Title
The Center of Olfactory Bulb-Seeded α-Synucleinopathy is the Limbic System and the Ensuing Pathology is Higher in Male than in Female Mice
Citation for published article
Mason DM, Wang Y, Bhatia TN, Miner KM, Trbojevic SA, Stolz JF, Luk KC, and Leak RK* (2019) The Center of Olfactory Bulb-Seeded α-Synucleinopathy is the Limbic System and the Ensuing Pathology is Higher in Male than in Female Mice. Brain Pathology (IF 6.2). Accepted and In Press
DOI
https://onlinelibrary.wiley.com/doi/abs/10.1111/bpa.12718
Peer Reviewed
1
Document Type
Article
School
School of Pharmacy
Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 International License.
Repository Citation
Mason DM, Wang Y, Bhatia TN, Miner KM, Trbojevic SA, Stolz JF, Luk KC, and Leak RK* (2019) The Center of Olfactory Bulb-Seeded α-Synucleinopathy is the Limbic System and the Ensuing Pathology is Higher in Male than in Female Mice. Brain Pathology (IF 6.2). Accepted and In Press
Fig1 - Logrank and weight DM.eps (2274 kB)
Fig. 1 Preformed α-synuclein fibril injections in the mouse OB/AON accelerate mortality in older mice without significant changes in body weight. Sixty-four mice were infused in the right olfactory bulb/anterior olfactory nucleus with either preformed α-synuclein fibrils (5 μg/1 μL) or an equivalent volume of PBS (1 μL) at the indicated ages. Logrank survival analyses were performed for all animals at 6 months post-infusion (a) and for the older males at 6 months (b) or 10.5 months (c) post-infusion. The two-tailed p values of the logrank survival analyses are shown. (d) The body weights of the older males at surgery (11 months old), 6 months post-surgery (17 months old), and 10.5 months post-surgery (21.5 months old) are displayed as scatter plots with mean and SEM bars. In panel d, crosses are placed next to mice that died before the planned time of assay. (e-f) Weight gained in both raw numbers (e) and percentage weight gain (f) six months after surgery in all experimental groups. Note the broken Y axis in panels a and d. For panels d-f, two-way ANOVAs were followed by the Bonferroni post hoc correction for multiple comparisons. In panels e-f, +++ p≤0.001 versus males that were infused at 3 months of age and perfused at 9 months of age (3-9 mo males). To view the original, higher resolution Adobe Illustrator or EPS files, please link to https://www.dropbox.com/sh/y656msmgwotgfy5/AACyqebjHFcGhy2bXtIcW_gDa?dl=0
Fig2 - peak density heatmap.eps (1505 kB)
Fig. 2 Heat map of α-synucleinopathy following preformed α-synuclein fibril injections in the mouse OB/AON. Qualitative assessments of the density of pSer129+ inclusions in the indicated brain regions of all fibril-infused animals that survived until the planned assay time (see Methods). Each row represents one animal. A blinded investigator systematically examined the entire 1-in-5 series of sagittal brain sections of the ipsilateral, right hemisphere following immunostaining of pathologically phosphorylated α-synuclein with the rabbit monoclonal EP1536Y pSer129 antibody (see Table S1). Boxes with a N/A label denote regions that were unavailable (see Methods). Brain regions with pSer129+ immunolabeling are arranged rostrocaudally and/or by anatomical/functional groupings. Most of the midbrain pathology was confined to the ventral tegmental area; only two male animals exhibited pSer129+ inclusions at the boundary zone between the substantia nigra and the ventral tegmental area at 6 months post-infusion. Abbreviations are defined in Table S2. To view the original, higher resolution Adobe Illustrator or EPS files, please link to https://www.dropbox.com/sh/y656msmgwotgfy5/AACyqebjHFcGhy2bXtIcW_gDa?dl=0
Fig3 - Rostral Rhinencephelon DM.eps (35790 kB)
Fig. 3 Impact of α-synuclein fibril infusions in the mouse OB/AON on pSer129+ inclusion counts, NeuN+ neuron counts, and Hoechst+ cell numbers in the rostral rhinencephalon. Mice were infused in the right olfactory bulb/anterior olfactory nucleus with either preformed α-synuclein fibrils (5 μg/1 μL) or an equivalent volume of PBS (1 μL). A blinded observer manually counted the number of pSer129+ structures (monoclonal 81A pSer129 Ab; see Table S1) per field of view (200× magnification) and used cellSens software to count the number of NeuN+ and Hoechst+ nuclei in the olfactory bulb (OB; a), the anterior olfactory nucleus (AON; b, d), and the nucleus of the lateral olfactory tract (LOT; c) in the same field of view. Shown are the mean and SD of raw, unnormalized data. N = 3-8 mice per group (see Methods and Fig. 1 for animal numbers). Two-way ANOVAs were followed by the Bonferroni post hoc correction. * p≤0.05, ** p≤0.01, *** p≤0.001 PBS vs fibrils; + p≤0.05 ++ p≤0.01 vs 3-9 month males; ~ p≤0.05 vs 11-17 month males. Abbreviations are defined in Table S2. To view the original, higher resolution Adobe Illustrator or EPS files, please link to https://www.dropbox.com/sh/y656msmgwotgfy5/AACyqebjHFcGhy2bXtIcW_gDa?dl=0
Fig4 - paleocortex amygdala striatum DM.eps (6008 kB)
Fig. 4 Impact of α-synuclein fibril infusions in the mouse OB/AON on pSer129+ inclusion counts, NeuN+ neuron counts, and Hoechst+ cell numbers in the paleocortices, amygdaloid complex, and the dorsal and ventral striata. Mice were infused in the right olfactory bulb/anterior olfactory nucleus with either preformed α-synuclein fibrils (5 μg/1 μL) or an equivalent volume of PBS (1 μL). A blinded observer manually counted the number of pSer129+ structures (monoclonal 81A pSer129 Ab; see Table S1) per field of view (200× magnification) and used cellSens software to count the number of NeuN+ and Hoechst+ nuclei in the rostromedial piriform cortex (Pir Rm; a), caudolateral piriform cortex (Pir Cl; b), amygdalopiriform transition area (APir; c), posteromedial cortical amygdala (PMCo; d), caudoputamen (CPu; e), and nucleus accumbens (Acb; f). Shown are the mean and SD of raw, unnormalized data. N = 3-8 mice per group (see Methods and Fig. 1 for animal numbers). Two-way ANOVAs were followed by the Bonferroni post hoc correction. * p≤0.05, ** p≤0.01, *** p≤0.001 PBS vs fibrils; + p≤0.05, ++ p≤0.01 vs 3-9 month males; ~~ p≤0.01 vs 11-17 month males. Abbreviations are defined in Table S2. To view the original, higher resolution Adobe Illustrator or EPS files, please link to https://www.dropbox.com/sh/y656msmgwotgfy5/AACyqebjHFcGhy2bXtIcW_gDa?dl=0
