Modification of Serial Pattern Learning by Designer Tryptamine Exposure during Adolescence: Comparison with Damage to the Dorsal Hippocampus or Prefrontal Cortex

Foxy or Methoxy Foxy (5-MeO-DIPT) is one of a series of new “club drugs” that within the past decade has gained in popularity among recreational users as an alternative to MDMA (Ecstasy). Unlike MDMA, not much is known about the neurobiological consequences of 5-MeO-DIPT use. Little is known about the effects of either compound on learning in a nonspatial appetitive task. In the present study, adolescent rats were given repeated injections of 10 mg/kg of 5-MeO-DIPT, MDMA, or a corresponding volume of isotonic saline. In serial learning tasks, depending on task demands, there is a growing body of evidence suggesting that multip le memory systems play a critical role, with each system playing a more o r less dominant role depending on the available stimuli and task demands. Therefore, for comparison purposes, the drug-treated rats were compared with that of h ippocampusor prefrontal cortex-lesioned rats. After adolescent drug exposure or lesions during adolescence, adult animals were trained All animals were trained for 30 days on a three-element, nonmonotonic pattern consisting of 21, 0, and 7 food pellets, respectively. Control rats were capable of d istinguishing among the elements of the series, as indexed by running times. As expected, the tracking performance of the lesioned rats was impaired. Performance in both the 5-MeO-DIPTand the MDMA-treated rats improved with training but after 30 days was not markedly d ifferent than the lesioned animals. The results are discussed in terms of measured alterat ions in serotonin activity in the fo rebrain and the consequences of compromised serotoninergic systems on the cognitive processes involved in appetitive serial learning tasks.


Introduction
Like other mammalian species, rats are capable of tracking the elements of a stimu lus series consisting of differing rein forcement quantities[see 1, for a review]. Although other methods have been employed[e.g., 2], demonstrations of serial learn ing in rats involves exposure to a three-to seven-element series consisting of differing numbers of food pellets. Anticipatory responding is inferred through the demonstration of different running t imes to a goal box as a function of the given reinforcement quantity.
Wh ile a nu mb er o f th eo retical mo d els hav e b een d eveloped [3], the bu lk o f th e ev idence s uppo rts t he existence of two associative mechanis ms that can explain rodent serial-pattern learn ing -(1) the develop ment of stimulus-stimulus associations [4,5] or (2) the ordinal position of each element of the stimu lus series comes to function as a differential cue [6,7]. In a variat ion of the latter theoretical view, a series of reinfo rcement events is converted to a spatial array [8].
A consensus has emerged that a hippocampus-dependent memo ry system, normally labelled as declarative memory, is crit ical for learning the mult iple relationships among stimuli [9,10]. Th is system is considered essential in order for the organis m to learn in formation about and then flexib ly utilize information about relat ionships between mu ltip le external cues and events [9,10]. A second dorsal striatum-dependent system has been described as necessary for the formation of reinforced stimu lus-response associations [10][11][12][13]. Both systems may be essential for serial learn ing but their relative importance is driven by task requirements [1].
Serial pattern learn ing involves flexible responding in the face of anticipated changes in the environ ment. A large body of research has implicated the prefrontal cortex (PFC) 264 David M . Compton et al.: M odification of Serial Pattern Learning by Designer Tryptamine Exposure during Adolescence: Comparison with Damage to the Dorsal Hippocampus or Prefrontal Cortex in many aspects of cognition as well as executive processes [14]. Specifically, the PFC appears to be an essential co mponent in learn ing and memory, decision making, and cognitive control over behaviour [15][16][17][18]. Last, past research has consistently indicated the involvement of the PFC in behavioural flexib ility [18][19][20].
Many of the MDMA-induced deficits have been lin ked to observed reductions in brain serotonin (5-HT) levels [31] and this effect has been observed across species (see 32, for a review). Of part icular interest here, 5-HT reductions are seen in a number of regions involved in different types of learning and memory, and include such critical brain regions as the hippocampus, the dorsal striatum, and the prefrontal cort ices [31]. Further, the effect is observed in adult rats [21] as well as rats exposed when they are young [24][25][26]. Of considerable import, alterations in 5-HT function have been reported to continue long after the MDMA exposure period [32][33][34][35][36].
