We thank Prof. Kelvin Luk for his generous gift of &#945;-synuclein PFFs, Conjung Zheng for establishing and culturing neuronal cells, and the Light Microscopy Unit at the Institute of Biotechnology, University of Helsinki, for imaging immunostained cells. This work was supported by grants from 3i-Regeneration by Business Finland (Finnish Funding Agency for Innovation), Academy of Finland #309489, #293392, #319195; Sigrid Juselius Foundation, and the statutory funds of the Maj Institute of Pharmacology, PAS, Poland.

<ol>
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The authors have nothing to disclose.

Spreading Lewy pathology, of which pS129-&#945;syn is a major constituent, is a histopathological hallmark of PD. Stopping or slowing down the accumulation of aggregated pS129-&#945;syn may slow down the degeneration of dopamine neurons and the progression of alpha-synucleinopathy. However, a mechanistic understanding of how pS129-&#945;syn aggregation contributes to the demise of dopamine neurons still has to be established. Evidence from human postmortem studies on brain samples from patients at different stages of the disease as well as observation of pS129-&#945;syn positive inclusion in transplanted fetal neurons strongly suggests the spreading of Lewy pathology between cells16,17,33. Consequently, prion-like spreading of pS129-&#945;syn was recently recapitulated by using &#945;-synuclein PFFs9,10. Establishing a robust, cost-effective, and relatively high- or medium-throughput model of pS129-&#945;syn spreading and accumulation, specifically in dopamine neurons, can considerably speed up the search for novel treatments and compounds modifying this process.
Because loss of dopamine neurons is the main cause of motor symptoms in PD and these cells possess many unique properties2,34,35, modeling of prion-like spreading of pS129-&#945;syn in dopamine neurons is the most relevant type of model from the translational perspective. Protocols utilizing micro island cultures of embryonic midbrain neurons on 4 well plates and semiautomatic quantification have been described previously18. The protocol described here was adapted to 96 well plates and provides less laborious preparation of micro islands, allowing for the preparation of up to four plates containing 60 wells each by an experienced researcher during one workday. Culturing dopamine neurons in 96 well plates allows for testing drugs at lower amounts and enables high transduction rates with lentivirus vectors. It is also possible to combine different treatments to perform more complex experiments.
Before applying any treatments (including PFFs), the quality of the culturing should be checked with bright-field microscopy. If the microscopy system does not utilize a CO2 chamber with heating, the cells should not be kept outside of the incubator for more than couple of minutes, because primary mouse dopamine neurons are delicate and easily stressed. For the same reason, it is advised that the first imaging should be done after 24 h of incubation (between DIV1-DIV3). The cells should appear to be alive with present cell bodies and homogenously spread inside the micro island. Primary neurons would have settled on the PO-coated ground and started to establish neuronal projections. It is possible to observe small clump of cells (i.e., diameter smaller than 150&#8211;200 &#956;m) that can be formed if the trituration process is not done properly or plating density is higher than recommended. These small clumps would not affect the experiment, unless they are more than a few per well and/or larger. Clumped cells make it very difficult to identify immunohistochemical markers and individual cells during the image analysis. It is essential to avoid such clumps by careful coating, triturating, and controlling plating density. If the uniformity of these conditions cannot be observed at certain wells, do not include these defective wells in the experiment. Such exclusion should be done before the execution of any treatments.&#160;
Moreover, utilization of 96 well plates allows for convenient multichannel pipette use during staining procedures and direct visualization with automatic plate microscopes, further increasing throughput. Utilization of automated image quantification is indispensable for the analysis of the data from high-content imaging platforms. In addition to the capability to process thousands of images obtained from each experiment, it ensures unbiased, identical quantification of all treatment groups. The workflow proposed for the image analysis is based on simple principles of segmenting dopamine neurons, filtering correctly segmented cells by supervised machine learning, and subsequently quantification of phenotypes (pS129-&#945;syn positive and pS129-&#945;syn negative), again by supervised machine learning. Although several different approaches for this task can be envisioned, we have found the combination of segmentation with machine learning to be the most robust for dopamine cultures due to high plating density, the diverse shapes of dopamine neurons, and the presence of strongly stained neurites. The proposed image analysis algorithm was implemented in CellProfiler and CellProfiler Analyst, open source, freely available high-content image analysis software26,27. The algorithm could also be implemented with other image analysis software, either open source (e.g., ImageJ/FIJI, KNIME) or proprietary. However, in our experience these often sacrifice customization capabilities for ease of use, and therefore might not perform well in complicated analyses. We have found that the CellProfiler and CellProfiler Analyst software packages give particularly reliable results by combining a substantial number of implemented algorithms, extreme flexibility in designing workflow, and simultaneously handling and efficiently processing of high-content imaging data.