Fig5 - hippocampal formation and perirhinal telencephalon DM.eps (6555 kB)
Fig. 5 Impact of α-synuclein fibril infusions in the mouse OB/AON on pSer129+ inclusion counts, NeuN+ neuron counts, and Hoechst+ cell numbers in the hippocampal formation and perirhinal telencephalon. Mice were infused in the right olfactory bulb/anterior olfactory nucleus with either preformed α-synuclein fibrils (5 μg/1 μL) or an equivalent volume of PBS (1 μL). A blinded observer manually counted the number of pSer129+ structures (monoclonal 81A pSer129 Ab; see Table S1) per field of view (200× magnification) and used cellSens software to count the number of NeuN+ and Hoechst+ nuclei in the pyramidal cell layers of hippocampal fields CA1 (a), CA2 (b) and CA3 (c), the stratum granulosum of the dentate gyrus (DG; d), the subiculum (Sub; e), and entorhinal cortex (Ent; f), and the ectorhinal cortex (Ect; g). Shown are the mean and SD of raw, unnormalized data. N = 3-8 mice per group (see Methods and Fig. 1 for animal numbers). Two-way ANOVAs were followed by the Bonferroni post hoc correction. * p≤0.05, ** p≤0.01, *** p≤0.001 PBS vs fibrils, + p≤0.05 vs 3-9 month males, ~ p≤0.05 vs 11-17 month males. Abbreviations are defined in Table S2. To view the original, higher resolution Adobe Illustrator or EPS files, please link to https://www.dropbox.com/sh/y656msmgwotgfy5/AACyqebjHFcGhy2bXtIcW_gDa?dl=0
Fig6 - representative histology.eps (764569 kB)
Fig. 6 Preformed α-synuclein fibril injections in the mouse OB/AON elicit α-synucleinopathy in olfactory/limbic regions of the temporal lobe. Mice were infused in the right olfactory bulb/anterior olfactory nucleus with either preformed α-synuclein fibrils (5 μg/1 μL) or an equivalent volume of PBS (1 μL). Sagittal brain sections were immunostained for pathologically phosphorylated α-synuclein (EP1536Y rabbit monoclonal pSer129 antibody; see Table S1). The Hoechst reagent was used to stain nuclei and identify anatomical boundaries. Representative images were captured at 40 and 200 (insets) magnification in the anterior olfactory nucleus (a), tenia tecta (b), dentate gyrus (c), hippocampal CA1 (d), the posteromedial cortical amygdala (e), and the nucleus accumbens (f). Abbreviations are defined in Table S2. To view the original, higher resolution Adobe Illustrator or EPS files, please link to https://www.dropbox.com/sh/y656msmgwotgfy5/AACyqebjHFcGhy2bXtIcW_gDa?dl=0
Fig7 - aggresome.eps (42769 kB)
Fig. 7 Preformed α-synuclein fibril injections in the mouse OB/AON induce the formation of aggresome-like structures. Mice were infused in the right olfactory bulb/anterior olfactory nucleus with either preformed α-synuclein fibrils (5 μg/1 μL) or an equivalent volume of PBS (1 μL). Sagittal brain sections from young male mice were immunostained for pathologically phosphorylated α-synuclein (monoclonal 81A pSer129 antibody; see Table S1). The Proteostat detection reagent was applied to the tissue and the Hoechst reagent was used to stain cellular nuclei. Images were captured with a 100× objective under oil immersion. (a) Perinuclear pSer129+ inclusions were stained with the Proteostat dye in all affected brain regions, including the anterior olfactory nucleus (AON), piriform cortex (Pir), posteromedial cortical nucleus of the amygdala (PMCo), hippocampal CA1, and entorhinal cortex (Ent), in young male mice six months after fibril infusions. See text for details on background staining in PBS group. (b) The arrow points to a pSer129+ neuritic inclusion that does not colocalize with the Proteostat dye, in contrast with the dual-labeled perinuclear inclusion in the soma (arrowhead). (c) Mild pSer129 but dense Proteostat label was observed in the tenia tecta. Abbreviations are defined in Table S2. To view the original, higher resolution Adobe Illustrator or EPS files, please link to https://www.dropbox.com/sh/y656msmgwotgfy5/AACyqebjHFcGhy2bXtIcW_gDa?dl=0
Fig8 - Cell layer widths DM.eps (72650 kB)
Fig. 8 Preformed α-synuclein fibril injections in the OB/AON induce atrophy of pyramidal and granule cell layers of the limbic allocortex in young male mice. Mice were infused in the right olfactory bulb/anterior olfactory nucleus with either preformed α-synuclein fibrils (5 μg/1 μL) or an equivalent volume of PBS (1 μL). A blinded observer employed the “arbitrary line” tool in cellSens software to measure the width of the compact Hoechst+ pyramidal cell layer in the rostromedial piriform cortex, caudolateral piriform cortex, CA1, CA2, and CA3 of the hippocampus, or the stratum granulosum of the dentate gyrus. Representative grayscale micrographs for the caudolateral piriform cortex and dentate gyrus are displayed in panel a and the quantifications of pyramidal and granule cell layers width (b) and Hoechst+ nuclear area (c) are graphed below. Shown are the mean and SD of raw, unnormalized data. N =3-8 mice per group (see Methods section and Fig. 1 for animal numbers). Two-way ANOVAs were followed by the Bonferroni post hoc correction. * p≤0.05, ** p≤0.01, *** p≤0.001 PBS vs fibrils; + p≤0.05, ++ p≤0.01 +++ p≤0.001 versus vs 3-9 month males; ~ p≤0.05, ~~ p≤0.01, ~~~ p≤0.001 vs 11-17 month males. Abbreviations are defined in Table S2. To view the original, higher resolution Adobe Illustrator or EPS files, please link to https://www.dropbox.com/sh/y656msmgwotgfy5/AACyqebjHFcGhy2bXtIcW_gDa?dl=0
Fig9 - M43 Stitch Series pSer129.eps (485731 kB)
Fig. 9 α-synucleinopathy remains centered in the limbic allocortex six months following infusions of preformed α-synuclein fibrils in the OB/AON. A series of stitched images of sagittal brain sections from two 17-month-old animals sacrificed six months after infusion of 1 µL PBS (left) or 5 µg/1 µL fibrils (right) into the OB/AON. All sections were stained in parallel with the monoclonal rabbit EP1536Y pSer129 antibody for pathologically phosphorylated α-synuclein (see Table S1). Anatomical labels are based on cytoarchitectonic details provided by the Hoechst nuclear marker (not shown). All sections were processed in parallel and photographed at the same exposure and intensity scaling. Abbreviations are defined in Table S2. To view the original, higher resolution Adobe Illustrator or EPS files, please link to https://www.dropbox.com/sh/y656msmgwotgfy5/AACyqebjHFcGhy2bXtIcW_gDa?dl=0