As is the case for many other tryptaminergic drugs, 5-Metho xy-N,N-di(iso)propyltryptamine hydrochloride (5-MeO-DIPT; FOXY) has become popular among recreational users. FOXY has properties very similar to that of other tryptaminergic hallucinogens [37]. As a consequence, recreational users of MDMA and other similar co mpounds have experimented with this drug. However, since it is similar to other tryptamine co mpounds of abuse, there have been reports of the negative consequences associated with FOXY use as a recreational drug[e.g., 38,39]. As reports of its use accumulated, in the United States FOXY was classified as a Schedule I drug [40]. Although some recent work has elucidated some of the effects of this compound [38][39][40][41][42][43], unlike M DMA, our knowledge of the consequences associated with the use of FOXY on the behaviour and neurobiology of mammalian systems remains limited.
Adolescence in rats includes the period fro m the 21st day following birth (postnatal day; PND) until PND 60 [44,45]. In addition, adolescence can be subdivided into mid adolescence (PND 34 to 46) and late adolescence (PND 46 to 59). According to Tirelli et al. [45], these two developmental periods are analogous to periadolescence and late adolescence/early adulthood. Rodent models of adolescence models are useful for co mparative assessments and for extrapolat ion to humans [46]. Specifically, the use of adolescent animals allows for a valuable experimental framework for testing the developmental effects associated with drugs of abuse at various time points in biological and cognitive development.
Of the published investigations, only a select few[e.g., 41,43] have specifically examined the effects that FOXY may have on cognition or explored the long-term consequences associated with exposure at different points in brain develop ment. So mewhat mo re is known about MDMA. However, no one has explored the effects of these compounds on a nonspatial cognitive learning task such as serial pattern learn ing. Unfortunately, the popularity of these drugs remains high and, as a consequence, the possible risks to development in vulnerable adolescents could be seen as an emerging societal health problem. Therefore, the present study was conducted to provide further elucidation into the consequences of developmental exposure to MDMA and FOXY.

Subjects
The subjects consisted of 33 male Long-Evans rats (Charles River, Boston, MA) 35 days of age at the beginning of the study. After arrival in the v ivariu m at 21 days of age, the animals were allowed to acclimate to the facility and were randomly assigned to one of five groups hereafter designated as follows: a control group (CON) consisting of n = 8 rats, two groups of drug-treated animals exposed to (±)-3,4-methylenedio xy methamphetamine (MDMA, n = 7) or 5-MeO-DIPT (FOXY, n = 7) and, t wo lesion groups receiving bilateral lesions of the dorsal hippocampus (HIP, n = 6) or the prefrontal cortex (PFC, n = 5). Four of the eight control rats received sham lesions where the electrode was lowered into the target area but no current was passed.
The rats were individually housed and maintained on a 12-hr light/12-hr dark cycle, with all testing conducted during the light phase. With the exception of serial learning training, the animals were maintained with ad lib access to food and water. The research protocol was reviewed and approved by the Institutional Animal Care and Use Co mmittee of Palm Beach Atlantic University and the animals were treated in accordance with the princip les of animal care outlined in the Guide for the Care and Use of Laboratory Animals [47].

Apparatus
The apparatus consisted of a wooden enclosed runway 185 cm long, 10 cm wide, and 14 cm high, with each section covered with h inged Plexig las. The start and goal bo xes (20 & 35 cm in length) were separated from the runway by two manually operated guillotine doors. The start box was painted flat white, the main runway flat grey, and the goal box flat black. Raising a guillotine door located between the start box and the main runway activated one digital t imer (Lafayette, Model 20225) wh ich was stopped when the rat interrupted a photobeam located 15 cm within the goal bo x. The goal box contained a removable ceramic dish with walls high enough to obscure the food reinforcement until the rat was physically inside the goal bo x. All food reinforcement consisted of a predetermined number o f 0.045-g Noyes food pellets. To min imize the presence of odor cues that might have influenced performance [48], the floor of the apparatus was swabbed with a wet sponge and dried with a paper towel before presentation of each element of the series.

