Animals and conditions
Male Sprague-Dawley rats (250–275 g; Charles River Laboratories, Raleigh, NC) were allowed to acclimate to housing conditions (19 ± 1°C, 50 ± 10% humidity, and 14 h light: 10 h dark cycle with lights on at 600 h) for 2 days prior to either MA or forced swim treatment. Rats were first housed in the colony room in pairs in cages measuring 46 × 24 × 20 cm, but separated on the day of testing to 28 × 16 × 12 cm polycarbonate cages in a different room maintained at an ambient temperature of 24°C during drug or forced swim administration. Separation of animals was done to ensure a consistent environment rather than have the potential for aggression when MA was administered, especially since this would influence any stress-related parameters. Food and water were provided ad libitum except during treatment. All procedures were conducted in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals, and were approved by the Institutional Animal Care and Use Committee of Cincinnati Children's Research Foundation. The vivarium was accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC).
(+)-Methamphetamine-HCl (expressed as the freebase, from the National Institute on Drug Abuse and greater than 95% pure) or isotonic saline (SAL) was injected subcutaneously to animals in 4 doses with a 120 min inter-dose interval beginning at 900 h. Each dose consisted of 10 mg/kg in a volume of 3 ml/kg for a total daily dose of 40 mg/kg over the 6 h period. Each animal was weighed prior to the first injection. For Experiment-1, the treatment groups were MA and SAL and were run in two separate cohorts. Four post-treatment time points were assessed: 1, 7, 24, and 72 h after the first injection (n = 8, 8, 17, and 10, respectively). The 72 h time point was included to demonstrate neurotoxicity with this dosing regimen, although it was the early time points that were of interest. Injections of both MA and SAL were delivered in the dorsum and injection sites were varied to prevent skin irritation.
Forced swim has previously been demonstrated to increase corticosterone levels more than other stressors . Forced swim for 30 min was performed by placing each rat in a 15 cm (diameter) by 46 cm tall PVC cylinder filled with 35 cm of water (22 ± 1°C) either once or four times; as with MA treatment. FS was administered beginning at 900 h for the single stress group and beginning at 900 h × 4 at 2 h intervals for the multiple FS group. Animals were weighed prior to the beginning of the FS regimen. Three time points were examined: 1, 7, and 24 h after the beginning of the first FS. Group sizes were FS = 8, control = 8 for each of the 3 time points, or 48 animals total. Between FS trials, all animals were maintained in 28 × 16 × 12 cm polycarbonate cages in a separate room from the colony, exactly as in Experiment-1.
On the day of arrival, animals were lightly anesthetized with isofluorane and injected with implantable temperature transponders (IPTT-300: Bio Medic Data Systems, Seaford, DE). The subcutaneous probes were used to alleviate the hyperthermia and stress of rectal temperature measurements during dosing  and the physical manipulation of the animal that occurs with such recordings. Previous data have demonstrated that handling and rectal temperature measurements increase corticosterone and body temperature .
During the period of MA or FS treatment, body temperatures were monitored every 30 min beginning with the first treatment and for the next 8 h. In order to prevent excessive hyperthermia during MA treatment, a cooling protocol was followed. If an animal's temperature reached 40°C it was placed in a shallow bath of room temperature water and its temperature was then monitored every 5–10 min. Once an animal's temperature fell below 40°C it was removed from the water and returned to its holding cage. After 8 h (2 h after the last dose), animals were returned to the colony room. A comparison of cooled animals versus non-cooled animals demonstrated that cooling did not significantly attenuate the depletions in monoamines.
Animals were weighed on the day of drug or FS administration prior to the first treatment. For experiment-1, animals were also weighed 24, 48, and 64 h after MA dosing.
At the designated time points, animals were transported individually to an adjacent suite (< 30 s after removal from cage), decapitated , and blood collected in polyethylene tubes (12 × 75 mm) containing 2% EDTA (0.05 ml/tube). The brain was rapidly removed, placed over ice, and the neostriatum and hippocampus were dissected as described previously  as well as the prefrontal cortex and cerebellum. All tissues were frozen on dry ice and stored at -80°C until assayed.
The adrenal glands and thymus from each animal were removed, freed of fatty tissue, and weighed. The data for tissue were expressed as absolute tissue weight or as a percentage of body weight. For experiments-1 and 2, initial body weights for percentage of body weight analysis were utilized except for experiment-1 the 64 h body weight was used for the 72 h time point. These tissues were collected since they are known to be affected by hormones of the HPA axis, particularly during increased activation of this pathway [39–42].
Corticosterone, creatinine and glucose determinations
Blood was collected in ice-chilled polypropylene tubes containing EDTA, centrifuged at 4°C for 15 min, plasma aliquoted, and stored at -80°C until assayed for corticosterone and creatinine (Crn). For the assessment of corticosterone, plasma was diluted 5:1 in assay buffer and assayed in duplicate using a commercially available EIA kit for corticosterone (IDS, Fountain Hills, AZ). Glucose in whole blood was determined using a commercially available glucometer (Precision Xtra, Abbott Laboratories, Bedford, MA).
For creatinine, 300 μl of methanol were added to 100 μl of plasma in microcentrifuge tubes, vortex-mixed for 1 min, and centrifuged at 9,400 × g for 10 min. The supernatant was transferred to an autosampler vial, and 50 μl was automatically injected into a high pressure liquid chromatography (HPLC) system. A Rainin Microsorb-MV reversed-phase column (4.6 mm × 15 cm, 5-μm bead size) was used. The mobile phase consisted of potassium phosphate monobasic (40 mmol/l), sodium dodecyl sulfate (10 mmol/l), 190 ml methanol, and 180 ml acetonitrile (adjusted to a pH of 3 with phosphoric acid). The flow rate was 0.9 ml/min and the retention time for creatinine and cimetidine (internal standard) were 6.9 min and 11.4 min, respectively. The absorbance detector was set at 236 nm. The analysis was run at 30°C.