Fig10 - confocal TH pSer129 Hoechst montage DM.eps (472755 kB)
Fig. 10 Mesencephalic α-synucleinopathy is centered medially in the limbic-related ventral tegmental area and does not colocalize with dopaminergic markers. A fibril-injected 17 month-old mouse with the most widespread Lewy-related pathology of all cases studied is displayed in this figure. (a) Stitched image of an entire brain section, with genuine α-synucleinopathy at the boundary zone between the ventral tegmental area and substantia nigra, pars compacta (true boundaries were identified with antibodies against the dopaminergic marker tyrosine hydroxylase on the same section—see panel d). Dense pSer129+ inclusions were observed in the anterior olfactory nucleus (AON) and nucleus accumbens (Acb), and sparse inclusions were observed in the olfactory bulb (OB), bed nucleus of stria terminalis (BST), orbitofrontal (VO) and cingulate cortices (A30), and ventral midbrain. A few sparse inclusions were observed in association neocortices (M2) of this one animal. Merged, higher-magnification images of Hoechst+ cells (pseudocolored blue) and pSer129 immunostaining (pseudocolored red; EP1536Y rabbit monoclonal pSer129 antibody; see Table S1) in the AON (b) and accumbens (c). (d) Tyrosine hydroxylase (TH; pseudocolored green) and pSer129 (pseudocolored red) immunostaining in the ventral midbrain of the same animal. (e-h) Lack of convincing colocalization of pSer129 and tyrosine hydroxylase in confocal images of the same animal. A movie of the rotating confocal Z-stack for this animal can be viewed in Figure S2. The lack of convincing colocalization of pSer129 and tyrosine hydroxylase in additional animals is presented in Figure S3. Abbreviations are defined in Table S2. To view the original, higher resolution Adobe Illustrator or EPS files, please link to https://www.dropbox.com/sh/y656msmgwotgfy5/AACyqebjHFcGhy2bXtIcW_gDa?dl=0
Fig11 - Odyssey Scan DM.eps (9271 kB)
Fig. 11 Preformed α-synuclein fibril injections in the OB/AON increase the expression of dopaminergic markers in the nigrostriatal and mesolimbic pathways of older male mice. Mice were infused in the right olfactory bulb/anterior olfactory nucleus with either preformed α-synuclein fibrils (5 μg/1 μL) or an equivalent volume of PBS (1 μL). Sagittal brain sections were immunostained with antibodies against the dopaminergic markers tyrosine hydroxylase (TH) and the dopamine transporter (DAT) and scanned on an ultrasensitive infrared imager (LiCor Odyssey). A blinded observer traced the anatomical boundaries of the dorsal striatum (caudoputamen), ventral striatum (nucleus accumbens), the substantia nigra, and the ventral tegmental area in LiCor ImageStudio software. (a) The shape areas drawn around the regions of interest are shown as raw data (unnormalized, arbitrary units) from ImageStudio software. (b) TH signal is displayed after normalization to area, reflecting TH signal density. (c) DAT signal per unit area is also shown, reflecting DAT signal density. (d) Representative pseudocolored images of DAT immunostaining in all groups. Shown are the mean and SD. N = 3-8 mice per group (see Methods and Fig. 1 for animal numbers). Two-way ANOVAs were followed by the Bonferroni post hoc correction. * p≤0.05, ** p≤0.01, *** p≤0.001 PBS vs fibrils; + p≤0.05 vs 3-9 month males; ~ p≤0.05, ~~ p≤0.01, ~~~ p≤0.001 vs 11-17 month males. Abbreviations are defined in Table S2. To view the original, higher resolution Adobe Illustrator or EPS files, please link to https://www.dropbox.com/sh/y656msmgwotgfy5/AACyqebjHFcGhy2bXtIcW_gDa?dl=0
Fig12 - Ventral midbrain DM.eps (1055022 kB)
Fig. 12 Impact of preformed α-synuclein fibril injections in the OB/AON on dopaminergic cell numbers in the ventral mesencephalon. Mice were infused in the right olfactory bulb/anterior olfactory nucleus with either preformed α-synuclein fibrils (5 μg/1 μL) or an equivalent volume of PBS (1 μL). Sagittal brain sections were stained with antibodies against the dopaminergic marker tyrosine hydroxylase (TH) and the nuclear marker Hoechst and 200× images were stitched together to form large, high-resolution photomontages of the ventral mesencephalon from every section of a 1-in-5 sagittal series. A blinded observer traced the anatomical boundaries of the ventral tegmental area and the anterior, posterior, and lateral subregions of the substantia nigra, pars compacta in cellSens software. (a) The areas of the shapes drawn around the regions of interest are shown as raw data (unnormalized, arbitrary units per stitched section). (b) Every single visible TH+ cell encompassing a Hoechst+ nucleus in all the ventral midbrain-containing sections was manually counted by a blinded observer. In the medial substantia nigra, pars compacta, counts were made separately in the anterior and posterior nigra based on boundaries observed in the TH staining (see top row of panel e). The lateral substantia nigra was counted separately, also based on boundaries observed in the TH staining (see bottom row of panel e). Data are presented as area of the region of interest in arbitrary units (a), TH+ cell counts per unit area (b), and Hoechst+ cell counts per unit area (c), within the boundaries of the dotted lines, as shown in panels d-e. Note that two of the panels in d-e are from fibril-infused mice exhibiting no pSer129 pathology in the ventral midbrain, to illustrate the lack of background staining in this region. Raw TH+ cell counts per animal (see Fig.S4 for raw data) were divided by the total TH signal generated on the LiCor imager for the ventral tegmental area (f) and substantia nigra, pars compacta (g) as a measure of “TH expression per cell”. Two-way ANOVAs were followed by the Bonferroni post hoc correction. Shown are the mean and SD from an n of 3-8 mice per group (see Methods and Fig. 1 for animal numbers). * p≤0.05 for PBS vs fibrils; + p≤0.05 vs 3-9 month male group; ~ p≤0.05, ~~ p≤0.01, ~~~ p≤0.001 vs 11-17 month male group. Abbreviations are defined in Table S2. To view the original, higher resolution Adobe Illustrator or EPS files, please link to https://www.dropbox.com/sh/y656msmgwotgfy5/AACyqebjHFcGhy2bXtIcW_gDa?dl=0
