The authors would like to thank all the staff at the Advanced Optical Microscopy Facility (AOMF) at University Health Network for their time and assistance in developing this protocol.

All procedures were approved by the University Health Network Animal Care Committee and performed in accordance with guidelines and regulations set by the Canadian Council on Animal Care.
1. Stereotactic injection
<ol>
	<li>Pair-house adult female Sprague-Dawley rats (250-280 g) in cages with wood bedding and ad lib access to food and water. Maintain the animal colony in a regular 12 h light/dark cycle (lights on 06:30) with constant temperature and humidity.</li>
	<li>Perform unilateral stereotactic injection of AAV directly to the SNpc on the right side of the brain (right or left side, according to the preferences of each lab) as previously described8,10. Inject 2 &#956;L of AAV1/2 at a final titer of 3.4 x 1012 genomic particles/mL.</li>
</ol>
2. Brain sectioning and immunohistochemistry (IHC)
<ol>
	<li>Anaesthetize the rat with 5% isoflurane by placing in an anaesthetizing chamber for 3 min. Other approved methods may be used for this step after appropriate institutional review.</li>
	<li>Once the rat has reached a surgical plane of deep anesthesia, transfer it to a nose cone firmly affixed to a necropsy table. Secure the rat&#39;s fore-paws using tape and use toe pinch-response method to determine the depth of the anesthesia. The animal must be unresponsive before continuing.</li>
	<li>Make a lateral incision below the sternum and cut through the diaphragm along the entire length of the rib cage to expose the pleural cavity. Lift and clamp the sternum with a hemostat and place above the head.</li>
	<li>Clamp the heart using forceps and insert a butterfly needle connected to a perfusion pump into the posterior end of the left ventricle. Perfuse rat transcardially with 150 mL of heparinized saline, or until the eyes and skin are clear. Perfusion with 4% paraformaldehyde (PFA), instead of saline, may be preferred to facilitate immunostaining with certain antibodies or thinner brain sectioning.</li>
	<li>Once perfusion is complete, decapitate with a guillotine and extract the brain to a brain matrix, ventral surface facing up.</li>
	<li>Using a fresh razor blade, make a cut in the coronal plane 2 mm rostral to the optic chiasm. Slide the blade from side to side to avoid warping the brain while slicing.</li>
	<li>Immerse the posterior portion of the brain in a pre-labeled vial containing approximately 20 mL of 4% PFA for 48 h of post-fixation at room temperature. The anterior portion of the brain may be flash frozen in 2-methylbutane chilled to -42 &#176;C before storage at -80 &#176;C.</li>
	<li>After 48 h, transfer the fixed brains to a labeled vial containing 30% sucrose in phosphate buffered saline (PBS) and store at 4 &#176;C until they sink (48-72 h).</li>
	<li>Prepare a microtome by placing dry ice in the trough of the specimen stage, followed by 100% ethanol. Once the stage has cooled, squeeze optimal cutting temperature (OCT) compound onto the stage until it forms a circle 2 cm in diameter and 0.5 cm thick. Once it has partially frozen, carefully lower the brain onto the mound of OCT, ensuring the striatal cutting surface remains parallel with the stage.</li>
	<li>Add more dry ice to the stage to help the brain to freeze. Once the brain has turned a cream color, clear the stage of dry ice.</li>
	<li>Poke a hole into the right side of the brain with a 25G needle to distinguish the right and left hemispheres. Take care not to pass the needle through anatomical structures of interest.</li>
	<li>Serially cut 40 &#956;m sections in the coronal plane beginning at bregma -3.8 and ending at bregma -6.8.</li>
	<li>Store six series in labeled tubes with anti-freeze solution (40% PBS, 30% 2-ethoxyethanol, 30% glycerol). Each series should contain 12 brain sections.</li>
	<li>Select one set of sections for immunohistochemical staining, and wash off anti-freeze solution with 3 x 10 min washes in 0.2% PBS-T.</li>
	<li>Block for 1 h at RT with gentle nutation in blocking solution (10% normal goat serum (NGS), 2% bovine serum albumin (BSA) in 0.2% PBS-T). Follow this with incubation with rabbit anti-tyrosine hydroxylase (TH) antibody (1:500) and mouse anti-&#945;-syn antibody (1:500) in 2% NGS in 0.2% PBS-T overnight at room temperature.</li>
	<li>Wash off primary antibody with 3 x 5 min washes in 0.2% PBS-T, followed by 1 h incubation with goat anti-rabbit Alexa Fluor 488 secondary antibody (1:500) and goat anti-mouse Alexa Fluor 555 secondary antibody in 2% NGS in 0.2% PBS-T. Ensure the sections are protected from light and nutating gently.</li>
	<li>Wash off secondary antibody with 3 x 5 min washes in 0.2% PBS-T and mount the complete set of sections on slides protected from light and dust using a narrow paintbrush. Coverslip with fluorescence mounting medium and seal with clear nail varnish.</li>