Behavioural Procedure
Four days before pretraining began, each rats was weighed and reduced to 85% of their free-feeding weight. This 15% reduction was maintained throughout the experiment by feeding a daily maintenance ration (about 14 g) of Mazuri Rodent Chow. Water was availab le ad lib.
After the rats' weights were stabilized at the targeted 15% reduction, a two-day pretraining program began. During this period, individual rats were hand tamed for a two-minute period, followed by a five-minute period where the rats were free to explore the apparatus. Both guillotine doors were elevated during this phase. Last, each rat was permitted to consume 21 food pellets located in the food dish located in the goal box.
Behavioural train ing began when the rats were approximately 100 days old. Fo llo wing the t wo days of pretraining, the experimental train ing began and continued for 30 days. The rats were transported in their home cages in squads of three animals with the training order randomized daily. A ll rats received two daily trials consisting of a three-element ordered sequence of 21, 0, and 7 food pellets (Noyes: 0.045-g). W ithin each three-animal squad, all rats completed trial one before any rat experienced trial t wo. The intertrial interval was appro ximately 15 minutes with within -trial inter-element intervals of appro ximately 1 minute. Once the rat successfully traversed the runway, it was confined in the goal box until all of the food pellets were consumed or for 30 seconds on 0-pellet elements. When a rat did not reach the goal box within 60 seconds, it was gently pushed toward the goal box, confined there until all food pellets, if any, were consumed, and a 60 second running time was recorded. After consumption of all of the pellets and following each element of the series, the rat was returned to its holding cage while the experimenter swabbed the floor of the apparatus, baited the food-well, and reset the timer and guillot ine doors. After the completion of both daily trials, the rats were returned to the rat colony, where they received their maintenance ration of Mazu ri Lab Chow not less than 90 minutes later.

Surgical Procedures
All surgeries were performed under anesthesia consisting of pretreatment with .25 mg/ kg atropine follo wed 10 minutes later by 40 mg/kg of Nembutal. Behavioral pret rain ing began no less than 10 days following a postoperative recovery period.
Following the appropriate plane of anesthesia, lesions of the dorsal hippocampus were created as follo ws. Bilateral openings were made in the craniu m with a trephine. Electrode placement was determined on the basis of stereotaxic coordinates determined fro m the atlas of Paxinos and Watson [49]. Following placement o f a stainless steel electrode insulated except at the tip, bilateral electrolyt ic lesions were created in the appropriate site. The coordinates calculated fro m breg ma and current parameters were AP = -3.8 mm, M L = ±1.5 mm, DV = -3.3 mm, 2 mA for 20 seconds and AP = -3.8 mm. M L = ±2.5 mm. DV = -3.4 mm for 20 seconds. Prefrontal cortex ab lations involved removal of areas of the cortex that correspond to those described as the medial precentral, anterior cingulate, and prelimb ic cortex as defined by Krettek and Price [50]. Last, as noted earlier, four animals were placed in the stereotaxic device and prepared similarly for lesions, but did not receive lesions (i.e., shams).

Histological Analysis & Biochemical Anal ysis
For animals who received lesions, following data collection the subjects were deeply anesthetized (Nembutual, 50 mg/kg) and perfused intracardially with 40 cm 3 of isotonic saline, followed by a 10% formalin solution. The brains were removed and stored in a 30% sucrose-100% formalin mixture for 48 hours before being frozen. All brains were sectioned in the coronal plane at 60-µm intervals, mounted on slides, and stained with cresyl violet acetate.
