Kowsky et al. 10.1073/pnas.0706177104. |
Fig. 6. Quantification of nigrostriatal fiber tract close to its origin. The number of (A) and total area covered by (B) nigrostriatal fibers were quantified in cryosections stained for TH, 120-240 mm rostral to the first substantia nigra soma.
Fig. 7. DAT protein expression in striatum of mice with or without DAT-Cre. Western blot analysis of DAT expression in the striatum of WT mice (-) and mice carrying the DAT-Cre construct (+) at 3, 12, and 24 months of age. The exposure times vary among different age groups to detect DAT also in mice carrying the DAT-Cre construct. In 3-month-old mice, as used in this study, there were comparable levels of DAT in both mouse groups and only in aging mice do the DAT levels decrease in mice carrying the DAT-Cre construct. Western blots for a-tubulin are shown as loading controls.
Fig. 8. Influence of Ret genotype on TH mRNA and protein expression. (A) Relative gene expression of TH by using real-time PCR normalized to GAPDH expression. The first three bars represent the relative TH expression in the striatum, the second three in the SNpc, and the last three in the liver as negative control for the genotypes Retlx, DAT-Cre, DAT-Retlx/lx (n = 4). TH expression is represented as mean ± SEM. For all three tissues, there were no significant differences for TH mRNA expression among genotypes; ANOVA followed by Tukey's post hoc test. (B and C) Relative protein expression and representative Western blot of TH normalized to b-actin. The first three bars and columns represent the relative TH expression in the striatum, the second three in the SNpc, and the last three in the liver as negative control for the genotypes Retlx, DAT-Cre, DAT-Retlx/lx (n = 4). TH expression is represented as mean ± SEM. For all three tissues, there were no significant differences in TH protein expression among genotypes; ANOVA followed by Tukey's post hoc test.
SI Text
Behavioral Analysis. Methods.
For open-field analysis, animals were surveyed in a 50 ´ 50 cm arena for 10 min by using a video tracking system (Videomot2, TSE Systems). Rearing was detected by infrared beams. Animals were placed into the arena before treatment, then 7, 14, 28, and 90 days after completion of the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or saline treatment.Results
. Before MPTP or saline treatment, dopamine transporter (DAT)-Cre, Retlx, and DAT-Retlx/lx mice did not show differences in the number of rearings (47 ± 23, 49 ± 21 and 44 ± 19 over the 10 min of tracking) and/or in the total running distance (3,972 ± 1,117, 3,168 ± 1,353 and 3,372 ± 837 cm in 10 min, respectively). Although we observed a reduction of both parameters after MPTP treatment, these reductions were not significantly different from those of saline-treated animals and therefore were not used for further analysis.Discussion.
Ideally, the observed changes in synaptic markers should be reflected by behavioral changes. In the literature, various paradigms have been described to assay behavioral consequences of MPTP treatment. Most of them have not been used repeatedly by the same or taken over by other groups. One exception is open-field analysis in a novel environment 14 days after MPTP treatment in untrained animals without repeated measurements (1-3). Because we were interested in the recovery from MPTP-induced defects, we chose repeated measurements of running distance and rearing. Most importantly, we did not observe differences among the three genotypes at baseline, which is consistent with our results of striatal dopaminergic fibers and catecholamines. Changes in subsequent measurements, however, exceeded differences among treatments, thus questioning their validity as markers for dopaminergic function, and most likely reflect the consequences of repeated handling, i.p. injections, and fading novelty of the arena.Fiber outgrowth from substantia nigra pars compacta. Methods.
Nigrostriatal fibers were quantified in cryosections stained for tyrosine hydroxylase (TH) (by using diaminobenzidine for stereology), 120-240 mm rostral to the first soma of dopaminergic substantia nigra neurons. Images were acquired by using the ×20 objective of a Leica DMI 6000 B microscope. Fibers were detected by thresholding, blinded for treatment and genotype. We determined the number of fibers and the total area covered by fibers by using ImageJ 1.38v (NIH) and the plug-in Threshold_Colour by Gabriel Landini (School of Dentistry, University of Birmingham).Results and discussion.
