This work was supported by the IMPACT Award from the Department of Radiation Oncology, University of Alabama-Birmingham Comprehensive Cancer Center, the Fighting Children's Cancer Foundation, and the Gabrielle's Angel Foundation (to ESY.).

1. Cell Culture
 <ol>
 <li>Culture neuronal cells and treat them as needed.</li>
 <li>Harvest cells into 15 ml tubes: aspirate media, rinse with phosphate buffered saline (PBS, calcium and magnesium free), add trypsin, collect in 15 ml tubes and neutralize trypsin with appropriate serum containing media.</li>
 <li>Spin at 1,000 x g for 5 min.</li>
 <li>Aspirate media.</li>
 <li>Resuspend cells in PBS.</li>
 <li>Spin at 1,000 x g for 5 min.</li>
 <li>Aspirate media and resuspend cells in fresh PBS.</li>
 <li>Count cells using a hemacytometer or your preferred cell counter.</li>
 <li>Cell samples should be prepared immediately before starting the assay.</li>
 <li>All samples should be handled in the dark or yellow light to prevent DNA damage from ultraviolet light.</li>
 </ol>
 2. Comet Assay
 1. Slide Preparation
 <ol>
 <li>Melt 1% agarose (1 g / 100 ml in 1X Tris Base, Boric acid, EDTA, TBE) in a microwave for 3 min until all granules disappear.</li>
 <li>Dip slides into the molten agarose and wipe a side clean with a kimwipe. Allow the agarose to air-dry to a transparent film. This can be done in advance and slides can be stored.</li>
 </ol>
 2. Neutral Comet Assay
 <ol>
 <li>Chill lysis solution (2.5 M NaCl, 100 mM EDTA pH 10, 10 mM Tris Base, 1% sodium lauryl sarcosinate, and 1% Triton X-100, pH 10) at 4 &deg;C for at least 1 hr before use.</li>
 <li>2.2.2. Melt 1% low melting point agarose (1 g / 100ml in 1X Tris Base, Boric acid, EDTA) in a microwave for 3 min until all granules disappear. The agarose needs to be cooled to 37 &deg;C in a water bath to avoid artificial induction of comet tail. Ideally, the agarose needs to be cooled for half an hour before use.</li>
 <li>2.2.3. Dilute the cell suspension so that there are 100,000 cells per ml. Combine the cell suspension with the low melting point agarose (at 37 &deg;C) at a ratio of 1:10 (v/v), vortex briefly, and immediately pipette 50 &mu;l onto the Comet Slide. Use the side of the pipette tip to spread the cell suspension evenly over the sample area. Each treatment group should be at least in triplicate.</li>
 <li>Place slides flat in refrigerator for 30 min until a circle appears in the periphery of the slide.</li>
 <li>Prechill the lysis solution and submerge slides in this solution for 30 min in the dark at 4 &deg;C (or in a refrigerator).</li>
 <li>Pour off or aspirate the lysis solution and add 1X neutral electrophoresis buffer (Tris base, Boric acid, EDTA, 1X TBE). Leave slides in this buffer for half an hour in the refrigerator.</li>
 <li>Add prechilled 1X Neutral Electrophoresis Buffer (TBE) in electrophoresis chamber, place slides in electrophoresis slide tray. Align slides so that they are equidistant from electrodes.</li>
 <li>Pour 1X neutral electrophoresis buffer up to 0.2 inches above slides. Excess buffer will interfere with electrophoresis.</li>
 <li>Set power supply voltage to 1 V per cm (measured electrode to electrode) and run for 30 min at 4 &deg;C (or the cold room) in the dark.</li>
 </ol>
 3. Alkaline Comet Assay
 <ol>
 <li>Chill lysis solution (2.5 M NaCl, 100 mM EDTA pH 10, 10 mM Tris Base, 1% sodium lauryl sarcosinate, and 1% Triton X-100, pH 10) at 4 &deg;C for at least 1 hr before use.</li>
 <li>Melt 1% low melting point agarose (1 g / 100 ml in 1X Tris Base, Boric acid, EDTA) in a microwave for 3 min until all granules disappear. Then cool in a 37 &deg;C water bath for at least 30 min.</li>
 <li>Combine cells at 1 x 105 / ml with molten low melting point agarose (at 37 &deg;C) at a ratio of 1:10 (v/v), vortex briefly, and immediately pipette 50 &mu;l onto the Comet Slide. Use the side of the pipette tip to spread the cell suspension evenly over the sample area. Each treatment group should be at least in triplicate.</li>
 <li>Place slides flat in refrigerator for 30 min until a circle appears in the periphery of the slide.</li>
 <li>Immerse slides in prechilled lysis solution and leave at 4 &deg;C for 1 hr to overnight in the dark.</li>