Fig13 - Behavior Graphs DM.eps (3272 kB)
Fig 13 α-Synuclein fibril injections in the OB/AON elicit sex and age-dependent behavioral deficits. Mice were infused in the right olfactory bulb/anterior olfactory nucleus with either preformed α-synuclein fibrils (5 μg/1 μL) or an equivalent volume of PBS (1 μL). The buried pellet test for olfactory capacity was conducted at 3 months post-infusion in all groups, and again at 10 months post-infusion in only the oldest group of mice (the only survivors at that time). A blinded observer calculated the latency to approach an exposed peanut (a) and the latency to approach a buried peanut (b). The cylinder test was performed at 4.5 months post-infusion (c-d). Shown are the number of rears (c) and forelimb contacts (d) with the walls of the cylinder in 10 minutes. The novel object/place recognition tests were performed at 5.5 months post-infusion (e-h). Note that the oldest group of mice were not subjected to any behavior assay after 6 months post-infusion, other than the buried pellet test. Two-way ANOVAs were followed by the Bonferroni post hoc correction. Shown are the mean and SD from an n of 3-16 mice per group (see Methods and Fig. 1 for animal numbers). * p≤0.05, *** p≤0.001 PBS vs fibrils; + p≤0.05, ++ p≤0.01 versus vs 3 month male group. ~ p≤0.05 vs 11 month male group. Abbreviations are defined in Table S2. To view the original, higher resolution Adobe Illustrator or EPS files, please link to https://www.dropbox.com/sh/y656msmgwotgfy5/AACyqebjHFcGhy2bXtIcW_gDa?dl=0
FigS1 - male female stitched images.eps (1619088 kB)
Figure S1: Preformed α-synuclein fibril injections in the olfactory peduncle induce a similar topographic extent of α-synucleinopathy in males and females. Mice were infused in the right olfactory bulb/anterior olfactory nucleus with either preformed α-synuclein fibrils (5 μg/1 μL) or an equivalent volume of PBS (1 μL). Sagittal sections were immunostained for pathologically phosphorylated α-synuclein (EP1536Y rabbit monoclonal pSer129 antibody; see Table S1). The nuclear marker Hoechst (pseudocolored blue) was used to denote cytoarchitectonic boundaries. Stitched images of sagittal brain sections from 9 month-old male (top) and female (bottom) mice perfused 6 months post-infusion are displayed from medial to lateral. All sections were processed in parallel and images were captured at the same camera and exposure settings. Note that you must zoom in on the computer screen to view the pathology. Abbreviations are defined in Table S2. To view the original, higher resolution Adobe Illustrator or EPS files, please link to https://www.dropbox.com/sh/y656msmgwotgfy5/AACyqebjHFcGhy2bXtIcW_gDa?dl=0
FigS2 confocal movie 60X oil.mp4 (6556 kB)
Figure S2: Neurons with α-synucleinopathic inclusions at the boundary zone between the substantia nigra and the ventral tegmental area are not tyrosine hydroxylase+. Mice were infused in the right rear olfactory bulb with either preformed α-synuclein fibrils (5 μg/1 μL) or an equivalent volume of PBS (1 μL). After 6 months, sagittal brain sections were stained with the rabbit monoclonal EP1536Y pSer129 antibody (pseudocolored red) for pathologically phosphorylated α-synuclein (see Table S1) and tyrosine hydroxylase (pseudocolored green). Nuclei were counterstained with the Hoechst reagent (pseudocolored blue). This confocal Z-stack video is of the same brain section as displayed in Figure 10. No colocalization of the two markers was observed. To view the original, higher resolution Adobe Illustrator or EPS files, please link to https://www.dropbox.com/sh/y656msmgwotgfy5/AACyqebjHFcGhy2bXtIcW_gDa?dl=0
FigS3 - montage VTA pSer129 TH.eps (75980 kB)
Figure S3: α-synucleinopathic inclusions in the ventral tegmental area are not tyrosine hydroxylase+. Mice were infused in the right rear olfactory bulb with either preformed α-synuclein fibrils (5 μg/1 μL) or an equivalent volume of PBS (1 μL). After 6 months, sagittal brain sections were stained with the rabbit monoclonal EP1536Y pSer129 antibody (pseudocolored red) for pathologically phosphorylated α-synuclein (see Table S1) and tyrosine hydroxylase (TH; pseudocolored green). No robust colocalization of the two markers was observed, and the arrow in the bottom row points to a single pSer129-positive structure that may be lightly TH-positive. Abbreviations are defined in Table S2. To view the original, higher resolution Adobe Illustrator or EPS files, please link to https://www.dropbox.com/sh/y656msmgwotgfy5/AACyqebjHFcGhy2bXtIcW_gDa?dl=0
FigS4 Ventral midbrain counts.eps (4280 kB)
Figure S4: Impact of preformed α-synuclein fibril injections in the OB/AON on dopaminergic cell numbers in the ventral mesencephalon. Mice were infused in the right olfactory bulb/anterior olfactory nucleus with either preformed α-synuclein fibrils (5 μg/1 μL) or an equivalent volume of PBS (1 μL). Sagittal brain sections were stained with antibodies against the dopaminergic marker tyrosine hydroxylase (TH) and the nuclear marker Hoechst and 200× images were stitched together to form large, high-resolution photomontages of the ventral mesencephalon from every section of a 1-in-5 sagittal series. A blinded observer traced the anatomical boundaries of the ventral tegmental area and the anterior, posterior, and lateral subregions of the substantia nigra, pars compacta in cellSens software. (a) Every Hoechst+ cell (within the TH+ area) and every TH+ cell encompassing a Hoechst+ nucleus was manually counted by a blinded observer. These raw data were then used for the cell density measurements presented in Fig.12. Data are presented as (a) raw, unnormalized TH+ cell counts, and (b) raw, unnormalized Hoechst+ cell counts. Two-way ANOVAs were followed by the Bonferroni post hoc correction. Shown are the mean and SD from an n of 3-8 mice per group (see Methods and Fig. 1 for animal numbers). * p≤0.05, ** p≤0.01 for PBS vs fibrils; + p≤0.05 vs 3-9 month male group; ~ p≤0.05 vs 11-17 month male group. Abbreviations are defined in Table S2. To view the original, higher resolution Adobe Illustrator or EPS files, please link to https://www.dropbox.com/sh/y656msmgwotgfy5/AACyqebjHFcGhy2bXtIcW_gDa?dl=0
