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>

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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#8482;</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

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4.3K Views.  University Health Network. Here we present an automated method for semi-quantitative determination of dopaminergic neuron number in the rat substantia nigra pars compacta.

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

Automated Sholl Analysis of Digitized Neuronal Morphology at Multiple Scales

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3. Specifically, it affects the ability of neurons to receive inputs from other cells2 and contributes to the propagation of action potentials4,5. The morphology of the neurites also affects how information is processed. The diversity of dendrite morphologies facilitate local and long range signaling and allow individual neurons or groups of neurons to carry out specialized functions within the neuronal network6,7. Alterations in dendrite morphology, including fragmentation of dendrites and changes in branching patterns, have been observed in a number of disease states, including Alzheimer's disease8, schizophrenia9,10, and mental retardation11. The ability to both understand the factors that shape dendrite morphologies and to identify changes in dendrite morphologies is essential in the understanding of nervous system function and dysfunction.
Neurite morphology is often analyzed by Sholl analysis and by counting the number of neurites and the number of branch tips. This analysis is generally applied to dendrites, but it can also be applied to axons. Performing this analysis by hand is both time consuming and inevitably introduces variability due to experimenter bias and inconsistency. The Bonfire program is a semi-automated approach to the analysis of dendrite and axon morphology that builds upon available open-source morphological analysis tools. Our program enables the detection of local changes in dendrite and axon branching behaviors by performing Sholl analysis on subregions of the neuritic arbor. For example, Sholl analysis is performed on both the neuron as a whole as well as on each subset of processes (primary, secondary, terminal, root, etc.) Dendrite and axon patterning is influenced by a number of intracellular and extracellular factors, many acting locally. Thus, the resulting arbor morphology is a result of specific processes acting on specific neurites, making it necessary to perform morphological analysis on a smaller scale in order to observe these local variations12.
The Bonfire program requires the use of two open-source analysis tools, the NeuronJ plugin to ImageJ and NeuronStudio. Neurons are traced in ImageJ, and NeuronStudio is used to define the connectivity between neurites. Bonfire contains a number of custom scripts written in MATLAB (MathWorks) that are used to convert the data into the appropriate format for further analysis, check for user errors, and ultimately perform Sholl analysis. Finally, data are exported into Excel for statistical analysis. A flow chart of the Bonfire program is shown in Figure 1.

We have developed a computer program to analyze neuronal morphology. In combination with two existing open source analysis tools, our program performs Sholl analysis and determines the number of neurites, branch points, and neurite tips. The analyses are performed so that local changes in neurite morphology can be observed.

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3.  Specifically, it affects the ability of neurons to ...

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 ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

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 ...

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 ...

The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. Structural changes at the NMJ can result in neurotransmission failure, resulting in weakness, atrophy and even death of the muscle fiber. Many studies have investigated how genetic modifications or disease can alter the structure of the mouse NMJ. Unfortunately, it can be difficult to directly compare findings from these studies because they often employed different parameters and analytical methods. Three protocols are described here. The first uses maximum intensity projection confocal images to measure the area of acetylcholine receptor (AChR)-rich postsynaptic membrane domains at the endplate and the area of synaptic vesicle staining in the overlying presynaptic nerve terminal. The second protocol compares the relative intensities of immunostaining for synaptic proteins in the postsynaptic membrane. The third protocol uses Fluorescence Resonance Energy Transfer (FRET) to detect changes in the packing of postsynaptic AChRs at the endplate. The protocols have been developed and refined over a series of studies. Factors that influence the quality and consistency of results are discussed and normative data are provided for NMJs in healthy young adult mice.

The neuromuscular junction (NMJ) is altered in a variety of conditions that can sometimes culminate in synaptic failure. This report describes fluorescence microscope-based methods to quantify such structural changes.

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. ...

Long-term Continuous EEG Monitoring in Small Rodent Models of Human Disease Using the Epoch Wireless Transmitter System

Many progressive neurologic diseases in humans, such as epilepsy, require pre-clinical animal models that slowly develop the disease in order to test interventions at various stages of the disease process. These animal models are particularly difficult to implement in immature rodents, a classic model organism for laboratory study of these disorders. Recording continuous EEG in young animal models of seizures and other neurological disorders presents a technical challenge due to the small physical size of young rodents and their dependence on the dam prior to weaning. Therefore, there is not only a clear need for improving pre-clinical research that will better identify those therapies suitable for translation to the clinic but also a need for new devices capable of recording continuous EEG in immature rodents. Here, we describe the technology behind and demonstrate the use of a novel miniature telemetry system, specifically engineered for use in immature rats or mice, which is also effective for use in adult animals.

Here we demonstrate the use of a wireless enabling technology for electroencephalogram (EEG) in neonatal rodent models of human disease. With telemetry, there are no encumbering connections, thus allowing natural behaviors.

Many progressive neurologic diseases in humans, such as epilepsy, require pre-clinical animal models that slowly develop the disease in order to test ...

High-density Electroencephalographic Acquisition in a Rodent Model Using Low-cost and Open-source Resources

Advanced electroencephalographic analysis techniques requiring high spatial resolution, including electrical source imaging and measures of network connectivity, are applicable to an expanding variety of questions in neuroscience. Performing these kinds of analyses in a rodent model requires higher electrode density than traditional screw electrodes can accomplish. While higher-density electroencephalographic montages for rodents exist, they are of limited availability to most researchers, are not robust enough for repeated experiments over an extended period of time, or are limited to use in anesthetized rodents.1-3 A proposed low-cost method for constructing a durable, high-count, transcranial electrode array, consisting of bilaterally implantable headpieces is investigated as a means to perform advanced electroencephalogram analyses in mice or rats.

Procedures for headpiece fabrication and surgical implantation necessary to produce high signal to noise, low-impedance electroencephalographic and electromyographic signals are presented. While the methodology is useful in both rats and mice, this manuscript focuses on the more challenging implementation for the smaller mouse skull. Freely moving mice are only tethered to cables via a common adapter during recording. One version of this electrode system that includes 26 electroencephalographic channels and 4 electromyographic channels is described below.

Instructions for the low-cost construction and surgical implantation of a chronic transcranial high-density electroencephalographic montage into mice are provided. Signal recording, extraction, and processing techniques are also described.

Advanced electroencephalographic analysis techniques requiring high spatial resolution, including electrical source imaging and measures of network ...

Low-Density Primary Hippocampal Neuron Culture

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility in genetic and chemical manipulation of individual cells or defined networks. Such ease of manipulation is simpler in the reduced culture system when compared to the intact brain tissue. While many methods for the isolation and growth of these primary neurons exist, each has its own limitations. This protocol describes a method for culturing low-density and high-purity rodent embryonic hippocampal neurons on glass coverslips, which are then suspended over a monolayer of glial cells. This &#39;sandwich culture&#39; allows for exclusive long-term growth of a population of neurons while allowing for trophic support from the underlying glial monolayer. When neurons are of sufficient age or maturity level, the neuron coverslips can be flipped-out of the glial dish and used in imaging or functional assays. Neurons grown by this method typically survive for several weeks and develop extensive arbors, synaptic connections, and network properties.

This article describes the protocol for culturing low-density primary hippocampal neurons growing on glass coverslips inverted over a glial monolayer. The neuron and glial layers are separated by paraffin wax beads. The neurons grown by this method are suitable for high-resolution optical imaging and functional assays.

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility ...

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) ...