Temperature measurements. Temperature was determined by the use of a rectal probe (YSI telethermometer from Yellow Springs Instruments, Yellow Spring, OH). Temperature was recorded once before and at 30-min intervals during the initial 2 h after the methamphetamine (METH) injection and at 1 h intervals thereafter for a total period of 7 h.

cDNA array preparation and analysis. Striatal tissues were taken from rats killed at 1 h after METH or saline injection. Tissues were processed using the Qiagen RNeasy Midi kit (Qiagen, Valencia, CA) to isolate total RNA. The experiments were done in three replicates [i.e., each time point has three RNA samples, with two animals per RNA sample (METH or saline), and each sample was hybridized to one cDNA array membrane]. The confirmation of the integrity of total RNA, RNA labeling, probing, and hybridization was according to our previously published procedures (1). Radiolabeled ³³P cDNA probes were hybridized to Atlas Plastic Rat 4-k Microarray by using ExpressHyb Plastic Hybridization solution according to the manufacturer’s instructions (BD Biosciences, San Diego). After a high-stringency wash, the membranes were exposed to a phosphor screen and scanned by using a Storm 840 Phosphoimager (Molecular Dynamics). Further analysis of cDNA arrays and hierarchical clustering is also similar to the published protocol (1).

RT-PCR analysis. To confirm the data obtained from the arrays, RT-PCR was performed on RNA isolated from striatal tissues according to protocols published by us (2). Briefly, Oligo dT primers and Advantage RT for PCR Kit (BD Biosciences) were used to reverse-transcribe the total RNA isolated. LightCycler FastStart DNA Master SYBR Green 1 kits (Roche Molecular Biochemicals) were used for the PCR experiments. Primers specific for the genes of interest were obtained from Synthesis and Sequencing Facility of Johns Hopkins University (Baltimore). The sequences are listed in Table 1. Clathrin was used as control.

HPLC analysis. Striatal tissues from the experimental rats (3 days after METH/saline administration) were weighed, ultrasonicated in 10% perchloric acid containing 10 ng/ml of the internal standard dihydroxybenzilamine, and centrifuged at 20,000 ´ g for 10 min. Concentrations of dopamine (DA) and homovanellic acid (HVA) were measured by HPLC with electrochemical detection as we described previously (3). Contents of DA and HVA were calculated as picograms per milligram of tissue.

Double-label immunofluorescence. Briefly, at 24 h after METH injection, the animals were perfused transcardially, under deep pentobarbital anesthesia, first with saline followed by 250 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (PB) at 4°C. The brains were removed and postfixed overnight in 4% paraformaldehyde. On the next day, 30-m m coronal sections were cut by using a cryostat. Free-floating sections were incubated for 30 min in 1% BSA/0.3% Triton X-100, followed by incubation with Fas ligand (FasL)/NeuN, FasL/GAD, FasL/ENK, or FasL/SP for 1 h at 37°C, then 4°C overnight. Subsequently, they were incubated with antibodies including biotinylated anti-mouse and fluorescein anti-rabbit IgG. Texas Red Avidin DCS was used to detect monoclonal antibodies. After immunostaining, images were processed by using a Zeiss LSM 410 microscope. Excitation and emission wavelengths were selected according to the suggested index.

METH administration causes induction of various transcription factors in the rat striatum. To confirm the array results (Fig. 6), we used quantitative PCR to measure transcript levels of the members of the Jun, Fos, Egr, and Nurr77 families of transcription factors. Transcript levels for c-Jun increased very early, reaching 3- to 5-fold increases during the course of the experiments (Fig. 7A). The changes in JunB mRNA also occurred very early, reaching close to 10-fold increases within the first hour after drug administration; these values had decayed toward normal by 4 h after drug administration (Fig. 7A). The increases in JunD mRNA, which was not identified as changed in the array experiments (Fig. 6), were of much lower magnitude than those observed for c-Jun or JunB, reaching only » 1.8-fold increases (Fig. 7A).

Because members of the Jun family of transcription factors (TFs) can form heterodimers with the fos members, we investigated the possibility that the latter were also affected by METH administration. To that end, we measured the expression of c-fos, fosB, Fra1, and Fra2 after METH injection. As can be seen in Fig. 7B, METH caused significant changes in their expression, with fosB showing the highest increases (» 50-fold) at 2 h after drug injection. c-Fos showed » 20-fold increases at 1 h postdrug, whereas Fra1 and Fra2 showed up to 5- and 6-fold increases, respectively (Fig. 7B).

We also confirmed the changes in Egr-1 and Egr-2 expression and investigated the effects of METH on the expression of the two members (Egr-3 and Egr-4) of this family of TFs (4, 5), because a literature search using Bibliosphere (Genomatix, Munich) revealed that the Egr transcription factors usually cluster together and exhibit similar responses to various stimuli including neurotransmitters, growth factors, and others (5). Fig. 7C shows that METH-induced changes in Egr-1, -2, and -3 occurred in a time-dependent manner in the striatum, with Egr-2 showing the greatest magnitude of changes (15- to 17-fold increases). These values had normalized at 4 h postinjection. Egr-4 showed only minimal changes (data not shown).

The array experiments had also documented METH-induced increases in the expression of Nur77 (Nr4a1, NGFI-B), which is an immediate early gene that codes for an orphan steroid receptor (6). We confirmed these results and extended them by measuring the expression of the other two members of the Nur77/NGFI-B subfamily of TFs, namely Nr4a2 (Nurr1) (7) and Nr4a3 (NOR-1) (8), because these TFs have been implicated in causing cell death (9). Fig. 7D shows that METH caused increases in all three members.

The DA antagonist, SCH23390, blocks METH-induced neurotoxicity in the rat striatum. To more directly assess the involvement of the calcineurin/nuclear factor of activated T cells (NFAT) cascade in the mediation of METH-induced neuronal apoptosis, we opted to use the DA D1 receptor antagonist, SCH23390, that has been reported to block METH toxicity on the striatal dopaminergic system (10). Thus, we measured the effects of pretreatment with the D1 antagonist on METH-induced cell death, monoamine contents, as well as body temperature. The effects of the combination of SCH23390 with METH on striatal concentration of DA and HVA are shown in Fig. 8A. Compared with saline-treated controls, METH caused 50% reduction (P< 0.001) in levels of both DA and HVA measured at 3 days after injecting the drug. Pretreatment with SCH23390 completely blocked the toxic effects of METH on DA and HVA (Fig. 8A). Similar results were observed for DOPAC (3,4-dihydroxyphenylacetic acid) (data not shown). Other investigators have reported similar findings in METH-treated rats (10). As can be seen in Fig. 8B, pretreatment with SCH23390 before METH injection also caused significant decreases in the number of TUNEL-positive cells in comparison to the METH alone group. Fig. 8C shows the results of these drug combinations on METH-induced increases in body temperature recorded over a 7 h period. Baseline body temperature averaged 39.5°C and did not differ among experimental groups. METH injection caused increases in body temperature, which approximated 1.5°C above basal levels. These numbers reverted to normal levels after 5 h. In contrast, the combination of SCH23390 and METH caused decreases that approximated 1.5-2°C below basal levels for that group. These decreases occurred during the first 3 h after drug injection and then reverted to normal. Bronstein and Hong (11) also showed SCH23390 in METH-treated rats decreased body temperature below basal levels.

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