The authors would like to thank all colleagues at Ksilink for their valuable help and discussions that lead to the design of the presented protocol.

1. Preparation of medium and plates for neuron seeding (Day 1)
<ol>
	<li>To prepare the plates for neuron seeding on Day-1, warm Laminin to room temperature (RT) just before use. Prepare the Laminin solution by diluting the Laminin stock solution (0.1 mg/mL) 1/10 in cold PBS+/+ (with Ca2+ and Mg2+). 
	NOTE: All reagents are listed in the Table of Materials. The compositions of solutions and buffers are described in Tables 1-4.</li>
	<li>Then, add 25 &#181;L of the Laminin solution to each well of a Poly-D-Lysine (PDL) precoated 384-well plate, and incubate overnight at 4 &#176;C. The coated plates can be stored at 4 &#176;C for up to one week. 
	NOTE: The protocol can be paused here for up to one week. Seal plates using plastic film.</li>
	<li>Prepare Complete Maintenance Media and store at 4 &#176;C for up to one month (Table 1).</li>
</ol>
2. Thawing of neurons (Day 0)
<ol>
	<li>To thaw the neurons on Day 0, prewarm a water bath to 37 &#176;C, and equilibrate the Complete Maintenance Media to RT, protected from light.</li>
	<li>Take the vial containing the commercially obtained frozen neurons (see Table of Materials) out of the liquid nitrogen tank and place it on dry ice. Then place the vial in the water bath for 2 min. Once the liquid is completely thawed, disinfect the vial with 70% ethanol.</li>
	<li>Aspirate the thawed neurons with a P1000 pipette (around 370 &#181;L) and transfer them in a 50 mL centrifuge tube without up-and-down pipetting. Next, rinse the vial with 630 &#181;L of Complete Maintenance Medium, dispense drop by drop (45&#176; angle) in the 50 mL tube. Agitate slowly while dispensing. 
	NOTE: It is critical to dispense the medium slowly, drop by drop, to avoid osmotic rupture of the cells. A 45&#176; angle and light agitation minimizes high local osmotic pressure during pipetting.</li>
	<li>Similarly, add 1 mL of Complete Maintenance Medium in the 50 mL centrifuge tube with a P1000 pipette. Then, slowly add 2 mL of Complete Maintenance Medium. Agitate carefully while dispensing.</li>
	<li>Count the cells. Prepare a microtube with 10 &#181;L Trypan Blue and 10 &#181;L cell suspension and add to a counting chamber slide (10 &#181;L). Perform counting using an automated cell counter (see Table of Materials) or manually.</li>
	<li>After counting, centrifuge the 50 mL tube containing the neurons at 400 x g for 5 min at RT, and remove the supernatant. Carefully resuspend the pellet using a P1000 pipette and 1 mL of Complete Maintenance Medium. Then add the necessary volume to reach the desired concentration (300,000 cells/mL, see also next step).</li>
</ol>
3. Seeding of neurons on prepared plates (Day 0)
<ol>
	<li>To seed neurons on the prepared plates on Day 0, take the coated plates out of the refrigerator, place them under the cell culture hood and let them equilibrate to RT for about 30 min.</li>
	<li>Just before seeding, aspirate 15 &#181;L coating solution with an automated liquid handler or a 16-channel pipette. Leave approximately 10 &#181;L per well to prevent damage to the coating.</li>
	<li>Next, dispense 50 &#181;L of the cell solution containing 300,000 cells/mL (prepared in step 2.6) per well with a 16-channel pipette, resulting in 15,000 seeded neurons per well and a final volume per well of 60 &#181;L.</li>
	<li>In a 384-well plate, avoid using columns 1, 2, 23, 24, and rows A, B, O, and P to minimize possible edge effects which can impact the phenotypic profiles. Fill the unused empty wells with 80 &#181;L of PBS. Incubate the plates at 37 &#176;C and 5% CO2.</li>
</ol>
4. Medium change or compound treatment (Day 3)
<ol>
	<li>Depending on the number wells, pre-warm an appropriate volume of Complete Maintenance Medium at RT. Protect from light.</li>
	<li>If a medium change is required, proceed to step 4.4. If compound treatment is desired, verify the compound stock concentration and the used solvent (water, DMSO, methanol, etc.).</li>
	<li>Prepare a 1.5x concentrated compound solution for all desired concentrations to be tested. Prepare the compound dilutions using Complete Maintenance Medium. 
	NOTE: As a neutral control, use the respective solvent at the same concentration as the tested compound. If multiple or unknown compounds at different concentrations are used, it is advisable to perform a separate dose-response experiment to measure solvent effects on the phenotypic profile.</li>
	<li>If an automatic pipetting system is used, add 60 &#181;L of the 1.5x compound solution to a 384-well storage plate. Alternatively, use a 16-channel pipette. If a medium change is required, add 60 &#181;L of Complete Maintenance Medium instead.</li>
	<li>Using the automatic pipetting system, aspirate and discard 40 &#181;L of medium per well from the neuron-containing plate to keep 20 &#181;L/well. Then add 40 &#181;L/well of 1.5x compound solution from the 384-well storage plate to each well to obtain the desired final concentration. 
	NOTE: It is critical not to perform full medium changes but to always leave residual medium in the well to prevent damage to the neuronal carpet or the coating.</li>
	<li>In case neuronal culture is performed for more than the 6 days described in this protocol, change the medium every 2-3 days.</li>
</ol>
5. Neuron fixation and staining (Days 6 to 7)
<ol>
	<li>To fix and stain the neurons on Days 6 and 7, make use of an automated liquid handler for all dispensing and washing steps. Alternatively, use a 16-channel pipette.</li>
	<li>Prepare a 10% Triton X-100 solution by diluting Triton X-100 in 1x PBS. Vortex until the solution is homogeneous. Store at 4 &#176;C.</li>
	<li>To fix the neurons, dispense 20 &#181;L/well of 16% PFA resulting in a 4% final concentration. Incubate the plate for 30 min at RT, and wash it three times with 1x PBS. Leave 20 &#181;L/well of PBS after the last wash. 
	CAUTION: PFA is recognized as a hazardous substance known to cause oral, dermal, and respiratory toxicity. It also poses a threat to the eyes and may lead to genetic mutations and cancer. Proper handling of PFA requires the use of suitable personal protective equipment, such as eye and face protection, in addition to ensuring proper ventilation. It is important to prevent the release of PFA into the environment.</li>