 <li>Remove the slides from the lysis solution, drain the slides and rinse once with cold neutralization buffer for 5 min to remove residual detergent and salts prior to the alkali-unwinding step.</li>
 <li>Place slides in a gel electrophoresis chamber filled with prechilled freshly made electrophoresis Buffer (300 mM NaOH, 1 mM EDTA, pH&gt;13) not to exceed 0.5 cm above slides. Align slides so that they are equidistant from electrodes.</li>
 <li>Let slides sit in the alkaline buffer for 30 min in the dark to allow for unwinding of the DNA and the expression of alkali-liable damage.</li>
 <li>Set power supply voltage to 1 V per cm (measured electrode to electrode) and run for 30 min at 4 &deg;C (or the cold room).</li>
</ol>
 4. Fixing and Staining Cells
 <ol>
 <li>Drain excess Electrophoresis Buffer</li>
 <li>Immerse slides in pre-chilled distilled water for 5 min at RT.</li>
 <li>Immerse slides in pre-chilled 70% ethanol for 5 min at RT.</li>
 <li>Dry samples overnight. Do not expose slides to bright light. Samples may be stored for months at room temperature prior to scoring at this stage.</li>
 <li>Stain slides by immersing in Sybr green (1X diluted in PBS) for 20 min in the refrigerator.</li>
 <li>Remove slides and allow them to dry completely in the dark. The agarose will become transparent when completely dry.</li>
</ol>
4. Image Acquisition and Analysis
 <ol>
 <li>Acquire images using fluorescent microscope set to the green filter (Zeiss AxioVision) and analyze using Comet Assay software (Perceptive Instruments).</li>
 <li>Click on the "comet head" (the nucleus) and the software calculates the parameters including the mean tail moment and amount of DNA in the nucleus.</li>
 <li>Analyze at least 200 cells per treatment.</li>
 <li>Export data to Microsoft Excel.</li>
 <li>Calculate mean tail moment for each treatment group and plot data as appropriate.</li>
 </ol>
 5. Representative Results
 An example of comet assay analysis on neuronal cells is shown in Figure 2 and Figure 3. In this case, irradiation of the neuronal cells induces DNA damage. As the cells are subjected to the electrical field, the DNA migrates at different rates due to differences in size which is subsequently analyzed using the Comet Assay software. The more the DNA damage, the farther the DNA migrates out of the nucleus. This decreases the fluorescent intensity in the nucleus which is subsequently picked up by the software and results in higher tail moment. Table 1 depicts a representative table from comet assay analysis and Figure 4 shows a representative graph comparing DNA damage in neuronal cells following radiation as measured by the comet assay.
 <img src="/files/ftp_upload/50049/50049fig1.jpg" alt="Figure 1"/> 
Figure 1. Flow chart of the comet assay. <a href="https://www.jove.com/files/ftp_upload/50049/50049fig1large.jpg" target="_blank"> Click here to view larger figure</a>.
 <img src="/files/ftp_upload/50049/50049fig2.jpg" alt="Figure 2"/> 
Figure 2. Representative images of neuronal cells (A) without and (B) with comet tail.
 <img src="/files/ftp_upload/50049/50049fig3.jpg" alt="Figure 3"/> 
Figure 3. Comet assay analysis using Comet Assay software. (A) Representative screen shots of image acquired using Carl Zeiss fluorescent microscope and analyzed using Comet Assay software. (B) Clicking on the nucleus or the "comet head" (circled in green) generates a fluorescent map and graph (circled in yellow) and a (C) data table (circled in red). <a href="https://www.jove.com/files/ftp_upload/50049/50049fig3large.jpg" target="_blank"> Click here to view larger figure</a>.
 <img src="/files/ftp_upload/50049/50049fig4.jpg" alt="Figure 4"/> 
Figure 4. Representative graph obtained by plotting the mean tail moment obtained by analyzing DNA damage in irradiated and non-irradiated neuronal cells using neutral comet assay. As expected, 3Gy radiation (x-ray) induces DNA damage as depicted by the higher mean tail moment in the irradiated neuronal cells. Shown is the mean tail moment (+/- Standard error), **P&lt;0.01.
 <img src="/files/ftp_upload/50049/50049table1.jpg" alt="Table 1"/> 
Table 1. Representative table obtained by comet assay analysis on irradiated and non-irradiated neuronal cells. <a href="https://www.jove.com/files/ftp_upload/50049/50049table1large.jpg" target="_blank"> Click here to view larger figure</a>.

