In 2006, 4.5 metric tons of MDMA, popularly known as Ecstasy was seized, worldwide (World Drug Report, 2008). Such vast quantities of drugs are being manufactured to meet a considerably large demand -- recreational drugs for human consumption. In the same year, New Zealand had a prevalence rate of MDMA use for those aged 15-64, of 2.6% (United Nations Office on Drugs and Crime, 2008). Considering studies have indicated ecstasy use in 60-80% of rave attendees (Branigan et al., 1997 as cited in Winstock, Griffiths & Stewart, 2001) but possibly up to 96% (Winstock, Griffiths & Stewart, 2001), it can be assumed that the sensory stimuli and engaging environment of a rave (a large group of people, typically young, who gather to dance to techno or dance music) is considered particularly inviting for drug use. In fact, poly-drug use is common in this environment, as is taking multiple or particularly high doses of MDMA to achieve the desired effect (Winstock, Griffiths & Stewart, 2001). It is for these reasons, that careful examination of the effects of 'club drugs', both short-term and long-term may be so crucial to the health of multiple generations. A transporter-mediated releaser of serotonin, and to a lesser degree dopamine, an acute dose of MDMA may release up to 80% of the brain's central serotonin stores (Vollenweider et al., 2003; Green, 1995; as cited in Schifano, 2004), which may account for the drugs short term enhanced mood effects, and delayed effects of depression, anxiety (Hando, Topp & Hall, 1997; Parrot, Sisk & Turner, 2000, as cited in Schifano, 2004). Other studies have also indicated that MDMA promotes hyperactivity in animals via indirect stimulation of the 5-HT1B receptor (Rempel, Callaway & Geyer, 1993, as cited in Schifano, 2004), with restlessness, tremor and excitation appearing as possible corroborating effects in humans (Liechti et al., 2000, as cited in Schifano). Similar stimulant-like effects in humans may be a result of noradrenaline release that has also been seen in studies, with a sympathomimetic effect on the body (Baumann, Wang & Rothman, 2007; Rothman et al., 2001, as cited in Schifano, 2004). In addition to possible NA-induced effects of arousal, MDMA has been shown to release acetylcholine in rat brain slices (Fischer et al., 2001) and also has affinity for histaminic receptors (Bataglia et al, 1988, as cited in Schifano 2004) which could potentially induce further physiological arousal, motor and memory effects (Schifano et al., 2004). Though anxiety was not reported explicitly, Schifano et al. (2004) claimed use of a scale to reveal some low to moderate anxiety in human users, with women scoring higher on these items. This would be consistent with animal studies indicating anxiogenic effects of MDMA (Ho, Pawlak et al.; Navarro, 1999, Gurtman et al., 2002;)(Gamm et al., 2000; McGuire, 2000; Parrott et al., 2000; Schifano et al., 1998; Verkes et al., 2001, as cited in Gurtman et al., 2002). However, the nature of MDMA and its effect on anxiety is not entirely clear, and there is also considerable evidence to the contrary, suggesting an anxiolytic effect (Navarro, Rivera et al., 2004) as well as numerous studies of subjective effects in humans citing pleasant and mood-enhancing effects (Baylen & Rosenberg, 2006). A number of variables may be responsible for the contention, including dosages and a possible change from anxiogenic effects at low doses, to anxiolytic at high doses (Lin et al., 1999). Additional confounding factors may include the choice of method for testing and measuring anxiety, number of consecutive doses, lack of MDMA purity control and the fact that many of the studies were retrospective (Lin et al., 1999).