	<li>For permeabilization and blocking, prepare a 2x blocking solution (Table 2).</li>
	<li>Add 20 &#181;L/well of 2x blocking solution (1x final concentration), incubate for 1 h at RT, and wash once with PBS. Keep 20 &#181;L/well of PBS after washing.</li>
	<li>For primary antibody (see Table of Materials) staining, prepare a 2x primary staining buffer (Table 3).</li>
	<li>Add 20 &#181;L/well of 2x primary staining buffer (1x final concentration) and incubate overnight at 4 &#176;C. The next morning on Day 7, wash three times with PBS. Leave 20 &#181;L/well of PBS after washing.</li>
	<li>For secondary antibody (see Table of Materials) staining, prepare a 2x secondary staining buffer (Table 4).</li>
	<li>Add 20 &#181;L/well of 2x secondary staining buffer (1x final concentration), incubate for 2 h at RT away from light, and wash three times with PBS. Leave 100 &#181;L PBS/well after the last wash.
	<ol>
		<li>Add aluminum sealing on the plate to minimize evaporation. Alternatively, cover the plate using plastic film and aluminum foil. Proceed to image acquisition, or store the plate at 4 &#176;C. 
		&#8203;NOTE: The protocol can be paused here for up to one week. If background fluorescence is observed, consider increasing the blocking time. If the staining is insufficient, try increasing the primary antibody concentration or incubation time.</li>
	</ol>
	</li>
</ol>
6. Imaging of fluorescently stained neurons (Day 7)
<ol>
	<li>Acquire images of the plated, cultured, and stained neurons on Day 7. Ideally, use an automated confocal fluorescence microscope (see Table of Materials). Alternatively, acquire images manually.</li>
	<li>Acquire the Hoechst, TH, &#945;-synuclein, and MAP2 channels using 405 nm, 488 nm, 561 nm, and 647 nm lasers, respectively (Figure 2). 
	NOTE: To generate sufficient amount of detailed data for phenotypic profiling, use a 40x objective and acquire 16 fields/well using Z-stacks consisting of 3 Z-slices separated by 2 &#181;m. In case there are pipetting-related damages in the neuronal carpet, try to avoid imaging these areas.</li>
	<li>Depending on the microscope and camera, adjust the exposure times and excitation intensities for each of the four fluorescent channels separately to obtain an optimal dynamic range of the fluorescent intensities. 
	&#8203;NOTE: Imaging software often provides a histogram to determine the ideal exposure time. If the histogram is shifted too much to the left in the low signal range, then the exposure time is too short or the excitation intensity is too low. If there is a sharp cliff at the maximum signal level on the right, then the signal value is saturated. In this case, reduce the excitation intensity or shorten the exposure time.</li>
	<li>Store images in a loss-free and open format such as .tif.</li>
</ol>
7. Image processing (Day 8)
<ol>
	<li>Image segmentation and phenotypic feature extraction are required for the creation of quantitative phenotypic profiles. Use the PhenoLink software for image segmentation and feature extraction (Table of Materials). Instructions for installing PhenoLink can be found in the GitHub repository (https://github.com/Ksilink/PhenoLink). 
	NOTE: Image segmentation is used to identify and separate different objects or regions in an image, while phenotypic feature extraction is used to analyze and extract relevant information from those regions. Several alternative software solutions, such as CellProfiler10, ImageJ/FIJI11, Napari12, or the Knime Analytics Platform13 are available to extract quantitative information from multichannel fluorescence images.</li>
	<li>Perform image segmentation on illumination-corrected raw images. Determine the respective fluorescent channel intensity thresholds empirically per plate so that the background signal is minimal, and the desired segmented signal corresponds to the signal in the raw image. Plates processed and stained on the same day typically require comparable channel intensity thresholds for segmentation.</li>
	<li>Define the nucleus size and intensity to separate living from dead cells. When using 40x images, keep all other default parameters and run the software. One hundred twenty-six quantitative image features will be calculated per well (Supplementary Table 1).</li>
	<li>Use the resulting tabular quantitative data to construct phenotypic profiles and compare phenotypic profiles from different cell lines or treatment conditions. Each row corresponds to a biological condition (well) and each column corresponds to a determined phenotypic feature. 
	&#8203;NOTE: We provide an example output file together with the data analysis pipeline to illustrate its use (see Table of Materials). Additionally, Figure 3 shows the composition of a phenotypic profile.</li>
</ol>
8. Phenotypic profile generation and visualization (Day 8)
<ol>
	<li>If you do not have Python and Jupyter installed on your computer, install the Anaconda Distribution and open the Jupyter software. Download the provided Jupyter notebook and all other provided files and save them in the same directory (see Table of Materials). Open the Jupyter notebook file using the Jupyter software. 
	NOTE: Anaconda is a free and open-source platform for programming languages such as Python. This platform comes with the Python interpreter Jupyter which can execute the provided Jupyter notebook to create and analyze phenotypic profiles (https://github.com/Ksilink/Notebooks/tree/main/Neuro/DopaNeuronProfiling).</li>
	<li>Execute the Jupyter notebook cell by cell using the Jupyter software. The provided sample data .fth and requirements .txt files need to be located in the same directory as the Jupyter notebook. Each Jupyter notebook cell is annotated to explain its functionality. 
	NOTE: It is crucial to use the Jupyter notebook in the correct order starting with data loading and scaling and to proceed cell by cell to the end. All data and graphics output will be stored in a newly created folder in the Jupyter notebook source directory. Figure 4 depicts the workflow and the workflow output.</li>
</ol>

