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model to date may be that of Sprague and Nichols [ 11, although it does not offer a complete accounting of the cascade of events that lead to MDMA-induced neurotoxicity. The most essential question in this field of research may be the role of the serotonin (5HT) 2A/2Creceptors, and whether activity at these receptors leads to increased or decreased release of in target structures. MDMA is a popular of abuse that is most commonly associated with “rave” parties [2], and recent articles have reported the mixing of d-lysergic acid diethylamide (LSD) with MDMA to increase the hallucinogenic and euphoric properties of the drug (“candy flipping” [3,4]). There has been much research exploring the neurotoxic effects following doses of MDMA in a multitude of species, and these data suggest that MDMA causes increased release of 5-HT, which in turn tonically increases dopamine release [5]. It has also been shown that 5-HT agonists such as I-(2,5-dimethoxy-4-iodophenyl)- aminopropane (DOI), melatonin, and 5-hydroxytriptamine increase this release following MDMA resulting in an increase in neurotoxicity [6]. There is disagreement as to whether 5-HT release is antagonistic [7], or agonistic [8,5] to dopamine release in brain. If 5-HT stimulates dopamine release then a partial 5-HT agonist such as LSD when given in combination with MDMA, may increase MDMA-induced neurotoxicity, and if not true then LSD may in fact provide protection. The final question is whether or not the effects of 5-HT on dopamine release are mediated primarily by the ~-HT~A and/or 5-HT2c receptors. A recent theory of MDMA-induced neurotoxicity is that abnormally high levels of dopamine are present following doses of MDMA, which is taken up via the 5-HTT and is responsible for the toxicity [9, lo]. In fact, dopamine itself has been shown to be taken up via 5-HTT and to be toxic to 5-HT terminals [ 11,12,13], and others have shown that 5-HT reuptake inhibitors (SSRIs) will block the degeneration of these terminals [5]. There have been numerous studies examining both physiological and behavioral effects in rats and humans [ 14,15,16] that suggest that the hippocampus is particularly vulnerable to MDMA-induced neurotoxicity. Therefore, we chose this structure as the focus of our immunohistological examination. The compound LSD has been characterized for many years as to its actions on 5-HT receptors, and there is a high correlation between the affinity for the ~-HT~A receptor subtype and its hallucinogenic qualities [ 171. In drug discrimination studies it has been shown that LSD transferred to that were agonists at 5-HT 2~ but not 2~ receptor subtypes [ 181. Although there is evidence that suggests that LSD has actions on dopamine and other neurotransmitters in the brain, the majority of NEUROSCIENCE RESEARCH COMMUNICATIONS, VOL. 35, NO. 2 85 evidence points to LSD as being a rather powerful and selective 5-HT partial agonist, primarily at the 5- HT~A receptor site.