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Parrott (2004) points out that animal research has suggested that administration of MDMA under hot, crowded or noisy conditions may be utilized to boost the arousal effects of MDMA, as seen in increased reinforcing properties of MDMA and methamphetamine under high temperature conditions (Cornish et al., as cited in Parrott, 2004; Cornish et al., 2008; Banks, Sprague, Czoty & Nader, 2008) auditory stimuli (Feduccia & Duvauchelle, 2008), the aggregate toxicity phenomenon (Gunn & Gurd, as cited in Parrott, 2004) and O'Shea et al's 2005 study finding ambient temperature to increase MDMA-mediated extracellular DA and 5-HT in the nucleus accumbens, a region which figures prominently in incentive properties of drugs. Parrott also postulates that other stimulating properties of raves, such as bright lights and loud noise may increase the state of hyperarousal by MDMA-using attendees, but also that such conditions may lead to increased negative effects as well. It is therefore possible, that the combination of stimulating environmental conditions, such as loud music, may increase the anxiogenic or anxiolytic effects of MDMA. This report aims to help elucidate the effects of MDMA and related drugs on anxiety, particularly in the presence of a strong environmental stimulus, in this instance, a loud techno dance track selected for its similarity to those played at raves, on the anxiogenic or anxiolytic effects of the included drugs.
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Awareness of the dangers of MDMA consumption, as well as initiatives to find legal alternatives with similar subjective effects may have led to the use of N-substituted piperazine analogues such as 1-benzylpiperazine (BZP) in similar settings as MDMA (Baumann et al., 2005). Studies comparing BZP with amphetamine and methamphetamines showed similarities regarding induction of turning behaviour in rats with nigrostriatal lesions of the dopamine pathway, suggesting BZP may contribute to dopamine release (Oberlander, 1979). Baumann et al., (2005) found that BZP stimulated extracellular 5-HT and DA increases in vivo, and appeared to result in dose-dependent behavioural changes such as increased sniffing, head-bobbing and ambulation. The authors also notice an apparent predominance of effect on DA systems over 5-HT. Such behavioural findings are consistent with prior research indicating methamphetamine and amphetamine-like properties in rats (Jones et al., 1980 as cited in Baumann, 2005; Hayase et al., 2005 as cited in Herbert & Hughes, 2009; Oberland, 1979; Herbert & Hughes, 2009; Lin, Bangs, Lee, Kidd & Russell, 2009, Yarosh, Katz, Coop & Fantegrossi, 2007). Other studies in rodents have confirmed an increased locomotor activity with BZP administration at high doses (30mg/kg and 100mg/kg), also consistent with findings of BZP as a behavioural stimulant (Fantegrossi et al., 2005). As a stimulant, considered to have similar subjective properties as MDMA, it is reasonable to suggest that BZP may also be susceptible to environmentally enhanced effects when administered in highly-stimulating conditions, and as such, makes an interesting comparison for the effects of MDMA.
Methylone, a Î²-ketone analogue of MDMA has also been known to have amphetamine-like and MDMA-like properties, despite sparse research examining its effects empirically. Acting upon the synaptic cleft to increase 5-HT, DA and NA concentrations via inhibition of monoamine reuptake transporters (Cozzi, et al., 1998, 1999 as cited in Kamata et al., 2006), methylone is being increasingly abused as a designer drug in Japan, the U.S and Europe (Katagi & Tsuchihashi, 2002; Uchiyama et al., 2008; Zaitsu et al., 2008; Becker et al., 2003; Johansen, 2003; Casale, Hays, Spratley & Smith, 2006; Bossong, van Dijk & Niesink, 2005, as cited in Shima, Katagi & Tsuchihashi, 2009). As the molecule itself and its main metabolites retain a similar structure to MDMA, it is proposed it may have similar health risks and adverse effects (Kamata, 2006; Shima, Katagi & Tsuchihashi, 2009). Although little is known about methylone as yet, the increase in its use a possible substitute for MDMA, and structural similarities are sound reason for inclusion as an MDMA-like drug in order to observe effects of said drugs on anxiety under certain environmental conditions.
Eighty PVGc hooded rats (male n=40; female n=40), bred and raised to adulthood (4-5 months) in the University of Canterbury's breeding facilities, were divided into groups of 8 rats of each sex in 5 drug conditions: Saline, Saline and music, MDMA and music, methylone and music, and BZP and music. The saline condition is used as a control to compare with Saline and music, to differentiate an effect of music without drugs. Drug and music conditions can be used as indicators of the effect of drugs under auditory stimulation, compared with the controls, and also to compare between drugs. All rats were housed in standard conditions, with food and water available ad libitum.