Fig. 1 Preformed α-synuclein fibril injections in the mouse OB/AON accelerate mortality in older mice without significant changes in body weight. Sixty-four mice were infused in the right olfactory bulb/anterior olfactory nucleus with either preformed α-synuclein fibrils (5 μg/1 μL) or an equivalent volume of PBS (1 μL) at the indicated ages. Logrank survival analyses were performed for all animals at 6 months post-infusion (a) and for the older males at 6 months (b) or 10.5 months (c) post-infusion. The two-tailed p values of the logrank survival analyses are shown. (d) The body weights of the older males at surgery (11 months old), 6 months post-surgery (17 months old), and 10.5 months post-surgery (21.5 months old) are displayed as scatter plots with mean and SEM bars. In panel d, crosses are placed next to mice that died before the planned time of assay. (e-f) Weight gained in both raw numbers (e) and percentage weight gain (f) six months after surgery in all experimental groups. Note the broken Y axis in panels a and d. For panels d-f, two-way ANOVAs were followed by the Bonferroni post hoc correction for multiple comparisons. In panels e-f, +++ p≤0.001 versus males that were infused at 3 months of age and perfused at 9 months of age (3-9 mo males). To view the original, higher resolution Adobe Illustrator or EPS files, please link to https://www.dropbox.com/sh/y656msmgwotgfy5/AACyqebjHFcGhy2bXtIcW_gDa?dl=0
Fig2 - peak density heatmap.eps (1505 kB)
Fig. 2 Heat map of α-synucleinopathy following preformed α-synuclein fibril injections in the mouse OB/AON. Qualitative assessments of the density of pSer129+ inclusions in the indicated brain regions of all fibril-infused animals that survived until the planned assay time (see Methods). Each row represents one animal. A blinded investigator systematically examined the entire 1-in-5 series of sagittal brain sections of the ipsilateral, right hemisphere following immunostaining of pathologically phosphorylated α-synuclein with the rabbit monoclonal EP1536Y pSer129 antibody (see Table S1). Boxes with a N/A label denote regions that were unavailable (see Methods). Brain regions with pSer129+ immunolabeling are arranged rostrocaudally and/or by anatomical/functional groupings. Most of the midbrain pathology was confined to the ventral tegmental area; only two male animals exhibited pSer129+ inclusions at the boundary zone between the substantia nigra and the ventral tegmental area at 6 months post-infusion. Abbreviations are defined in Table S2. To view the original, higher resolution Adobe Illustrator or EPS files, please link to https://www.dropbox.com/sh/y656msmgwotgfy5/AACyqebjHFcGhy2bXtIcW_gDa?dl=0
Fig3 - Rostral Rhinencephelon DM.eps (35790 kB)
Fig. 3 Impact of α-synuclein fibril infusions in the mouse OB/AON on pSer129+ inclusion counts, NeuN+ neuron counts, and Hoechst+ cell numbers in the rostral rhinencephalon. Mice were infused in the right olfactory bulb/anterior olfactory nucleus with either preformed α-synuclein fibrils (5 μg/1 μL) or an equivalent volume of PBS (1 μL). A blinded observer manually counted the number of pSer129+ structures (monoclonal 81A pSer129 Ab; see Table S1) per field of view (200× magnification) and used cellSens software to count the number of NeuN+ and Hoechst+ nuclei in the olfactory bulb (OB; a), the anterior olfactory nucleus (AON; b, d), and the nucleus of the lateral olfactory tract (LOT; c) in the same field of view. Shown are the mean and SD of raw, unnormalized data. N = 3-8 mice per group (see Methods and Fig. 1 for animal numbers). Two-way ANOVAs were followed by the Bonferroni post hoc correction. * p≤0.05, ** p≤0.01, *** p≤0.001 PBS vs fibrils; + p≤0.05 ++ p≤0.01 vs 3-9 month males; ~ p≤0.05 vs 11-17 month males. Abbreviations are defined in Table S2. To view the original, higher resolution Adobe Illustrator or EPS files, please link to https://www.dropbox.com/sh/y656msmgwotgfy5/AACyqebjHFcGhy2bXtIcW_gDa?dl=0
Fig4 - paleocortex amygdala striatum DM.eps (6008 kB)
Fig. 4 Impact of α-synuclein fibril infusions in the mouse OB/AON on pSer129+ inclusion counts, NeuN+ neuron counts, and Hoechst+ cell numbers in the paleocortices, amygdaloid complex, and the dorsal and ventral striata. Mice were infused in the right olfactory bulb/anterior olfactory nucleus with either preformed α-synuclein fibrils (5 μg/1 μL) or an equivalent volume of PBS (1 μL). A blinded observer manually counted the number of pSer129+ structures (monoclonal 81A pSer129 Ab; see Table S1) per field of view (200× magnification) and used cellSens software to count the number of NeuN+ and Hoechst+ nuclei in the rostromedial piriform cortex (Pir Rm; a), caudolateral piriform cortex (Pir Cl; b), amygdalopiriform transition area (APir; c), posteromedial cortical amygdala (PMCo; d), caudoputamen (CPu; e), and nucleus accumbens (Acb; f). Shown are the mean and SD of raw, unnormalized data. N = 3-8 mice per group (see Methods and Fig. 1 for animal numbers). Two-way ANOVAs were followed by the Bonferroni post hoc correction. * p≤0.05, ** p≤0.01, *** p≤0.001 PBS vs fibrils; + p≤0.05, ++ p≤0.01 vs 3-9 month males; ~~ p≤0.01 vs 11-17 month males. Abbreviations are defined in Table S2. To view the original, higher resolution Adobe Illustrator or EPS files, please link to https://www.dropbox.com/sh/y656msmgwotgfy5/AACyqebjHFcGhy2bXtIcW_gDa?dl=0
Fig5 - hippocampal formation and perirhinal telencephalon DM.eps (6555 kB)