MATERIALS AND METHODS Adult Sprague-Dawley rats bred at UCLA that weighed between 200-250 grams, were individually housed, and maintained on a reverse 12 hour light cycle at 54% humidity, and had food and water available ad-libitum throughout. Care was taken to ensure that the ambient temperature never deviated from 20°C. Animals were treated in accordance with the UCLA Animal Research Committee guidelines. Rats were administered the drugs for 4 days, were allowed to recover for 3 days, and then were perfused with 4% paraformaldehyde (for immunohistochemistry only) and sacrificed. Rats were assigned to treatment groups that received drug twice daily via subcutaneous (s.c.) injections. The treatment groups received: saline, MDMA, LSD, MDL 11,939, MDMA + LSD, and MDMA + MDL 11,939. Doses were chosen in order to provide consistency with current literature and were: MDMA; 20mg/kg/day, LSD; 25, 50, and lOOpg/kg/day, and MDL 11,939; 2.5, 5, and 7.5mglkglday. For each treatment group 6 animals were used with 4 being used for IHC and 2 for Northern blotting. Dl-3,4-methylenedioxymethamphetamine (MDMA) and d-lysergic acid diethylamide (LSD)(supplied by The National Institute of Drug Abuse) were dissolved in 0.9% saline, and animals received approximately 0.25mls per injection. The compound a-Phenyl- 1-(2-phenylethyl)-4- piperidinemethanol (MDL 11,939; supplied by Aventis Pharmaceuticals) was dissolved with 1M HCl, then 0.9% saline. The control group received 0.9% saline vehicle twice daily. The damage to pre- synaptic 5-HT terminals was assessed with antibodies that label proteins that comprise the (5-HTT). The mRNA signal for serotonin transporters was quantified using cDNA probes in a Northern blotting technique. Brains used for immunohistochemistry were submerged in 4% paraformaldehyde at 4°C for 24 hours then in a 20% sucrose solution for 24 hours at 4’C before being embedded in mounting medium. Sections through the hippocampus were cut on a cryostat at 2Opm and thaw mounted onto Superfrost slides, and stored at -70°C until utilized. Slides were warmed to 20°C for 30 min. and the tissue was circled with a PAP pen, quenched with 3% Hz02 x 10 min. washed with buffer (50mM TBS) 3x5 min., treated with 3% normal serum for 1 hour, and then primary antibody over night (5-HTT; ST (C-20): ~~-1458; supplied by Santa Cruz Biotechnology Inc.). Slides were then washed in buffer 3x5 min. and treated with secondary antibody for 1 hour (ABC Vectastain Elite Kit purchased from Vector Labs). Slides were then washed in buffer and incubated in ABC substrate solution for 30 min. Slides were then washed in buffer and developed in 1XDAB for 5 min, washed in DDH20 and then buffer, dehydrated in successive EtOH baths for 2 min. each, cleared in xylene x3, and then coverglass was applied using Permount. The 5-HTT antibody that was supplied had never before been used for an immunohistochemical experiment so we first performed fixation and titration analyses to assess the proper fixative and dilution for this antibody (4% PFA; and 1: 100, respectively). Images of tissue sections were taken with a SPOT camera linked to a Zeiss microscope and a computer using Photoshop software. The region of the hippocampus was traced at the interaural level of -6.20mm, using the atlas of Paxinos and Watson [ 191, and optical densitometry measurements were taken using the NIH Image (Scion) p ro g ram. Significant differences were calculated using the all pairwise multiple comparison 86 NEUROSCIENCE RESEARCH COMMUNICATIONS, VOL. 35, NO. 2 procedures using Student Newman Keuls method, and for differences from control, Dunnet’s method. Brains (without the olfactory bulbs and cerebellum) were isolated from control and treated rats, then homogenized in 4M guanidium isothiocyanate solution using an ultraturax (polytron) at 28K rpm. Total RNA was extracted as previously described [20]. RNA concentrations were estimated by spectrophotometry (Beckman DU 640B) and 60 pg of each sample were transferred into eppendorf tubes prior to evaporation under vaccum (speedvac). Pellets were reconstituted in 30 ~1 of 1X BOH- DNB loading buffer containing p-mercaptoethanol. Heat-denaturated RNA samples were loaded into a 0.8 % agarose / 2% formaldehyde gel. Migration was conducted at 4’C using 80V for 2.5 hrs. Gels were photographed using a digital gel documentation system (Lighthouse Research Speedlight) then transferred overnight onto nylon membranes (Turboblotter, Schliecher & Schull). Blots were rinsed, baked, and UV-crosslinked prior to hybridization. Membranes were first prehybridized for 2 hrs, and then hybridized with 400,000 cpm/~l [32P]-random labeled probe (Invitrogen) for 16 hrs in UltrahybTM hybridization solution (Ambion) at 44OC. Hybridizations were performed sequentially (SHTT and cyclophillin, respectively). For 5-HTT, a 1040bp selective cDNA probe encoding for the 5HTT transcript was excised using Xho-1 restriction enzyme from Bluescript SK- plasmid (generously supplied by Dr. Randy Blakely at Vanderbilt University).The rat cyclophillin 650bp probe was PCR- generated and inserted in TOPO-PCR4 vector prior to amplification in TOP10 cells (Invitrogen). Probe was prepared by excision using EcoRl restriction enzyme. Nature of the insert was confirmed by sequencing. Membranes were washed twice in 2X SSC, 0.1% SDS solution at 44OC for 5 min., and then washed twice in 0.1X SSC, 0.1% SDS for 15 min. at 44OC. After a 3 day-exposure, signals were detected by a Phosphorimager (Molecular Dynamics Inc.) and analyzed using ImageQuant software. In order to combine the data obtained from the blots the values were expressed as percent of control, so that measures were normalized against the cyclophillin signal in each group. Statistical analysis was accomplished using Sigma Stat (Jandel Scientific), and graphs were generated using Graph Pad (Prism) software.

