NRSA: Enrichment and Methamphetamine Neurotoxicity

Specific Aim 1: Determine if environmental enrichment protects against methamphetamine-induced dopamine and serotonin neurotoxicity. Specific Aim 2: Determine if environmental enrichment protects against methamphetamine-induced loss of functional activity of the mesolimbic dopamine system as measured by in vivo microdialysis. Both Specific Aims 1 and 2 are currently being conducted and data collection will be completed by January 2004. For Specific Aim 1, enriched, isolated, and social reared rats have been raised in their respective environments, and they have received a treatment regimen of either saline or methamphetamine (10 mglkg) every 2 hours for a total of 4 injections. Fifteen days after treatment, rats were killed and the frontal cortex, striatum, and nucleus accumbens were dissected and frozen immediately. The samples are being kept at -70°C until all samples are collected for assay. Approximately 40 of the 60 rats required for the study have treated and the brains dissected. Once all samples are collected, then the tissue concentrations of monoamines will be assessed using HPLC. For Specific Aim 2, enriched and isolated rats have been raised in their respective environment and they have received a treatment regimen of either saline or methamphetamine (10 mglkg) every 2 hours for a total of 4 injections. Rats were allowed to recover for 5 days following treatment. Stereotaxic surgery was then conducted to implant cannula into the right striatum of each rat. After a second recovery period of at least 5 days, rats wer~ tethered and habituated to a liquid swivel system (BAS Beekeeper system for freely moving animals). On the next day, a microdialysis probe was inserted in the awake rat and there was 1 hour of habituation before any samples were collected. The probes were perfused at a rate of 1.2 Ill/min using a microdialysis pump with normal, artificial CSF consisting of 145 mM NaCl, 2.7 mM KCl, 1.2mM CaCh, 1.0 mM MgCh, 0.2 mM ascorbic acid, and 2.0 mM NaH2P04 (pH 7.4). All probes were calibrated for recovery of dopamine, DOPAC, HVA, 5-HIAA, and serotonin at 37°C and pH 7.4 prior to their use. For potassium stimulation, the CSF solution was switched to high potassium artificial CSF consisting of 47.7 mM NaCl, 100 mM KCl, 1.2mM CaCh, 1.0mM MgCh, 0.2 mM ascorbic acid, and 2.0 mM NaH2P04 (pH 7.4) for a single 20 minute fraction. Two hours later, 100 11Mmethamphetamine was included in the perfusate for a single 20 minute fraction. Five additional fractions were collected after the methamphetamine stimulation. Dialysate samples were immediately frozen at -70°C until the time of the assay. After the last sample had been collected, rats were killed by rapid decapitation. The right side of the brain was frozen and kept to verify the probe location using a cryostat. The frontal cortex, striatum and nucleus accumbens on the left side of the brain were dissected and frozen at -70°C until assayed. Currently, all dialysate and tissue samples have been collected. Approximately 75% of the samples have been assayed and the remaining 25% will be assayed by January 2004. Specific Aim 3: Determine if environmental enrichment prevents the methamphetamineinduced alteration of subsequent methamphetamine reward as measured by conditioned place preference. In the next year I plan to complete Specific Aim 3. For Specific Aim 3, enriched and isolated rats will be raised in their environments and will then receive a treatment regimen of either saline or methamphetamine (10 mg/kg) every 2 hours for a total of 4 injections. Rats will be allowed to rest for 5 days following treatment. The conditioned place preference (CPP) procedure will consist of one pretest day, 8 days of conditioning (4 trials), and one CPP test day. For the pretest, rats will be placed in the center compartment of a 3-compartment CPP chamber and will be allowed to explore all three compartments for 15 minutes. The time spent in each compartment will be measured. For the conditioning phase, a 4-trial conditioning procedure will be used. There will be four days of methamphetamine conditioning and four days of saline treatment. Methamphetamine conditioning days will alternate with saline treatment days. Rats in the saline groups will receive saline on all eight days. On each of the eight conditioning days, rats will be administered an injection of saline or methamphetamine (0.3 or 1.0 mg/kg, s.c.) immediately prior to being placed in one of the end compartments for 30 min with the solid partitions in place. One half of the rats in each group will be conditioned to the white compartment and the other half will be conditioned to the black compartment. Rats in the saline groups will be administered saline before being placed in the white and black compartments on alternating days. On the day following the eighth day of conditioning, each animal will be tested for CPP. For the test, rats will again be placed in the center compartment and will be allowed to explore all three compartments for 15 minutes. The time spent in each compartment will be measured. Following the CPP test, rats receivin,g saline during CPP will be killed and the frontal cortex, the striatum, and nucleus accumbens will be dissected and frozen immediately. The samples will be kept at -70 °c until all samples are collected for assay.