Culturing Human Midbrain Dopaminergic Neurons for Phenotypic Profiling

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All authors are employed by Ksilink.

Phenotypic profiling is a technique to measure a large number of phenotypes in cells by applying fluorescent stainings, microscopy, and image analysis3. Phenotypic profiles can be obtained and compared across cell lines or other experimental conditions to understand complex changes in cellular biology that might go unnoticed when using a single readout. Here we describe the application of phenotypic profiling to human iPSC-derived mDA neurons, a cell type frequently used to model PD cellular biolo...

A variety of cell biological processes are disturbed in Parkinson&#39;s disease (PD). For example, mitochondrial dysfunction, oxidative stress, protein degradation defects, disruption of vesicular trafficking and endolysosomal function have been associated with midbrain dopaminergic (mDA) neuron loss, are commonly observed in PD1. Therefore, PD appears to involve multiple disease mechanisms that can interact with and worsen each other. One useful way to investigate this mechanistic interplay is the creation of a comprehensive phenotypic fingerprint or profile of midbrain dopaminergic (mDA) neurons.
Phenotypic profiling is an approach that involves creating a profile of a sample based on a collection of measurable characteristics, and second, it involves making predictions about a sample based on this profile2,3. The goal of profiling is to capture a diverse range of features, some of which may not have been previously associated with a disease or treatment3. As a result, profiling can reveal unexpected biological processes. Phenotypic profiling typically relies on fluorescently stained cells, and standardized assays, such as Cell Painting, have been developed to create phenotypic profiles4. Recently, phenotypic profiling has, for example, been applied for the characterization of small molecules or the accurate prediction of PD-subtypes solely based on patient-derived fibroblasts5,6. Despite these advances, phenotypic profiling has rarely been applied to post-mitotic differentiated cells, such as human induced pluripotent stem cell (iPSC)-derived mDA neurons that express PD-linked mutations such as LRRK2 G2019S. Significant challenges of iPSC-derived models include the presence of subtle or variable pathological features across differentiation batches or genotypes, and the fact that isolated PD phenotypes do not capture the full complexity of the disease. Furthermore, while iPSC neuronal models are physiologically relevant, they are rarely used in PD drug discovery processes due to concerns about technical complexity7,8.
We previously developed a robust methodology to measure multiple PD-related pathophysiological phenotypes in human mDA neurons that are both sensitive to genetic and chemical compound-induced phenotypic changes9. This article describes in detail a further optimized version of this methodology to create phenotypic profiles from mDA neurons (Figure 1). This protocol has several advantages over the previously described phenotypic profiling approaches, such as the use of high-quality mDA neurons and technical reproducibility. For the first time, this protocol enables phenotypic profiling in physiologically relevant post-mitotic mDA neurons after chemical perturbations in a highly scalable fashion. Fully differentiated and cryopreserved mDA neurons are commercially available, significantly decreasing batch-to-batch differentiation variability. Secondly, technical variability can be further reduced by using a well-defined experimental design (i.e., culture duration or avoiding edge wells), automated liquid handling and automated microscopy. Additionally, the initial steps of phenotypic profile analysis using unsupervised clustering or supervised classification approaches are outlined here, indicating how phenotypic profiling data can be analyzed. This protocol will be of use for researchers interested in phenotypic changes of mDA neurons induced by genetic or chemical perturbations, specifically when a highly scalable study setup is required, for example, during screening campaigns or when the effects of a smaller number of compounds are to be studied, for example, to determine toxic effects. In summary, it is anticipated that the application of phenotypic profiling of human neurons is a valuable technique to study complex disease-related phenotypes and characterize the cellular effects of drug candidates.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65570/65570fig01.jpg" /> 
