
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DPAT: Locomotion. As the locomotor effects elicited by DPAT are short-lived, we added the 15-min time point to the analysis of motor activity. Microinjections of either PBS or 0.5 nmol of WAY 100635 followed by the systemic injection of saline (PBS/DPAT and WAY/DPAT) had no effect on locomotor activity (Fig. 4B). All of the groups given i.p. injections of DPAT (PBS/DPAT and WAY/DPAT) demonstrated significant and similar increases in locomotion 15 min postinjection.
Microinjection of WAY 100635: MDMA
There were no differences in baseline values of any measured parameter among any of the treatment groups (Fig. 5).
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MDMA: Locomotion. Systemic injections of MDMA significantly increased locomotion at all time points after its injection (Fig. 5B). Unlike the systemic administration of WAY 100635 (Fig. 2B), which attenuated MDMA-mediated locomotion, microinjections of WAY 100635 had no affect on locomotion induced by MDMA.
MDMA: Heart Rate. As seen with temperature (Fig. 5A), the microinjection procedure caused a stress response in the animals manifested as transient increases in heart rate in both the PBS/saline and WAY/saline groups (Fig. 5C). Microinjections of PBS (100 nl) followed by systemic administration of MDMA (PBS/MDMA) caused a gradual increase in heart rate that was significantly different from baseline by 60 min (+42 ± 17 beats/min, p = 0.02) and from then on. As with the systemic administration of WAY 100635 followed by MDMA (Fig. 2B), microinjections of WAY 100635 (0.5 nmol) into the rRP resulted in significant increases in MDMA-mediated tachycardia at 30 min and from that point on compared with all other groups, except the WAY/MDMA (OR) group in which numbers of animals were too small (n = 4) for comparison. In animals with microinjection cannulae misplaced outside the rRP (OR), microinjections of WAY 100635 failed to alter significantly the effect of systemically administered MDMA on heart rate compared with the PBS/MDMA group.
MDMA: Mean Arterial Pressure. Except for the transient increases in mean arterial pressure seen after microinjections, consistent and sustained elevations in mean arterial pressure did not occur in any of the groups (Fig. 5D). This result is different from that seen after the systemic administration of WAY 100635 followed by MDMA (Fig. 2) for which mean arterial pressure increased in a manner similar to heart rate.
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Discussion |
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A potential confounder in our results was that when administered systemically, WAY 100635 alone increased temperature and heart rate (Fig. 2, A and C). That WAY 100635 alone can increase temperature has been reported previously (Saadat et al., 2005). We do not, however, believe that the differences seen between animals treated with WAY/MDMA and saline/MDMA occurs simply via a competitive physiologic mechanism. First, the degree to which WAY 100635 alone increases temperature and heart rate cannot by itself account for the changes seen between WAY/MDMA and saline/MDMA animals. Second, the effects WAY 100635 alone had on temperature and heart rate were transient, whereas its effects on changes in temperature and heart rate induced by MDMA were more sustained.
Serotonin has long been implicated in the regulation of body temperature with initial studies showing that its microinjection into the anterior hypothalamus caused hypothermia (Cox et al., 1980). Contrary to this fact, however, the systemic injection of drugs such as MDMA that increase the central release of serotonin typically causes hyperthermia (Gordon et al., 1991). These seemingly conflicting findings may be due to the opposing effects of serotonin on 5-HT1A and 5-HT2A receptors. Agonists of 5-HT1A receptors, such as DPAT, cause dose-dependent hypothermia (Gudelsky et al., 1986) by increasing heat loss through the dilation of cutaneous vessels and by decreasing heat generation through inhibition of IBAT (Ootsuka and Blessing, 2006a,b). Activation of 5-HT2A receptors on the other hand, causes hyperthermia through cutaneous vasoconstriction and by increasing heat production in brown adipose tissue (Ootsuka et al., 2004; Ootsuka and Blessing, 2006b). Therefore, drugs such as MDMA, which cause a large release of serotonin may affect both 5-HT1A and 5-HT2A receptors. Because serotonin has >1000 times the affinity for 5-HT1A receptors than for 5-HT2A receptors (Peroutka et al., 1981), a low dose of MDMA might affect 5-HT1A receptors to a greater extent, thereby causing hypothermia. Conversely, higher doses of MDMA could conceivably result in the activation of enough 5-HT2A or other nonserotonin receptors to cause hyperthermia. We found evidence supporting this hypothesis, as a 7.5 mg/kg dose of MDMA caused pure