Fig. 5 Impact of α-synuclein fibril infusions in the mouse OB/AON on pSer129+ inclusion counts, NeuN+ neuron counts, and Hoechst+ cell numbers in the hippocampal formation and perirhinal telencephalon. Mice were infused in the right olfactory bulb/anterior olfactory nucleus with either preformed α-synuclein fibrils (5 μg/1 μL) or an equivalent volume of PBS (1 μL). A blinded observer manually counted the number of pSer129+ structures (monoclonal 81A pSer129 Ab; see Table S1) per field of view (200× magnification) and used cellSens software to count the number of NeuN+ and Hoechst+ nuclei in the pyramidal cell layers of hippocampal fields CA1 (a), CA2 (b) and CA3 (c), the stratum granulosum of the dentate gyrus (DG; d), the subiculum (Sub; e), and entorhinal cortex (Ent; f), and the ectorhinal cortex (Ect; g). Shown are the mean and SD of raw, unnormalized data. N = 3-8 mice per group (see Methods and Fig. 1 for animal numbers). Two-way ANOVAs were followed by the Bonferroni post hoc correction. * p≤0.05, ** p≤0.01, *** p≤0.001 PBS vs fibrils, + p≤0.05 vs 3-9 month males, ~ p≤0.05 vs 11-17 month males. Abbreviations are defined in Table S2. To view the original, higher resolution Adobe Illustrator or EPS files, please link to https://www.dropbox.com/sh/y656msmgwotgfy5/AACyqebjHFcGhy2bXtIcW_gDa?dl=0
Fig6 - representative histology.eps (764569 kB)
Fig. 6 Preformed α-synuclein fibril injections in the mouse OB/AON elicit α-synucleinopathy in olfactory/limbic regions of the temporal lobe. Mice were infused in the right olfactory bulb/anterior olfactory nucleus with either preformed α-synuclein fibrils (5 μg/1 μL) or an equivalent volume of PBS (1 μL). Sagittal brain sections were immunostained for pathologically phosphorylated α-synuclein (EP1536Y rabbit monoclonal pSer129 antibody; see Table S1). The Hoechst reagent was used to stain nuclei and identify anatomical boundaries. Representative images were captured at 40 and 200 (insets) magnification in the anterior olfactory nucleus (a), tenia tecta (b), dentate gyrus (c), hippocampal CA1 (d), the posteromedial cortical amygdala (e), and the nucleus accumbens (f). Abbreviations are defined in Table S2. To view the original, higher resolution Adobe Illustrator or EPS files, please link to https://www.dropbox.com/sh/y656msmgwotgfy5/AACyqebjHFcGhy2bXtIcW_gDa?dl=0
Fig7 - aggresome.eps (42769 kB)
Fig. 7 Preformed α-synuclein fibril injections in the mouse OB/AON induce the formation of aggresome-like structures. Mice were infused in the right olfactory bulb/anterior olfactory nucleus with either preformed α-synuclein fibrils (5 μg/1 μL) or an equivalent volume of PBS (1 μL). Sagittal brain sections from young male mice were immunostained for pathologically phosphorylated α-synuclein (monoclonal 81A pSer129 antibody; see Table S1). The Proteostat detection reagent was applied to the tissue and the Hoechst reagent was used to stain cellular nuclei. Images were captured with a 100× objective under oil immersion. (a) Perinuclear pSer129+ inclusions were stained with the Proteostat dye in all affected brain regions, including the anterior olfactory nucleus (AON), piriform cortex (Pir), posteromedial cortical nucleus of the amygdala (PMCo), hippocampal CA1, and entorhinal cortex (Ent), in young male mice six months after fibril infusions. See text for details on background staining in PBS group. (b) The arrow points to a pSer129+ neuritic inclusion that does not colocalize with the Proteostat dye, in contrast with the dual-labeled perinuclear inclusion in the soma (arrowhead). (c) Mild pSer129 but dense Proteostat label was observed in the tenia tecta. Abbreviations are defined in Table S2. To view the original, higher resolution Adobe Illustrator or EPS files, please link to https://www.dropbox.com/sh/y656msmgwotgfy5/AACyqebjHFcGhy2bXtIcW_gDa?dl=0
Fig8 - Cell layer widths DM.eps (72650 kB)
Fig. 8 Preformed α-synuclein fibril injections in the OB/AON induce atrophy of pyramidal and granule cell layers of the limbic allocortex in young male mice. Mice were infused in the right olfactory bulb/anterior olfactory nucleus with either preformed α-synuclein fibrils (5 μg/1 μL) or an equivalent volume of PBS (1 μL). A blinded observer employed the “arbitrary line” tool in cellSens software to measure the width of the compact Hoechst+ pyramidal cell layer in the rostromedial piriform cortex, caudolateral piriform cortex, CA1, CA2, and CA3 of the hippocampus, or the stratum granulosum of the dentate gyrus. Representative grayscale micrographs for the caudolateral piriform cortex and dentate gyrus are displayed in panel a and the quantifications of pyramidal and granule cell layers width (b) and Hoechst+ nuclear area (c) are graphed below. Shown are the mean and SD of raw, unnormalized data. N =3-8 mice per group (see Methods section and Fig. 1 for animal numbers). Two-way ANOVAs were followed by the Bonferroni post hoc correction. * p≤0.05, ** p≤0.01, *** p≤0.001 PBS vs fibrils; + p≤0.05, ++ p≤0.01 +++ p≤0.001 versus vs 3-9 month males; ~ p≤0.05, ~~ p≤0.01, ~~~ p≤0.001 vs 11-17 month males. Abbreviations are defined in Table S2. To view the original, higher resolution Adobe Illustrator or EPS files, please link to https://www.dropbox.com/sh/y656msmgwotgfy5/AACyqebjHFcGhy2bXtIcW_gDa?dl=0
Fig9 - M43 Stitch Series pSer129.eps (485731 kB)
Fig. 9 α-synucleinopathy remains centered in the limbic allocortex six months following infusions of preformed α-synuclein fibrils in the OB/AON. A series of stitched images of sagittal brain sections from two 17-month-old animals sacrificed six months after infusion of 1 µL PBS (left) or 5 µg/1 µL fibrils (right) into the OB/AON. All sections were stained in parallel with the monoclonal rabbit EP1536Y pSer129 antibody for pathologically phosphorylated α-synuclein (see Table S1). Anatomical labels are based on cytoarchitectonic details provided by the Hoechst nuclear marker (not shown). All sections were processed in parallel and photographed at the same exposure and intensity scaling. Abbreviations are defined in Table S2. To view the original, higher resolution Adobe Illustrator or EPS files, please link to https://www.dropbox.com/sh/y656msmgwotgfy5/AACyqebjHFcGhy2bXtIcW_gDa?dl=0
Fig10 - confocal TH pSer129 Hoechst montage DM.eps (472755 kB)
