In a first experiment, normal male C57Bl6 mice (Charles River, Stockholm) of 8 weeks of age were administered with saline and increasing doses of S33138. In a second experiment, male WT or D3R KO mice (C57bl6/129SV background; The Jackson Laboratory, Bar Harbor, ME, USA) were used. Mice were of 6 weeks at the time of first injections. All animals were group housed in air-conditioned rooms (12-hour dark/light cycle) at 20°C with humidity of 53%. Experiments were performed in agreement with the European Communities Council (86/609/ECC) and efforts were made to minimize pain or discomfort. They were approved by the ethical committee at Karolinska Institute (N351/08).
Experimental design, drug treatments, and fixation
In the first experiment, WT mice were divided into groups which were injected subcutaneously (s.c.) for 21 days with either 33138 (0.16, 0.64, or 2.5 mg/kg), Haloperidol (2 mg/kg), or vehicle (saline) (Fig. 1). In the second experiment, WT and D3R KO mice were injected subcutaneously (s.c.) for 21 days with a single concentration of S33138 (0.64 mg/kg) or vehicle (saline) (Fig. 1). To measure cell survival, BrdU (Sigma, Stockholm, Sweden) was injected intraperitoneally (i.p.) twice daily (75 mg/kg) for three consecutive days before commencing chronic treatment with S33138 (Fig. 1). On the final day of injections, brains were fixed via transcardial perfusion. Briefly, mice were anesthetized with Ketamine/Xylazine (80/10 mg/kg; Intervet/Bayer, Stockholm, Sweden) and then perfused with cold phosphate-buffered saline (PBS) (0.1 M; pH 7.4) followed by 4% paraformaldehyde. Mouse brains were then removed, post-fixed overnight at 4°C, and cryoprotected in 30% sucrose for several days. Brains were snap frozen and sectioned at 40μm using a cryostat (Leica CM1850; Sollentuna, Sweden) and stored at 4°C in 0.1 M PBS containing sodium azide (Sigma) until stained.
Figure 1. Experimental Design. In experiment 1, normal mice were injected for 21 days with either 33138 (0.16, 0.64, or 2.5 mg/kg), Haloperidol (2 mg/kg) or vehicle (saline) and striatal delta-FosB was measured. In experiment 2, WT and D3R KO mice were injected twice daily with BrdU for three consecutive days before commencing subcutaneous (s.c.) treatment for 21 days with S33138 (0.64 mg/kg) or vehicle (saline). Striatal delta-FosB was measured along with hippocampal Ki-67, DCX and BrdU.
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Original immunohistochemistry stainings to visualize the dose response of delta-FosB expression were performed using 3,3-diaminobenzidine. Briefly, sections were permeabilized (15 min in 1% Triton X-100/0.1 M PBS) and blocked against endogenous peroxidases (20 min with 3% H2O2/0.1 M PBS). Sections were then incubated with blocking solution for 1 h with serum followed by the primary antibody, rabbit anti-delta-FosB (1 : 500; Santa Cruz Biotechnology; Santa Cruz,CA, USA; overnight at 4°C), and the secondary biotinylated antibody (goat anti-rabbit, 1 : 200, Vector Laboratories, Burlingame, CA, USA; 2 h at 20°C). A signal amplification step was then performed by incubation in ABC reagent (ABC kit Vector Laboratories; for 1 h) followed by a reaction with 0.05% 3,3-diaminobenzidine-tetrahydrochloride/0.01% hydrogen peroxide in phosphate buffer. Finally, sections were dehydrated and mounted on Polylysine-coated slides (Histolab), dried, and coverslipped using VECTASHIELD® HardSet™ anti-fade mounting medium (Vector Laboratories). For improved confocal visualization, subsequent examination of delta-FosB in WT and KO mice were performed using fluorescent labeling as described below.
For fluorescent immunohistochemical staining, sections were incubated for 1 h in blocking solution (3% Bovine Serum Albumin (Sigma), 0.3% Triton X-100 (Sigma) in 0.1 M PBS) followed by incubation overnight in primary antibody. For BrdU staining, DNA in sections was denatured with HCl (15 min in 2 M HCl (Sigma) at 37°C) before incubation with primary antibodies. The primary antibodies used were rat anti-BrdU (1 : 500; Accurate Chemical; Trichem AB, Skandeborg, Denmark); rabbit anti-Ki-67 (1 : 1000; Novocastra, Newcastle upon Tyne, UK), rabbit anti-DCX (1 : 500; Cell Signaling Technology; In vitro AB, Stockholm, Sweden), mouse anti-NeuN (1 : 1000; Chemicon; Millipore AB, Stockholm, Sweden), and rabbit anti-delta-FosB(1 : 500; Santa Cruz Biotechnology). Sections were then incubated for 2 h with respective fluorescent secondary antibodies: anti-rat IgG Alexa Fluor™ -488, anti-rabbit IgG Alexa Fluor™-568, anti-mouse IgG Alexa Fluor™ -488 or 568 (1 : 200–500; Invitrogen, Lidingö, Sweden). For double immunostainings, sections were incubated simultaneously in two primary antibodies followed by simultaneous incubation in two secondary antibodies. For stainings with 4′,6-diamidino-2-phenylindole (DAPI), sections were also incubated for 20 min in 300 nM DAPI (Invitrogen). Sections were finally mounted on Poly-Lysine-coated slides (Histolab, Stockholm, Sweden), dried overnight, and mounted using FluorSave mounting medium (Calbiochem, San Diego, CA, USA). Confocal images were obtained using a Zeiss 710LSM laser scanning microscope (Carl Zeiss HB, Stockholm, Sweden) using a 40 × W lens and Zen 2009 software (Carl Zeiss HB).
For quantifications of delta-FosB, six striatal sections from each mouse were quantified. For each region, an image was captured from both hemispheres of each section using a Nikon Eclipse E600 light microscope with appropriate fluorescent filters connected to a Nikon digital sight DS-U1 camera with the NIS-Elements F 2.20 software (NIS-Elements; Melville, New York). The number of delta-FosB-positive cells for each image was then quantified using Image J. Briefly, the image was converted into an 8-bit image, and thereafter the intensity threshold was restricted (lower threshold 20, upper threshold 75). To differentiate adjacent touching nuclei, an Image J watershed function was used. Finally, the nucleus counter function (plug-in from WCIF- http://www.uhnresearch.ca/facilities/wcif/fdownload.html) was used with a specific threshold for size (min: 40, max: 1000). The resulting values from the quantifications were transferred to MS office EXCEL and the total number of positive cells averaged for each region in each brain. For quantification of neurogenesis, immunostainings were quantified by an observer blind to experimental conditions using a modified stereological method (Malberg et al. 2000). Briefly, analysis of each parameter was performed on a set of sections consisting of every sixth section throughout the hippocampus (roughly six to eight sections per set), which was then stained with the appropriate antibody. Labeled cells were manually quantified in the SGZ along the granule cell layer of the hippocampus using a 40 × W lens. To estimate the total number of cells in the entire dentate gyrus, the number of positive cells was divided by the number of sections counted to get an average and then multiplied by 60, the average number of sections in the hippocampus.