RESULTS It was hypothesized that the MDMA-treated animals would show 5-HT terminal loss, and that the MDMA + LSD group would show more damage to the brain than those given MDMA alone. We also expected that the blocking action of the 5-HT 2~2~ receptor antagonist MDL 11,939 would decrease the toxicity seen in the MDMA + MDL 11,939 group in comparison to the MDMA-alone treated group. The damage to the brain was defined as the loss of 5-HT terminals, primarily in the hippocampus, as toxic effects in this structure are among the most consistent findings among researchers [ 14,151. It is evident from the results that LSD had a synergistic effect when given in conj unction with MDMA. The immunohistochemical staining shows a dramatic decrease in 5-HTTs in the hippocampus (Fig. 1.). It is also evident that there is less staining in the MDMA + LSD treated groups over the MDMA alone treated one, and that the staining progressively decreases as the LSD dose increases. NEUROSCIENCE RESEARCH COMMUNICATIONS, VOL. 35, NO. 2 81 There was little effect on 5-HTTs from the drug LSD when given alone. When optical density was quantified for the hippocampus it was found that MDMA, as well as all of the MDMA + LSD groups, were significantly different from the control group (p < .05, all pairwise multiple comparison procedure using Dunnett’s method). Moreover; the MDMA + LSD 100 group had significantly less staining than the MDMA group (p< .05, all pairwise multiple comparison Student Newman Keuls method; see Fig 3).

B

E

Fig. 1 Immunohistochermcal (MC) treated sections of the hippocampus of the LSD group at a magnification of 25X. A = Control, B = LSD lOOug/kgiday, C = MDMA 20mglkgiday (same as in ), D = MDMA 20mglkgiday + LSD 25&kg/day, E = MDMA 20mg/kg/day + LSD 50ug/kg/day, F = MDMA 20mg/kg/day + LSD lOO&kg/day. NEUROSCIENCE RESEARCH COMMUNICATIONS, VOL. 35, NO. 2

Fig. 2 IHC treated sections of the hippocampus of the MDL 11,939 group at a magnification of 25X. A = Control, B = MDL 7Smg/kglday, C = MDMA 20mglkgiday (same as in lC), D = MDMA 20mg/kg/day + MDL 2.5mg/kg/day, E = MDMA 20mgikglday + MDL Smgikglday, F = MDMA 20mglkgiday + MDL 7.5mgkg/day. NEUROSCIENCE RESEARCH COMMUNICATIONS, VOL. 35, NO. 2 89

Drug Treatments

Fig. 3 The NIH Image program was used to quantify the optical density of the hippocampus. The MDMA and MDMA + LSD-treated groups were all different from control (* = p < .05, all pairwise multiple comparison procedure using Dunnett’s method). The MDMA group was also different from the MDMA + LSD 100 group (t = p < .05, all pairwise multiple comparison using Student Newman Keuls method). Means are shown with error bars generatedusing S.E.M.

