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  • All animal work was performed according to national and international guidelines following approved animal procedures by the Beltsville Area Animal Care Committee, United States Department of Agriculture (Protocol no. 09-010–Toxoplasmosis in mice; approved June 4, 2009). This protocol is reviewed annually, and any amendments are approved separately. Growth of parasites and host cells: T. gondii strains were maintained in human foreskin fibroblasts (HFFs) as previously described [9]. PC-12 cells obtained from ECACC (Salisbury) were maintained as described by the supplier. Female Swiss Webster mice infected with T. gondii VEG strain were used for histology. Immunofluorescence assay of brain sections: Immunofluorescence against multiple targets was performed on paraformaldehyde-fixed, paraffin-embedded mouse brain sections. Female Swiss Webster mice were infected with T. gondii VEG strain oocysts 6–8 weeks prior to processing. Tissues were collected, formalin-fixed and paraffin-embedded using standard protocols and following approved guidelines. Slides were deparaffinized and rehydrated with an alcohol descending row, which was then followed by epitope retrieval in 10 mM sodium citrate buffer (pH 6.0) overnight at 60°C following sectioning. Slides were blocked with 2% normal goat sera for 1 h at room temperature. TRITC-conjugated lectin from Dolichos biflorus (Cat # L9658, Sigma, St. Louis) was introduced to the slides for 4 h at room temperature, diluted 1∶200 in primary staining solution (1% BSA, 0.1% cold fish skin gelatine, 0.5% Triton X-100 in 0.1 M PBS pH 7.2). Next, samples were washed (3×10 min) in wash buffer (TBS pH 8.4 with 0.1% Triton X-100 and 1% fish skin gelatin) and blocked using a biotin-streptavidin blocking kit (Cat # SP-2002, Vector Labs, Peterborough) according to the manufacturer's protocol. Samples were incubated with primary antibody (raised in rabbit) against dopamine (Cat # ab8888, Abcam, Cambridge, MA) (diluted 1∶200) or tyrosine hydroxylase (Cat # ab112, Abcam) (diluted 1∶500) overnight at 4°C. Samples were rinsed with wash buffer and incubated for 1 h with biotinylated anti-rabbit IgG secondary antibody (Cat # B-1000, Vector Labs) diluted 1∶500 in secondary antibody solution (0.05% Tween in 0.1 M PBS pH 7.2). Sections were rinsed and incubated with FITC-conjugated streptavidin (Cat # SA-5001, Vector Labs) diluted 1∶100 according to the manufacturer's guidelines in secondary antibody buffer at room temperature. Finally, slides were rinsed in wash buffer containing DAPI and double-distilled water prior to mounting in Fluoromount G (Southern Biotech, Birmingham). All incubation and blocking steps were carried out in a wet chamber. All slides were kept at 4°C in the dark before imaging using a Zeiss LSM510 META laser scanning inverted AxioVert 200M confocal microscope with DIC optics. 3D reconstructions of serial sections were generated with the same equipment using the LSM imaging software for the 3D deconvolution. To assess the specificity of dopamine staining, sections were incubated either without primary antibody or with primary anti-dopamine antibody in the presence of freshly prepared dopamine or serotonin for 30 min prior to and for overnight following addition to the sections. A T. gondii tyrosine hydroxylase antibody custom antibody (Genscript, Piscataway) was developed to assess the parasite enzyme in animals. The affinity purified antibody is directed against a unique sequence (CIRSSPDPLDLRKMT) in the amino terminal domain that is not found in mammalian tyrosine hydroxylase and has no significant similarity to any protein in the predicted mammalian proteome or other proteins of the T. gondii proteome. The specificity of the antibody for T. gondii tyrosine hydroxylase was confirmed by Western analysis. Total protein from half mouse brains was isolated in 20 volumes (wt/vol)lysis buffer (20 mM Tris-HCl pH 8; 137 mM NaCl; 10% glycerol; 1% Triton X; 2 mM EDTA and protease inhibitors (cOmplete Mini EDTA-free cocktail, Roche)) and quantified using Bradford reagent (Sigma) as per manufacturer's instructions. Expression and purification of T. gondii tyrosine hydroxylase was as previously described [9]. SDS-PAGE was following standard protocols with 2–20 µg protein separated on a 12% sodium dodecylsulphate- polyacrylamide gel. The proteins were transferred to nitrocellulose membrane, blocked with 5% non-fat dried milk in PBS containing 0.05% Tween-20 (vol/vol) for 1 hour. Incubation with the custom antibody (1∶2500) 4°C overnight was followed by washing in PBS-Tween (0.05%) and incubation with an anti-rabbit (1∶5000) conjugated horseradish peroxidase antibody (Sigma) at room temperature for 1 hour. Blots were then washed as above and developed using Supersignal West Pico Chemiluminescent kit (Pierce, Rockford, IL). Bands were visualised with an X-Omat film system. The membrane was stripped and re-probed