Free creatine was measured in the gastrocnemius muscle and in several brain regions including: cerebellum, neostriatum, prefrontal cortex, and hippocampus. Gastrocnemius muscle was chosen because MA-treated animals display hyperactivity and FS induces vigorous paddling, both behaviors require the use of the gastrocnemius muscle. Free creatine was measured using a fluorimetric technique described previously  with modification. Briefly, this method combines ninhydrin, at alkaline pH, with guanidine, and monosubstituted guanidines to form a fluorescent product that is specific for creatine. The assay has an effective range from 1.0 × 10-7 M to 2.5 × 10-5 M. The creatine standard was made from reagent grade creatine (MP Biomedicals, Inc.). Each sample was weighed and 200 μl of RIPA (50 mM Tris, 1% NP-40, 25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA, Na3VO4, 1 mM NaF along with 1 μg/ml of each: aprotinin, leupeptin, pepstatin) for each 100 mg of tissue was added prior to tissue homogenization. Homogenates were heated for 5 min at 103°C and then centrifuged at 32,091 × g (4°C) for 10 min. The supernatant was then collected.
Creatine determination involved adding 120 μl of the supernatant with 240 μl of Ba(OH)2 and 240 μl of ZnSO4. After the formation of a precipitate the samples were centrifuged at 32,091 × g (4°C) for 3 min. A total of 150 μl of the supernatant was placed in a 96-well, black-bottomed plate (ISC Bioexpress) and each sample was run in triplicate. Ninhydrin (75 μl) was added to each well while keeping the plate protected from external light during and after the addition of 75 μl of KOH. Eight min following the addition of the KOH, the plate was read using a fluorimeter (Spectramax M2; Molecular Devices) at an absorbance of 410 nm excitation and 525 nm emission. Flourimetric readings were recorded and creatine concentrations were calculated from standard curves generated from each plate and expressed as μmol/mg tissue.
Brain tissue concentrations of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), serotonin (5-HT), and 5-hydroxyindolacetic acid (5-HIAA) in the neostriatum and 5-HT and 5-HIAA in the hippocampus were quantified using HPLC with electrochemical detection as described previously . Tissues were homogenized in 50 volumes of 0.2 M perchloric acid and centrifuged for 6 min at 10,000 × g. Aliquots of 20 μl were injected onto a C18-column (MD-150, 3 × 150 mm; ESA, Chelmsford, MA) connected to either a LC-4B amperometric detector (Bioanalytical Systems, West Lafayette, IN) or a Coulochem (25A, Chemsford, MA) detector and an integrator recorded the peak heights that followed each injection. The potential for the LC-4B was 0.6 V and the potentials of E1 and E2 on the analytical cell (model 5014B) of the Coulochem were -150 and -160 mV, respectively and Ag/AgCl reference electrodes were used. The mobile phase consisted of 35 mM citric acid, 54 mM sodium acetate, 50 mg/l of disodium ethylenedeamine tetraacetate, 70 mg/l of octanesulfonic acid sodium salt, 6% (v/v) methanol, 6% (v/v) acetonitrile, pH 4.0, and pumped at a flow rate of 0.4 ml/min. Quantities of the analytes were calculated on the basis of known standards. Retention times for DOPAC, DA, 5-HIAA, and 5-HT were approximately 3.0, 5.0, 7.0, and 20.0 min, respectively.
Neostriata from Experiment-1 (72 h) were homogenized in cold RIPA buffer. Protein concentrations were determined using Pierce BCA Protein Assay Reagent Kit (Rockford, IL) according to the manufacturer's specifications and homogenates were diluted to a concentration of 1 μg protein/ml with PBS. Samples (10 μl) were diluted 1:1 with 2× loading buffer, boiled for 5 min and loaded on 8–16% polyacrylamide gels (ISC BioExpress, Kaysville, UT) with control and experimental animals equally distributed on each gel. Proteins separated by electrophoresis were transferred in a buffer of 25 mM bicine, 20 mM Tris, and 10% MeOH at 40 V for 1.5 h to Immobilon-PVDF membranes (Millipore, Bedford, MA). Membranes were blocked by incubating at 4°C with 3% BSA in PBS with 0.1% Tween-20 (PBST) overnight to prevent nonspecific binding of antibodies. Membranes were then incubated for 1 h with a 1:500 dilution of GFAP (Fitzgerald, Concord, MA) or actin (Chemicon International, Temecula, CA) antibody. Following washing (3 × 5 min with PBST), membranes were incubated for 30 min at room temperature with 1:5000 dilution of goat anti-mouse conjugated to alkaline phosphatase for both GFAP and actin. Membranes were washed (3 × 5 min with PBS), followed by incubation with CDP Star chemiluminescent (KPL, Gaithersburg, MD) substrate for 5 min. Membranes were exposed to film until adequate signal development was achieved; films were then scanned and protein bands were quantified using ImageJ software (NIH, Bethesda, MD). The density of the GFAP or actin band of the treated samples was divided by the density of the control sample in each gel and GFAP values were divided by actin values to control for protein concentration for each sample.
Data were analyzed using factorial analysis of variance (ANOVA), general linear model (GLM; SAS Institute, Cary, NC) or t-tests (GFAP only). Treatment (MA or SAL, Exp-1; FS or Naïve, Exp-2) and Time (1, 7, 24, or 72 h) were between-subject factors. Significance was set at a level of p ≤ 0.05. Data are presented as group means ± SEM.