The described protocol could also be adapted for quantification of other cellular phenotypes characterized by immunostaining with different antibodies, such as markers of other neuronal populations (e.g., DAT, GAD67, 5-HT etc.) and protein aggregates (e.g., phospo-Tau, ubiquitin). Multiple fluorescent markers could also be combined to distinguish multiple phenotypes (e.g., cells with inclusions at different stages of maturation). Automated classification of multiple phenotypes should also be easy to implement in the described image analysis pipelines by merely adding a channel containing immunofluorescent images of additional markers to measurement steps and sorting cells into multiple bins. Utilization of multiple markers at the same time would, however, require the optimization of immunostaining and imaging conditions. Additionally, for better quality in immunofluorescence imaging, the use of special black-walled 96 well plates explicitly designed for the fluorescent microscope is recommended. However, these can be considerably more expensive than standard cell culture plates, which are sufficient for the analysis described in our protocol.
The type and quality of utilized PFFs are critical for the outcome of the experiments. PFFs can both affect the robustness of the assay and the interpretation of results. Preparation conditions might affect the seeding efficiency of PFFs and, indeed, PFF &#34;strains&#34; with different physiological properties have been reported36. Nonetheless, the preparation and validation of PFFs are beyond the scope of this article and have been described in several publications11,28,29,30. In addition to the preparation protocol, the species of origin of &#945;-synuclein in PFFs (e.g., mouse, human) and the usage of wild type or mutated protein (e.g., human A53T &#945;-synuclein) should be considered, depending on the particular experimental conditions. Induction of pS129-&#945;syn accumulation by PFFs was shown to be dependent on age of the culture (i.e., days in vitro), with more mature cultures showing more pronounced induction11. This is probably due to the increased number of neuronal connections in more mature cultures, and increased &#945;-synuclein protein levels. In our hands, treatment with PFFs at DIV8 gave the most robust results, with pronounced accumulation of pS129-&#945;syn in dopamine neuron soma, while not compromising neuronal survival. The described protocol is well suited to study treatments modifying early events leading to aggregation of endogenous &#945;-synuclein because we quantify pS129-&#945;syn positive inclusions at a relatively early time point, 7 days after inoculation with PFFs. At this time point, intrasomal inclusions are present in a significant fraction of cells and can be easily distinguished by immunostaining while no PFF-induced cell death is observed, simplifying the interpretation of the results. Importantly, as the morphology and composition of PFF-induced inclusions can change over time12,13, the described protocol could, in principle, be modified to study more mature inclusions by fixation and immunostaining at later time points. However, keeping dopamine neurons in culture for longer than 15 days requires extreme care, and might induce additional variation because of cells failing to survive independently from PFF inoculation. Additionally, more extended cultures complicate drug treatment schedules. Many compounds have limited or not poorly characterized stability in the cell culture medium, and replenishment of a drug is not trivial because complete exchange of medium compromises the survival of the dopamine cultures.
Phosphorylation of &#945;-synuclein at Ser129 is consistently reported in PFF-based models of &#945;-synuclein aggregation and colocalizes with markers of misfolding and aggregation such as Thioflavin S, ubiquitin, or conformation-specific antibodies11,12. In our hands, immunostaining for pS129-&#945;syn also gives the strongest signal with the lowest background and is most straightforward to analyze, giving robust results when multiple treatments are screened. Importantly, immunostaining with pS129-&#945;syn antibody does not detect PFFs that remain outside of the cells, significantly reducing the background. However, it is important to remember that Ser129 phosphorylation is probably one of the earliest processes linked with the misfolding of &#945;-synuclein and might be differently regulated under specific conditions. Therefore, any findings that show positive effects on pS129-&#945;syn should be confirmed by other markers.
Statistical analysis should be tailored correspondingly to experimental design. It is essential to perform experiments in at least three independent biological replicates (i.e., separate primary neuronal cultures). These replicates should be plated on different plates and treated independently. We analyze the data obtained from replicates on different plates with random block design ANOVA37 to take into account the pairing of data for different experimental plates.&#160;