Drug dosages for MDMA (3,4-methylenedioxymethamphetamine) were set at 10mg/kg for female rats. Male rats were initially administered with 10mg/kg of the appropriate drug also, but following the illness and premature death of 5 of the 8 males in the MDMA condition around Day 2, male drug dosages were reduced to 5mg/kg for the remainder of the experiment. The male rats which died prematurely were replaced with males of similar age and weight. Dosage levels for methylone (3,4-methylenedioxy-N-methylcathinone, or bk-MDMA) were 8mg/kg for males and females, dosages for BZP (benzylpiperazine) were 20mg/kg for both sexes.
Apparatus used for each drug condition included a black, wooden 4x4 open field maze with a small camera attached to sit directly overhead; a wooden and plastic Y maze with a small attached holding compartment and sheet metal sleeve inserts painted black (3 sleeves) and white (1 sleeve); acoustic startle cages, placed inside darkened compartments in a quiet room.
Day 1 of the experiment consisted of intra-peritoneal drug administration of the appropriate drugs to the designated rats in each condition, then placement of the saline and music (henceforth referred to as Music), MDMA and music, methylone and music, and BZP and music rats (all to be henceforth referred to by the drug name only) in individual open-field mazes in a room containing a sound system playing a single techno track loudly on loop. Rats were given 20 minutes free exploration of the open-field maze to allow for drugs to take effect, then the experimenters observed the rat behaviours for 5 minutes, recording the presence of any of the following behaviours at 3 second intervals: rearing, grooming, head-weaving, tremor, fore-paw treading, circling, backing and ataxia. Observation also included noting which square the rat occupied at each interval, from which number of transitions, as well as time spent in centre and corner squares were obtained. Additionally, faecal boluses were counted after completion of the 5 minute observation. Mazes were cleaned after each round of testing. Saline rats were treated using the same procedure, with the exception of their location in a separate, quiet room.
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Immediately following the open-field test, rats were moved into a non-music room and run through a standard Y-maze test. Rats were placed in the holding compartment for one minute, before being allowed to explore the maze with 1 white and 1 black sleeve in place (placement of each colour arm determined prior) for 6 minutes. Rats were then returned to the holding compartment while 2 fresh black inserts replaced the explored ones, then released again. Position of the rat in the central stem, left arm or right arm was recorded, to obtain 180 seconds of behaviour data regarding rat position in the Y-maze. Following the Y-maze testing, rats were placed into one of 5 MED startle boxes for an acoustic startle test consisting of 100ms 95-dB white noise bursts.
All tests are conducted with one rat, identified by colour-coding the tail, from each drug condition simultaneously, then a second round of one rat from each drug condition and so forth in a specified order, thus each rat had drugs administered and was tested approximately 24 hours apart each day. Additionally, all female rats were tested for Days 1-11, with male rats being run through procedure for 11 days following a short time after completion of female rats.
Days 2-9 only the open-field maze was used to collect data, with procedure remaining the same as on Day 1. Day 10 procedure was identical to Day 1, in order to quantify the effects of chronic drug administration, including the Y-maze and acoustic startle procedures following the open-field maze observation. Day 11 also followed the same procedure as Day 1, however all rats received only a saline injection. Day 1 results provide all Acute condition data, as a measure of the effects of an initial drug dose, Day 10 data is used for the Chronic condition, as an indicator of the effect of consecutive days of drug administration and Day 11 data is used for the Saline condition results, as an indicator of residual effects after chronic drug administration.
Startle responses for drug and administration conditions were obtained from the acoustic startle test, and is being used as an indication of anxiety levels and physiological arousal.
Results of a repeated measures ANOVA indicates significant main effects for time on startle F(2, 150) = 45.445, p<0.001, and a Tukey HSD test showing both Acute and Chronic conditions to have significantly higher responses than Saline, at a p<0.001 level. However, a significant main effect did not occur for drug , F(4, 75) = 1.14, p = 0.34.
Figure 2. Mean Startle for Phases.
Figure 1. Mean Startle for Drug Conditions.
Figure 3. Mean Startle for Drugs Across All Conditions.
A significant interaction effect was also found for administration and drug F(8, 150) = 2.85, p < 0.01 (see Figures 1-3). Examination of post-hoc Tukey HSD tests for the startle data reveals MDMA Chronic responses are significantly higher than MDMA Saline at a p<0.001 level, Methylone Saline at a p<0.05 level and BZP Saline at a p<0.01 level.