hypothermia and a 15 mg/kg dose of MDMA caused predominantly hyperthermia. On closer inspection of Fig. 1, the initial increase in temperature after 15 mg/kg MDMA is not evident until 30 min after injection, suggesting that 5-HT1A receptor activation may have delayed the development of hyperthermia. We do not think this delay is secondary to drug absorption as decreases in temperature after 7.5 mg/kg MDMA are evident within 15 min of injection. These results are similar to those of other studies in which doses of 40, 20, 15, and 10 mg/kg MDMA produced either an initial hypothermia followed by hyperthermia or a delay in the development of hyperthermia (Gordon et al., 1991; Malberg et al., 1996). As doses of MDMA increase, the decrease in the initial hypothermia was likewise less (Malberg and Seiden, 1998). Data reported by Abdel-Fattah et al. (1997) support the idea that lower concentrations of serotonin may preferentially activate 5-HT1A receptors, causing hypothermia. In their study they demonstrated that when rats were systemically administered the serotonin precursor tryptophan and the monoamine oxidase inhibitor pargyline developed biphasic temperature responses and that hypothalamic levels of 5HT were lower during the hypothermic phase and higher during the hyperthermic phase. Furthermore these authors showed that administering the 5-HT1A antagonist pindolol prevented the hypothermic phase caused by tryptophan and pargyline (Abdel-Fattah et al., 1995). In a similar fashion, the systemic administration of WAY 100635 prevented hypothermia evoked by the serotonin-releasing agent dexfenfluramine (Cryan et al., 2000). As hyperthermia correlates with neurotoxicity induced by MDMA, the finding in our study that 5-HT1A antagonists increase hyperthermia and the corollary that 5-HT1A agonists might prevent hyperthermia could lead to better selection of drugs to treat life-threatening hyperthermia. An example is that the 
-blocker propranolol has, on occasion, been suggested as a treatment for toxicity from MDMA or in cases of serotonin syndrome (Jones and Story, 2005). As propranolol is a 5-HT1A antagonist, its usage could actually result in worsening hyperthermia.
The fact that other authors have not consistently reported MDMA-induced hypothermia may be due in part to the dose of MDMA used and the method of temperature measurement. In most of the studies to date reporting MDMA-induced hypothermia at room temperature radiotelemetry was used to measure body temperature (Berkey et al., 1990; Bexis and Docherty, 2006). As handling and rectal temperature measurements significantly increase body temperature and serotonin release (Berkey et al., 1990; Adell et al., 1997), animals given MDMA in studies in which these methods of temperature measurement were used may have increased amounts of serotonin release over animals in which biotelemetry was used to measure temperature. As we have previously discussed, higher concentrations of serotonin may result in the activation of 5-HT2A or nonserotoninergic receptors and hence less hypothermia. Although we saw very reproducible reductions in body temperature after 7.5 mg/kg MDMA in both the dose-response experiment and the experiment in which WAY 100635 was systemically administered, the degree of hypothermia seen when microinjections preceded systemic MDMA administration (compare Figs. 1 and 2A with Fig. 5A) was less. Possible explanations for this result may be that microinjection of PBS into the rRP causes local stimulation of neurons resulting in elevations in temperature and heart rate (see Figs. 4 and 5) or the stress of the procedure itself. As the procedure requires the placement of a needle injector into a guide cannula followed several minutes later by its removal, it requires the investigator to be in proximity of the animal for a longer period of time than for a simple i.p. injection. Lending support to either of these ideas is the fact that control animals (PBS/saline in Fig. 5A) microinjected with PBS in the rRP followed by i.p. saline injection had larger increases from baseline in core temperatures at 30 min (+0.8 ± 0.2°C) compared with the control animals receiving two i.p. doses of saline (saline/saline in Fig. 2) and no microinjection (+0.2 ± 0.2°C). Another possible explanation for the reduced MDMA-mediated hypothermia when drugs were microinjected is that the placement of the microinjection cannula may have damaged neurons in the rRP, reducing the hypothermia induced by MDMA. We could account for this possibility in the experiments with DPAT by making sure that animals with microinjection cannula in the rRP were capable of developing hypothermia to DPAT and eliminating those that did not from the final analysis. We did not do a similar screening procedure with MDMA as we were concerned that the neurotoxicity of MDMA might potentially affect the responses to further doses of MDMA.
