Article title: Deuterium-substituted L-DOPA displays increased behavioral potency and dopamine output in an animal model of Parkinson´s disease: comparison with the effects produced by L-DOPA and an MAO-B inhibitor
Journal name: Journal of Neural Transmission
Author names: Torun Malmlöf, Kristin Feltmann, Åsa Konradsson-Geuken, Frank Schneider, Rudolf-Giesbert Alken, Torgny H. Svensson and Björn Schilström
Corresponding author: Torun Malmlöf, KarolinskaInstitutet, Department of Physiology and Pharmacology, Nanna Svartzväg 2, 171 77 Stockholm, Sweden. Email:
SUPPLEMENTARY DATA
Materials and methods
Transcardial perfusion
Following dialysis experiments, the rats were deeply anaesthetized by an injection of sodium pentobarbital (≈120 mg/kg i.p., Apoteket AB, Sweden), placed on ice and transcardially perfused with saline followed by ice cold 4% paraformaldehyde (Sigma-Aldrich, Sweden). The brain was dissected and placed in 4% paraformaldehyde for 2 hours and then transferred to 20 % sucrose for at least three days. The brains were frozen on dry-ice and stored in -20⁰C.
Histology
The brains were cut in 30 µM sections on a microtome; striatal sections were collected and stored in 0.1 M PBS at 4⁰C for further immunohistochemical analysis. Sections showing tissue damage from the dialysis probe were processed separately and mounted on superfrost slides, stained with neutral red and finally dehydrated. The position of the dialysis probe was verified under light microscopy and compared to the rat brain atlas by Paxinos & Watson, 6TH edition.
Tyrosine hydroxylase (TH) immunohistochemistry
Sections were rinsed in 0.05 M PBS and unspecific antibody binding was reduced by incubation in 10% BSA and 0.03% Triton in 0.05 M PBS for 1 hour. The tissue was incubated with the primary polyclonal antibody, rabbit anti-tyrosine hydroxylase (1/5000 diluted in carrier solution: 1% BSA and 3% Triton in 0.05 M PBS) (AB152, Millipore, USA), for 48 hours in 4⁰C. Sections were further rinsed in carrier solution and incubated with the secondary biotinylated antibody, sheep anti-rabbit (1/400 diluted in carrier solution, Vectastain Elite ABC, Vector Laboratories, United states), for 1 hour at room temperature. Following incubation, sections were rinsed in carrier solution and incubated in avidin-biotin-conjugated horse radish peroxidase (1/1000 in 0.05 M PBS, Vectastain Elite ABC, Vector Laboratories, United States) for 1 hour at room temperature. After rinsing in 0.05 M PBS a solution of 3´3´-diaminobenzidine (ImmPACT DAB, Vector Laboratories, United states) was applied for 5 minutes to stain immunopositive areas. Sections were rinsed in distilled water, mounted on superfrost slides and dehydrated. Digital pictures of the stained sections were captured by a Nikon D300 digital camera (Nikon, Sweden). The pictures were converted to grey scale in the Image J software (free download at and inverted. The mean grey value from the intact and the lesioned side were measured and the background value was subtracted from both.
In vitro radioligand binding assays
The binding of dopamine and deuterated dopamine to dopamine receptors D1, D2 (L) and D2 (S) as well as to the dopamine transporter (DAT) was evaluated using radioligand binding assays. Radioligand binding assays were performed according to a standard screening protocol (Chiu et al. 2004) by MDS Pharma Services, Taiwan Ltd. The receptors and transporters were expressed by human recombinant Chinese hamster ovary (CHO) cells.The test compounds were dopamine-Hbr, dopamine with double β-carbon substitutions (β,β-D2-dopamine) and dopamine with double α and β-carbon substitutions (α,α,β,β-D4-dopamine).The inhibition of the binding of a specific radioligand to dopamine receptors and theDAT was assessed at a single concentration of the test compounds and analyzed by means of liquid scintillation, for the specific experimental conditions see Table 1.
Statistics
Comparisons of group means were performed by one way ANOVA or the t-test when appropriate. All statistical calculations were performed in the STATISTICA software version 10 (StatSoft, Inc. United states) and the significance level was set to α=0.05.
Results
Tyrosine hydroxylase immunoreactivity
Fig. 1a shows a representative striatal section stained for TH, the lesioned hemispehere shows no immunoreactivity. The mean loss of TH immunoreactivity in the lesioned striatum was 97.6%±0.7 and there was no significant difference between treatment groups (Fig. 1b).
Radioligand binding
In order to verify if deuterated dopamine, which is formed following decarboxylation of administered deuterium-L-DOPA, interacts differently with dopamine receptors or the transporter radioligand assays were performed. The biding of dopamine and deuteriated dopamine to the different receptor subtypes and the DAT are shown in Fig1.There were no significant difference in the % inhibition between dopamine and deuterated dopamine in any of the assays.
References
Chiu PJ, Marcoe KF, Bounds SE, Lin CH, Feng JJ, Lin A, Cheng FC, Crumb WJ, Mitchell R (2004) Validation of a [3H]astemizole binding assay in HEK293 cells expressing HERG K+ channels. J Pharmacol Sci 95:311-319.
Figure legends
Table 1Experimental conditions of the different radioligand binding assays
Fig. 1a,Representative picture of a TH-stained section of the striatum. b, Loss of TH-immunoreactivity in the lesioned striatum.Data is presented as mean ±SEM. c,Radioligand binding assay of dopamine and deuterated dopamine towards the DAT and dopamine receptors D1, D2 (S) and D2 (L). Data is presented as mean ±SEM of two replicate assays