The group treated with Smglkg MDL 11,939 + MDMA appears to have a greater amount of staining in the hippocampus when compared to the MDMA treated group (Fig. 2.). It can also be seen that the group treated with MDL 11,939 did not have staining that was appreciably different from the control group (Fig. 3). The 5-HTT cDNA probe used for the Northern blot hybridized to the RNA in a pattern that was consistent with the results from the IHC portions of the experiment (see Fig. 4 for an image of one of the blots). There was less hybridization in the MDMA alone treated group compared to the control group, and the LSD alone treated group was not appreciably different from control (Fig. 4). There appears to be a relationship between the dose of LSD added to MDMA and the amount of binding seen on the blot, such that the MDMA + LSD 100 group showed the least amount of binding. There was a significant difference between the control group and all of the MDMA-treated groups (p < .Ol; Student’s “t” test). A Pearson’s correlation analysis revealed that there was a significant correlation between the dose of LSD added to MDMA (0, 25, 50, and lOOpg), and the amount of binding measured on the blot, r = - 395, p < .05, see Fig. 5. 90 NEUROSCIENCE RESEARCH COMMUNICATIONS, VOL. 35, NO. 2

Fig. 4 Picture of the Northern blot hybridized with the 5-HTT (top), and cyclophillin (bottom) probes. LSD Group

Drug Treatment Fig. 5 5-HTT expression as a percent of control for the ratios of 5-HTT/cyclophillin. CON = control (saline vehicle), MDMA 20mg/kg/day, LSD lOO&kg/day, MDMA+LSD groups received 20mg/kg/day of MDMA + 25, 50, or lOOpg/kg/day of LSD. For all groups n = 2, and all received twice daily S.C.injections of either drug cocktail or saline. Significant differences were found between the CON and MDMA-treated, as well as the MDMA+LSD-treated groups (* = p < .Ol, alpha-correctedt-tests). A significant correlation was also found between the dose of LSD given and the amount of expression (LSD alone, MDMA+LSD 25, MDMA+LSD 50, MDMA+LSD loo), r = -.895, p < .05. Means are shown with error bars generatedusing S.E.M.

The results from the MDL 11,939 group showed that this drug appeared to rescue the 5-HTTs from the toxic effects of MDMA (Fig. 6). The groups MDL alone, and MDMA + MDL 5 do not appear NEUROSCIENCE RESEARCH COMMUNICATIONS, VOL. 35, NO. 2 91 to differ from control. Therefore, the dose of Smg/kg appeared to be effective at attenuating MDMA- induced toxicity to the 5-HTTs as evidenced by the increased binding seen on the blot. However, because the doses of 2.5 and 7Smg/kg appear to be similar to the MDMA alone group, a dose response relationship does not appear to exist for MDL 11,939 in this experiment. MDL Group

0

Drug Treatment Fig. 6 5-HTT expression as a percent of control for the ratios of 5-HTT/cyclophillin. CON = control (saline vehicle), MAMA 20mg/kg/day, MDL alone 7Smg/kg/day. MDMA+MDL groups received 20mg/kg/day of MDMA + 2.5, 5, or 7.5 mg/kg/day of MDL 11,939. The MDMA, MDMA+MDL 2.5, and MDMA+MDL 7.5 were all found to be different from control (* = p < .Ol, alpha-correctedt-tests). Means are shown with error bars generatedusing S.E.M.