Figure 1: Schematic depiction of the experimental protocol to generate image-based phenotypic profiles from human iPSC-derived mDA neurons. <a href="https://www.jove.com/files/ftp_upload/65570/65570fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anti- chicken &#8211; Alexa 647</td>
			<td>Jackson ImmunoRearch</td>
			<td>703-605-155</td>
			<td>Immunofluorescence</td>
		</tr>
		<tr>
			<td>Anaconda</td>
			<td></td>
			<td>https://www.anaconda.com/download</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Map2</td>
			<td>Novus</td>
			<td>NB300-213</td>
			<td>Immunofluorescence</td>
		</tr>
		<tr>
			<td>Anti-mouse - Alexa 488</td>
			<td>Thermo Fisher</td>
			<td>A11001</td>
			<td>Immunofluorescence</td>
		</tr>
		<tr>
			<td>Anti-rabbit - Alexa 555</td>
			<td>Thermo Fisher</td>
			<td>A21429</td>
			<td>Immunofluorescence</td>
		</tr>
		<tr>
			<td>Anti-Tyrosine Hydroxylase</td>
			<td>Merck</td>
			<td>T2928</td>
			<td>Immunofluorescence</td>
		</tr>
		<tr>
			<td>Anti-&#945;-synuclein</td>
			<td>Abcam</td>
			<td>138501</td>
			<td>Immunofluorescence</td>
		</tr>
		<tr>
			<td>Bravo Automated Liquid Handling Platform with 384ST head</td>
			<td>Agilent</td>
			<td></td>
			<td>If no liquid handler is available, the use of an electronic multichannel pipette is recommended.</td>
		</tr>
		<tr>
			<td>Confocal microscope&#160;</td>
			<td>Yokogawa</td>
			<td>CV7000</td>
			<td>The use of an automated confocal fluorescence microscope is recommended to ensure image quality consistency.</td>
		</tr>
		<tr>
			<td>Countess Automated cell counter</td>
			<td>Invitrogen</td>
			<td></td>
			<td>Cell counting before seeding. Can also be done using a manual counting chamber.</td>
		</tr>
		<tr>
			<td>DPBS +/+</td>
			<td>Gibco</td>
			<td>14040-133</td>
			<td>Buffer for washing</td>
		</tr>
		<tr>
			<td>EL406 Washer Dispenser&#160;</td>
			<td>BioTek (Agilent)&#160;</td>
			<td></td>
			<td>If no liquid handler is available, the use of an electronic multichannel pipette is recommended.</td>
		</tr>
		<tr>
			<td>Formaldehyde Solution (PFA 16 %)</td>
			<td>Euromedex</td>
			<td>EM-15710-S</td>
			<td>Fixation before staining</td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Invitrogen</td>
			<td>H3570</td>
			<td>Nuclear staining</td>
		</tr>
		<tr>
			<td>iCell Base Medium 1</td>
			<td>Fujifilm</td>
			<td>M1010</td>
			<td>Base medium for neurons</td>
		</tr>
		<tr>
			<td>iCell DPN, Donor#01279, Phenotype AHN, lot#106339, 1M</td>
			<td>Fujifilm</td>
			<td>C1087</td>
			<td>Apparently healthy donor</td>
		</tr>
		<tr>
			<td>iCell DPN, Donor#11299, Phenotype LRRK2 G2019S, phenotype PD lot#106139</td>
			<td>Fujifilm</td>
			<td>C1149</td>
			<td>Donor carrying LRRK2 G2019S mutation&#160;</td>
		</tr>
		<tr>
			<td>iCell Nervous System Supplement</td>
			<td>Fujifilm</td>
			<td>M1031</td>
			<td>Supplement for base medium</td>
		</tr>
		<tr>
			<td>iCell Neural Supplement B</td>
			<td>Fujifilm</td>
			<td>M1029</td>
			<td>Supplement for base medium</td>
		</tr>
		<tr>
			<td>Jupyter Python Notebook</td>
			<td>In-house development</td>
			<td>https://github.com/Ksilink/Notebooks/tree/main/Neuro/DopaNeuronProfiling</td>
			<td>Notebook to perform phenotypic profile visualization and classification from raw data.</td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Biolamina</td>
			<td>LN521</td>
			<td>Plate coating</td>
		</tr>
		<tr>
			<td>PFE-360</td>
			<td>MedChemExpress</td>
			<td>HY-120085</td>
			<td>LRRK2 kinase inhibitor</td>
		</tr>
		<tr>
			<td>PhenoLink</td>
			<td>In-house development</td>
			<td>https://github.com/Ksilink/PhenoLink</td>
			<td>Software for image analysis</td>
		</tr>
		<tr>
			<td>PhenoPlate 384w, PDL coated</td>
			<td>Perkin Elmer</td>
			<td>6057500</td>
			<td>Pre-coated plate for cell culture and imaging. This plate allows imaging of all wells using all objectives of the Yokogawa CV7000 microscope.</td>
		</tr>
		<tr>
			<td>Storage plates Abgene 120 &#181;L</td>
			<td>Thermo Scientific</td>
			<td>AB-0781</td>
			<td>Necessary for compound dispensing using the Vprep pipetting system. If not available, the use of an electronic multichannel pipette is recommended.</td>
		</tr>
		<tr>
			<td>Triton</td>
			<td>Sigma</td>
			<td>T9284</td>
			<td>Permeabilization before lysis</td>
		</tr>
		<tr>
			<td>Trypan Blue</td>
			<td>Sigma</td>
			<td>T8154-20ML</td>
			<td>Determination of living cells</td>
		</tr>
		<tr>
			<td>Vprep Pipetting System&#160;</td>
			<td>Agilent</td>
			<td></td>
			<td>Medium change and compound dispensing. Alternatively, an electronic multichannel pipette can be used.</td>
		</tr>
	</tbody>
</table>

