Supporting Information - PNAS · 2017. 9. 12. · above). Confocal microscopy was performed with a...
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Supporting Information Thakur et al. 10.1073/pnas.1710442114 SI Materials and Methods Tissue Processing and Immunohistochemistry. Animals were per- fused under deep pentobarbital anesthesia (1.5 mL, i.p.) with 50 mL of saline (0.9% wt/vol) at room temperature, followed by ice- cold paraformaldehyde (4% wt/vol in 0.1 M PBS). Brains were postfixed in the same solution for at least 24 h and cryoprotected overnight in 25% sucrose (in 0.1 M PBS). Then, 35-μm-thick co- ronal sections were cut using a freezing microtome (Leica) and collected in eight series. Free-floating sections were incubated at 80 °C for 30 min in Tris·EDTA buffer [10 mM Tris base, 1 mM EDTA solution, 0.05% Tween 20 (pH 9.0)]. Staining was performed as described elsewhere (9) using antibodies listed in Table S1. For triple-fluorescence immunostaining, Alexa 488-, Alexa 568-, and Alexa 642-conjugated secondary antibodies were used. Thioflavin staining was performed as follows. Sections were dipped in water, washed, and then incubated successively in a 0.01% thioflavin S (Sigma) solution for 5 min and 70% ethanol for 1 min and then washed in PBS three times. For proteinase K di- gestions, mounted sections were incubated in proteinase K solution (5 μg/mL; QIAGEN) for 60 min at room temperature before pro- ceeding with DAB (3,3′-diaminobenzidine) staining (as described above). Confocal microscopy was performed with a Leica TCS SP8 confocal microscope under 20× or 63× objective magnifi- cation. Three-dimensional images were generated from z-stacks of pictures captured throughout the thickness of the 35-μm-thick sec- tions using Volocity v.5.4.2 image analysis software (PerkinElmer). Quantification of TH + Cell Numbers and Fiber Density. The total number of TH + neurons in the SN and VTA was determined by unbiased stereology according to the optical fractionator prin- ciple using a Stereo-Investigator System (MBF Biosciences). The border of SN or VTA was traced at 5× magnification, and counting was done at 100× magnification. Cell counting was performed separately on both sides of the brain. Final results were expressed as the percentage of remaining cells on the injected side compared with the contralateral side. TH + fiber density was measured bilaterally at three sites in the medial, central, and lateral part of the caudate-putamen and at two sites in the NAcc using the “sphere counting” technique of Mouton et al. (39) as described previously. For measurement of axonal swellings, a z-stack of pictures was captured throughout the thickness of the section using a 40× objective, and images were compiled using Volocity 5.4.2 software. TH + structures with a volume larger than 20 μm 3 were defined as pathological and quantified. Quantification of IBA1 + Microglia. The density of IBA1 + microglia was determined in the SN bilaterally using the same Volocity 5.4.2 software. A z-stack of pictures was captured throughout the thickness of the 35-μm-thick sections using a 40× objective, and 3D images were compiled. From each measured area, 338 × 330 μm, the total number of IBA1 + cells was recorded from pars compacta and pars reticulata at two levels through the SN, -5.2 mm and -5.3 mm behind bregma. Data are expressed as the total number per measuring field and as the percentage of the control side. Mean staining density was determined and expressed as the per- centage of density measured on the control side. Behavioral Testing. Cylinder test. Rats were placed in a transparent glass cylinder and allowed to move freely. A total of 20 paw touches were counted, and the percentage of touches made with the left paw (contra- lateral side) was used as a measure of the dopaminergic system. Stepping test. Rats were held in a manner that restrained one paw while the other paw was left freely moving, and were then allowed to walk over a length of 90 cm both in forehand and backhand directions. The total number of steps taken by the left and right paws was recorded. Rats were trained to perform this test a day before the test. To reduce the bias, animals were tested for three consecutive days, the median of the readings was used, and the same researcher performed the test blindly for the entire experiment. In Vivo Amperometry to Detect DA Release and Reuptake. Chro- noamperometric recordings were made with nafion-coated car- bon fiber electrodes (SF1A; Quanteon) (38, 40). A 10-Hz square wave potential was applied (0.55-V applied potential, 0-V resting potential), and the resulting oxidation and reduction current were recorded with a temporal resolution of 0.5 s. Each electrode was individually calibrated in vitro in 0.1 M PBS, and the linearity of the response was verified by three additions of 2 μM DA. Lin- earity of the response (r 2 ) was 0.999 ± 0.002, and the limit of detection was 45 nM ± 71 nM (SD). Selectivity of DA vs. ascorbic acid averaged 3,018:1. Bilateral recordings were made 10 d after PFF injection from striatum at two sites (from bregma: AP, +1; L, ±2.7; V, -4.5 and -5.0 mm) from 11 α-SYN + FIB, 10 FIB ONLY, and 9 α-SYN ONLY animals. Each electrode was mounted together with a micropipette (10–15 μm OD) 90–110 μm away from the recording electrode. DA release was induced by pressure ejection of 120– 150 nL of 120 mM KCl using a micropressure system (Parker picospritzer III) with three consecutive ejections spaced 5 min apart. DA release from the first KCl ejection was used in the data analysis. The first release event was considered to be release from the idle state, whereas the two following events were used to in- vestigate reloading capacity. No difference in reloading capacity was found between groups. Peak amplitude was calculated as the maximum DA concentration after KCl ejection minus baseline. Reuptake rate and the release rate were defined as the linear concentration change per time unit (μM·s -1 ) between 40% and 60% of the peak maximum portion of the reuptake part of the curve (post peak) and release part (prepeak), respectively. Thakur et al. www.pnas.org/cgi/content/short/1710442114 1 of 7
Transcript of Supporting Information - PNAS · 2017. 9. 12. · above). Confocal microscopy was performed with a...