Similar to other researchers, we showed that blocking 5-HT1A receptors significantly reduced MDMA-mediated locomotion (Callaway et al., 1992). That decreases in MDMA-mediated locomotion in animals pretreated with WAY 100635 occurred at 30 but not at 60 min is consistent with findings reported by Kehne et al. (1996). In their study, they suggested that early (0–30 min) locomotion induced by MDMA may be dependent on activation of 5-HT1A and dopamine D2 receptors, whereas later increases (30–60 min) depend on activation of 5-HT2A receptors (Kehne et al., 1996). Our results conflict, however, with those of McCreary et al. (1999), who reported that WAY 100635 had no effect on MDMA (3 mg/kg s.c.)-mediated locomotion. Differences in our studies may be explained by the lower dose of MDMA used or the method of analysis; McCreary et al. (1999) analyzed summed data over a 90-min period.
Although administration of MDMA increases heart rate and systolic blood pressure in human volunteers (Lester et al., 2000), few researchers have studied the cardiovascular effects of MDMA in animals. After administering 20 mg/kg MDMA s.c. to five Long-Evan-rats, Gordon et al. (1991) reported a steady rise in the heart rate of three animals and dramatic increases in both heart rate and temperature in two rats that subsequently died. Irvine et al. (2001) gave MDMA in a range of doses from 5 to 20 mg/kg s.c. to female Sprague-Dawley rats at a temperature of 20°C and did not report increases in heart rate and blood pressure. At a temperature of 30°C, however, all doses of MDMA tested increased heart rate and blood pressure (Irvine et al., 2001). Bexis and Docherty (2006) recently reported that a dose of MDMA (20 mg/kg s.c.) that caused a purely hypothermic response did not significantly affect heart rate but did increase mean arterial pressure. In our study, i.p. injections of saline followed by MDMA (7.5 mg/kg) at an ambient temperature of 24 to 25°C increased heart rate but not mean arterial pressure. Differences between the strains of rats used in our study (Sprague-Dawley) and that of Bexis and Docherty (Wistar) may explain these contrary findings (Gaudreault et al., 2001; van den Buuse and Wegener, 2005). In our study when WAY 100635 was administered, a subsequent dose of MDMA evoked a greater initial increase in heart rate and mean arterial pressure than a dose of MDMA alone. This result suggests that activation of 5-HT1A receptors initially suppresses the tachycardia and hypertension mediated by MDMA.
The results of our study reveal for the first time that activation of 5-HT1A receptors in the region of the rRP mediates, at least in part, the hypothermic response from the systemic administration of the 5HT1A receptor agonist DPAT. DPAT lowers body temperature in rats (Gudelsky et al., 1986) by inhibiting IBAT thermogenesis and decreasing tail blood flow (Ootsuka and Blessing, 2006), effects that are blocked by pretreatment with WAY 100635 (Ootsuka and Blessing, 2006b). The rRP is the principal location of sympathetic premotor neurons regulating activity of tail and pinna cutaneous vasoconstrictor nerves in rats and rabbits, respectively, and sympathetic innervation for IBAT in rats (Morrison et al., 1999; Morrison, 2004). Considerable evidence suggests that glutamate is the major excitatory neurotransmitter released by terminals of these sympathetic premotor neurons in the rRP onto thermoregulatory sympathetic preganglionic neurons in the spinal cord (Nakamura et al., 2005; Stornetta et al., 2005). However, serotonergic neurons in the rRP also project to the same sympathetic regions of the spinal cord, and markers for both glutamate and serotonin are found in some regions (Helke et al., 1997). Likewise microinjections of serotonin potentiate increases in brown adipose sympathetic nerve activity elicited by subsequent spinal microinjection of N-methyl-D-aspartate (Madden and Morrison, 2006). Serotonergic neurons in the rRP are transsynaptically labeled after injection of pseudorabies virus into the rat tail (Tóth et al., 2006) and are responsible, at least in part, for cutaneous vasoconstrictor responses in the pinna of the rabbit that are evoked from the rRP (Ootsuka and Blessing, 2006). Spinally projecting serotonergic and nonserotonergic neurons in the rRP possess 5-HT1A receptors (Helke et al., 1997). Microinjection of DPAT into the region of the rRP blocks leptin-induced thermogenesis in rats (Morrison, 2004) and cold-induced cutaneous vasoconstriction in rabbits (Ootsuka and Blessing, 2006). The location of neurons containing 5-HT1A receptors involved in the cardiovascular responses to leptin and chemical disinhibition of the dorsomedial hypothalamus has likewise been reported to be in the region of the rRP and medullary brainstem (Morrison, 2004; Horiuchi et al., 2005).