</ol>
3. Confocal microscopy and image acquisition
<ol>
	<li>Capture IHC images using software coupled to a confocal microscope at 10x magnification. Open the pinhole to 1.5 AU to capture a wide plane totaling ~1.5 &#956;m and set the focus on the injected side of the brain.</li>
	<li>On the Acquisition tab, check the Tile Scan imaging option and set the dimensions to 10 x 4.</li>
	<li>Under the Acquisition Mode panel, set the Zoom to 1.1. This helps to avoid any obvious stitching marks between tile scan images.</li>
	<li>Set the Frame Size to 1024 x 1024 pixels and the Averaging to 2 to ensure high quality image acquisition.</li>
	<li>In the Channels panel, set track 1 to Alexa488 and track 2 to Alexa555.</li>
	<li>Load the slide onto the stage and choose a section with strong TH staining. Click on Live on the Acquisition panel.</li>
	<li>In the Channels panel, set the Laser Strength and Gain to levels that maximize signal and limit noise from the background. Use the range indicator to ensure that the signals are not overexposed (as indicated by a dark red overlay).</li>
	<li>Repeat the above step with multiple slides to ensure staining is consistent between slides as the laser strength/gain cannot be adjusted between slides.</li>
	<li>On the Acquisition tab, check the Positions box.</li>
	<li>At this point, you are ready to begin imaging. Using the eyepiece, choose the first section showing positive TH staining, set the focus at the point of interest (i.e., SNpc) and then move the stage to the midline of the section. This saves the position in the x, y, and z axes and will image a tile scan capturing the whole section.</li>
	<li>Repeat the above step for all sections throughout the SNpc giving a complete set of images of the SNpc. If detailed analysis of the uninjected side is required, steps 3.10 to 3.11 should be repeated by setting the focus on the uninjected side.</li>
</ol>
4. Image analysis and quantitation
<ol>
	<li>Separate image files using appropriate software and import image files to automated image analysis software.</li>
	<li>Define a region of interest by selecting the Pen annotation tool to draw an annotation around the SNpc. 
	NOTE: In sections which have a large amount of dopaminergic neuron loss, temporarily increasing the emittance/absorption can help to clearly define the SNpc (Figure 2).</li>
	<li>Move to the Analysis tab and from the drop-down Analyze menu, select Real-Time Tuning. This opens a separate window on the section image allowing for real-time modification of analysis parameters (Figure 3).</li>
	<li>Under the Analysis Magnification section, select the appropriate image zoom.</li>
	<li>Under the Cell Detection section, select nuclear dye as the dye used for TH staining (Alexa Fluor 488).</li>
	<li>Adjust the Nuclear Contrast Threshold, Minimum Nuclear Intensity, Nuclear Segmentation Aggressiveness, and Nuclear Size settings while carefully watching the Real-Time Tuning window. 
	NOTE: Accurate representation of each individual cell as a single cell in the Real-Time Tuning window is vital for accuracy. These settings are on an arbitrary scale depending on the software used, but correct adjustment is needed to allow the software to accurately differentiate between individual cells, and between cells and the background (Figure 3).</li>
	<li>Repeat this process with a minimum of 10 separate samples to ensure a uniform agreement of what constitutes a cell across different sections. 
	NOTE: Additional cell markers (such as &#945;-syn or NeuN) can be identified within the same analysis platform using the Marker 1 or Marker 2 sections on the analysis tab.</li>
	<li>Once an appropriate number of images have been sampled and Real-Time Tuning has been adjusted accordingly, save the analysis settings in the Settings Actions drop-down menu.</li>
	<li>Select all images to be analyzed and click on Analyze.</li>
	<li>Choose the analysis setting you have just saved and in the Region of Analysis window, check the Annotation Layer(s) box. Then, check Layer 1 and click on Analyze. 
	NOTE: For a single brain, the analysis typically takes about 5 mins. The completed result will clearly show each item that has been counted as a cell (Figure 4).</li>
	<li>Once complete, export the summary analysis data for all sections. There is an option to export Object Analysis Data, which will give detailed data, including cell size of each individual cell detected. This dataset could be used to examine changes in cell size in response to a toxin/therapeutic.</li>
	<li>Add the Total Cells from each section analyzed per animal and the Total Analyzed Area (mm2). Divide the total number of cells by the total area analyzed to calculate the number of cells/mm2 in the SNpc for each rat</li>
</ol>