Supporting InformationThakur et al. 10.1073/pnas.1710442114SI Materials and MethodsTissue Processing and Immunohistochemistry. Animals were per-fused under deep pentobarbital anesthesia (1.5 mL, i.p.) with50mLof saline (0.9%wt/vol) at room temperature, followed by ice-cold paraformaldehyde (4% wt/vol in 0.1 M PBS). Brains werepostfixed in the same solution for at least 24 h and cryoprotectedovernight in 25% sucrose (in 0.1 M PBS). Then, 35-μm-thick co-ronal sections were cut using a freezing microtome (Leica) andcollected in eight series. Free-floating sections were incubated at80 °C for 30 min in Tris·EDTA buffer [10 mM Tris base, 1 mMEDTA solution, 0.05% Tween 20 (pH 9.0)]. Staining was performedas described elsewhere (9) using antibodies listed in Table S1. Fortriple-fluorescence immunostaining, Alexa 488-, Alexa 568-, andAlexa 642-conjugated secondary antibodies were used.Thioflavin staining was performed as follows. Sections were
dipped in water, washed, and then incubated successively in a0.01% thioflavin S (Sigma) solution for 5 min and 70% ethanol for1 min and then washed in PBS three times. For proteinase K di-gestions, mounted sections were incubated in proteinase K solution(5 μg/mL; QIAGEN) for 60 min at room temperature before pro-ceeding with DAB (3,3′-diaminobenzidine) staining (as describedabove). Confocal microscopy was performed with a Leica TCSSP8 confocal microscope under 20× or 63× objective magnifi-cation. Three-dimensional images were generated from z-stacksof pictures captured throughout the thickness of the 35-μm-thick sec-tions using Volocity v.5.4.2 image analysis software (PerkinElmer).
Quantification of TH+ Cell Numbers and Fiber Density. The totalnumber of TH+ neurons in the SN and VTA was determined byunbiased stereology according to the optical fractionator prin-ciple using a Stereo-Investigator System (MBF Biosciences). Theborder of SN or VTA was traced at 5× magnification, andcounting was done at 100× magnification. Cell counting wasperformed separately on both sides of the brain. Final resultswere expressed as the percentage of remaining cells on the injectedside compared with the contralateral side.TH+ fiber density was measured bilaterally at three sites in the
medial, central, and lateral part of the caudate-putamen and attwo sites in the NAcc using the “sphere counting” technique ofMouton et al. (39) as described previously. For measurement ofaxonal swellings, a z-stack of pictures was captured throughoutthe thickness of the section using a 40× objective, and imageswere compiled using Volocity 5.4.2 software. TH+ structureswith a volume larger than 20 μm3 were defined as pathologicaland quantified.
Quantification of IBA1+ Microglia. The density of IBA1+ microgliawas determined in the SN bilaterally using the same Volocity5.4.2 software. A z-stack of pictures was captured throughout thethickness of the 35-μm-thick sections using a 40× objective, and 3Dimages were compiled. From each measured area, 338 × 330 μm,
the total number of IBA1+ cells was recorded from pars compactaand pars reticulata at two levels through the SN, −5.2 mm and−5.3 mm behind bregma. Data are expressed as the total numberper measuring field and as the percentage of the control side.Mean staining density was determined and expressed as the per-centage of density measured on the control side.
Behavioral Testing.Cylinder test. Rats were placed in a transparent glass cylinder andallowed to move freely. A total of 20 paw touches were counted,and the percentage of touches made with the left paw (contra-lateral side) was used as a measure of the dopaminergic system.Stepping test.Rats were held in a manner that restrained one pawwhile the other paw was left freely moving, and were then allowedto walk over a length of 90 cm both in forehand and backhanddirections. The total number of steps taken by the left and rightpaws was recorded. Rats were trained to perform this test a daybefore the test. To reduce the bias, animals were tested for threeconsecutive days, the median of the readings was used, andthe same researcher performed the test blindly for the entireexperiment.