The results of our study demonstrate for the first time that activation of 5-HT1A receptors in the region of the rRP after a systemic dose of MDMA suppresses tachycardia but not hyperthermia. Blessing and colleagues likewise reported that inhibition of the rRP with 1 nmol of the GABA agonist muscimol did not prevent subsequent increases in sympathetic nerve activity in ear pinna of rabbits given MDMA (6 mg/kg i.v.) (Ootsuka et al., 2004). The systemic administration of the 5-HT2A antagonist SR 46349B (0.1 mg/kg i.v.), however, completely reversed both the sympathetic nerve activity and vasoconstriction of the rabbit ear pinna mediated by MDMA (Ootsuka et al., 2004). These data suggest that vasoconstriction mediated by MDMA occurs downstream of the rRP, probably by neurons expressing 5-HT2A receptors in the spinal cord. Whether 5-HT1A receptors involved in MDMA-mediated hypothermia are likewise in the spinal cord is unknown. In their article, Madden and Morrison (2006) demonstrated that microinjection of 5HT into the intermediolateral column of the spinal cord, a region containing preganglionic sympathetic neurons, increased sympathetic nerve activity to IBAT after a delay of approximately 20 min. The authors presented several plausible explanations for this delay, but one area not explored was whether activation of 5-HT1A receptors may have initially suppressed sympathetic nerve activity to IBAT with later activation occurring after 5-HT1A receptor desensitization (Araneda and Andrade, 1991).
In conclusion, we demonstrate that MDMA can cause hypothermia as well as hyperthermia and that unlike DPAT, hypothermia mediated by MDMA does not involved 5-HT1A receptors in the rostral raphe pallidus. We also demonstrate that activation of 5-HT1A receptors in the rRP suppress tachycardia mediated by MDMA. These results suggest that the thermogenic and cardiovascular effects caused by MDMA involve activation of both 5-HT1A and 5-HT2A receptors with their interactions having potential consequences for the development of hyperthermia and toxicity in humans.
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Part of this work was presented in abstract form: Rusyniak DE, Zaretsky DV, Zaretskaia MV, and DiMicco JA (2005) WAY 100635 reverses the initial hypothermia from MDMA in rats, in Neuroscience 2005; 2005 Nov 12–16; Washington, DC. Society for Neuroscience, Washington DC.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: MDMA, 3,4-methylenedioxymethamphetamine; 5HT, 5-hydroxytryptamine, serotonin; IBAT, interscapular brown adipose tissue; DPAT, 8-hydroxy-2-di-n-propylamino-tetralin; WAY 100635, N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinylcyclohexanecarboxamide, WAY; rRP, rostral raphe pallidus; PBS, phosphate-buffered saline; ANOVA, analysis of variance; LSD, least significant difference; au, units of activity; OR, outside the region; SR 46349B, 1(Z)-[2-(dimethylamino) ethoxyimino]-1(2-fluorophenyl)-3-(4-hydroxyphenyl)-2(E)-propene.