Parkinson&#8217;s disease (PD) is a neurodegenerative disorder characterized by the death of the midbrain dopamine neurons in the substantia nigra (SN), subsequent loss of dopamine tone in basal ganglia, and consequent motor impairments1,2. A major histopathological feature in the brains of PD patients are intracellular protein/lipid aggregates found in neuronal soma, called Lewy bodies (LB), or in neurites, Lewy neurites (LN), collectively known as Lewy pathology3. Lewy pathology in the brain appears to progress with advancing PD resembling the spread of pathogenic factors through neuronal connections. Abundant Lewy pathology is found in dopamine neurons in the SN and cells in other areas affected by neurodegeneration4. However, during disease progression, spread and onset of protein aggregation do not always correlate with neuronal death and the exact contribution of Lewy pathology to neuronal death is still unclear5.
LB and LN had been shown to consist of membranous and proteinaceous components3. The former are membrane fragments, vesicular structures (possibly lysosomes and autophagosomes) and mitochondria3. The latter consists of at least 300 different proteins6. A hallmark study by Spillantini et al.7 demonstrated that the major protein component of Lewy pathology is &#945;-synuclein. Highly expressed in neurons, and linked with membrane fusion and neurotransmitter release, &#945;-synuclein in Lewy pathology is present mostly in misfolded, amyloid fibril form, the bulk of which is phosphorylated at Ser129 (pS129-&#945;syn)4,8.
Importantly, it was also demonstrated that due to its prion-like properties, misfolded &#945;-synuclein might have a causative role in Lewy pathology formation4. The prion-like properties of misfolded &#945;-synuclein were shown with both midbrain extracts from patients and exogenously prepared &#945;-synuclein preformed fibrils (PFFs) to induce &#945;-synuclein aggregates in neurons in culture and in vivo9,10. PFFs present a reliable and robust model to study the progression of &#945;-synuclein pathology in dopamine neurons. When PFFs are applied to cultured primary neurons or injected into the animal brain, they lead to the formation of &#945;-synuclein-containing inclusions in neurites and cell soma11 that recapitulate many features seen in Lewy pathology. Observed inclusions are detergent-insoluble in Triton X, ubiquitinated, stained with the amyloid specific dye Thioflavin S, and contain &#945;-synuclein hyperphosphorylated at Ser12911,12. Importantly, these inclusions do not form in &#945;-synuclein knockout animals11, indicating the dependence of their formation on endogenous &#945;-synuclein.
Nonetheless, it is difficult to directly compare PFF-induced inclusions and Lewy pathology found in PD patients because human LBs and LNs are highly heterogeneous3. Observed heterogeneity of Lewy pathology might be caused by different stages of the formation, different anatomical location, or differences in the conformation of misfolded &#945;-synuclein initiating the aggregation process. The same factors might influence PFF-induced pS129-&#945;syn positive inclusions. Indeed, recently it was demonstrated that PFF-induced pS129-&#945;syn positive inclusions in primary neuronal cultures represent very early stages of pathology that can mature to structures closely resembling LB after prolonged incubation period12,13.
Modeling early spreading and accumulation of misfolded &#945;-synuclein with PFFs is valuable for drug development, as Lewy pathology spread is considered one of the early-stage disease markers. Therefore, aggregation-preventive treatments may be promising for stopping or slowing down the progression of PD at very early stages. Several clinical trials aimed at slowing or stopping &#945;-synuclein accumulation are ongoing14. For later-stage patients, transplantation of dopamine neuronal progenitors can be a better treatment alternative15. However, Lewy pathology was documented in transplanted embryonic neurons during the post-mortem analysis of PD patient brains16,17, also indicating the need for protection against &#945;-synuclein accumulation.
In vitro, &#945;-synuclein PFFs are known to induce aggregation in immortalized cell lines, or more commonly, in rodent primary hippocampal or cortical neurons. Neither of these are close to recapitulating dopamine neurons10. Culturing these neurons requires dense plating of certain numbers of neurons in vitro18. To achieve high plating density with limited material (e.g., primary dopamine neurons), the micro island culturing method is commonly utilized. In micro island culturing, cells are initially plated in a small drop of medium (usually a few microliters) kept together by surface tension in the middle of a large well18. After the neurons attach, the entire well is filled with the medium while the cells remain confined at high density in the small plating area. In addition to achieving high plating density, micro islands also prevent plating near the edges of wells, where variations in cell density and survival are frequent. Micro islands are often utilized in relatively large wells or dishes; however, establishing midbrain neuronal cultures in micro islands in 96 well plate format enables the study of Lewy pathology in bona fide dopamine neurons with medium-to-high-throughput power. In vitro experiments with these neurons allowed us to discover the glial cell line-derived neurotrophic factor (GDNF), which promotes survival of mature dopamine neurons in vitro and in vivo19,20,21,22 and also prevents the formation of &#945;-synuclein aggregates in dopamine neurons23. Human patient-induced pluripotent stem cell-derived dopamine neurons constitute a more accurate model due to their human origin and longer survival time in vitro. However, induction of &#945;-synuclein pathology in human neurons is observed after multiple months, compared to a week in mouse embryonic neurons, and/or with multiple stressors (e.g., combination of &#945;-synuclein overexpression and PFFs)24,25. In addition, maintenance of human dopamine neurons is more costly and laborious when compared to primary embryonic neurons, essentially limiting their use in high-throughput applications.
Further, primary dopamine neuronal cultures can be genetically modified (e.g., with CRISPR/Cas9) and/or treated with pharmacological agents23. They constitute a fast and reproducible platform for applications like molecular pathway dissection and drug library screening. Even though limited material can be obtained from these cultures, it is still possible to conduct small size genomics/proteomics analyses. Culturing primary neurons in 96 well format is better for immunocytochemistry and fluorescence microscopy techniques, followed by high-content phenotype analysis. Multispectral fluorescence images derived from automated imaging of 96 well plates can be converted into quantitative results (e.g., the number of LB-containing neurons per well). Such analyses can be done with free software, such as CellProfiler26,27. Overall, primary embryonic midbrain cultures plated in 96 well plates provide a robust and efficient platform to study dopamine neurons and &#945;-synuclein aggregation with the opportunity for high-throughput phenotype screening.