DISCUSSION Taken as a whole, the results show that MDMA induced neurotoxicity in these animals, and that there was a dramatic dose related increase in neurotoxicity when LSD was given concurrently with MDMA. Both the IHC and northern blot results confirm that the amount of 5-HTTs were decreased in these animals in the hippocampus. The drug LSD did not induce decreases in 5-HTTs. However, when LSD was given in conjunction with MDMA, the neurotoxic effects were increased considerably. The ~-HT~A/~c antagonist MDL 11,939 had a protective effect on 5-HTTs. However, the relationship between the dose of MDL 11,939 and the neurotoxicity seen was not as expected. It does appear from this experiment (and from previous ones; Schmidt et al. 1990a), that the most effective dose of this drug is Smg/kg in rats. 92 NEUROSCIENCE RESEARCH COMMUNICATIONS, VOL. 35, NO. 2 5-HTTs are ubiquitous in the brain and have been observed not only at terminals, but axons as well [23]. Moreover, it has been shown that 5-HT diffuses up to 20pm away from the terminals from which it is released and that 5-HTTs are present there. Therefore, the loss to 5-HTTs may provide evidence that there is damage to axons as well as terminals and may be more widespread than previously thought. The results support the model of Sprague and Nichols [l] in that it appears that the ~-HT~A partial agonist LSD potentiated the neurotoxicity of MDMA, whereas the ~-HT~A/~c antagonist MDL 11,939 protected the terminals and axons from this neurotoxicity. Although the doses of MDMA given in this experiment are large, they may approximate those taken by some human abusers [24], especially if inter-species scaling is taken into account [25]. MDMA seems to disrupt the function of the thermoregulatory system. When rats are given MDMA in an ambient temperature of 24’C or higher, then results, whereas when rats are in an ambient temperature of 10°C then hypothermia results [26]. There has been speculation that the neurotoxicity of MDMA is dependent upon hyperthermia. However, other studies using fluoxetine have shown that the toxicity of MDMA can be blocked without causing hypothermia [27]. Moreover, researchers have been able to show that with the use of ~-HT~A antagonists (ketanserin; [28], and MDL- 11,939 [22] they were able to block the toxicity resulting from MDMA without decreasing the hyperthermia of the animal. Although, Malberg et al., [27] showed that the blockade of toxicity by ketanserin was removed when the core temperature of the rat was increased. It is difficult to separate the toxic effects of a drug and its hyperthermic actions. Protection of toxicity by lowering body temperatures has been shown following a variety of drugs and may be because the neurochemical cascades that accompany the administration of a drug are slowed when the internal milieu of the animal is colder [29]. However, it does not appear that the toxicity of 5-HT axons and terminals following MDMA dosing is simply a result that is secondary to hyperthermia [30]. Moreover, in this study the ambient temperature was carefully monitored and did not deviate from 20°C. MDMA-induced neurotoxicity to 5-HT terminals may not be related to the release in dopamine [3 11. However, there is still convincing evidence that the release of dopamine is integral to the mechanisms of 5-HT neurotoxicity as , a dopamine uptake inhibitor, attenuates long-term depletion of dopamine which may contribute to long-term 5-HT depletion [32]. Moreover, it has been shown that prior depletion of dopamine does not induce reductions in plasmalemmal dopamine transport NEUROSCIENCE RESEARCH COMMUNICATIONS, VOL. 35, NO. 2 93 [33]. Therefore, the role of dopamine release in MDMA-induced toxicity to 5-HT terminals has not been eliminated. The ability of MDMA to exert some of its effects is thought to be linked to the ~-HT~A receptor, in part because 5-HT 2~ antagonists attenuate the toxicity [22], and it is known that ~-HT~A second messenger systems cause increases in intracellular Ca++ levels [34], and it has been shown that nimodipine (an L-type Ca” channel blocker) attenuates MDMA related toxicity [35,36]. It has been proposed by Kramer et al. [37] that the PKC translocation and activation that results from increased intracellular Ca* contributes to the toxicity of MDMA. However, Takei et al. [38] argue that this effect may be due to nimodipine’s action as an antioxidant (also see Zhou et al. [39]. Furthermore, ~-HT~A antagonists that decrease intracellular Ca” levels also significantly decrease dopamine levels following toxic doses of MDMA [22,39]. Finally, activation of the 5-HT 2~ receptor has been shown to increase dopamine release and synthesis [39,22], and it is suggested by Poblete and Azmitia [41] that activation of the ~-HT~A receptor results in an increase of glycogen phosphorylase activity, leading to glycogen breakdown, the depletion of the cell’s energy source, and eventually starvation (see also Darvesh and Gudelsky [42]). Therefore, the principal receptor responsible for the effects seen here could be the 5- HT~A receptor. Further experimentation will eventually tease out the differing roles of the ~-HT~A and 5- HT2c receptors in the brain.

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