Address correspondence to: Dr. Daniel E. Rusyniak, Regenstrief Health Center, Suite R2200, 1050 Wishard Blvd., Indianapolis, IN 46202-2859. E-mail: drusynia{at}iupui.edu
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Abdel-Fattah AF, Matsumoto K, el-Hady KA, and Watanabe H (1995) 5-HT1A and 5-HT2 receptors mediate hypo- and hyperthermic effects of tryptophan in pargyline-pretreated rats. Pharmacol Biochem Behav 52: 379–384.[CrossRef][Medline]
Abdel-Fattah AF, Matsumoto K, Murakami Y, Adel-Khalek Gammaz H, Mohamed MF, and Watanabe H (1997) Central serotonin level-dependent changes in body temperature following administration of tryptophan to pargyline- and harmaline-pretreated rats. Gen Pharmacol 28: 405–409.[Medline]
Adell A, Casanovas JM, and Artigas F (1997) Comparative study in the rat of the actions of different types of stress on the release of 5-HT in raphe nuclei and forebrain areas. Neuropharmacology 36: 735–741.[CrossRef][Medline]
Araneda R and Andrade R (1991) 5-Hydroxytryptamine2 and 5-hydroxytryptamine1A receptors mediate opposing responses on membrane excitability in rat association cortex. Neuroscience 40: 399–412.[CrossRef][Medline]
Berkey DL, Meeuwsen KW, and Barney CC (1990) Measurements of core temperature in spontaneously hypertensive rats by radiotelemetry. Am J Physiol 258: R743–R749.[Medline]
Bexis S and Docherty JR (2006) Effects of MDMA, MDA and MDEA on blood pressure, heart rate, locomotor activity and body temperature in the rat involve 
-adrenoceptors. Br J Pharmacol 147: 926–934.[CrossRef][Medline]
Blessing WW (2004) 5-Hydroxytryptamine 1A receptor activation reduces cutaneous vasoconstriction and fever associated with the acute inflammatory response in rabbits. Neuroscience 123: 1–4.[CrossRef][Medline]
Blessing WW, Seaman B, Pedersen NP, and Ootsuka Y (2003) Clozapine reverses hyperthermia and sympathetically mediated cutaneous vasoconstriction induced by 3,4-methylenedioxymethamphetamine (Ecstasy) in rabbits and rats. J Neurosci 23: 6385–6391.
Blessing WW, Zilm A, and Ootsuka Y (2006) Clozapine reverses increased brown adipose tissue thermogenesis induced by 3,4-methylenedioxymethamphetamine and by cold exposure in conscious rats. Neuroscience 141: 2067–2073.[CrossRef][Medline]
Callaway CW, Rempel N, Peng RY, and Geyer MA (1992) Serotonin 5-HT1-like receptors mediate hyperactivity in rats induced by 3,4-methylenedioxymethamphetamine. Neuropsychopharmacology 7: 113–127.[Medline]
Cox B, Kerwin RW, Lee TF, and Pycock CJ (1980) A dopamine-5-hydroxytryptamine link in the hypothalamic pathways which mediate heat loss in the rat. J Physiol 303: 9–21.
Cryan JF, Harkin A, Naughton M, Kelly JP, and Leonard BE (2000) Characterization of D-fenfluramine-induced hypothermia: evidence for multiple sites of action. Eur J Pharmacol 390: 275–285.[CrossRef][Medline]
Dafters RI (1994) Effect of ambient temperature on hyperthermia and hyperkinesis induced by 3,4-methylenedioxymethamphetamine (MDMA or "ecstasy") in rats. Psychopharmacology (Berl) 114: 505–508.[CrossRef][Medline]
Deussen A (2007) Hyperthermia and hypothermia: effects on the cardiovascular system. Anaesthesist, in press.
DiMicco JA, Sarkar S, Zaretskaia MV, and Zaretsky DV (2006) Stress-induced cardiac stimulation and fever: common hypothalamic origins and brainstem mechanisms. Auton Neurosci 126–127: 106–119.
Gaudreault N, Santure M, Pitre M, Nadeau A, Marette A, and Bachelard H (2001) Effects of insulin on regional blood flow and glucose uptake in Wistar and Sprague-Dawley rats. Metabolism 50: 65–73.[CrossRef][Medline]
Goodwin GM, De Souza RJ, Green AR, and Heal DJ (1987) The pharmacology of the behavioural and hypothermic responses of rats to 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT). Psychopharmacology (Berl) 91: 506–511.[CrossRef][Medline]
Gordon CJ (1990) Thermal biology of the laboratory rat. Physiol Behav 47: 963–991.[CrossRef][Medline]
Gordon CJ, Watkinson WP, O'Callaghan JP, and Miller DB (1991) Effects of 3,4-methylenedioxymethamphetamine on autonomic thermoregulatory responses of the rat. Pharmacol Biochem Behav 38: 339–344.[CrossRef][Medline]
Gough B, Imam SZ, Blough B, Slikker W Jr, and Ali SF (2002) Comparative effects of substituted amphetamines (PMA, MDMA, and METH) on monoamines in rat caudate: a microdialysis study. Ann N Y Acad Sci 965: 410–420.[Medline]
Gowing LR, Henry-Edwards SM, Irvine RJ, and Ali RL (2002) The health effects of ecstasy: a literature review. Drug Alcohol Rev 21: 53–63.[CrossRef][Medline]
Gudelsky GA, Koenig JI, and Meltzer HY (1986) Thermoregulatory responses to serotonin (5-HT) receptor stimulation in the rat: evidence for opposing roles of 5-HT2 and 5-HT1A receptors. Neuropharmacology 25: 1307–1313.[CrossRef][Medline]
Helke CJ, Capuano S, Tran N, and Zhuo H (1997) Immunocytochemical studies of the 5-HT1A receptor in ventral medullary neurons that project to the intermediolateral cell column and contain serotonin or tyrosine hydroxylase immunoreactivity. J Comp Neurol 379: 261–270.[CrossRef][Medline]
Horiuchi J, Wakabayashi S, and Dampney RA (2005) Activation of 5-hydroxytryptamine 1A receptors suppresses the cardiovascular response evoked from the dorsomedial hypothalamic nucleus. Hypertension 46: 173–179.