<table>
	<tbody>
		<tr>
			<td>0.4% Trypan Blue stain</td>
			<td>Gibco, Thermo Scientific, Waltham, Massachusetts, USA</td>
			<td>15250061</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml CELLSTAR Polypropylene tube</td>
			<td>Greiner Bio-One GmbH, Frickenhausen, Germany</td>
			<td>188261</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#8217;,6-diamidino-2-phenylindole (DAPI)</td>
			<td>Sigma-Aldrich, St. Louis, Missouri, USA</td>
			<td>10236276001</td>
			<td></td>
		</tr>
		<tr>
			<td>5 M HCl</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Media kitchen, Institute of Biotechnology, University of Helsinki</td>
		</tr>
		<tr>
			<td>5 M NaOH</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Media kitchen, Institute of Biotechnology, University of Helsinki</td>
		</tr>
		<tr>
			<td>96-well ViewPlate (Black)</td>
			<td>PerkinElmer, Waltham, Massachusetts, USA</td>
			<td>6005182</td>
			<td></td>
		</tr>
		<tr>
			<td>99.5% Ethanol (EtOH)</td>
			<td>Altia Oyj, Rajam&#228;ki, Finland</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>AquaSil Siliconizing Fluid</td>
			<td>Thermo Scientific, Waltham, Massachusetts, USA</td>
			<td>TS-42799</td>
			<td></td>
		</tr>
		<tr>
			<td>Autoclaved 1.5 ml microcentrifuge tube</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Media kitchen, Institute of Biotechnology, University of Helsinki</td>
		</tr>
		<tr>
			<td>Bioruptor sonication device</td>
			<td>Diagenode, Liege, Belgium</td>
			<td>B01020001</td>
			<td></td>
		</tr>
		<tr>
			<td>Ca2+, Mg2+ free Hank&#8217;s Balanced Salt Solution (HBSS)</td>
			<td>Gibco, Thermo Scientific, Waltham, Massachusetts, USA</td>
			<td>14175-053</td>
			<td></td>
		</tr>
		<tr>
			<td>CellProfiler and CellAnalyst software packages</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>free open-source software</td>
		</tr>
		<tr>
			<td>Centrifuge 5702</td>
			<td>Eppendorf AG, Hamburg, Germany</td>
			<td>5702000019</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td>HeraCell, Thermo Scientific, Waltham, Massachusetts, USA</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Counting chamber</td>
			<td>BioRad Inc., Hercules, California, USA</td>
			<td>1450015</td>
			<td></td>
		</tr>
		<tr>
			<td>DeltaVision Ultra High Resolution Microscope with air table and cabinet</td>
			<td>GE Healthcare Life Sciences, Boston, Massachusetts, USA</td>
			<td>29254706</td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxyribonuclease-I (Dnase I)</td>
			<td>Roche, Basel, Switzerland</td>
			<td>22098700</td>
			<td></td>
		</tr>
		<tr>
			<td>D-glucose</td>
			<td>Sigma-Aldrich, St. Louis, Missouri, USA</td>
			<td>G8769</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Gibco, Thermo Scientific, Waltham, Massachusetts, USA</td>
			<td>21331&#8211;020</td>
			<td></td>
		</tr>
		<tr>
			<td>donkey anti-mouse AlexaFluor 488</td>
			<td>Thermo Scientific, Waltham, Massachusetts, USA</td>
			<td>A21202</td>
			<td></td>
		</tr>
		<tr>
			<td>donkey anti-rabbit AlexaFluor 647</td>
			<td>Thermo Scientific, Waltham, Massachusetts, USA</td>
			<td>A31573</td>
			<td></td>
		</tr>
		<tr>
			<td>donkey anti-sheep AlexaFluor 488</td>
			<td>Thermo Scientific, Waltham, Massachusetts, USA</td>
			<td>A11015</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s buffer</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Media kitchen, Institute of Biotechnology, University of Helsinki</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Gibco, Thermo Scientific, Waltham, Massachusetts, USA</td>
			<td>10500056</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand Plain Economy PTFE Stir Bar</td>
			<td>fisher scientific, Thermo Scientific, Waltham, Massachusetts, USA</td>
			<td>11507582</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageXpress Nano Automated Imaging System</td>
			<td>Molecular Devices, San Jose, California, USA</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Gibco, Thermo Scientific, Waltham, Massachusetts, USA</td>
			<td>25030&#8211;032</td>
			<td></td>
		</tr>
		<tr>
			<td>mouse monoclonal anti-tyrosine hydroxylase</td>
			<td>Millipore, Merck KGaA, Darmstadt, German</td>
			<td>MAB318</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 supplement</td>
			<td>Gibco, Thermo Scientific, Waltham, Massachusetts, USA</td>
			<td>17502&#8211;048</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Horse Serum</td>
			<td>Vector Laboratories Inc., Burlingame, California, United States</td>
			<td>S-2000</td>
			<td></td>
		</tr>
		<tr>
			<td>paraformaldehyde (PFA)</td>
			<td>Sigma-Aldrich, St. Louis, Missouri, USA</td>
			<td>158127</td>
			<td></td>
		</tr>
		<tr>
			<td>paraformaldehyde powder, 95%</td>
			<td>Sigma-Aldrich, St. Louis, Missouri, USA</td>
			<td>158127</td>
			<td></td>
		</tr>
		<tr>
			<td>pH-Fix, color-fixed indicator strips</td>
			<td>Macherey-Nagel, D&#252;ren, Germany</td>
			<td>92110</td>
			<td></td>
		</tr>
		<tr>
			<td>Phospohate Buffer Saline (PBS)</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Media kitchen, Institute of Biotechnology, University of Helsinki</td>
		</tr>
		<tr>
			<td>poly-L-ornithine</td>
			<td>Sigma-Aldrich, St. Louis, Missouri, USA</td>
			<td>P4957</td>
			<td></td>
		</tr>
		<tr>
			<td>Primocin</td>
			<td>InvivoGen, San Diego, California, USA</td>
			<td>ant-pm-1, ant-pm-2</td>
			<td></td>
		</tr>
		<tr>
			<td>rabbit monoclonal anti-alpha-synuclein filament</td>
			<td>Abcam, Cambridge, United Kingdom</td>
			<td>ab209538</td>
			<td></td>
		</tr>
		<tr>
			<td>rabbit monoclonal anti-phospha-serine129-alpha-synuclein</td>
			<td>Abcam, Cambridge, United Kingdom</td>
			<td>ab51253</td>
			<td></td>
		</tr>
		<tr>
			<td>RCT Basic, IKA Magnetic Stirrer</td>
			<td>IKA&#174;-Werke GmbH, Staufen, Germany</td>
			<td>3810000</td>
			<td></td>
		</tr>
		<tr>
			<td>recombinant human glia-derived neurotrophic factor (hGDNF)</td>
			<td>PeproTech, Rocky Hill, NJ, USA or Prospec, Ness-Ziona, Israel</td>
			<td>450-10 (PeproTech), CYT-305 (Prospec)</td>
			<td></td>
		</tr>
		<tr>
			<td>recombinant mouse alpha synuclein Pre-Formed Fibrils (PFFs)</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Gift from collaborator, Prof. Kelvin C Luk</td>
		</tr>
		<tr>
			<td>Research Stereomicroscope System</td>
			<td>Olympus Corporation, Tokyo, Japan</td>
			<td>SZX10</td>
			<td></td>
		</tr>
		<tr>
			<td>sheep polyclonal anti-tyrosine hydroxylase</td>
			<td>Millipore, Merck KGaA, Darmstadt, German</td>
			<td>ab1542</td>
			<td></td>
		</tr>
		<tr>
			<td>TC 20 Automated Cell Counter</td>
			<td>BioRad Inc., Hercules, California, USA</td>
			<td>1450102</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich, St. Louis, Missouri, USA</td>
			<td>11332481001</td>
			<td></td>
		</tr>
		<tr>
			<td>trypsin</td>
			<td>MP Biomedicals, Valiant Co., Yantai, Shandong, China</td>
			<td>2199700</td>
			<td></td>
		</tr>
	</tbody>
</table>