In Vivo Amperometry to Detect DA Release and Reuptake. Chro-noamperometric recordings were made with nafion-coated car-bon fiber electrodes (SF1A; Quanteon) (38, 40). A 10-Hz squarewave potential was applied (0.55-V applied potential, 0-V restingpotential), and the resulting oxidation and reduction current wererecorded with a temporal resolution of 0.5 s. Each electrode wasindividually calibrated in vitro in 0.1 M PBS, and the linearity ofthe response was verified by three additions of 2 μM DA. Lin-earity of the response (r2) was 0.999 ± 0.002, and the limit ofdetection was 45 nM ± 71 nM (SD). Selectivity of DA vs.ascorbic acid averaged 3,018:1.Bilateral recordings were made 10 d after PFF injection from
striatum at two sites (from bregma: AP, +1; L, ±2.7; V, −4.5 and−5.0 mm) from 11 α-SYN + FIB, 10 FIB ONLY, and 9 α-SYNONLY animals. Each electrode was mounted together with amicropipette (10–15 μm OD) 90–110 μm away from the recordingelectrode. DA release was induced by pressure ejection of 120–150 nL of 120 mM KCl using a micropressure system (Parkerpicospritzer III) with three consecutive ejections spaced 5 minapart. DA release from the first KCl ejection was used in the dataanalysis. The first release event was considered to be release fromthe idle state, whereas the two following events were used to in-vestigate reloading capacity. No difference in reloading capacitywas found between groups. Peak amplitude was calculated as themaximum DA concentration after KCl ejection minus baseline.Reuptake rate and the release rate were defined as the linearconcentration change per time unit (μM·s−1) between 40% and60% of the peak maximum portion of the reuptake part of thecurve (post peak) and release part (prepeak), respectively.
Thakur et al. www.pnas.org/cgi/content/short/1710442114 1 of 7
Fig. S1. α-Syn spread and impact on motor behavior. (A) Human α-syn staining shows PFF injected in SN and VTA 3 h or 3, 12, and 24 wk (w) after injection.Distribution of human α-syn derived from the AAV vector injected in SN and VTA (B) or SN (C) is shown, as indicated by the asterisks, 5–7 wk after vectorinjection. [Scale bars: A, B (striatum), B (SN), 1 mm.] (D and E) Changes in performance in the cylinder and stepping tests. Cylinder and stepping data bothshowed a significant group effect: F(2, 21) = 5.719, P = 0.0104, and F(2, 27) = 25.74, P < 0.0001, respectively.
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Fig. S2. Impact on DA neuron survival. TH immunostaining shows the extent of DA neuron cell loss in animals that received a combination of AAV vector andPFF (A), AAV vector alone (B), or PFF alone (C). Asterisks in A show the injection sites. (Scale bar: 1 mm.) w, week(s).
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Fig. S3. Development of Lewy-like synucleinopathy in nigral DA neurons over time. Triple-immunofluorescent staining shows the expression of the AAV-derived human α-syn protein (red) and the morphological appearance of the PFF-induced pSyn+ aggregates (white) in the TH+ neurons (green) in the SN.Representative photographs of individual neurons were selected from the α-SYN + FIB (Left), α-SYN ONLY (Center), and FIB ONLY (Right) groups at fourdifferent time points after PFF injection: 10 d (A–C), 3 wk (D–F), 12 wk (G–I), and 24 wk (J–L). Images were acquired as collated, 3D rendered images in theconfocal microscope.
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Fig. S4. Characterization of the aggregates. The pSyn-immunostained aggregates (green) observed in the SN express features characteristic of Lewy bodies (ar-rowheads) and Lewy neurites (arrows), staining positively for ubiquitin (red) (A–C) and thioflavin (green in E), and being resistant to proteinase K (ProtK) digestion(D and D′). Images were captured from an α-SYN + FIB–treated animal at 3 wk post-PFF injection. (Scale bars: A–C, 50 μm; D, 100 μm; D′, 50 μm; E, 10 μm.)
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Fig. S5. Inflammatory response in SN pars reticulata. Total number of IBA1+ cells (A) and their staining density (B) were quantified in the SN pars reticulata ofanimals injected with α-SYN + FIB (purple), α-SYN ONLY (red), FIB ONLY (green), or monomers (mono; white). The differences between the injected andnoninjected sides were significant in both total number, F(1, 55) = 48.61, P < 0.001, and density, F(1, 55) = 14.28, P < 0.001. All data are mean ± SD. *P < 0.05;**P < 0.01; ***P < 0.001, compared with the contralateral side. (C–K) Representative images of IBA1 staining are presented for each group at the differenttime points. (Scale bars: 20 μm.)
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Table S1. List of antibodies used in the study
Antigen Company Host Dilution
α-Syn BD Biosciences Mouse 1:1,000α-Syn EMD Millipore Rabbit 1:1,000α-Syn 211(human-specific) Abcam Mouse 1:1,000pSyn 5038 (Ser129) EMD Millipore Rabbit 1:1,000pSyn 81A (Ser129) Abcam Mouse 1:10,000CD4 AbD Serotec Mouse 1:500CD8 AbD Serotec Mouse 1:500DARPP-32 Abcam Rabbit 1:1,000Hu Abcam Mouse 1:200IBA-1 Wako Rabbit 1:1,000NeuN EMD Millipore Mouse 1:1,000TH EMD Millipore Sheep 1:1,000TH Abcam Rabbit 1:1,000Ubiquitin Dako Rabbit 1:200VMAT-2 Abcam Rabbit 1:5,000
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