Irvine RJ, Toop NP, Phillis BD, and Lewanowitsch T (2001) The acute cardiovascular effects of 3,4-methylenedioxymethamphetamine (MDMA) and p-methoxyamphetamine (PMA). Addict Biol 6: 45–54.[CrossRef]
Jones D and Story DA (2005) Serotonin syndrome and the anaesthetist. Anaesth Intensive Care 33: 181–187.
Kehne JH, Ketteler HJ, McCloskey TC, Sullivan CK, Dudley MW, and Schmidt CJ (1996) Effects of the selective 5-HT2A receptor antagonist MDL 100,907 on MDMA-induced locomotor stimulation in rats. Neuropsychopharmacology 15: 116–124.[CrossRef][Medline]
Lester SJ, Baggott M, Welm S, Schiller NB, Jones RT, Foster E, and Mendelson J (2000) Cardiovascular effects of 3,4-methylenedioxymethamphetamine: a double-blind, placebo-controlled trial. Ann Intern Med 133: 969–973.
Madden CJ and Morrison SF (2006) Serotonin potentiates sympathetic responses evoked by spinal NMDA. J Physiol 577: 525–537.
Malberg JE, Sabol KE, and Seiden LS (1996) Co-administration of MDMA with drugs that protect against MDMA neurotoxicity produces different effects on body temperature in the rat. J Pharmacol Exp Ther 278: 258–267.
Malberg JE and Seiden LS (1998) Small changes in ambient temperature cause large changes in 3,4-methylenedioxymethamphetamine (MDMA)-induced serotonin neurotoxicity and core body temperature in the rat. J Neurosci 18: 5086–5094.
McCreary AC, Bankson MG, and Cunningham KA (1999) Pharmacological studies of the acute and chronic effects of (+)-3,4-methylenedioxymethamphetamine on locomotor activity: role of 5-hydroxytryptamine1A and 5-hydroxytryptamine1B/1D receptors. J Pharmacol Exp Ther 290: 965–973.
Morrison SF (2004) Activation of 5-HT1A receptors in raphe pallidus inhibits leptinevoked increases in brown adipose tissue thermogenesis. Am J Physiol 286: R832–R837.
Morrison SF, Sved AF, and Passerin AM (1999) GABA-mediated inhibition of raphe pallidus neurons regulates sympathetic outflow to brown adipose tissue. Am J Physiol 276: R290–R297.[Medline]
Nakamura K, Matsumura K, Kobayashi S, and Kaneko T (2005) Sympathetic premotor neurons mediating thermoregulatory functions. Neurosci Res 51: 1–8.[CrossRef][Medline]
Ootsuka Y and Blessing WW (2003) 5-Hydroxytryptamine 1A receptors inhibit cold-induced sympathetically mediated cutaneous vasoconstriction in rabbits. J Physiol 552: 303–314.
Ootsuka Y and Blessing WW (2006a) Activation of 5-HT1A receptors in rostral medullary raphe inhibits cutaneous vasoconstriction elicited by cold exposure in rabbits. Brain Res 1073–1074: 252–261.