Methylone Acute also has significantly higher responses than MDMA and BZP Saline at a p<0.01 level, and methylone Saline at a p<0.001 level. Additionally, methylone Chronic results are significantly higher than Saline and Music Saline conditions at a p<0.01 level, and MDMA, methylone and BZP Saline conditions at a p<0.001 level.
BZP Acute shows significantly higher responses than BZP Saline at a p<0.05 level, as well as BZP Chronic being significantly higher than MDMA Saline a p<0.05 and BZP Saline at p<0.001.
Transitions indicates the number of times a rat moved between the delineated squares in the open-field maze grid, and acts as a measure of activity. Repeated measures ANOVA results of number of transitions between squares in the open-field maze grid indicate a significant main effect of drug, F(4,75)=32.298, p<0.001. Post-hoc analyses indicated MDMA transitions to be significantly higher than all other drugs, at p<0.001, and BZP to be significantly higher than the two controls, music and saline at a p<0.05 level.
A significant effect of administration was also found, F(2,150)=97.667, p<0.001, with the acute condition showing significantly higher transitions than both chronic and saline conditions, at p<0.001 (see Figures 4-6).
Figure 5. Mean Transitions for All Phases.
Figure 4. Mean Transitions for All Drugs.
Figure 6. Mean Transitions for Drugs Across All Conditions.
The interaction of drug and administration was also significant, F(8,150)=41.734, p<0.001. Post-hoc analyses indicate the number of transitions for MDMA Acute is significantly higher than all other drug/administration conditions at a p<0.001 level.
Transitions for methylone Acute are significantly greater than Saline and Music Acute, Music Chronic and MDMA Saline at a p<0.05 level. Methylone Acute transitions are also significantly higher than methylone Chronic, methylone Saline and BZP Saline at a p<0.01 level.
BZP Acute transitions are significantly higher than transitions for Saline and Music in the Acute, Chronic and Saline conditions at a p<0.001 level. Transitions are also significantly greater than MDMA Chronic and Saline, methylone Chronic and Saline and BZP Saline at the p<0.001 level, as well as BZP Chronic at a p<0.01 significant level. BZP Saline transitions are also significantly lower than those of the Saline Acute condition at a p<0.001 level.
Time Spent in Corners
Repeated measures ANOVA revealed significant main effects for the drug condition , F(4,75)=6.9281, p<0.001, with post-hoc tests indicating significantly less time spent in corners by MDMA rats and all other drug conditions, including controls, at a p<0.05 level.
A significant main effect was also found for administration conditions , F(2,150)= 13.248, p<0.001, with Tukey HSD tests showing significant differences between the Acute condition and both Chronic and Saline conditions at a p<0.001 level.
A significant interaction effect, F(8,150)=23.427, p<0.001 was also uncovered, with significant differences between the MDMA Acute time spent in corners and all other conditions at a p<0.001 level (see Figures 7-9).
Figure 8. Percentage Time Spent in Corners for All Phases.
Figure 7. Percentage Time Spent in Corners for All Drugs.
Figure 9. Percentage Time Spent in Corners Across All Conditions.
Time spent in novel arm
Time spent in the novel arm of the Y-maze can be interpreted in a number of ways; it can be used as a memory test, or in this case, as a possible indicator of anxiety.
Repeated measure ANOVA indicated a significant main effect for the drug condition, F(4,75)=4.3533, p<0.01. Tukey post-hoc analyses show that the methylone condition rats spent a significantly lower proportion of time in the novel arm than rats in the MDMA condition, at a p<0.01 level. No significant main effect was seen for the administration condition, F(2,150)=.65225, p=.52. However, a significant effect was seen for the interaction of drug and administration conditions, F(8,150)=2.3797, p<.05 (see Figures 10-12). Further analyses highlight that the methylone Chronic condition rats spent significantly less time in the novel arm than the saline Chronic and MDMA Chronic rats at a p<0.05 level, and significantly less time than music Acute (p<0.01) and MDMA Acute (p<0.001).