Fig. 10 Mesencephalic α-synucleinopathy is centered medially in the limbic-related ventral tegmental area and does not colocalize with dopaminergic markers. A fibril-injected 17 month-old mouse with the most widespread Lewy-related pathology of all cases studied is displayed in this figure. (a) Stitched image of an entire brain section, with genuine α-synucleinopathy at the boundary zone between the ventral tegmental area and substantia nigra, pars compacta (true boundaries were identified with antibodies against the dopaminergic marker tyrosine hydroxylase on the same section—see panel d). Dense pSer129+ inclusions were observed in the anterior olfactory nucleus (AON) and nucleus accumbens (Acb), and sparse inclusions were observed in the olfactory bulb (OB), bed nucleus of stria terminalis (BST), orbitofrontal (VO) and cingulate cortices (A30), and ventral midbrain. A few sparse inclusions were observed in association neocortices (M2) of this one animal. Merged, higher-magnification images of Hoechst+ cells (pseudocolored blue) and pSer129 immunostaining (pseudocolored red; EP1536Y rabbit monoclonal pSer129 antibody; see Table S1) in the AON (b) and accumbens (c). (d) Tyrosine hydroxylase (TH; pseudocolored green) and pSer129 (pseudocolored red) immunostaining in the ventral midbrain of the same animal. (e-h) Lack of convincing colocalization of pSer129 and tyrosine hydroxylase in confocal images of the same animal. A movie of the rotating confocal Z-stack for this animal can be viewed in Figure S2. The lack of convincing colocalization of pSer129 and tyrosine hydroxylase in additional animals is presented in Figure S3. Abbreviations are defined in Table S2. To view the original, higher resolution Adobe Illustrator or EPS files, please link to https://www.dropbox.com/sh/y656msmgwotgfy5/AACyqebjHFcGhy2bXtIcW_gDa?dl=0
Fig11 - Odyssey Scan DM.eps (9271 kB)
Fig. 11 Preformed α-synuclein fibril injections in the OB/AON increase the expression of dopaminergic markers in the nigrostriatal and mesolimbic pathways of older male mice. Mice were infused in the right olfactory bulb/anterior olfactory nucleus with either preformed α-synuclein fibrils (5 μg/1 μL) or an equivalent volume of PBS (1 μL). Sagittal brain sections were immunostained with antibodies against the dopaminergic markers tyrosine hydroxylase (TH) and the dopamine transporter (DAT) and scanned on an ultrasensitive infrared imager (LiCor Odyssey). A blinded observer traced the anatomical boundaries of the dorsal striatum (caudoputamen), ventral striatum (nucleus accumbens), the substantia nigra, and the ventral tegmental area in LiCor ImageStudio software. (a) The shape areas drawn around the regions of interest are shown as raw data (unnormalized, arbitrary units) from ImageStudio software. (b) TH signal is displayed after normalization to area, reflecting TH signal density. (c) DAT signal per unit area is also shown, reflecting DAT signal density. (d) Representative pseudocolored images of DAT immunostaining in all groups. Shown are the mean and SD. N = 3-8 mice per group (see Methods and Fig. 1 for animal numbers). Two-way ANOVAs were followed by the Bonferroni post hoc correction. * p≤0.05, ** p≤0.01, *** p≤0.001 PBS vs fibrils; + p≤0.05 vs 3-9 month males; ~ p≤0.05, ~~ p≤0.01, ~~~ p≤0.001 vs 11-17 month males. Abbreviations are defined in Table S2. To view the original, higher resolution Adobe Illustrator or EPS files, please link to https://www.dropbox.com/sh/y656msmgwotgfy5/AACyqebjHFcGhy2bXtIcW_gDa?dl=0
Fig12 - Ventral midbrain DM.eps (1055022 kB)
Fig. 12 Impact of preformed α-synuclein fibril injections in the OB/AON on dopaminergic cell numbers in the ventral mesencephalon. Mice were infused in the right olfactory bulb/anterior olfactory nucleus with either preformed α-synuclein fibrils (5 μg/1 μL) or an equivalent volume of PBS (1 μL). Sagittal brain sections were stained with antibodies against the dopaminergic marker tyrosine hydroxylase (TH) and the nuclear marker Hoechst and 200× images were stitched together to form large, high-resolution photomontages of the ventral mesencephalon from every section of a 1-in-5 sagittal series. A blinded observer traced the anatomical boundaries of the ventral tegmental area and the anterior, posterior, and lateral subregions of the substantia nigra, pars compacta in cellSens software. (a) The areas of the shapes drawn around the regions of interest are shown as raw data (unnormalized, arbitrary units per stitched section). (b) Every single visible TH+ cell encompassing a Hoechst+ nucleus in all the ventral midbrain-containing sections was manually counted by a blinded observer. In the medial substantia nigra, pars compacta, counts were made separately in the anterior and posterior nigra based on boundaries observed in the TH staining (see top row of panel e). The lateral substantia nigra was counted separately, also based on boundaries observed in the TH staining (see bottom row of panel e). Data are presented as area of the region of interest in arbitrary units (a), TH+ cell counts per unit area (b), and Hoechst+ cell counts per unit area (c), within the boundaries of the dotted lines, as shown in panels d-e. Note that two of the panels in d-e are from fibril-infused mice exhibiting no pSer129 pathology in the ventral midbrain, to illustrate the lack of background staining in this region. Raw TH+ cell counts per animal (see Fig.S4 for raw data) were divided by the total TH signal generated on the LiCor imager for the ventral tegmental area (f) and substantia nigra, pars compacta (g) as a measure of “TH expression per cell”. Two-way ANOVAs were followed by the Bonferroni post hoc correction. Shown are the mean and SD from an n of 3-8 mice per group (see Methods and Fig. 1 for animal numbers). * p≤0.05 for PBS vs fibrils; + p≤0.05 vs 3-9 month male group; ~ p≤0.05, ~~ p≤0.01, ~~~ p≤0.001 vs 11-17 month male group. Abbreviations are defined in Table S2. To view the original, higher resolution Adobe Illustrator or EPS files, please link to https://www.dropbox.com/sh/y656msmgwotgfy5/AACyqebjHFcGhy2bXtIcW_gDa?dl=0
Fig13 - Behavior Graphs DM.eps (3272 kB)