phenotypic profiling of human stem cell-derived midbrain dopaminergic neurons

Parkinson&#39;s disease (PD) is linked to a range of cell biological processes that cause midbrain dopaminergic (mDA) neuron loss. Many current in vitro PD cellular models lack complexity and do not take multiple phenotypes into account. Phenotypic profiling in human induced pluripotent stem cell (iPSC)-derived mDA neurons can address these shortcomings by simultaneously measuring a range of neuronal phenotypes in a PD-relevant cell type in parallel. Here, we describe a protocol to obtain and analyze phenotypic profiles from commercially available human mDA neurons. A neuron-specific fluorescent staining panel is used to visualize the nuclear, &#945;-synuclein, Tyrosine hydroxylase (TH), and Microtubule-associated protein 2 (MAP2) related phenotypes. The described phenotypic profiling protocol is scalable as it uses 384-well plates, automatic liquid handling and high-throughput microscopy. The utility of the protocol is exemplified using healthy donor mDA neurons and mDA neurons carrying the PD-linked G2019S mutation in the Leucine-rich repeat kinase 2 (LRRK2) gene. Both cell lines were treated with the LRRK2 kinase inhibitor PFE-360 and phenotypic changes were measured. Additionally, we demonstrate how multidimensional phenotypic profiles can be analyzed using clustering or machine learning-driven supervised classification methods. The described protocol will particularly interest researchers working on neuronal disease modeling or studying chemical compound effects in human neurons.

Phenotypic profiling in mDA neurons is an efficient way to quantify multiple aspects of cellular biology and their changes during the experimental modulation. To exemplify this methodology, this study made use of cryopreserved LRRK2 G2019S and healthy donor mDA neurons. These neurons have been differentiated for approximately 37 days, are post-mitotic and express neuronal markers (TUBB3 and MAP2) and dopaminergic neuron markers, including tyrosine hydroxylase (TH) in combination with FOXA2, while the glial marker Glial F...

Watch this Scientific Journal Video about Generating Neuronal Phenotypic Profiles - A Protocol to Culture and Image Human Midbrain Dopaminergic Neurons at JoVE.com

Author Spotlight: Generating Neuronal Phenotypic Profiles - A Protocol to Culture and Image Human Midbrain Dopaminergic Neurons 

This protocol describes the cell culturing of human midbrain dopaminergic neurons, followed by immunological staining and the generation of neuronal phenotypic profiles from acquired microscopic high-content images allowing the identification of phenotypic variations due to genetic or chemical modulations.

Phenotypic Profiling of Human Stem Cell-Derived Midbrain Dopaminergic Neurons

Parkinson&#39;s disease (PD) is linked to a range of cell biological processes that cause midbrain dopaminergic (mDA) neuron loss. Many current in vitro ...

phenotypic-profiling-human-stem-cell-derived-midbrain-dopaminergic

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Neuroscience

1.4K Views.  Ksilink. This protocol describes the cell culturing of human midbrain dopaminergic neurons, followed by immunological staining and the generation of neuronal phenotypic profiles from acquired microscopic high-content images allowing the identification of phenotypic variations due to genetic or chemical modulations.