studying pre-formed fibril induced &#945;-synuclein accumulation in primary embryonic mouse midbrain dopamine neurons

The goal of this protocol is to establish a robust and reproducible model of &#945;-synuclein accumulation in primary dopamine neurons. Combined with immunostaining and unbiased automated image analysis, this model allows for the analysis of the effects of drugs and genetic manipulations on &#945;-synuclein aggregation in neuronal cultures. Primary midbrain cultures provide a reliable source of bona fide embryonic dopamine neurons. In this protocol, the hallmark histopathology of Parkinson&#8217;s disease, Lewy bodies (LB), is mimicked by the addition of &#945;-synuclein pre-formed fibrils (PFFs) directly to neuronal culture media. Accumulation of endogenous phosphorylated &#945;-synuclein in the soma of dopamine neurons is detected by immunostaining already at 7 days after the PFF addition. In vitro cell culture conditions are also suitable for the application and evaluation of treatments preventing &#945;-synuclein accumulation, such as small molecule drugs and neurotrophic factors, as well as lentivirus vectors for genetic manipulation (e.g., with CRISPR/Cas9). Culturing the neurons in 96 well plates increases the robustness and power of the experimental setups. At the end of the experiment, the cells are fixed with paraformaldehyde for immunocytochemistry and fluorescence microscopy imaging. Multispectral fluorescence images are obtained via automated microscopy of 96 well plates. These data are quantified (e.g., counting the number of phospho-&#945;-synuclein-containing dopamine neurons per well) with the use of free software that provides a platform for unbiased high-content phenotype analysis. PFF-induced modeling of phosphorylated &#945;-synuclein accumulation in primary dopamine neurons provides a reliable tool to study the underlying mechanisms mediating formation and elimination of &#945;-synuclein inclusions, with the opportunity for high-throughput drug screening and cellular phenotype analysis.