with mouse anti-β-actin (1∶25,000; Sigma) overnight at 4°C followed by anti-mouse (1∶10,000) conjugated horseradish peroxidase antibody (AutogenBioclear, Wiltshire, UK) at room temperature for 1 hour and subsequent visualisation. The anti-T. gondii tyrosine hydroxylase antibody was used for immunofluorescence (diluted 1∶500) following a similar protocol as described above. Immunofluorescence of tyrosine hydroxylase in cultured parasites was performed with paraformaldehyde-fixed cell cultures. Cultures of T. gondii stably expressing RFP-conjugated GRASP protein (kindly donated by Manami Nishi from David Roos laboratory, University of Philadelphia, USA) in human foreskin fibroblasts grown on polylysine-coated coverslips were alkaline induced for differentiation as published and differentiation monitored by counting the number of parasites in the vacuoles in the normal and pH shifted cultures. These conditions yielded bradyzoite forms as shown by RT-qPCR with the bradyzoite markers BAG1 and SAG4 [9]. After five days, coverslips were paraformaldehyde-fixed and probed with tyrosine hydroxylase antibody (Cat # ab112, Abcam) (diluted 1∶500) with visualisation as described above. Immunohistochemical assay of brain sections: For immunohistochemical assays, sections were treated as described above, except for the following steps: for washing and dilution buffers, 0.1 M PBS supplemented with 0.1% Tween was used. After antigen retrieval, slides were incubated in 0.3% H2O2 (in 0.1 M PBS) to quench endogen peroxidases. Following secondary antibody treatment, 5 µg/ml HRP-conjugated streptavidin (Cat # SA-5004, Vector Labs) was applied, and next, the peroxidase substrate kit (Vector Labs, ImmPACT™DAB, Cat # SK-4105) was used according to the manufacture's protocol. Prior to mounting, sections were stained with haematoxylin to visualize cell nuclei. Imaging of slides was performed using a Zeiss Axioplan microscope equipped with DIC optics. Photomicrographs were collected with a Photometrics CoolSNAP camera and Improvision Openlab software. A cytochemical method was used to assess the dopamine staining of tissue cyst-containing neural cells in infected mice brain. Glyoxylic acid reacts with catecholamines in a gaseous reaction to form fluorescent products. Dopamine reacts with glyoxylic acid to form a product that specifically emits at 478–480 nm [17]. Dopamine accumulation and release from dopaminergic cells: The dopaminergic cell line PC12 (ECACC) was infected with Prugniard strain of T. gondii tachyzoites that had been alkaline shocked to induce bradyzoite differentiation. Liberated tachyzoites were incubated in RPMI media at pH 8 with 1% FCS at 37°C and ambient CO2 for 16–18 h, then rinsed with DMEM and returned to standard PC12 cell culturing conditions. PC12 cultures were infected 2.5×105–7.5×105 parasites and cultured for five days prior to assay. The cultures infected with higher numbers of parasites had parasitemia of 40–50%. Prior to assay, samples were normalized to equivalent numbers of cells (2.5×106) per assay. One set of cultures was harvested by centrifugation and lysed by sonication in 0.1 M perchlorate for total dopamine measurement by HPLC with electrochemical detection. A parallel set of cultures were equilibrated with wash buffer with low KCl containing buffer (140 mM NaCl, 4.7 mM KCl, 1.2 mM MgCl2, 2.5 mM CaCl2, 11 mM dextrose, 10 mM HEPES, pH 7.4) for 30 min followed by incubation with two volumes high KCl containing buffer (40 mM NaCl, 100 mM KCl, 1.2 mM MgCl2, 2.5 mM CaCl2, 11 mM dextrose, 10 mM HEPES, pH 7.4) for 2 min to induce dopamine release as previously described [18]. During the assay, samples were taken from the media, washing buffer, and high KCl containing buffer and immediately supplemented with 0.3 volumes 0.1 M perchlorate. Three independent experiments were performed with a representative experiment shown. Following centrifugation, cell homogenates and media were assayed by HPLC-ED. Reverse phase chromatography, combined with electrochemical detection, was performed with a Dionex HPLC system consisting of a P580 Pump (Dionex) and Ultimate 3000 Autosampler Column Compartment with a C18 Acclaim 120 column (5 µm, 4.6×150 mm) and an ESA Coulochem III cell, equipped with a glassy carbon electrode used at 700 mV versus Ag/AgCl reference electrode for detection of monoamines. The mobile phase consisted of degassed 57 mM anhydrous citric acid (Fisher Scientific, Loughborough), 43 mM sodium acetate (Dionex, Sunnyvale) buffer containing 0.1 mM EDTA (Sigma Aldrich), 1 mM sodium octanesulphonate monohydrate, and 10% methanol. The pH was adjusted to 4. The mobile phase was delivered at a flow rate of 1.5 ml/min, and the column temperature was set at 40°C. Applied standards (dopamine, L-DOPA) were dissolved in 0.1 M perchlorate for chromatography. The concentration of compounds was determined using Chromeleon software.
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