Figure 11. Mean Percentage Time Spent in Novel Arm for All Phases.
Figure. 10 Mean Percentage Time Spent in Novel Arm for All Drugs.
Figure 12. Mean Percentage Time Spent in Novel Arm Across All Conditions.
Examination of Saline drug condition rats indicates only one significant difference between administration conditions, with rats in the Chronic condition spending a smaller proportion of time in the corners of the open-field maze, however the significance level for this difference was borderline (p=0.05). It is possible that this occurred as a result of the rats becoming accustomed to the testing process, but in any case, it is not considered problematic. As Saline drug rats only ever received saline injections for 11 consecutive days no differences were expected other than those which could be accounted for by chance variation, and other than time spent in corners, no significant differences were found. A similar result was expected for Music condition rats, with the possible exception of a slight decline in anxiety as subjects became accustomed to the auditory stimuli they were exposed to for the 11 consecutive days. As the results indicated, no significant differences were seen in Music rat results.
A comparison of the Saline drug condition rats with Music condition rats on all administration condition reveals no significant differences, from which we can infer that the presence of the techno music track alone was not sufficient to affect any of the measures being examined. Though it may be noted that startle responses for Music rats were slightly, though not statistically significantly, higher than Saline rats, which may be a result of the auditory stimuli, and that this difference is ameliorated by Day 11, which could be a result of habituation to the stimuli over repeated exposures.
Examining the results for the MDMA condition, we can see a notably high number of transitions being made in the open-field test in the acute administration phase, coupled with a low proportion of time spent in the corner squares. It seems reasonable to conclude that this is a result of the hyperactivity that has been shown to occur with MDMA use, likely due to the physiologically arousing properties of the drug (Rempel, Callaway & Geyer, 1993, as cited in Schifano, 2004; Baumann, Wang & Rothman, 2007; Rothman et al., 2001, as cited in Schifano, 2004; Fischer et al., 2001; Bataglia et al, 1988, as cited in Schifano 2004; Schifano et al., 2004). While it is possible that the hyper-locomotion could be attributable to restlessness or a desire to escape from the open-field maze, this wouldn't be consistent with the startle response results, which put the MDMA acute condition well within the range of the controls, even on the lower side of the Music Acute group. Further investigation shows the highest observed proportion of time spent in the novel arm for the Y-maze. These results all suggest a fairly low level of anxiety, compared with the other drug conditions, and even somewhat compared with the controls, which serves as evidence for MDMA as having some anxiolytic properties (Navarro, Rivera et al., 2004). However, when we then look at the results for the MDMA group in the Chronic phase, after 10 consecutive days of MDMA administration, a different picture emerges. The startle response has increased, to the point of being significantly higher than is seen by the same group when no longer under the influence of MDMA. We can also see that the hyperactivity initially seen in the Acute phase has dropped significantly to the point of no longer differentiating the group from any of the other drug or control conditions for either transitions or time spent in corner squares. Results for the Y-maze also show some decrease in time spent in the novel arm. Examining the results for the MDMA Acute and changes in the Chronic phase, it appears that repeated administration on consecutive days appears to detract from possible anxiolytic effects or increase anxiogenic aspects. This could potentially be related to the demonstrated dose-dependent effects of anxiolysis and anxiogenesis with MDMA (Lin et al., 1999), as well as MDMA's nonlinear pharmacokinetics (Schifano, 2004; Baumann, Wang & Rothman, 2007) as given the effects of MDMA on the body, more than 24 hours may be required in order for the neurotransmitter levels in the brain to return to their prior levels. For example, assuming MDMA may in fact release up to 80% of the brain's central 5-HT stores with one use( Vollenweider et al., 2003; Green, 1995; as cited in Schifano, 2004) resulting in longer-term negative effects such as depression and anxiety (Hando, Topp & Hall, 1997; Parrot, Sisk & Turner, 2000, as cited in Schifano, 2004; Gurtman et al., 2003; Ludwig, Mikhov & Schwarting, 2008), after multiple administrations on consecutive days, it is unlikely that the drug will be capable of having the same initial anxiolytic effects and possibly showing compounded negative sequelae (Britt & McCance-Katz, 2005).