A few days after the plating (DIV1&#8211;DIV3), bright-field microscopy was done to assess the health and homogenous spread of the cultured cells, and uniformity of these conditions at the individual wells (Figure 3). Cultured midbrain cells were spread homogenously within the micro island created before the plating (Figure 3A,B). Primary neurons had settled on the coated ground homogenously and established neuronal projections (Figure 3B). A small clump of cells (diameter smaller than 150 &#956;m) was observed at the well and shown as an example (Figure 3C).
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61118/61118fig03.jpg" /> 
Figure 3: A few days after the plating (e.g., DIV3), the condition of primary midbrain cultures were observed with bright-field microscopy. (A) Cultured cells spread across the middle of the well within the PO-coated micro island with an approximate radius of 4.4 mm (shown with white arrows) created by scratching PO from approximately 1 mm well perimeter (shown with black arrows). (B) Cultured primary neurons homogenously spread within the area and neuronal projections (marked with red arrowheads) were observed. (C) A cell clump with a diameter smaller than 150 &#956;m is marked with blue arrowheads. Scale bars = 100 &#956;m. <a href="https://www.jove.com/files/ftp_upload/61118/61118fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Primary mouse midbrain cultures were immunostained with anti-TH and anti-pS129-&#945;syn antibodies and imaged with an automated microscope after 15 days in vitro. Coated micro islands provided restricted area for the attachment of cells in the middle of wells (Figure 4A,B). Dopamine neurons immunolabeled with TH marker were spread around the micro island in a monolayer, separated from each other, without any clumping (Figure 4A&#8217; and 4B&#8217;).
<img alt="Figure 4" class="xfigimg bigfig" src="/files/ftp_upload/61118/61118fig4v2.jpg" /> 
Figure 4: At DIV15, 800&#8211;1,000 dopamine cells were quantified from each micro island, with or without PFF treatment. Representative images of embryonic midbrain cultures immonustained with anti-TH and anti-pS129-&#945;syn antibodies. (A, A&#8217;, A&#8217;&#8217;) Control cells without PFF treatment. (B, B&#8217;, B&#8217;&#8217;) PFF-treated cells with pS129-&#945;syn inclusions. (C) Quantification of TH-positive cell numbers in wells treated with vehicle (control), PFFs, or PFFs with 50 ng/mL GDNF. (D) Quantification of pS129-&#945;syn aggregates in TH-positive cells treated with vehicle (control), PFFs, or PFFs with 50 ng/mL GDNF. Statistical significance was calculated with random block design ANOVA. **p &#60; 0.01, n = 4 individual plates (biological replicates), each with 3&#8211;6 wells per treatment group (technical repeats). Scale bars = 300 &#956;m for A, B; 50 &#956;m for A&#8217;, B&#8217;; 25 &#956;m for A&#8217;&#8217;, B&#8217;&#8217;. <a href="https://www.jove.com/files/ftp_upload/61118/61118fig4v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
While cultures without PFF treatment did not have any pS129- &#945;syn signal (Figure 4A&#8217; and 4A&#8217;&#8217;), cultures treated with &#945;-synuclein PFFs developed pS129-&#945;syn positive inclusions (Figure 4B&#8217; and 4B&#8217;&#8217;). In vitro PFF treatment for 7 days did not cause any significant decrease in numbers of TH-positive neurons, compared to other experimental groups (Figure 4C). PFF-treated cultures had a population of ~40% of pS129-&#945;syn positive TH-positive dopamine neurons. Treatment with positive control, GDNF, reduced the percentage of TH-positive dopamine neurons with pS129-&#945;syn positive inclusions (Figure 4D, see also the raw data and example images in the Supplementary Files).
Supplementary Files: Example pipelines for high-content image analysis with CellProfiler and the CellAnalyst software packages, example images, and raw data for Figure 4E, F. (1-9) Example_Images. Open these images with ImageJ or CellProfiler. (10) Fig_4_raw_data_Er_et_al.xlxs. (11) TH_LB_V1.cpipe (Steps 6.2&#8211;6.8), (12) TH_LB_V2.cpipe (Steps 6.11&#8211;6.18)&#160;<a href="https://cloudflare.jove.com/files/ftp_upload/61118/Supplementary_Files.zip" target="_blank">Please click here to download this file.</a>

Watch this Scientific Journal Video about Studying Pre-formed Fibril Induced ÃŽÂ±-Synuclein Accumulation in Primary Embryonic Mouse Midbrain Dopamine Neurons at JoVE.com

Studying Pre-formed Fibril Induced &#945;-Synuclein Accumulation in Primary Embryonic Mouse Midbrain Dopamine Neurons

Here, we present a detailed protocol to study neuronal &#945;-synuclein accumulation in primary mouse dopamine neurons. Phosphorylated &#945;-synuclein aggregates in neurons are induced with pre-formed &#945;-synuclein fibrils. Automated imaging of immunofluorescently labeled cells and unbiased image analysis make this robust protocol suitable for medium-to-high throughput screening of drugs that inhibit &#945;-synuclein accumulation.

The goal of this protocol is to establish a robust and reproducible model of &#945;-synuclein accumulation in primary dopamine neurons. Combined with ...

studying-pre-formed-fibril-induced-synuclein-accumulation-primary

Research

JoVE Journal

Neuroscience

10.3K Views.  University of Helsinki. Here, we present a detailed protocol to study neuronal α-synuclein accumulation in primary mouse dopamine neurons. Phosphorylated α-synuclein aggregates in neurons are induced with pre-formed α-synuclein fibrils. Automated imaging of immunofluorescently labeled cells and unbiased image analysis make this robust protocol suitable for medium-to-high throughput screening of drugs that inhibit α-synuclein accumulation.

Video: Studying Pre-formed Fibril Induced α-Synuclein Accumulation in Primary Embryonic Mouse Midbrain Dopamine Neurons

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect neuronal process outgrowth, shRNA or cDNA constructs can be introduced into primary neurons via chemical transfection or viral transduction. However, with primary cortical cells, a heterogeneous pool of cell types (glutamatergic neurons from different layers, inhibitory neurons, glial cells) are transfected using these methods. The use of in utero electroporation to introduce DNA constructs in the embryonic rodent cortex allows for certain subsets of cells to be targeted: while electroporation of early embryonic cortex targets deep layers of the cortex, electroporation at late embryonic timepoints targets more superficial layers. Further, differential placement of electrodes across the heads of individual embryos results in the targeting of dorsal-medial versus ventral-lateral regions of the cortex. Following electroporation, transfected cells can be dissected out, dissociated, and plated in vitro for quantitative analysis of neurite outgrowth. Here, we provide a step-by-step method to quantitatively measure neuronal process outgrowth in subsets of cortical cells.
The basic protocol for in utero electroporation has been described in detail in two other JoVE articles from the Kriegstein lab 1, 2. We will provide an overview of our protocol for in utero electroporation, focusing on the most important details, followed by a description of our protocol that applies in utero electroporation to the study of gene function in neuronal process outgrowth.