HIP lesions were examined under microscopic magnificat ion against stereotaxic atlas temp lates using the atlas of Paxinos and Watson [49]. Viewed fro m a dorsal perspective, all cort ically ab lated brains were photographed. Subsequent analysis of cortical surface lesions proceeded with a dot grid method [51]. The dot grid method permits placement of a digital grid containing 256 dots per square inch over a Lashley diagram with the accompanied ablat ion. The number of dots contained within the area of the traced cortical lesion is counted, thus providing an estimate of the amount of cortical surface area destroyed.
Approximately, five weeks after the completion of training on the serial learning task, the animals exposed to MDMA or FOXY were euthanized. Serotonin levels were assessed in a manner described elsewhere [42]. Briefly, we determined 5-HT levels in the hippocampus, striatum, and the prefrontal cortex using high performance liquid chromatography (HPLC; a Waters Model 600 with electrochemical detection). Procedures were consistent with that outlined by Chapin, Lookingland, and Moore [52] with modification. The system used a Waters C 18 reverse phase analytical co lu mn (3.9 X 300 mm; 4 µm). Concentrations in the amounts of 0.04% sodium octyl sulfate, 0.1 mM disodiumethylenediamine-tetraacetate, 0.05 M sodium phosphate were dissolved in laboratory-grade H20 using 0.03 M citric acid as a buffer. The aqueous portion of the mobile phase was held within pH levels between 2.7 and 2. 9. The mobile phase consisted of 20% methanol and 80% aqueous phase. 5-HT levels were calculated and reported as ng/g tissue.

Data Analysis
In order to normalize the data, the running times were transformed using the reciprocal (X= 1/ X) data transformation [53]. The data were co llapsed into three blocks consisting of days 1through 6, 7 through 18, and 19 through 30, respectively and were analyzed, using a three-way ANOVA : 3 (groups) x 3 (b locks) x 4 (elements). We treated groups as a between-subjects factor, whereas blocks and elements were treated as within -subject factors. We used Tukey HSD tests to analyze the within-group differences in running times to the series elements.

Histological Analysis
Visual examination of the HIP-lesioned animals revealed the following (see Figure 1). Substantial damage to the overlying cortex, corpus callosum, and cingulum was seen in all an imals as was considerable damage to the fimb ria. All an imals had minor damage to the laterodorsal area of the thalamus and the stria terminalis. One animal received minor damage to the anterodorsal region of the thalamus. In addition, extra-h ippocampal structures with minor damage included the dorsal lateral geniculate nucleus (one animal), the anterior pretectal nucleus (one animal), and the paraventricular nucleus (one animal).
Examination of the PFC lesions indicated that they generally involved the area fro m the frontal pole to the genu of the corpus callosum. With the exception of one rat, the tissue along the medial wall o f the medial walls of the saggital sulcus, including the majority of the cingulate gyrus, was undamaged. Conversely, some involvement of the anterior cingulate gyrus was found in all five animals. Using the dot grid method described previously, suggested that when the lesions were considered fro m the dorsal perspective that they were uniform in size (M = 0.21, SD = 0.013).

Neurochemical Anal ysis of Brain 5-HT Le vels
The mean levels of 5-HT in the hippocampus, prefrontal cortex, and striatum are reported in  001. Post hoc examination of these results using Tukey HSD tests revealed the following differences among groups. When compared to the CON (NaCl-treated) animals, an examination of the 5-HT levels in the hippocampus indicated significant reductions in 5-HT (63.66% & 50.11%) in both the MDMA and FOXY d rug groups. However, when the two drug groups were compared, the 5-HT levels were comparable (i.e., p >.05).
Reductions in prefrontal 5-HT levels were observed in both the MDMA-treated rats (67.85%) and the FOXY-treated rats (60.57%). Ho wever, the d ifference in 5-HT levels in both drug-treated groups was nonsignificant. Last, when compared to control animals, significant reductions in striatal 5-HT were also observed (39.24% for MDMA & 32.47% for the FOXY rats). The pattern of differences between the CON group and two drug groups but not the two drug groups as measured 5-HT levels in these target areas is consistent with previous work [42].