Fig 13 α-Synuclein fibril injections in the OB/AON elicit sex and age-dependent behavioral deficits. Mice were infused in the right olfactory bulb/anterior olfactory nucleus with either preformed α-synuclein fibrils (5 μg/1 μL) or an equivalent volume of PBS (1 μL). The buried pellet test for olfactory capacity was conducted at 3 months post-infusion in all groups, and again at 10 months post-infusion in only the oldest group of mice (the only survivors at that time). A blinded observer calculated the latency to approach an exposed peanut (a) and the latency to approach a buried peanut (b). The cylinder test was performed at 4.5 months post-infusion (c-d). Shown are the number of rears (c) and forelimb contacts (d) with the walls of the cylinder in 10 minutes. The novel object/place recognition tests were performed at 5.5 months post-infusion (e-h). Note that the oldest group of mice were not subjected to any behavior assay after 6 months post-infusion, other than the buried pellet test. Two-way ANOVAs were followed by the Bonferroni post hoc correction. Shown are the mean and SD from an n of 3-16 mice per group (see Methods and Fig. 1 for animal numbers). * p≤0.05, *** p≤0.001 PBS vs fibrils; + p≤0.05, ++ p≤0.01 versus vs 3 month male group. ~ p≤0.05 vs 11 month male group. Abbreviations are defined in Table S2. To view the original, higher resolution Adobe Illustrator or EPS files, please link to https://www.dropbox.com/sh/y656msmgwotgfy5/AACyqebjHFcGhy2bXtIcW_gDa?dl=0
FigS1 - male female stitched images.eps (1619088 kB)
Figure S1: Preformed α-synuclein fibril injections in the olfactory peduncle induce a similar topographic extent of α-synucleinopathy in males and females. Mice were infused in the right olfactory bulb/anterior olfactory nucleus with either preformed α-synuclein fibrils (5 μg/1 μL) or an equivalent volume of PBS (1 μL). Sagittal sections were immunostained for pathologically phosphorylated α-synuclein (EP1536Y rabbit monoclonal pSer129 antibody; see Table S1). The nuclear marker Hoechst (pseudocolored blue) was used to denote cytoarchitectonic boundaries. Stitched images of sagittal brain sections from 9 month-old male (top) and female (bottom) mice perfused 6 months post-infusion are displayed from medial to lateral. All sections were processed in parallel and images were captured at the same camera and exposure settings. Note that you must zoom in on the computer screen to view the pathology. Abbreviations are defined in Table S2. To view the original, higher resolution Adobe Illustrator or EPS files, please link to https://www.dropbox.com/sh/y656msmgwotgfy5/AACyqebjHFcGhy2bXtIcW_gDa?dl=0
FigS2 confocal movie 60X oil.mp4 (6556 kB)
Figure S2: Neurons with α-synucleinopathic inclusions at the boundary zone between the substantia nigra and the ventral tegmental area are not tyrosine hydroxylase+. Mice were infused in the right rear olfactory bulb with either preformed α-synuclein fibrils (5 μg/1 μL) or an equivalent volume of PBS (1 μL). After 6 months, sagittal brain sections were stained with the rabbit monoclonal EP1536Y pSer129 antibody (pseudocolored red) for pathologically phosphorylated α-synuclein (see Table S1) and tyrosine hydroxylase (pseudocolored green). Nuclei were counterstained with the Hoechst reagent (pseudocolored blue). This confocal Z-stack video is of the same brain section as displayed in Figure 10. No colocalization of the two markers was observed. To view the original, higher resolution Adobe Illustrator or EPS files, please link to https://www.dropbox.com/sh/y656msmgwotgfy5/AACyqebjHFcGhy2bXtIcW_gDa?dl=0
FigS3 - montage VTA pSer129 TH.eps (75980 kB)
Figure S3: α-synucleinopathic inclusions in the ventral tegmental area are not tyrosine hydroxylase+. Mice were infused in the right rear olfactory bulb with either preformed α-synuclein fibrils (5 μg/1 μL) or an equivalent volume of PBS (1 μL). After 6 months, sagittal brain sections were stained with the rabbit monoclonal EP1536Y pSer129 antibody (pseudocolored red) for pathologically phosphorylated α-synuclein (see Table S1) and tyrosine hydroxylase (TH; pseudocolored green). No robust colocalization of the two markers was observed, and the arrow in the bottom row points to a single pSer129-positive structure that may be lightly TH-positive. Abbreviations are defined in Table S2. To view the original, higher resolution Adobe Illustrator or EPS files, please link to https://www.dropbox.com/sh/y656msmgwotgfy5/AACyqebjHFcGhy2bXtIcW_gDa?dl=0
FigS4 Ventral midbrain counts.eps (4280 kB)
Figure S4: Impact of preformed α-synuclein fibril injections in the OB/AON on dopaminergic cell numbers in the ventral mesencephalon. Mice were infused in the right olfactory bulb/anterior olfactory nucleus with either preformed α-synuclein fibrils (5 μg/1 μL) or an equivalent volume of PBS (1 μL). Sagittal brain sections were stained with antibodies against the dopaminergic marker tyrosine hydroxylase (TH) and the nuclear marker Hoechst and 200× images were stitched together to form large, high-resolution photomontages of the ventral mesencephalon from every section of a 1-in-5 sagittal series. A blinded observer traced the anatomical boundaries of the ventral tegmental area and the anterior, posterior, and lateral subregions of the substantia nigra, pars compacta in cellSens software. (a) Every Hoechst+ cell (within the TH+ area) and every TH+ cell encompassing a Hoechst+ nucleus was manually counted by a blinded observer. These raw data were then used for the cell density measurements presented in Fig.12. Data are presented as (a) raw, unnormalized TH+ cell counts, and (b) raw, unnormalized Hoechst+ cell counts. Two-way ANOVAs were followed by the Bonferroni post hoc correction. Shown are the mean and SD from an n of 3-8 mice per group (see Methods and Fig. 1 for animal numbers). * p≤0.05, ** p≤0.01 for PBS vs fibrils; + p≤0.05 vs 3-9 month male group; ~ p≤0.05 vs 11-17 month male group. Abbreviations are defined in Table S2. To view the original, higher resolution Adobe Illustrator or EPS files, please link to https://www.dropbox.com/sh/y656msmgwotgfy5/AACyqebjHFcGhy2bXtIcW_gDa?dl=0
Comments
Rehana Leak is the corresponding author at 407 Mellon Hall, 600 Forbes Ave, Duquesne University, Pittsburgh, PA 15282, United States.