Video: Author Spotlight: Generating Neuronal Phenotypic Profiles - A Protocol to Culture and Image Human Midbrain Dopaminergic Neurons 

Application of a NMDA Receptor Conductance in Rat Midbrain Dopaminergic Neurons Using the Dynamic Clamp Technique

Neuroscientists study the function of the brain by investigating how neurons in the brain communicate. Many investigators look at changes in the electrical activity of one or more neurons in response to an experimentally-controlled input. The electrical activity of neurons can be recorded in isolated brain slices using patch clamp techniques with glass micropipettes. Traditionally, experimenters can mimic neuronal input by direct injection of current through the pipette, electrical stimulation of the other cells or remaining axonal connections in the slice, or pharmacological manipulation by receptors located on the neuronal membrane of the recorded cell.
Direct current injection has the advantages of passing a predetermined current waveform with high temporal precision at the site of the recording (usually the soma). However, it does not change the resistance of the neuronal membrane as no ion channels are physically opened. Current injection usually employs rectangular pulses and thus does not model the kinetics of ion channels. Finally, current injection cannot mimic the chemical changes in the cell that occurs with the opening of ion channels.
Receptors can be physically activated by electrical or pharmacological stimulation. The experimenter has good temporal precision of receptor activation with electrical stimulation of the slice. However, there is limited spatial precision of receptor activation and the exact nature of what is activated upon stimulation is unknown. This latter problem can be partially alleviated by specific pharmacological agents. Unfortunately, the time course of activation of pharmacological agents is typically slow and the spatial precision of inputs onto the recorded cell is unknown.
The dynamic clamp technique allows an experimenter to change the current passed directly into the cell based on real-time feedback of the membrane potential of the cell (Robinson and Kawai 1993, Sharp et al., 1993a,b; for review, see Prinz et al. 2004). This allows an experimenter to mimic the electrical changes that occur at the site of the recording in response to activation of a receptor. Real-time changes in applied current are determined by a mathematical equation implemented in hardware.
We have recently used the dynamic clamp technique to investigate the generation of bursts of action potentials by phasic activation of NMDA receptors in dopaminergic neurons of the substantia nigra pars compacta (Deister et al., 2009; Lobb et al., 2010). In this video, we demonstrate the procedures needed to apply a NMDA receptor conductance into a dopaminergic neuron.

In this video, we demonstrate how to apply a conductance into a dopaminergic neuron recorded in the whole cell configuration in rat brain slices. This technique is called the dynamic clamp.

Neuroscientists study the function of the brain by investigating how neurons in the brain communicate. Many investigators look at changes in the ...

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

The mouse is an excellent model organism to study mammalian brain development due to the abundance of molecular and genetic data. However, the developing mouse brain is not suitable for easy manipulation and imaging in vivo since the mouse embryo is inaccessible and opaque. Organotypic slice cultures of embryonic brains are therefore widely used to study murine brain development in vitro. Ex-vivo manipulation or the use of transgenic mice allows the modification of gene expression so that subpopulations of neuronal or glial cells can be labeled with fluorescent proteins. The behavior of labeled cells can then be observed using time-lapse imaging. Time-lapse imaging has been particularly successful for studying cell behaviors that underlie the development of the cerebral cortex at late embryonic stages 1-2. Embryonic organotypic slice culture systems in brain regions outside of the forebrain are less well established. Therefore, the wealth of time-lapse imaging data describing neuronal cell migration is restricted to the forebrain 3,4. It is still not known, whether the principles discovered for the dorsal brain hold true for ventral brain areas. In the ventral brain, neurons are organized in neuronal clusters rather than layers and they often have to undergo complicated migratory trajectories to reach their final position. The ventral midbrain is not only a good model system for ventral brain development, but also contains neuronal populations such as dopaminergic neurons that are relevant in disease processes. While the function and degeneration of dopaminergic neurons has been investigated in great detail in the adult and ageing brain, little is known about the behavior of these neurons during their differentiation and migration phase 5. We describe here the generation of slice cultures from the embryonic day (E) 12.5 mouse ventral midbrain. These slice cultures are potentially suitable for monitoring dopaminergic neuron development over several days in vitro. We highlight the critical steps in generating brain slices at these early stages of embryonic development and discuss the conditions necessary for maintaining normal development of dopaminergic neurons in vitro. We also present results from time lapse imaging experiments. In these experiments, ventral midbrain precursors (including dopaminergic precursors) and their descendants were labeled in a mosaic manner using a Cre/loxP based inducible fate mapping system 6.

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

A method to generate organotypic slices from the E12.5 murine embryonic midbrain is described. The organotypic slice cultures can be used to observe the behavior of dopaminergic neurons or other ventral midbrain neurons.

The  mouse is an excellent model organism to study mammalian brain development due  to the abundance of molecular and genetic data. However, the ...

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 ...