We measured the density of TH-positive nigrostriatal fibers close to their origin in the direct proximity of the substantia nigra pars compacta (SNpc). The number of TH-positive fibers, as well as the area covered by these fibers, was substantially reduced at 14 days after MPTP treatment and did not recover after 90 days. For both parameters and at both time points, there was no difference between genotypes. Together, these data suggest that there is no outgrowth of new axons but likely a sprouting response in the terminal field of the nigrostriatal pathway (SI Fig. 6).TH mRNA and protein expression. Methods.
Total RNA was isolated out of the striatum, SNpc, and liver of four PBS-perfused Retlx, DAT-Cre, and DAT-Retlx/lx mice by using the RNeasy Mini Kit (Qiagen) following the instructions of the manufacturer. DNA was digested by RQ1 RNase Free DNase (Promega) and protected against RNases by adding 20 units of RNase Inhibitor RNasin (Promega). Total RNA (2.5 mg) was reverse transcribed with 250 units of M-MLV reverse transcriptase (Promega). Two microliters of a 1:5 diluted cDNA sample was amplified by real-time PCR by using SYBR Green (Thermo Scientific, Epsom) and PCR primers for tyrosine hydroxylase (TH sense primer, 5′-GGTATACGCCACGCTGAAGG-3′; antisense primer, 5′-TAGCCACAGTACCGTTCCAGA-3′) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH sense primer, 5′-TGGCAAAGTGGAGATTGTTGCC-3′; antisense primer, 5′-AAGATGGTGATGGGCTTCCCG-3′) as a reference gene. Reactions were performed in a Mx3000P sequence detection system (Stratagene). Quantitative real-time PCR analysis was carried out by using the 2-DDCt method. Data are represented as means with SEM, and ANOVA was performed by using the Prism software (GraphPad). In all analyses, differences were considered statistically significant at P < 0.05.Total protein was isolated out of the striatum, SNpc, and liver of four PBS-perfused Retlx, DAT-Cre, and DAT-Retlx/lx mice via acetone precipitation from RLT buffer lysates by using the RNeasy Mini Kit (Qiagen) and following the instructions of the manufacturer. Total protein was resuspended in 0.23 M Tris•HCl (pH 6.8), 50% glycerol, and 5% SDS. Protein concentration was measured and calculated by using the DC Protein Assay (Bio-Rad), a Mithras LB 940 plate reader, and Mikro Win software 2000 (Berthold Technologies). Laemmli buffer (5×) was added, and the samples were boiled for 5 min. Twenty micrograms of total protein per sample were subjected to SDS/PAGE (10%), transferred to nitrocellulose membranes (Whatman), and immunoblotted by using a TH rabbit antibody (Chemicon, Chandlers Ford, UK, 1:3,000), a mouse b-actin antibody (Sigma-Aldrich; 1:6,000), and adequate secondary antibodies conjugated to HRP (GE Healthcare; 1:10,000).
Bound proteins were visualized by enhanced chemiluminescence (Chemi Glow, Alpha Innotech) by using the AlphaImager technique and Fluor Chem 8900 software (Alpha Innotech). TH expression was quantified as a ratio to b-actin expression and represented as mean with SEM.
Results.
DAT-Retlx/lx mice showed no alteration in the expression of TH mRNA and protein as compared with DAT-Cre and Retlx mice in the striatum and SNpc (SI Fig. 8).Discussion.
The lack of Ret has no influence on mRNA and protein expression of TH. Therefore, the differences observed in the dopamine concentrations were unlikely to be influenced by differences of TH expression that directly depend on Ret signaling.1. Crocker SJ, Smith PD, Jackson-Lewis V, Lamba WR, Hayley SP, Grimm E, Callaghan SM, Slack RS, Melloni E, Przedborski S, et al. (2003) J Neurosci 23:4081-4091.
2. Hayley S, Crocker SJ, Smith PD, Shree T, Jackson-Lewis V, Przedborski S, Mount M, Slack R, Anisman H, Park DS (2004) J Neurosci 24:2045-2053.
3. Smith PD, Crocker SJ, Jackson-Lewis V, Jordan-Sciutto KL, Hayley S, Mount MP, O'Hare MJ, Callaghan S, Slack RS, Przedborski S, et al. (2003) Proc Natl Acad Sci USA 100:13650-13655.