animal: Rat strain: Sprague-Dawley gender: Male age: 8 weeks weight: 330-370 g tissue: substantia nigra (SN) and ventral tegmental area (VTA) treatment: injected with increasing doses of methamphetamine, challenged 72 hours later with saline
Biomaterial provider
Charles Rivers Laboratories, Raleigh, NC
Treatment protocol
Following habituation, rats were injected intraperitoneally with either (+-)-METH-hydrochloride (NIDA, Baltimore, MD) or an equivalent volume of 0.9% saline for a period of three weeks as described in Table S1 in supplemental data. The saline- or METH-pretreated animals received either saline or METH (5 mg/kg x 8 at 1 h intervals) challenges 72 hours after the preconditioning period (See Below). Similar doses of METH are known to cause significant decreases in the levels of monoamines in the rat striatum (Graham et al., J. Neurochem. 2008; 105:1873-1885, Cadet et al, Neurotox. Res. 2009; 15:252-259) which received dopaminergic terminals from midbrain dopaminergic cell bodies located in the substantia nigra (SN) and ventral tegmental area (VTA) (Lindvall and Björklund, Biochem Psychopharmacol 1978; 19:1-23, Domesick, Ann N Y Acad Sci. 1988; 537:10-26). Thus, the four groups of animals were: saline/saline (SS), saline/METH (SM), METH/saline (MS), and METH/METH (MM). The animals were euthanized 24 h later by decapitation. Their brains were quickly removed, striatal and SN/VTA tissues were dissected on ice, snap frozen on dry ice, and stored at -80°C until used. Table S1. Schedule of METH pretreatment and challenges Monday Tuesday Wednesday Thursday Friday Week 1 9:00 0.5 mg/kg 1 mg/kg 1 mg/kg 1.5 mg/kg 10:00 11:00 1 mg/kg 1.5 mg/kg 12:00 13:00 1 mg/kg 1.5 mg/kg 14:00 15:00 0.5 mg/kg 1 mg/kg 1 mg/kg 1.5 mg/kg 16:00
Initially the rats were divided into two groups, with one group receiving saline and the other group getting METH pretreatment according to the schedule described below during first and second weeks as well as Monday of the third week. The saline pretreatment was followed by with either saline (SAL/SAL) or METH challenges (SAL/METH), the METH pretreatment was followed by METH challenges (METH/METH) or METH followed by Saline (METH/SAL) on Wednesday of the third week and killed 24 h later.
Growth protocol
Animals were housed in a humidity- and temperature-controlled room and were given free access to food and water.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated using Qiagen RNeasy Midi kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. RNA integrity was assessed using an Agilent 2100 Bioanalyzer (Agilent, Palo Alto, CA) and showed no degradation.
Label
biotin
Label protocol
600 ng aliquot of total RNA from each striatal sample was amplified using Ambion’s Illumina RNA Amplification kit (cat. no. IL1791; Ambion, Austin, TX). Single-stranded RNA (cRNA) was generated and labeled by incorporating biotin-16-UTP (Roche Diagnostics GmbH, Mannheim, Germany, cat. no. 11388908910)
Hybridization protocol
750 ng of each cRNA sample were hybridized to Illumina arrays at 55 oC overnight according to the Illumina Whole-Genome Gene Expression Protocol for BeadStation (Illumina Inc., San Diego, CA, cat. # 11201828). Hybridized biotinylated cRNA was detected with Cyanine3-streptavidin (Amersham Biosciences, Piscataway, NJ, cat. #146065).
Scan protocol
Signals quantified using Illumina's BeadStation 500GX Genetic Analysis Systems scanner.
Description
The four groups of animals were: saline/saline (SS), saline/METH (SM), METH/saline (MS), and METH/METH (MM).
Data processing
The Illumina Inc. BeadStudio background corrected data is processed by the standard one-color GeneSpring normalized protocol. Briefly, in the data transformation process, values less than 0.01 become 0.01, per-chip procedure normalized the data to the median of the array, and per-gene procedure normalized the gene probes to their median across all arrays.
