84 NEUROSCIENCE RESEARCH COMMUNICATIONS, VOL. 35, NO. 2 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 dopamine in target structures. MDMA is a popular drug 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 drugs 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 serotonin transporter (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 gram. 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.