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

Dopaminergic (DA) neurons in the substantia nigra pars compacta (also known as A9 DA neurons) are the specific cell type that is lost in Parkinson&#8217;s disease (PD). There is great interest in deriving A9 DA neurons from human pluripotent stem cells (hPSCs) for regenerative cell replacement therapy for PD. During neural development, A9 DA neurons originate from the floor plate (FP) precursors located at the ventral midline of the central nervous system. Here, we optimized the culture conditions for the stepwise differentiation of hPSCs to A9 DA neurons, which mimics embryonic DA neuron development. In our protocol, we first describe the efficient generation of FP precursor cells from hPSCs using a small molecule method, and then convert the FP cells to A9 DA neurons, which could be maintained in vitro for several months. This efficient, repeatable and controllable protocol works well in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) from normal persons and PD patients, in which one could derive A9 DA neurons to perform in vitro disease modeling and drug screening and in vivo cell transplantation therapy for PD.

We, based on knowledge from developmental biology and published research, developed an optimized protocol to efficiently generate A9 midbrain dopaminergic neurons from both human embryonic stem cells and human induced pluripotent stem cells, which would be useful for disease modeling and cell replacement therapy for Parkinson&#8217;s disease.

Dopaminergic (DA) neurons in the substantia nigra pars compacta  (also known as A9 DA neurons) are the specific cell type that is lost in ...

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 ...

Functional Evaluation of Biological Neurotoxins in Networked Cultures of Stem Cell-derived Central Nervous System Neurons

Therapeutic and mechanistic studies of the presynaptically targeted clostridial neurotoxins (CNTs) have been limited by the need for a scalable, cell-based model that produces functioning synapses and undergoes physiological responses to intoxication. Here we describe a simple and robust method to efficiently differentiate murine embryonic stem cells (ESCs) into defined lineages of synaptically active, networked neurons. Following an 8 day differentiation protocol, mouse embryonic stem cell-derived neurons (ESNs) rapidly express and compartmentalize neurotypic proteins, form neuronal morphologies and develop intrinsic electrical responses. By 18 days after differentiation (DIV 18), ESNs exhibit active glutamatergic and &#947;-aminobutyric acid (GABA)ergic synapses and emergent network behaviors characterized by an excitatory:inhibitory balance. To determine whether intoxication with CNTs functionally antagonizes synaptic neurotransmission, thereby replicating the in vivo pathophysiology that is responsible for clinical manifestations of botulism or tetanus, whole-cell patch clamp electrophysiology was used to quantify spontaneous miniature excitatory post-synaptic currents (mEPSCs) in ESNs exposed to tetanus neurotoxin (TeNT) or botulinum neurotoxin (BoNT) serotypes /A-/G. In all cases, ESNs exhibited near-complete loss of synaptic activity within 20 hr. Intoxicated neurons remained viable, as demonstrated by unchanged resting membrane potentials and intrinsic electrical responses. To further characterize the sensitivity of this approach, dose-dependent effects of intoxication on synaptic activity were measured 20 hr after addition of BoNT/A. Intoxication with 0.005 pM BoNT/A resulted in a significant decrement in mEPSCs, with a median inhibitory concentration (IC50) of 0.013 pM. Comparisons of median doses indicate that functional measurements of synaptic inhibition are faster, more specific and more sensitive than SNARE cleavage assays or the mouse lethality assay. These data validate the use of synaptically coupled, stem cell-derived neurons for the highly specific and sensitive detection of CNTs.

A custom protocol is described to differentiate mouse ES cells into defined populations of highly pure neurons exhibiting functioning synapses and emergent network behavior. Electrophysiological analysis demonstrates the loss of synaptic transmission following exposure to botulinum neurotoxin serotypes /A-/G and tetanus neurotoxin.

Therapeutic and mechanistic studies of the presynaptically targeted clostridial neurotoxins (CNTs) have been limited by the need for a scalable, ...

Reliable Identification of Living Dopaminergic Neurons in Midbrain Cultures Using RNA Sequencing and TH-promoter-driven eGFP Expression

In Parkinson&#39;s Disease (PD) there is widespread neuronal loss throughout the brain with pronounced degeneration of dopaminergic neurons in the SNc, leading to bradykinesia, rigidity, and tremor. The identification of living dopaminergic neurons in primary Ventral Mesencephalic (VM) cultures using a fluorescent marker provides an alternative way to study the selective vulnerability of these neurons without relying on the immunostaining of fixed cells. Here, we isolate, dissociate, and culture mouse VM neurons for 3 weeks. We then identify dopaminergic neurons in the cultures using eGFP fluorescence (driven by a Tyrosine Hydroxylase (TH) promoter). Individual neurons are harvested into microcentrifuge tubes using glass micropipettes. Next, we lyse the harvested cells, and conduct cDNA synthesis and transposon-mediated &#34;tagmentation&#34; to produce single cell RNA-Seq libraries1,2,3,4,5. After passing a quality-control check, single-cell libraries are sequenced and subsequent analysis is carried out to measure gene expression. We report transcriptome results for individual dopaminergic and GABAergic neurons isolated from midbrain cultures. We report that 100% of the live TH-eGFP cells that were harvested and sequenced were dopaminergic neurons. These techniques will have widespread applications in neuroscience and molecular biology.