Ootsuka Y and Blessing WW (2006b) Thermogenesis in brown adipose tissue: increase by 5-HT2A receptor activation and decrease by 5-HT1A receptor activation in conscious rats. Neurosci Lett 395: 170–174.[CrossRef][Medline]
Ootsuka Y, Nalivaiko E, and Blessing WW (2004) Spinal 5-HT2A receptors regulate cutaneous sympathetic vasomotor outflow in rabbits and rats; relevance for cutaneous vasoconstriction elicited by MDMA (3,4-methylenedioxymethamphetamine, "Ecstasy") and its reversal by clozapine. Brain Res 1014: 34–44.[CrossRef][Medline]
Paxinos G and Watson C (1998) The Rat Brain in Stereotaxic Coordinates, Academic Press, New York.
Pedersen NP and Blessing WW (2001) Cutaneous vasoconstriction contributes to hyperthermia induced by 3,4-methylenedioxymethamphetamine (Ecstasy) in conscious rabbits. J Neurosci 21: 8648–8654.
Peroutka SJ, Lebovitz RM, and Snyder SH (1981) Two distinct central serotonin receptors with different physiological functions. Science 212: 827–829.
Rothman RB, Baumann MH, Dersch CM, Romero DV, Rice KC, Carroll FI, and Partilla JS (2001) Amphetamine-type central nervous system stimulants release norepinephrine more potently than they release dopamine and serotonin. Synapse 39: 32–41.[CrossRef][Medline]
Saadat KS, O'Shea E, Colado MI, Elliott JM, and Green AR (2005) The role of 5-HT in the impairment of thermoregulation observed in rats administered MDMA (`ecstasy') when housed at high ambient temperature. Psychopharmacology (Berl) 179: 884–890.[CrossRef][Medline]
Salmi P and Ahlenius S (1998) Evidence for functional interactions between 5-HT1A and 5-HT2A receptors in rat thermoregulatory mechanisms. Pharmacol Toxicol 82: 122–127.[Medline]
Schmidt CJ, Black CK, Abbate GM, and Taylor VL (1990) Methylenedioxymethamphetamine-induced hyperthermia and neurotoxicity are independently mediated by 5-HT2 receptors. Brain Res 529: 85–90.[CrossRef][Medline]
Spanos LJ and Yamamoto BK (1989) Acute and subchronic effects of methylenedioxymethamphetamine [(+/–) MDMA] on locomotion and serotonin syndrome behavior in the rat. Pharmacol Biochem Behav 32: 835–840.[CrossRef][Medline]
Sprague JE, Yang X, Sommers J, Gilman TL, and Mills EM (2007) Roles of norepinephrine, free fatty acids, thyroid status, and skeletal muscle uncoupling protein 3 expression in sympathomimetic-induced thermogenesis. J Pharmacol Exp Ther 320: 274–280.
Stornetta RL, Rosin DL, Simmons JR, McQuiston TJ, Vujovic N, Weston MC, and Guyenet PG (2005) Coexpression of vesicular glutamate transporter-3 and 
-aminobutyric acidergic markers in rat rostral medullary raphe and intermediolateral cell column. J Comp Neurol 492: 477–494.[CrossRef][Medline]
Tóth IE, Tóth DE, Boldogkoi Z, Hornyak A, Palkovits M, and Blessing WW (2006) Serotonin-synthesizing neurons in the rostral medullary raphe/parapyramidal region transneuronally labelled after injection of pseudorabies virus into the rat tail. Neurochem Res 31: 277–286.[CrossRef][Medline]
van den Buuse M and Wegener N (2005) Involvement of serotonin1A receptors in cardiovascular responses to stress: a radio-telemetry study in four rat strains. Eur J Pharmacol 507: 187–198.[CrossRef][Medline]
Zaretsky DV, Zaretskaia MV, and DiMicco JA (2003) Stimulation and blockade of GABAA receptors in the raphe pallidus: effects on body temperature, heart rate, and blood pressure in conscious rats. Am J Physiol 285: R110–R116.
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, significantly different compared with all other groups at the specified time point. ANOVA and LSD, p < 0.05. n = 6 for each group.
, injections of WAY 100635 or PBS followed by systemic injection of DPAT; 
, injections of WAY 100635 or PBS outside the region of the rRP followed by the systemic injection of DPAT; 
, injections of PBS followed by the systemic injections of MDMA; 
, WAY 100635 followed by the systemic injections of MDMA; 
, injections of WAY 100635 outside the region of the rRP followed by systemic injections of MDMA. py, pyramids; bas, basilar artery.