<ol><li>Kalia, L. V., Lang, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Parkinson%27s+disease%5BTitle%5D)+AND+%22Lancet%22%5BJournal%5D)">Parkinson's disease.</a> Lancet. 386 (9996), 896-912 (2015).</li>
<li>West, M. J., Slomianka, L., Gundersen, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Unbiased+stereological+estimation+of+the+total+number+of+neurons+in+thesubdivisions+of+the+rat+hippocampus+using+the+optical+fractionator%5BTitle%5D)+AND+%22The+Anatomical+Record%22%5BJournal%5D)">Unbiased stereological estimation of the total number of neurons in thesubdivisions of the rat hippocampus using the optical fractionator.</a> The Anatomical Record. 231 (4), 482-497 (1991).</li>
<li>Nair-Roberts, R. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stereological+estimates+of+dopaminergic%2C+GABAergic+and+glutamatergic+neurons+in+the+ventral+tegmental+area%2C+substantia+nigra+and+retrorubral+field+in+the+rat%5BTitle%5D)+AND+%22Neuroscience%22%5BJournal%5D)">Stereological estimates of dopaminergic, GABAergic and glutamatergic neurons in the ventral tegmental area, substantia nigra and retrorubral field in the rat.</a> Neuroscience. 152 (4), 1024-1031 (2008).</li>
<li>Golub, V. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neurostereology+protocol+for+unbiased+quantification+of+neuronal+injury+and+neurodegeneration%5BTitle%5D)+AND+%22Frontiers+in+Aging+Neuroscience%22%5BJournal%5D)">Neurostereology protocol for unbiased quantification of neuronal injury and neurodegeneration.</a> Frontiers in Aging Neuroscience. 7, 196(2015).</li>
<li>Schmitz, C., Hof, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Design-based+stereology+in+neuroscience%5BTitle%5D)+AND+%22Neuroscience%22%5BJournal%5D)">Design-based stereology in neuroscience.</a> Neuroscience. 130 (4), 813-831 (2005).</li>
<li>Penttinen, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Implementation+of+deep+neural+networks+to+count+dopamine+neurons+in+substantia+nigra%5BTitle%5D)+AND+%22European+Journal+of+Neuroscience%22%5BJournal%5D)">Implementation of deep neural networks to count dopamine neurons in substantia nigra.</a> European Journal of Neuroscience. 48 (6), 2354-2361 (2018).</li>
<li>Yousef, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neuron+loss+and+degeneration+in+the+progression+of+TDP-43+in+frontotemporal+lobar+degeneration%5BTitle%5D)+AND+%22Acta+Neuropathologica+Communications%22%5BJournal%5D)">Neuron loss and degeneration in the progression of TDP-43 in frontotemporal lobar degeneration.</a> Acta Neuropathologica Communications. 5 (1), 68(2017).</li>
<li>Koprich, J. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Expression+of+human+A53T+alpha-synuclein+in+the+rat+substantia+nigra+using+a+novel+AAV1%2F2+vector+produces+a+rapidly+evolving+pathology+with+protein+aggregation%2C+dystrophic+neurite+architecture+and+nigrostriatal+degeneration+with+potential+to+model+the+pathology+of+Parkinson%27s+disease%5BTitle%5D)+AND+%22Molecular+Neurodegeneration%22%5BJournal%5D)">Expression of human A53T alpha-synuclein in the rat substantia nigra using a novel AAV1/2 vector produces a rapidly evolving pathology with protein aggregation, dystrophic neurite architecture and nigrostriatal degeneration with potential to model the pathology of Parkinson's disease.</a> Molecular Neurodegeneration. 5, 43(2010).</li>
<li>Koprich, J. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Progressive+neurodegeneration+or+endogenous+compensation+in+an+animal+model+of+Parkinson%27s+disease+produced+by+decreasing+doses+of+alpha-synuclein%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Progressive neurodegeneration or endogenous compensation in an animal model of Parkinson's disease produced by decreasing doses of alpha-synuclein.</a> PLoS One. 6 (3), 17698(2011).</li>
<li>McKinnon, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Early-onset+impairment+of+the+ubiquitin-proteasome+system+in+dopaminergic+neurons+caused+by+alpha-synuclein%5BTitle%5D)+AND+%22Acta+Neuropathologica+Communications%22%5BJournal%5D)">Early-onset impairment of the ubiquitin-proteasome system in dopaminergic neurons caused by alpha-synuclein.</a> Acta Neuropathologica Communications. 8 (1), 17(2020).</li>
<li>Henderson, M. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spread+of+alpha-synuclein+pathology+through+the+brain+connectome+is+modulated+by+selective+vulnerability+and+predicted+by+network+analysis%5BTitle%5D)+AND+%22Nature+Neuroscience%22%5BJournal%5D)">Spread of alpha-synuclein pathology through the brain connectome is modulated by selective vulnerability and predicted by network analysis.</a> Nature Neuroscience. 22 (8), 1248-1257 (2019).</li>
<li>Ip, C. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((AAV1%2F2-induced+overexpression+of+A53T-alpha-synuclein+in+the+substantia+nigra+results+in+degeneration+of+the+nigrostriatal+system+with+Lewy-like+pathology+and+motor+impairment%3A+a+new+mouse+model+for+Parkinson%27s+disease%5BTitle%5D)+AND+%22Acta+Neuropathologica+Communications%22%5BJournal%5D)">AAV1/2-induced overexpression of A53T-alpha-synuclein in the substantia nigra results in degeneration of the nigrostriatal system with Lewy-like pathology and motor impairment: a new mouse model for Parkinson's disease.</a> Acta Neuropathologica Communications. 5 (1), 11(2017).</li>
<li>Webster, J. D., Dunstan, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Whole-slide+imaging+and+automated+image+analysis%3A+considerations+and+opportunities+in+the+practice+of+pathology%5BTitle%5D)+AND+%22Veterinary+Pathology%22%5BJournal%5D)">Whole-slide imaging and automated image analysis: considerations and opportunities in the practice of pathology.</a> Veterinary Pathology. 51 (1), 211-223 (2014).</li>
</ol>