Behavioural
The goals of the present investigation were to determine (1) if prior exposure of M DMA or FOXY disrupted the ability of rodents to learn a non monotonic serial pattern and (2) compare the results to that of hippocampal or PFC lesioned rats. Therefore, learn ing performance was considered at points within the 30 days of training. To do this, the data were collapsed into three blocks consisting of days 1 through 6, 7 through 18, and 19 through 30. In o rder to meet the assumptions associated with the analysis of variance (ANOVA), all running times were transformed using the reciprocal transformation.
The serial learning performance of each group collapsed into the three training period blocks is presented in Figure 2. A one between (drug or lesion groups), two-between (blocks & elements) ANOVA revealed the fo llo wing. A main effect of group was found, F(4, 28) = 6.71, p < .01, indicat ing that running times differed across the training period. As the International Journal of Psychology and Behavioral Sciences 2012, 2(6): 263-273 267 main effect of blocks, F(2, 56) = 64.25, p < .05, suggests, the animals improved across the training period and running times differed, F(2, 56) = 74.57, p < .05, as a function of run within the three-element series. The group X blocks and group x elements interactions were both significant as well (Fs(8, 56) = 3.30 & 8.96, ps < .01, respectively). Here, the results suggest that group differences emerged both across the three blocks associated with the early, midd le, and late training periods as well as within indiv idual elements of the series.  [49] However, the results must be interpreted in light of the significant group x b locks X elements interaction, F(16, 112) = 3.19, p <. 01. To assess the tracking ability o f each group, the three-way interaction was decomposed using post hoc tests for repeated measures (p < .05). Within the three-element series, accurate tracking was defined as differential running times to each element of the series in anticipation of each quantity of reinfo rcement [54]. That is, accurate tracking of the elements of the series involved running significantly mo re slowly to the 0-pellet element than to the two rewarded elements of the series, and significantly mo re slowly to the 7-than to the 21-pellet element.
For the control group, differences among the elements of the series first appeared in the second block of train ing with the rats running faster to the 21-than the 0-pellet element. As reflected in their running times, by the third block of training the control animals were able to distinguish between all three elements of the series. HIP-lesioned animals were able to d istinguish between the 21-pellet and 0-pellet elements by the second block of training and between the 21-pellet and the other t wo elements of the series by the last block of training. However, unlike the control animals, the performance o f HIP-lesioned animals was impaired on the second and third elements of the series (ps > .05). PFC-lesioned animals performed worse with significant differences in running times only detected on the 21-and 7-pellet elements in block three of training.
Examination of the animals exposed to the drugs in adolescence revealed the fo llo wing. MDMA animals showed litt le evidence of tracking throughout training until the third block. Ho wever, in the third block of training, the rats did have significantly lower running times to the 21-pellet element but only when it was co mpared with the 7-pellet element. The running times to the 0-pellet element were significantly faster than to the 7-pellet element but did not differ fro m the 21-pellet element of the series. The wo rst tracking performance of all was observed among the animals exposed in adolescence to FOXY. These animals were unable to distinguish among the elements of the series (see Figure 2). Additional confirmation of the observed lesion and drug associated deficits through the use of regression analyses. Figure 3 illustrates the linear relationship between the quantity of reinforcement and the b lock three running times used in the previous analysis. In comparison with the other groups, the control rats demonstrated high levels of anticipatory responding, R 2 = .730, F( l, 22) = 59. 44. p < . 001. Consistent with the previous section, anticipatory tracking performance was impaired in both lesion groups, with PFC rats displaying a mo re significant impairment (R 2 s = .578 & .218, HIPs & PFCs, respectively). When the adolescent-treated drug rats were considered, similar impairments consistent with above were observed R 2 s = .221 & .108, MDMA-& FOXY-treated rats, respectively). In fact, the amount of variance in running times observed in the FOXY-treat rats, 10.8%, was nonsignificant. Last, trend analyses revealed that, with the exception of the control group, a quadratic equation provided the best fit of the data when the elements considered in their order within the series. Conversely, a linear equation provided the best fit of the data for the remaining groups where the third element of the series, 7 pellets, was associated with the slowest running times. As Botwinick [55] noted, when considering instrumental learning tasks, motor deficits can impact the results, suggesting a cognitive impairment when the changes are not related to alterations in cognition [56]. To potentially ru le this out, the data from the last block were co llapsed and the groups compared. Th is analysis yielded a significant effect of group, F(4, 28) = 3.90, p < .05. However, post hoc comparison of each group versus the control group (Dunnett, two-tailed ) revealed only a significant d ifference between the control animals and the HIP-lesioned animals. Thus, although not unequivocal, the results suggest that the groups were capable of similar running speeds.

