cell type: spiny projection neurons lesion: 6-OHDA trap line: CP101 drug treatment: Chronic high levodopa genetic background: C57BL/6J genotype: Drd2::EGFP-L10a
Treatment protocol
A hemi-parkinsonian condition was induced in mice by unilateral injection of 6-OHDA in the right medial forebrain bundle (MFB). Animals were anesthetized with a mixture of ketamine (Putney Inc., Portland, ME) and xylazine (Lloyd Inc., Shenandoah, IA) and mounted on a Kopf stereotaxic frame in a flat-skull position. 6-Hydroxydopamine (6- OHDA) hydrochloride (Sigma Chemical Co., St. Louis, MO) was dissolved at a concentration of 3.2 μg/μl free-base in 0.02% ice-cold ascorbate/saline and used within 2 hrs. One microliter of this solution was injected at the following coordinates (in mm relative to bregma, sagittal suture and dural surface): AP= -1.2, L= -1.3, DV= -4.75. Two weeks after the lesions, mice were evaluated with tests of spontaneous ipsilateral turning behavior and limb use asymmetry (cylinder test) in order to select animals with successful lesions (1). For TRAP studies of the effect of dopamine depletion alone, we compared animals that received an MFB lesion to animals that received a mock lesion (ascorbate/saline alone injected). Both of these groups went on to receive chronic saline injections, so that the MFB group could also be used as a comparison group for those additional MFB-lesioned mice that received chronic levodopa injections. L-DOPA methyl ester and the peripheral DOPA decarboxylase inhibitor benserazide- hydrochloride (Sigma Chemical Co., St. Louis, MO) were dissolved in physiological saline immediately prior to use. The drugs were injected at the volume of 0.1 ml/10 g body weight in a single intraperitoneal (i.p.) injection per day. Benserazide was always administered at a fixed dose (12 mg/kg per injection). Three weeks after lesion surgery, mice with successful lesions were randomly allocated to receive treatment with ‘high- dose L-DOPA’, ‘low-dose L-DOPA’, or saline. The ‘high-dose’ L-DOPA regimen (3 mg/kg on days 1-3, followed by 6 mg/kg on days 4-9) was expected to induce severe dyskinesia in all MFB-lesioned mice (1, 3). The low-dose L-DOPA regimen (1 mg/kg on days 1-3, followed by 2 mg/kg on days 4-9) was expected to reverse limb use asymmetry without causing conspicuous dyskinesias. To equalize the effects of stress and handling across all groups, including control groups, all mice were equally handled and thus received saline injections when not receiving levodopa injections. Abnormal involuntary movements (AIMs) were rated using a validated scale, as previously described (1). Each mouse was observed individually for 1 min every 20 min for 3 hr, starting 20 min after L- DOPA/benserazide (or saline) administration. Only hyperkinetic and dystonic movements that could be clearly distinguished from naturally occurring behaviors (i.e. grooming, sniffing, rearing and gnawing) were considered in the ratings. The AIMs were classified into three subtypes: (i) axial AIMs, i.e. twisting of the neck and upper body towards the side contralateral to the lesion; (ii.) orolingual AIMs, i.e. jaw movements and contralateral tongue protrusion; (iii) forelimb AIMs, i.e. fluttering movements of the forelimb. Each AIMs subtype was rated on a severity scale from 0 to 4 (0, absent; 1, present during less than 50% of the observation time; 2, present during more than 50% of the observation time; 3, present all the time but suppressible by light visual-auditory stimuli; 4, continuous, severe and not suppressible).
Growth protocol
Mice were housed with food and water provided ad libitum. Experiments were conducted with Drd1::EGFP-L10a or Drd2::EGFP-L10a BAC transgenic, adult (9-14 weeks) male mice of the C57BL/6J strain background.
Extracted molecule
total RNA
Extraction protocol
Immediately following the last session of behavioral testing, mice were sacrificed by decapitation and the dorsal striatum ipsilateral to the lesion of each mouse was manually dissected for TRAP purification of mRNA. As noted above, to equalize the effects of stress and handling across all groups, including control groups, all mice were equally handled and thus received saline injections when not receiving levodopa injections. TRAP purifications were carried out as described in (Heiman, 2008), with the following modifications: purifications were conducted from single animal tissue; the affinity matrix consisted of anti-GFP monoclonal antibodies 19F7 and 19C8 (Memorial Sloan-Kettering Monoclonal Antibody Facility, New York, NY) bound to biotinylated Protein L (Thermo Scientific, Rockford IL)-coated Streptavidin MyOne T1 Dynabeads (Invitrogen, Carlsbad CA); affinity matrix was bound to clarified tissue lysate for 16-18 hours; TRAP-purified RNA was loaded directly onto Absolutely RNA Nanoprep Kit purification columns (Agilent Technologies, Santa Clara CA) and purified with on-column DNase digestion.
Label
Biotin
Label protocol
Standard Affymetrix protocol for mouse 430 2.0 platform.
Hybridization protocol
Standard Affymetrix protocol for mouse 430 2.0 platform.
Scan protocol
Standard Affymetrix protocol for mouse 430 2.0 platform.
Description
Lesion_CH_67_CP101_1597
Data processing
Affymetrix CEL files were processed and normalized (across all samples) using the RMA algorithm from the 'affy' package in Bioconductor 2.13