The authors report no competing interests.

The reliable assessment of the integrity of the dopaminergic system in pre-clinical models of PD is critical to determine the effectiveness of potential disease-modifying therapeutics. Therefore, it is important to control and minimize potential confounds that may reduce the reliability and reproducibility of histopathological data. Careful quantitative outcomes can provide more information than qualitative or semi-quantitative descriptions alone. At the same time, we must recognize that constraints in time and resources...

Parkinson&#39;s disease (PD) is a prevalent neurodegenerative movement disorder characterized by the presence of protein aggregates containing &#945;-synuclein (&#945;-syn) and the preferential loss of dopaminergic neurons in the substantia nigra pars compacta&#160;(SNpc)1. Quantification of dopaminergic neuron number is a vital part of PD research as it permits the evaluation of the integrity of the nigrostriatal system, thus, providing an important endpoint to assess the effectiveness of potential disease-modifying therapeutics. Currently, the standard for quantification of cell number is unbiased stereological counting, which utilizes two-dimensional (2D) cross-sections of tissue to estimate volumetric features in three-dimensional (3D) structures2,3,4. Modern design-based stereological methods employ comprehensive random sampling procedures and apply counting protocols (known as probes) to avoid potential artifacts and systematic errors, allowing for reliable detection of differences only slightly greater than inter-animal variation5. While stereology provides a powerful analytical tool for in vivo histological studies, it is time intensive, assumes uniform specimen preparation, and requires validation at several steps, which can impact the efficiency increasingly required for pre-clinical translational investigation.
Recent technological advances in digital science make it possible to adopt novel applications for more efficient assessments of pathology without a stereomicroscope, while filling a need as a surrogate of unbiased stereology. These methods increase speed, reduce human error, and improve the reproducibility of stereological techniques6,7. HALO is one such image analysis platform for quantitative tissue analysis in digital pathology. It comprises a variety of different modules and reports morphological and multiplexed expression data on a cell-by-cell basis across entire tissue sections using pattern recognition algorithms. The cytonuclear FL module measures the immunofluorescent positivity of fluorescent markers in the nucleus or cytoplasm. This allows for reporting of the number of cells positive for each marker, and the intensity score for each cell. The module can be adapted to provide individual cell sizes and intensity measurements, although this feature is not required for quantification of dopaminergic neurons.
The aim of this study is to verify this method with a previously validated viral vector-based &#945;-syn rat model of nigral neurodegeneration8,9,10. In this model, human mutant A53T &#945;-syn is expressed in the SNpc by stereotactic injection of adeno-associated virus hybrid serotype 1/2 (AAV1/2), resulting in significant neurodegeneration over a period of 6 weeks. The contralateral uninjected SNpc may, in some studies, serve as an internal control for the injected side. More commonly, injection of AAV-Empty Vector (AAV-EV) in a control cohort of animals is used as a negative control. We present a step-by-step guide to estimate the density of dopaminergic neurons remaining in the injected SNpc after 6 weeks using an automated image analysis software (Figure 1).

