Supplemental Materials and Methods
Generation of transgenic mice
Recombineering reagents were generously provided by the Biological Resources Branch, National
Cancer Institute-Frederick (Maryland, USA). The DTR-GFP construct was generously provided by Dr
Jung (Jung et al. 2002). The PCR primers used to amplify the homology arms were as follows:
homology region 5’ATG: TCC TGC GAG TCT CGA GCT CCG CCA CGG CGG GGT ACG GCG GGG CCG GCG
CGA TGA AGC TGC TGC CGT CGG TGG; homology region 3’ATG: TGC GCG ATC GCC ACC GAG ATG
CCC AGC ACG CGG CCC CGG CAC AGC TGC CGG TTA TAT TAT GTA CCT GAC TGA TGA AGT TCC.
Quantitative RT-PCR
After extraction, RNA concentration was determined with a nanodrop ND-1000 spectrophotometer
(Nanodrop technologies) and the quality was evaluated with an Agilent bioanalyzer (Agilent
technologies). The RNA samples selected for real-time PCR experiments had an >8.0 RNA integrity
number on a scale of 10. Primer pairs were tested for specificity with the melting curve analysis (1°C
increment from 55°C to 92°C). Real-time qPCRs were carried out in a total volume of 25 µl using 1/40
of the cDNA produced by reverse transcription, using SYBR GreenER qPCR Supermix for iCycler
Instrument (Invitrogen) and primer pair of the gene of interest (Eurofins operon).
Data analysis
For quantitative in situ hybridization and dopamine transporter autoradiography, analysis was
performed from film autoradiograms using Densirag software (BIOCOM). mRNA levels were
converted from film autoradiograms to relative optical densities after subtracting the background
signal. For in situ hybridization cellular analysis (GAD 67 in SNr and GP; TH in SNc), the number of
silver grains per cell was estimated under polarized light on emulsion-coated sections, by measuring
optical density with respect to a standard curve of a defined number of silver grains (Visioscan image
analysis software, BIOCOM). The number of striatal interneurons was determined using Mercator
System (Explora Nova, v4.52) combined with a DMR Leica microscope coupled to a DXC-990P color
video camera (Sony). For some experiments, striatonigral MSNs ablation was evaluated by calculating
the fluorescence intensity of anti-GFP staining with an automated procedure run into ImageJ (v1.48t)
after image acquisition with a Macroconfocal microscope AZ-C2 (Nikon) equipped with AZ Plan Fluor
5x objective and NIS software (v4.13). All other images were collected with an AxioImager Z1
equipped with the Apotome system (Zeiss) using Zen software (Zen 2011, blue edition). The following
objectives (Zeiss) were used: Plan-Apochromat 10×/0.45 and 20×/0.8 objectives or Plan-Neofluar
40x/1.3 for the high magnification.
1. Jung S, Unutmaz D, Wong P, Sano G, De los SK, Sparwasser T, et al. (2002). In vivo depletion of
CD11c(+) dendritic cells abrogates priming of CD8(+) T cells by exogenous cellassociated antigens. Immunity 17: 211-220.
Supplementary Figure Legends
Supplementary Figure 1: BAC construction (A) DT incubation (10 μg/ml for 8 hours) of cultured
hippocampal neurons transfected with the DTR-GFP cDNA triggered apoptosis in GFP positive cells as
illustrated by the DNA fragmentation revealed with DAPI staining. (B) Schematic drawing of the
Slc35d3 BAC transgene design. The cDNA of DTR-GFP was cloned into a BAC (RP23-344M6) centered
on the Slc35d3 gene. Pr: promoter, PA: Poly-A. (C) Immunofluorescence detection of GFP on coronal
brain sections of DTR- and DTR+ mice. Scale bar, 20 μm. Cx: cortex, St: striatum.
Supplementary Figure 2: Characterization of striatonigral MSNs ablation after DT injection into the
dorsal striatum (A) Immunofluorescence detection of GFP on coronal brain sections in DTR+ mouse
injected unilaterally with DT in the dorsal striatum. Sections cover the striatum in the rostrocaudal
axis from AP=+1.54 to +0.14 according to the mouse stereotaxic atlas of Paxinos and Franklin. Scale
bar, 200 μm. D. St: dorsal striatum, NAc: nucleus accumbens, Cx: cortex, V: ventricle, dotted line:
anterior commissure. (B) Time course of striatonigral MSNs ablation after DT injection in DTR+ mice.
GFP intensity level in the dorsal striatum was quantified at different days after DT injection (n=4 mice
at each time, 4 sections per mouse). Data are expressed as percentage ± SEM. Student’s t test: *** p
< 0.001 vs. uninjected side.