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In utero electroporation is a valuable method for transfecting neuronal progenitor cells in vivo. Depending upon the placement of the electrodes and the developmental timepoint of electroporation, certain subsets of cortical cells can be targeted. Targeted cells can then be analyzed in vivo or in vitro for effects of genetic alteration.

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect ...

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and synapse formation and function. An advantage of culturing these neurons is that they readily polarize, forming distinctive axons and dendrites, on a two dimensional substrate at very low densities. This property has made them extremely useful for determining many aspects of neuronal development. Furthermore, by providing glial conditioning for these neurons they will continue to develop, forming functional synaptic connections and surviving for several months in culture. In this protocol we outline a technique to dissect, culture and transfect embryonic mouse hippocampal and cortical neurons. Transfection is accomplished by electroporating DNA into the neurons before plating via nucleofection. This protocol has the advantage of expressing fluorescently-tagged fusion proteins early in development (~4-8hrs after plating) to study the dynamics and function of proteins during polarization, axon outgrowth and branching. We have also discovered that this single transfection before plating maintains fluorescently-tagged fusion protein expression at levels appropriate for imaging throughout the lifetime of the neuron (&gt; 2 months in culture). Thus, this methodology is useful for studying protein localization and function throughout CNS development with little or no disruption of neuronal function.

This protocol outlines the steps required to dissect, transfect via electroporation and culture mouse hippocampal and cortical neurons. Short-term cultures may be used for studies of axon outgrowth and guidance, while long-term cultures can be used for studies of synaptogenesis and dendritic spine analysis.

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and ...

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain dopaminergic neurons is implicated in Parkinson's disease, Schizophrenia, depression, and dementia1-5. Thus, studying the regulation of midbrain dopaminergic neuron differentiation could not only provide important insight into mechanisms regulating midbrain development and neural progenitor fate specification, but also help develop new therapeutic strategies for treating a variety of human neurological disorders.
Dopaminergic neurons differentiate from neural progenitors lining the ventricular zone of embryonic ventral midbrain. The development of neural progenitors is controlled by gene expression programs6,7. Here we report techniques utilizing electroporation to express genes specifically in the midbrain of Hamburger Hamilton (HH) stage 11 (thirteen somites, 42 hours) chick embryos8,9. The external development of chick embryos allows for convenient experimental manipulations at specific embryonic stages, with the effects determined at later developmental time points10-13. Chick embryonic neural tubes earlier than HH stage 13 (nineteen somites, 48 hours) consist of multipotent neural progenitors that are capable of differentiating into distinct cell types of the nervous system. The pCAG vector, which contains both a CMV promoter and a chick &beta;-actin enhancer, allows for robust expression of Flag or other epitope-tagged constructs in embryonic chick neural tubes14. In this report, we emphasize special measures to achieve regionally restricted gene expression in embryonic midbrain dopaminergic neuron progenitors, including how to inject DNA constructs specifically into the embryonic midbrain region and how to pinpoint electroporation with small custom-made electrodes. Analyzing chick midbrain at later stages provides an excellent in vivo system for plasmid vector-mediated gain-of-function and loss-of-function studies of midbrain development. Modification of the experimental system may extend the assay to other parts of the nervous system for performing fate mapping analysis and for investigating the regulation of gene expression.

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

To assess the function and the regulation of genes during the development of midbrain dopaminergic neurons, we describe a method that involves in ovo electroporation of plasmid DNA constructs into embryonic chick ventral midbrain dopaminergic neuron progenitors. This technique can be used to achieve efficient expression of genes of interest to study different aspects of midbrain development and dopaminergic neuron differentiation.

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain ...

Primary Culture of Mouse Dopaminergic Neurons

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions such as motor control, motivation, and working memory. Nigrostriatal dopaminergic neurons selectively degenerate in Parkinson&#39;s disease (PD). This progressive neuronal loss is unequivocally associated with the motors symptoms of the pathology (bradykinesia, resting tremor, and muscular rigidity). The main agent responsible of dopaminergic neuron degeneration is still unknown. However, these neurons appear to be extremely vulnerable in diverse conditions. Primary cultures constitute one of the most relevant models to investigate properties and characteristics of dopaminergic neurons. These cultures can be submitted to various stress agents that mimic PD pathology and to neuroprotective compounds in order to stop or slow down neuronal degeneration. The numerous transgenic mouse models of PD that have been generated during the last decade further increased the interest of researchers for dopaminergic neuron cultures. Here, the video protocol focuses on the delicate dissection of embryonic mouse brains. Precise excision of ventral mesencephalon is crucial to obtain neuronal cultures sufficiently rich in dopaminergic cells to allow subsequent studies. This protocol can be realized with embryonic transgenic mice and is suitable for immunofluorescence staining, quantitative PCR, second messenger quantification, or neuronal death/survival assessment.

Dopaminergic neurons play a vital regulatory role in the brain. Their loss is associated with Parkinson's disease. In this video, we show how to generate primary cultures of central dopaminergic neurons from embryonic mouse mesencephalon. Such cultures are useful to study the extreme vulnerability of these neurons to various stresses.

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions ...