<table><tbody><tr><td>A-Syn Antibody</td><td>ThermoFisher Scientific</td><td>32-8100</td><td></td></tr><tr><td>ABC Elite</td><td>Vector Labs</td><td>PK-6102</td><td></td></tr><tr><td>Alexa Fluor 488 secondary antibody</td><td>ThermoFisher Scientific</td><td>A-11008</td><td></td></tr><tr><td>Alexa Fluor 555 secondary antibody</td><td>ThermoFisher Scientific</td><td>A-28180</td><td></td></tr><tr><td>Alkaline phosphatase-conjugated anti-rabbit igG</td><td>Jackson Immuno</td><td>111-055-144</td><td></td></tr><tr><td>Biotinylated anti-mouse IgG</td><td>Vector Labs</td><td>BA-9200</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma</td><td>A2153</td><td></td></tr><tr><td>DAKO fluorescent mouting medium</td><td>Agilent</td><td>S3023</td><td></td></tr><tr><td>HALO&amp;trade;</td><td>Indica Labs</td><td></td><td></td></tr><tr><td>Histo-Clear II</td><td>Diamed</td><td>HS202</td><td></td></tr><tr><td>ImmPACT DAB Peroxidase substrate</td><td>Vector Labs</td><td>SK-4105</td><td></td></tr><tr><td>LSM880 Confocal Microscope</td><td>Zeiss</td><td></td><td></td></tr><tr><td>NeuN Antibody</td><td>Millipore</td><td>MAB377</td><td></td></tr><tr><td>Normal Goat Serum</td><td>Vector Labs</td><td>S-1000-20</td><td></td></tr><tr><td>OCT</td><td>Tissue-Tek</td><td></td><td></td></tr><tr><td>Paraformaldehyde</td><td>BioShop</td><td>PAR070.1</td><td></td></tr><tr><td>Sliding microtome</td><td>Leica</td><td>SM2010 R</td><td></td></tr><tr><td>Stereo Investigator</td><td>MBF Bioscience</td><td></td><td></td></tr><tr><td>Sucrose</td><td>BioShop</td><td>SUC700</td><td></td></tr><tr><td>TH Antibody</td><td>ThermoFisher Scientific</td><td>P21962</td><td></td></tr><tr><td>VectaMount mounting medium</td><td>Vector Labs</td><td>H-5000</td><td></td></tr><tr><td>Vector Blue Alkaline Phosphatase substrate</td><td>Vector Labs</td><td>SK-5300</td><td></td></tr><tr><td>Zen Black Software</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Zen Blue Software</td><td>Zeiss</td><td></td><td></td></tr></tbody></table>

semi-quantitative determination of dopaminergic neuron density in the substantia nigra of rodent models using automated image analysis

Estimation of the number of dopaminergic neurons in the substantia nigra is a key method in pre-clinical Parkinson's disease research. Currently, unbiased stereological counting is the standard for quantification of these cells, but it remains a laborious and time-consuming process, which may not be feasible for all projects. Here, we describe the use of an image analysis platform, which can accurately estimate the quantity of labeled cells in a pre-defined region of interest. We describe a step-by-step protocol for this method of analysis in rat brain and demonstrate it can identify a significant reduction in tyrosine hydroxylase positive neurons due to expression of mutant &#945;-synuclein in the substantia nigra. We validated this methodology by comparing with results obtained by unbiased stereology. Taken together, this method provides a time-efficient and accurate process for detecting changes in dopaminergic neuron number, and thus is suitable for efficient determination of the effect of interventions on cell survival.

By applying the above methods to brain tissue collected 6 weeks after AAV injections, we demonstrated that stereotactic injection of AAV expressing mutant A53T &#945;-syn (AAV-A53T) in the SNpc of rat brain results in a significant reduction in the density of dopaminergic neurons compared to injection of empty vector AAV (AAV-EV) as a control (Figure 5A,B). The mean number of TH-positive neurons/mm2 in the SNpc of rats injected with AAV-EV was 276.2 &#177; 34.7, a...

Watch this Scientific Journal Video about Semi-Quantitative Determination of Dopaminergic Neuron Density in the Substantia Nigra of Rodent Models using Automated Image Analysis at JoVE.com

Semi-Quantitative Determination of Dopaminergic Neuron Density in the Substantia Nigra of Rodent Models using Automated Image Analysis

Here we present an automated method for semi-quantitative determination of dopaminergic neuron number in the rat substantia nigra pars compacta.

Estimation of the number of dopaminergic neurons in the substantia nigra is a key method in pre-clinical Parkinson's disease research. Currently, unbiased ...

semi-quantitative-determination-dopaminergic-neuron-density

Research

JoVE Journal

Neuroscience

4.4K Views.  University Health Network. Schatting van dopaminerge neuron nummer in de substantia nigra is een belangrijke uitkomstmaat in de ziekte van Parkinson onderzoek. Dit protocol verkort de werklast die nodig is om het neuronnummer te schatten aanzienlijk. Deze methode biedt een tijdsefficiënt en nauwkeurig proces voor het detecteren van veranderingen in het aantal dopaminerge neuronen en is geschikt voor het bepalen van het effect van interventies op de celoverleving.Deze methode kan eenvoudig worden aangepast om na...