Discussion
The purpose of the present experiment was to examine the impact of adolescent exposure of MDMA and FOXY on the acquisition of appetitive serial learning task. A second goal was to compare the performance of the drug-treated rats with that of HIP-and PFC-lesioned animals, two regions implicated in serial pattern learning [1] and processes such as rule-learn ing and response flexib ility [57]. Exposure to either drug during adolescence produced a marked learning impairment in the serial learning task in (d rug-free) adulthood. The deficits were as severe as that of the brain lesioned groups, with the performance of the drug-treated animals all but indistinguishable fro m that of the PFC animals. Only the CON an imals demonstrated accurate tracking performance. In related work [42], no d ifferences in performance on the rotating balance beam or in levels of general activ ity have been found suggesting that the deficits were not simply a result of motor deficits.
There is a growing body of evidence suggesting that mu ltip le brain areas are involved in sequential learn ing and me mo ry [58]. In hu mans, this is supported from work with drug-treated [59] or brain damaged individuals on serial reaction time tasks [60,61]. The work of Nissen and colleagues supported the proposal that serial learning involves activity within both declarative and nondeclarative (procedural) me mory systems [59,60; see also, 62]. Individuals with A lzheimer's disease or experimental participants treated with the acetylcholine antagonist scopolamine imp roved across trials with a 10-element sequence but had no explicit recognition for the learning experience [59,60]. As such, the results suggested that serial learning relies on a nondeclarative memory system. Conversely, Huntington's disease patients, with a compro mised nondeclarative memory system that includes but is not limited to the basal ganglia showed no improvement. Last, much like Huntington's patients, individuals with Parkinson's disease have sequence learning deficits that are independent of more general impairments in motor performance [58,63,64].
As Muller & Fountain [58] noted, the available evidence suggests that rats may rely on at least three cognitive processes in serial pattern learn ing. The use of memory for items in a series may involve any comb ination of (1) the processing of external discriminative cues, (2) counting time encoding processes for each position of the elements of the series and/or, abstraction of a rule or rules for encoding an internal representation of the structure of the pattern [58].
Consistent with the earlier d iscussion, in rats mastery of an appetitive serial-pattern learn ing task is influenced by at least two dissociable neural systems. First, animals are capable of learning the serial pattern using a declarative memory system that includes the hippocampus, incorporat ing stimulus-stim ulus and rule learning [1,58,65]. If this system is compro mised or if the salience of stimu lus elements is skewed, then a nondeclarative memory system that includes the dorsal striatum will permit the rat to learn the series [1]. In fact, the nondeclarative system appears to drive the formation of reinforced S-R responses, with a third system that includes the amygdala appearing to contribute to the affective aspects of the experience [66][67][68][69]. Which neural system is do minant is largely determined by the stimu li available to predict a given trial outcome and the complexity of the specific serial pattern [1].
The prefrontal cortex is one of a number of critical brain regions that are involved in the ability to respond in an effective manner when confronted with changing contingencies between a stimulus and response [15,70]. In serial pattern learn ing, such flexibility is critical as contingencies (trials) change. Indeed, there is evidence that depletion of prefrontal/orbitofrontal 5-HT is highly correlated with the perseverative impairments [71,72].