Environmental Modulations of the Number of Midbrain Dopamine Neurons in Adult Mice

Long-lasting changes in the brain or &#8216;brain plasticity&#8217; underlie adaptive behavior and brain repair following disease or injury. Furthermore, interactions with our environment can induce brain plasticity. Increasingly, research is trying to identify which environments stimulate brain plasticity beneficial for treating brain and behavioral disorders. Two environmental manipulations are described which increase or decrease the number of tyrosine hydroxylase immunopositive (TH+, the rate-limiting enzyme in dopamine (DA) synthesis) neurons in the adult mouse midbrain. The first comprises pairing male and female mice together continuously for 1 week, which increases midbrain TH+ neurons by approximately 12% in males, but decreases midbrain TH+ neurons by approximately 12% in females. The second comprises housing mice continuously for 2 weeks in &#8216;enriched environments&#8217; (EE) containing running wheels, toys, ropes, nesting material, etc., which increases midbrain TH+ neurons by approximately 14% in males. Additionally, a protocol is described for concurrently infusing drugs directly into the midbrain during these environmental manipulations to help identify mechanisms underlying environmentally-induced brain plasticity. For example, EE-induction of more midbrain TH+ neurons is abolished by concurrent blockade of synaptic input onto midbrain neurons. Together, these data indicate that information about the environment is relayed via synaptic input to midbrain neurons to switch on or off expression of &#8216;DA&#8217; genes. Thus, appropriate environmental stimulation, or drug targeting of the underlying mechanisms, might be helpful for treating brain and behavioral disorders associated with imbalances in midbrain DA (e.g. Parkinson&#8217;s disease, attention deficit and hyperactivity disorder, schizophrenia, and drug addiction).

This protocol describes two different environmental manipulations and a concurrent brain infusion protocol to study environmentally-induced brain changes underlying adaptive behavior and brain repair in adult mice.

Long-lasting changes in the brain or &#8216;brain plasticity&#8217; underlie adaptive behavior and brain repair following disease or injury. Furthermore, ...

Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.

The causes of degeneration of midbrain dopaminergic neurons during Parkinson&#8217;s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration.

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes ...

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their post-natal development, abundance, and accessibility, CGNs are an ideal model to study neuronal processes, including neuronal development, neuronal migration, and physiological neuronal activity stimulation. In addition, CGN cultures provide an excellent model for studying different modes of cell death including excitotoxicity and apoptosis. Within a week in culture, CGNs express N-methyl-D-aspartate (NMDA) receptors, a specific ionotropic glutamate receptor with many critical functions in neuronal health and disease. The addition of low concentrations of NMDA in conjunction with membrane depolarization to rodent primary CGN cultures has been used to model physiological neuronal activity stimulation while the addition of high concentrations of NMDA can be employed to model excitotoxic neuronal injury. Here, a method of isolation and culturing of CGNs from 6 day old pups as well as genetic manipulation of CGNs by adenoviruses and lentiviruses are described. We also present optimized protocols on how to stimulate NMDA-induced excitotoxicity, low-potassium-induced apoptosis, oxidative stress and DNA damage following transduction of these neurons.

This protocol describes a simple method for isolating and culturing primary mouse cerebral granule neurons (CGNs) from 6-7 day old pups, efficient transduction of CGNs for loss and gain of function studies, and modelling NMDA-induced neuronal excitotoxicity, low-potassium-induced cell death, DNA-damage, and oxidative stress using the same culture model.

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their ...

The Mouse Hindbrain As a Model for Studying Embryonic Neurogenesis

The mouse embryo forebrain is the most commonly employed system for studying mammalian neurogenesis during development. However, the highly folded forebrain neuroepithelium is not amenable to wholemount analysis to examine organ-wide neurogenesis patterns. Moreover, defining the mechanisms of forebrain neurogenesis is not necessarily predictive of neurogenesis in other parts of the brain; for example, due to the presence of forebrain-specific progenitor subtypes. The mouse hindbrain provides an alternative model for studying embryonic neurogenesis that is amenable to wholemount analysis, as well as tissue sections to observe the spatiotemporal distribution and behavior of neural progenitors. Moreover, it is easily dissected for other downstream applications, such as cell isolation or molecular biology analysis. As the mouse hindbrain can be readily analyzed in the vast number of cell lineage reporter and mutant mouse strains that have become available, it offers a powerful model for studying the cellular and molecular mechanisms of developmental neurogenesis in a mammalian organism. Here, we present a simple and quick method to use the mouse embryo hindbrain for analyzing mammalian neural progenitor cell (NPC) behavior in wholemount preparations and tissue sections.

This article demonstrates how the mouse embryonic hindbrain can be used as a model for studying developmental neurogenesis in both whole organ and tissue section preparations.

The mouse embryo forebrain is the most commonly employed system for studying mammalian neurogenesis during development. However, the highly folded ...

Primary Culture of Neurons Isolated from Embryonic Mouse Cerebellum

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model system is to provide a controlled microenvironment and maintain the high survival rate and the natural features of dissociated neuronal and nonneuronal cells as much as possible under in vitro conditions. In this article, we demonstrate a method of isolating primary neurons from the developing mouse cerebellum, placing them in an in vitro environment, establishing their growth, and monitoring their viability and differentiation for several weeks. This method is applicable to embryonic neurons dissociated from cerebellum between embryonic days 12&ndash;18.

Conducting in vitro experiments to reflect in vivo conditions as adequately as possible is not an easy task. The use of primary cell cultures is an important step toward understanding cell biology in a whole organism. The provided protocol outlines how to successfully grow and culture embryonic mouse cerebellar neurons.

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model ...