Video: Semi-Quantitative Determination of Dopaminergic Neuron Density in the Substantia Nigra of Rodent Models using Automated Image Analysis

Comprehensive Profiling of Dopamine Regulation in Substantia Nigra and Ventral Tegmental Area

Dopamine is a vigorously studied neurotransmitter in the CNS. Indeed, its involvement in locomotor activity and reward-related behaviour has fostered five decades of inquiry into the molecular deficiencies associated with dopamine regulation. The majority of these inquiries of dopamine regulation in the brain focus upon the molecular basis for its regulation in the terminal field regions of the nigrostriatal and mesoaccumbens pathways; striatum and nucleus accumbens. Furthermore, such studies have concentrated on analysis of dopamine tissue content with normalization to only wet tissue weight. Investigation of the proteins that regulate dopamine, such as tyrosine hydroxylase (TH) protein, TH phosphorylation, dopamine transporter (DAT), and vesicular monoamine transporter 2 (VMAT2) protein often do not include analysis of dopamine tissue content in the same sample. The ability to analyze both dopamine tissue content and its regulating proteins (including post-translational modifications) not only gives inherent power to interpreting the relationship of dopamine with the protein level and function of TH, DAT, or VMAT2, but also extends sample economy. This translates into less cost, and yet produces insights into the molecular regulation of dopamine in virtually any paradigm of the investigators' choice.
We focus the analyses in the midbrain. Although the SN and VTA are typically neglected in most studies of dopamine regulation, these nuclei are easily dissected with practice. A comprehensive readout of dopamine tissue content and TH, DAT, or VMAT2 can be conducted. There is burgeoning literature on the impact of dopamine function in the SN and VTA on behavior, and the impingements of exogenous substances or disease processes therein 1-5. Furthermore, compounds such as growth factors have a profound effect on dopamine and dopamine-regulating proteins, to a comparatively greater extent in the SN or VTA 6-8. Therefore, this methodology is presented for reference to laboratories that want to extend their inquiries on how specific treatments modulate behaviour and dopamine regulation. Here, a multi-step method is presented for the analyses of dopamine tissue content, the protein levels of TH, DAT, or VMAT2, and TH phosphorylation from the substantia nigra and VTA from rodent midbrain. The analysis of TH phosphorylation can yield significant insights into not only how TH activity is regulated, but also the signaling cascades affected in the somatodendritic nuclei in a given paradigm.
We will illustrate the dissection technique to segregate these two nuclei and the sample processing of dissected tissue that produces a profile revealing molecular mechanisms of dopamine regulation in vivo, specific for each nuclei (Figure 1).

Dopamine is distinctly regulated in the midbrain nuclei, which contain the cell bodies and dendrites of the dopamine neurons. Here we describe a dissection and sample-handling approach to maximize results, and thus conclusions and insights, on dopamine regulation in the midbrain nuclei of the substantia nigra (SN) and ventral tegmental area (VTA) in rodents.

Dopamine is a vigorously studied neurotransmitter in the CNS. Indeed, its involvement in locomotor activity and reward-related behaviour has fostered five ...

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, ...

Assessment of Dopaminergic Homeostasis in Mice by Use of High-performance Liquid Chromatography Analysis and Synaptosomal Dopamine Uptake

Dopamine (DA) is a modulatory neurotransmitter controlling motor activity, reward processes and cognitive function. Impairment of dopaminergic (DAergic) neurotransmission is strongly associated with several central nervous system-associated diseases such as Parkinson&#39;s disease, attention-deficit-hyperactivity disorder and drug addiction1,2,3,4. Delineating disease mechanisms involving DA imbalance is critically dependent on animal models to mimic aspects of the diseases, and thus protocols that assess specific parts of the DA homeostasis are important to provide novel insights and possible therapeutic targets for these diseases.
Here, we present two useful experimental protocols that when combined provide a functional read-out of the DAergic system in mice. Biochemical and functional parameters on DA homeostasis are obtained through assessment of DA levels and dopamine transporter (DAT) functionality5. When investigating the DA system, the ability to reliably measure endogenous levels of DA from adult brain is essential. Therefore, we present how to perform high-performance liquid chromatography (HPLC) on brain tissue from mice to determine levels of DA. We perform the experiment on tissue from dorsal striatum (dStr) and nucleus accumbens (NAc), but the method is also suitable for other DA-innervated brain areas.
DAT is essential for reuptake of DA into the presynaptic terminal, thereby controlling the temporal and spatial activity of released DA. Knowing the levels and functionality of DAT in the striatum is of major importance when assessing DA homeostasis. Here, we provide a protocol that allows to simultaneously deduce information on surface levels and function using a synaptosomal6 DA uptake assay.
Current methods combined with standard immunoblotting protocols provide the researcher with relevant tools to characterize the DAergic system.

Synaptosomal dopamine uptake and high-performance liquid chromatography analysis represent experimental tools to investigate dopamine homeostasis in mice by assessing the function of the dopamine transporter and levels of dopamine in striatal tissue, respectively. Here we present protocols to measure dopamine tissue content and assess the functionality of the dopamine transporter.

Dopamine (DA) is a modulatory neurotransmitter controlling motor activity, reward processes and cognitive function. Impairment of dopaminergic (DAergic) ...