Further, experimental man ipulations of 5-HT levels in the cerebral cortex leading to lower 5-HT levels are associated with an enduring increase in response impulsivity [62]. In the present experiment, determination of whether the observed deficits were a result of cognitive flexib ility or other processes, or both is not readily determined. As noted earlier, rodent serial learning involves mult iple brain structures and mu ltip le learning strategies that differ depending on task demands [1,58]. Last, it has been observed that genetic variations in the monoaminerg ic transporter protein SERT impact cognitive flexib ility [73]. Reductions in SERT binding follo wing MDMA exposure are considered indicative of serotonergic axonal damage[74].
In the present study, FOXY-treated rats were impaired relative to CON animals with the impairment approaching that of MDMA-treated rats. Although generally the effects of the former do not appear to be as severe as the latter [43,75], neither appear to diminish with age [42]. In a related study, we co mpared adolescent exposure of FOXY with MDMA and periodically tested the animals across the lifespan with the preliminary results indicating that the deficits largely remain throughout the lifespan [76].
In one recent investigation [43], rats treated with FOXY during postnatal day 11-20 were impaired relative to control animals in spatial learning but not tests of spatial memory or path integration. Ho wever, in related work with adult rats [77], a path integration deficit was observed. Of relevance here, the authors suggest that the difference is possibly a reflection of h ippocampal development[c. f., 78] that occurs during the exposure period used in their study.
When considering factors such as exposure to drugs of abuse, the period of exposure during biological development is a relevant variable [43]. For example, in one study of 5-HT turnover in the nucleus accumbens of rats [79], levels of 5-HT turnover in the nucleus accumbens were four times lower in adolescent rats than prepubescent rats (PND 10 to 15) or adult rats. Further, in rodents, 5-HT2A receptor in the cortex is at its peak just before the onset of adolescence, followed by a gradual decline to adult levels [80]. Thus, the timing of exposure of each of the compounds could have a variety of effects that differ marked ly depending on when exposure takes place during neural development, what other drugs are taken concurrently, and the length of exposure. Data fro m the neurochemical assessment of 5-HT revealed substantial reduction in 5-HT levels measured in the prefrontal cortex, hippocampus, and the striatum. This result is largely in accord with previous research with rats [41][42][43]75,76] and mice [81]. As noted above, the timing of exposure is important, with some reports suggesting more persistent 5-HT reductions if the drug exposure period includes early adolescence rather than during a later developmental period [81]. In addition, mult iple doses of MDMA can produce measurable 5-HT to xicity in periods greater than 100 days following exposure [82]. Nonetheless, the available evidence is mixed as there are some reports of an absence in the reduction of 5-HT levels following adolescent exposure [83] and some species differences (rats vs. mice) have been observed [82,84,85].
One additional caveat concerning the present results is noteworthy. While significant reductions in 5-HT levels were detected after exposure to both MDMA and FOXY, lin king 5-HT levels with that of neurotoxicity is still an area of debate[74]. Th is is true even if measured using different methods, including the methods employed here and elsewhere [43,74] and radioligand binding in SERT studies [86,87]. While the issue cannot be settled here, excellent discussions of the issues can be found in the literature [88][89][90]. At any rate, regardless of whether the drug-induced deficits were related to axonal damage or another process, on a functional level the animals were impaired when tested as adults, long after adolescent exposure and is consistent with previous reports [42,43].
The present study lends additional support to the suggestion that there is a developmental period of vulnerability to the effects of both MDMA and FOXY. Perusal of reco mmendations on the internet[e.g., 91,92] suggest that concurrent use of selective serotonin reuptake inhibitors with M DMA can ameliorate or even prevent the adverse side effects or even damage caused by MDMA[74]. In the present study as well as reports by others [42,43], exposure to MDMA and FOXY appear to produce lasting consequences. Since other neurotransmitter systems (e.g., dopamine, norepinephrine) may be co mpro mised by the use of these compounds as well [43,74,75,93,94], it is imperative in future research to examine the behavioural consequences in youth who use such drugs for recreational purposes. By doing so, more effective ameliorative and therapeutic strategies can be developed.