Compared with the MDMA results, anxiogenic effects may be more apparent, with startle responses in the Acute condition being the highest of all conditions, though not to the point of statistical significance when comparing with controls. Some increased locomotion is apparent in slightly elevated transitions, though not to the same degree as MDMA, which could possibly be due to the inhibiting effect of higher anxiety levels on the desire to explore or move around. Looking at the Y-maze results also suggests a lack of propensity to explore the novel arm of the Y-maze. From this we might extrapolate that methylone has less of an anxiolytic effect in low or initial doses than MDMA does. Furthermore, when considering the Chronic phase for methylone, it can be seen that, similar to the MDMA group, the startle response shows an increase, reaching significance when compared with the methylone groups own saline phase. Additionally, number of transitions remains low, and time spent in corners correspondingly high. Notably, the preference for the novel arm drops to a level significantly lower than controls also. From this data it is suggested that methylone may undergo a similar change in effect with repeated consecutive administration as suggested for MDMA above, which would be congruent with their chemical similarity (Kamata, 2006; Shima, Katagi & Tsuchihashi, 2009).
Finally, BZP startle responses for the acute phase suggests significantly higher anxiety compared with the saline phase, though not with controls. A stimulation effect does appear to be occurring, as evidenced by the significantly elevated number of transitions seen in the acute and chronic phases, which as with MDMA and methylone, drops to a non-significant level. Time spent in the corner squares fails to show significant difference from other conditions, though it is slightly low (though non-significantly) in the acute phase, corresponding somewhat with the greater number of transitions. Y-maze preferences show little change over different administration phases, with only slight, but non-significant, preference to avoiding the novel arm.
The focus of this study has been of a retrospective nature, and as such was not set up specifically for the purpose of examining measures of anxiety in MDMA, methylone and BZP exposure in rats. For this reason, the measures used may not have been optimal for measuring the variable in question. Had the focus been planned ahead of time, use of a test such as the elevated plus-maze would likely have been more appropriate. Other issues with procedure may also have contributed to a lack of clearly differentiating effects, such as the use of a large number of observers/coders of rat behaviour may have resulted in blurred criteria for behaviour or procedure which might produce confounding variation in the data. Also notable in the limitations was difficulty controlling the levels of ambient noise, both for the saline condition which theoretically required consistent quiet, and in determining the volume of the audio track, which was not specifically measured.
In conclusion, the data appears to support the idea of an initial small anxiolytic effect of MDMA, followed by a reversal of effect with repeated, consecutive administration for 10 days. It is postulated that this may be related to the neurochemical effects of the drug having a longer-lasting effect, and the non-linear pharmacokinetics which could create a compounded negative effect on subsequent exposures (Schifano, 2004; Baumann, Wang & Rothman, 2007; Britt & McCance-Katz, 2005, Gurtman et al., 2003). Further evidence for this may be apparent in the premature illness and expiry of a number of the male rats in the MDMA condition. Why this would occur only in the male sex is not apparent, though there have been suggestions of increased sensitivity to MDMA for male rodents (Allot & Redman, 2007), however the consecutive exposure effects may have contributed to this, as the subjects survived past initial exposures. The experiment may also provide further evidence for the similarity of the overall effects of methylone to MDMA, while suggesting possible differences in the initial exposure, which could be reasonably attributed to methylone's different effects on the neurotransmitters of the central nervous system (Cozzi, et al., 1998, 1999 as cited in Kamata et al., 2006). Some evidence for anxiety and hyperactivity with BZP administration can also be seen, though the results are less conclusive than those for MDMA and methylone. Further research into the differing effects of MDMA and methylone would be both fruitful and prudent, given the increasing interest in methylone consumption by designer drug users (Katagi & Tsuchihashi, 2002; Uchiyama et al., 2008; Zaitsu et al., 2008; Becker et al., 2003; Johansen, 2003; Casale, et al., 2006; Bossong, van Dijk & Niesink, 2005, as cited in Shima, Katagi & Tsuchihashi, 2009). Studies which look specifically at the effects of repeated, consecutive exposure to MDMA to elucidate changes in result would also be relevant, due to the immense popularity of the drug.