Stereological Estimation of Dopaminergic Neuron Number in the Mouse Substantia Nigra Using the Optical Fractionator and Standard Microscopy Equipment

In pre-clinical Parkinson&#39;s disease research, analysis of the nigrostriatal tract, including quantification of dopaminergic neuron loss within the substantia nigra, is essential. To estimate the total dopaminergic neuron number, unbiased stereology using the optical fractionator method is currently considered the gold standard. Because the theory behind the optical fractionator method is complex and because stereology is difficult to achieve without specialized equipment, several commercially available complete stereology systems that include the necessary software do exist, purely for cell counting reasons. Since purchasing a specialized stereology setup is not always feasible, for many reasons, this report describes a method for the stereological estimation of dopaminergic neuronal cell counts using standard microscopy equipment, including a light microscope, a motorized object table (x, y, z plane) with imaging software, and a computer for analysis. A step-by-step explanation is given on how to perform stereological quantification using the optical fractionator method, and pre-programmed files for the calculation of estimated cell counts are provided. To assess the accuracy of this method, a comparison to data obtained from a commercially available stereology apparatus was performed. Comparable cell numbers were found using this protocol and the stereology device, thus demonstrating the precision of this protocol for unbiased stereology.

This work presents a step-by-step protocol for the unbiased stereological estimation of dopaminergic neuronal cell numbers in the mouse substantia nigra using standard microscopy equipment (i.e., a light microscope, a motorized object table (x, y, z plane), and public domain software for digital image analysis.

In pre-clinical Parkinson&#39;s disease research, analysis of the nigrostriatal tract, including quantification of dopaminergic neuron loss within the ...

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in regulating neuronal growth and development. However, the mechanisms underlying Drd1&#945; receptor abnormalities mediating behavioral responses and modulating working memory function are still unclear. Using a novel RNA in situ hybridization assay, the current study identified dopamine Drd1&#945; receptor and tyrosine hydroxylase (TH) RNA expression from DA-related circuitry in the nucleus accumbens (NAc) area and substantia nigra region (SNR), respectively. Drd1&#945; expression in the NAc shows a &#34;discrete dot&#34; staining pattern. Clear sex differences in Drd1&#945; expression were observed. In contrast, TH shows a &#34;clustered&#34; staining pattern. Regarding TH expression, female rats displayed a higher signal expression per cell relative to male animals. The methods presented here provide a novel in situ hybridization technique for investigating changes in dopamine system dysfunction during the progression of central nervous system diseases.

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

Identification of dopamine D1-alpha receptor in the nucleus accumbens is critical for clarifying D1 receptor dysfunction during a central nervous system disease. We performed a novel RNA in situ hybridization assay to visualize single RNA molecules in a specific brain area.

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in ...

Stereological Estimation of Cholinergic Fiber Length in the Nucleus Basalis of Meynert of the Mouse Brain

The length of cholinergic or other neuronal axons in various brain regions are often correlated with the specific function of the region. Stereology is a useful method to quantify neuronal profiles of various brain structures. Here we provide a software-based stereology protocol to estimate the total length of cholinergic fibers in the nucleus basalis of Meynert (NBM) of the basal forebrain. The method uses a space ball probe for length estimates. The cholinergic fibers are visualized by choline acetyltransferase (ChAT) immunostaining with the horseradish peroxidase-diaminobenzidine (HRP-DAB) detection system. The staining protocol is also valid for fiber and cell number estimation in various brain regions using stereology software. The stereology protocol can be used for estimation of any linear profiles such as cholinoceptive fibers, dopaminergic/catecholaminergic fibers, serotonergic fibers, astrocyte processes, or even vascular profiles.

Neuronal fiber length within a three-dimensional structure of a brain region is a reliable parameter to quantify specific neuronal structural integrity or degeneration. This article details a stereological quantification method to measure cholinergic fiber length within the nucleus basalis of Meynert in mice as an example.

The length of cholinergic or other neuronal axons in various brain regions are often correlated with the specific function of the region. Stereology is a ...

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.

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 ...

Standardized Data Acquisition for Neuromelanin-Sensitive Magnetic Resonance Imaging of the Substantia Nigra

The dopaminergic system plays a crucial role in healthy cognition (e.g., reward learning and uncertainty) and neuropsychiatric disorders (e.g., Parkinson's disease and schizophrenia). Neuromelanin is a byproduct of dopamine synthesis that accumulates in dopaminergic neurons of the substantia nigra. Neuromelanin-sensitive magnetic resonance imaging (NM-MRI) is a noninvasive method for measuring neuromelanin in those dopaminergic neurons, providing a direct measure of dopaminergic cell loss in the substantia nigra and a proxy measure of dopamine function. Although NM-MRI has been shown to be useful for studying various neuropsychiatric disorders, it is challenged by a limited field-of-view in the inferior-superior direction resulting in the potential loss of data from the accidental exclusion of part of the substantia nigra. In addition, the field is lacking a standardized protocol for the acquisition of NM-MRI data, a critical step in facilitating large-scale multisite studies and translation into the clinic. This protocol describes a step-by-step NM-MRI volume placement procedure and online quality control checks to ensure the acquisition of good-quality data covering the entire substantia nigra.