In Parkinson&#39;s Disease (PD), Substantia Nigra (SNc) dopaminergic neurons degenerate, leading to motor dysfunction. Here we report a protocol for culturing ventral midbrain neurons from a mouse expressing eGFP driven by a Tyrosine Hydroxylase (TH) promoter sequence, harvesting individual fluorescent neurons from the cultures, and measuring their transcriptome using RNA-seq.

In Parkinson&#39;s Disease (PD) there is widespread neuronal loss throughout the brain with pronounced degeneration of dopaminergic neurons in the SNc, ...

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

The recent emergence of Zika virus (ZIKV) in susceptible populations has led to an abrupt increase in microcephaly and other neurodevelopmental conditions in newborn infants. While mosquitos are the main route of viral transmission, it has also been shown to spread via sexual contact and vertical mother-to-fetus transmission. In this latter case of transmission, due to the unique viral tropism of ZIKV, the virus is believed to predominantly target the neural progenitor cells (NPCs) of the developing brain.
Here a method for modeling ZIKV infection, and the resulting microcephaly, that occur when human cerebral organoids are exposed to live ZIKV is described. The organoids display high levels of virus within their neural progenitor population, and exhibit severe cell death and microcephaly over time. This three-dimensional cerebral organoid model allows researchers to conduct species-matched experiments to observe and potentially intervene with ZIKV infection of the developing human brain. The model provides improved relevance over standard two-dimensional methods, and contains human-specific cellular architecture and protein expression that are not possible in animal models.

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

This protocol describes a technique used to model Zika virus infection of the developing human brain. Using wildtype or engineered stem cell lines, researchers may use this technique to uncover the various mechanisms or treatments that may affect early brain infection and resulting microcephaly in Zika virus-infected embryos.

The recent emergence of Zika virus (ZIKV) in susceptible populations has led to an abrupt increase in microcephaly and other neurodevelopmental conditions ...

Method for High Speed Stretch Injury of Human Induced Pluripotent Stem Cell-derived Neurons in a 96-well Format

Traumatic brain injury (TBI) is a major clinical challenge with high morbidity and mortality. Despite decades of pre-clinical research, no proven therapies for TBI have been developed. This paper presents a novel method for pre-clinical neurotrauma research intended to complement existing pre-clinical models. It introduces human pathophysiology through the use of human induced pluripotent stem cell-derived neurons (hiPSCNs). It achieves loading pulse duration similar to the loading durations of clinical closed head impact injury. It employs a 96-well format that facilitates high throughput experiments and makes efficient use of expensive cells and culture reagents. Silicone membranes are first treated to remove neurotoxic uncured polymer and then bonded to commercial 96-well plate bodies to create stretchable 96-well plates. A custom-built device is used to indent some or all of the well bottoms from beneath, inducing equibiaxial mechanical strain that mechanically injures cells in culture in the wells. The relationship between indentation depth and mechanical strain is determined empirically using high speed videography of well bottoms during indentation. Cells, including hiPSCNs, can be cultured on these silicone membranes using modified versions of conventional cell culture protocols. Fluorescent microscopic images of cell cultures are acquired and analyzed after injury in a semi-automated fashion to quantify the level of injury in each well. The model presented is optimized for hiPSCNs but could in theory be applied to other cell types.

Here we present a method for a human in vitro model of stretch injury in a 96-well format on a timescale relevant to impact trauma. This includes methods for fabricating stretchable plates, quantifying the mechanical insult, culturing and injuring cells, imaging, and high content analysis to quantify injury.

Traumatic brain injury (TBI) is a major clinical challenge with high morbidity and mortality. Despite decades of pre-clinical research, no proven ...

Compartmentalization of Human Stem Cell-Derived Neurons within Pre-Assembled Plastic Microfluidic Chips

Use of microfluidic devices to compartmentalize cultured neurons has become a standard method in neuroscience. This protocol shows how to use a pre-assembled multi-compartment chip made in a cyclic olefin copolymer (COC) to compartmentalize neurons differentiated from human stem cells. The footprint of these COC chips are the same as a standard microscope slide and are equally compatible with high resolution microscopy. Neurons are differentiated from human neural stem cells (NSCs) into glutamatergic neurons within the chip and maintained for 5 weeks, allowing sufficient time for these neurons to develop synapses and dendritic spines. Further, we demonstrate multiple common experimental procedures using these multi-compartment chips, including viral labeling, establishing microenvironments, axotomy, and immunocytochemistry.

This protocol demonstrates the use of compartmentalized microfluidic chips, injection molded in a cyclic olefin copolymer to cultured neurons differentiated from human stem cells. These chips are preassembled and easier-to-use than traditional compartmentalized poly(dimethylsiloxane) devices. Multiple common experimental paradigms are described here, including viral labeling, fluidic isolation, axotomy, and immunostaining.