This protocol shows how to acquire neuromelanin-sensitive magnetic resonance imaging data of the substantia nigra.

The dopaminergic system plays a crucial role in healthy cognition (e.g., reward learning and uncertainty) and neuropsychiatric disorders (e.g., ...

Analyzing the Parkinson's Disease Mouse Model Induced by Adeno-associated Viral Vectors Encoding Human &#945;-Synuclein

Parkinson&#39;s disease is a neurodegenerative disorder that involves the death of the dopaminergic neurons of the nigrostriatal pathway and, consequently, the progressive loss of control of voluntary movements. This neurodegenerative process is triggered by the deposition of protein aggregates in the brain, which are mainly constituted of &#945;-synuclein. Several studies have indicated that neuroinflammation is required to develop the neurodegeneration associated with Parkinson&#39;s disease. Notably, the neuroinflammatory process involves microglial activation as well as the infiltration of peripheral T cells into the substantia nigra (SN). This work analyzes a mouse model of Parkinson&#39;s disease that recapitulates microglial activation, T-cell infiltration into the SN, the neurodegeneration of nigral dopaminergic neurons, and motor impairment. This mouse model of Parkinson&#39;s disease is induced by the stereotaxic delivery of adeno-associated viral vectors encoding the human wild-type &#945;-synuclein (AAV-h&#945;Syn) into the SN. The correct delivery of viral vectors into the SN was confirmed using control vectors encoding green fluorescent protein (GFP). Afterward, how the dose of AAV-h&#945;Syn administered in the SN affected the extent of h&#945;Syn expression, the loss of nigral dopaminergic neurons, and motor impairment were evaluated. Moreover, the dynamics of h&#945;Syn expression, microglial activation, and T-cell infiltration were determined throughout the time course of disease development. Thus, this study provides critical time points that may be useful for targeting synuclein pathology and neuroinflammation in this preclinical model of Parkinson&#39;s disease.

This work analyzes the vector dose and exposure time required to induce neuroinflammation, neurodegeneration, and motor impairment in this preclinical model of Parkinson&#39;s disease. These vectors encoding the human &#945;-synuclein are delivered into the substantia nigra&#160;to recapitulate the synuclein pathology associated with Parkinson&#39;s disease.

Parkinson&#39;s disease is a neurodegenerative disorder that involves the death of the dopaminergic neurons of the nigrostriatal pathway and, ...

Advanced Live-Imaging Techniques for Accurate Neurite Outgrowth Measurement

Standardization of a Novel Semi-Automatic Software for Neurite Outgrowth Measurement

Effective live-imaging techniques are crucial to assess neuronal morphology in order to measure neurite outgrowth in real time. The proper measurement of neurite outgrowth has been a long-standing challenge over the years in the neuroscience research field. This parameter serves as a cornerstone in numerous in vitro experimental setups, ranging from dissociated cultures and organotypic cultures to cell lines. By quantifying the neurite length, it is possible to determine if a specific treatment worked or if axonal regeneration is enhanced in different experimental groups. In this study, the aim is to demonstrate the robustness and accuracy of the Incucyte Neurotrack neurite outgrowth analysis software. This semi-automatic software is available in a time-lapse microscopy system which offers several advantages over commonly used methodologies in the quantification of the neurite length in phase contrast images. The algorithm masks and quantifies several parameters in each image and returns neuronal cell metrics, including neurite length, branch points, cell-body clusters, and cell-body cluster areas. Firstly, we validated the robustness and accuracy of the software by correlating its values with those of the manual NeuronJ, a Fiji plug-in. Secondly, we used the algorithm which is able to work both on phase contrast images as well as on immunocytochemistry images. Using specific neuronal markers, we validated the feasibility of the fluorescence-based neurite outgrowth analysis on sensory neurons in vitro cultures. Additionally, this software can measure neurite length across various seeding conditions, ranging from individual cells to complex neuronal nets. In conclusion, the software provides an innovative and time-effective platform for neurite outgrowth assays, paving the way for faster and more reliable quantifications.

Author Spotlight: Unveiling the Molecular Basis of Pain Perception and Neuropathic Pain

Neurite outgrowth assays provide a quantitative value about regenerative neuronal processes. The advantage of this semi-automatic software is that it segments cell bodies and neurites separately by creating a mask and measures various parameters such as neurite length, number of branch points, cell-body cluster area, and number of cell clusters.

Effective live-imaging techniques are crucial to assess neuronal morphology in order to measure neurite outgrowth in real time. The proper measurement of ...

Semi-Quantitative Determination of Dopaminergic Neuron Density in the Substantia Nigra of Rodent Models using Automated Image Analysis

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