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No conflicts of interest declared.

The comet assay has the unique capacity of analyzing individual cells. This is advantageous in identification of subpopulations of cells that demonstrate differential response to cytotoxic agents. A few practical limitations have to be taken into account. The number of cells that can be evaluated individually may vary depending on the individual. The sample size needs to be increased if there is variance in DNA damage within a population. Viable single-cell suspension is critical for this assay since predominant presence...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalog number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>1.5 ml tubes</td>
 <td>Santa Cruz Biotechnology, Inc</td>
 <td>Sc-200271</td>
 <td></td>
 </tr>
 <tr>
 <td>10X TBE (Tris base, boric acid, EDTA)</td>
 <td>Fisher Scientific</td>
 <td>BP13331</td>
 <td></td>
 </tr>
 <tr>
 <td>15 ml tube</td>
 <td>Fisher Scientific</td>
 <td>0553851</td>
 <td></td>
 </tr>
 <tr>
 <td>Agarose</td>
 <td>Sigma</td>
 <td>A5093</td>
 <td></td>
 </tr>
 <tr>
 <td>Aluminum foil</td>
 <td>Fisher Scientific</td>
 <td>01213101</td>
 <td></td>
 </tr>
 <tr>
 <td>Beaker</td>
 <td>Fisher Scientific</td>
 <td>FB102300</td>
 <td></td>
 </tr>
 <tr>
 <td>Centrifuge</td>
 <td>Thermo Scientific</td>
 <td>75004261PR</td>
 <td></td>
 </tr>
 <tr>
 <td>Comet Assay software</td>
 <td>Comet Assay IV Image Analysis System</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Cylinder</td>
 <td>Fisher Scientific</td>
 <td>08555F</td>
 <td></td>
 </tr>
 <tr>
 <td>EDTA</td>
 <td>GIBCO</td>
 <td>15575</td>
 <td></td>
 </tr>
 <tr>
 <td>Electrophoresis chamber</td>
 <td>Thermo Scientific</td>
 <td>09528101</td>
 <td></td>
 </tr>
 <tr>
 <td>Ethanol</td>
 <td>Fisher Scientific</td>
 <td>A407P4</td>
 <td></td>
 </tr>
 <tr>
 <td>Fluorescent microscope</td>
 <td>Zeiss Axio Vision</td>
 <td></td>
 <td>Any fluorescent microscope with green filter will suffice</td>
 </tr>
 <tr>
 <td>Hemacytometer</td>
 <td>Fisher Scientific</td>
 <td>0267152</td>
 <td></td>
 </tr>
 <tr>
 <td>Low melting point agarose</td>
 <td>Promega</td>
 <td>V2111</td>
 <td></td>
 </tr>
 <tr>
 <td>Microwave</td>
 <td>Sears</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Phosphate buffered saline, calcium free, magnesium free</td>
 <td>HyClone</td>
 <td>SH3025601</td>
 <td></td>
 </tr>
 <tr>
 <td>Power supply</td>
 <td>BioRad</td>
 <td>1645050</td>
 <td></td>
 </tr>
 <tr>
 <td>Refrigerator</td>
 <td>Sears</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Ruler</td>
 <td>Staples</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Slide</td>
 <td>Fisher Scientific</td>
 <td>12550143</td>
 <td></td>
 </tr>
 <tr>
 <td>Sodium chloride</td>
 <td>Sigma</td>
 <td>S7653</td>
 <td></td>
 </tr>
 <tr>
 <td>Sodium hydroxide</td>
 <td>Sigma</td>
 <td>S5881</td>
 <td></td>
 </tr>
 <tr>
 <td>Sodium lauryl sarcosinate</td>
 <td>Fisher Scientific</td>
 <td>S529</td>
 <td></td>
 </tr>
 <tr>
 <td>Sybr green</td>
 <td>Invitrogen</td>
 <td>S7585</td>
 <td></td>
 </tr>
 <tr>
 <td>Tray</td>
 <td>Fisher Scientific</td>
 <td>15242B</td>
 <td></td>
 </tr>
 <tr>
 <td>Tris Base</td>
 <td>Sigma</td>
 <td>T6066</td>
 <td></td>
 </tr>
 <tr>
 <td>Triton-X 100</td>
 <td>Sigma</td>
 <td>T8787</td>
 <td></td>
 </tr>
 <tr>
 <td>Trypsin</td>
 <td>HyClone</td>
 <td>SH3023601</td>
 <td></td>
 </tr>
 <tr>
 <td>Vortex</td>
 <td>Fisher Scientific</td>
 <td>02216108</td>
 <td></td>
 </tr>
 <tr>
 <td>Water bath</td>
 <td>Fisher Scientific</td>
 <td>154622Q</td>
 <td></td>
 </tr>
 </tbody>
 </table>

assaying dna damage in hippocampal neurons using the comet assay

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been extensively evaluated. Traditional methods are time consuming, expensive, resource intensive and require replicating cells. In contrast, the comet assay, a single cell gel electrophoresis assay, is a faster, non-invasive, inexpensive, direct and sensitive measure of DNA damage and repair. All forms of DNA damage as well as DNA repair can be visualized at the single cell level using this powerful technique.
	The principle underlying the comet assay is that intact DNA is highly ordered whereas DNA damage disrupts this organization. The damaged DNA seeps into the agarose matrix and when subjected to an electric field, the negatively charged DNA migrates towards the cathode which is positively charged. The large undamaged DNA strands are not able to migrate far from the nucleus. DNA damage creates smaller DNA fragments which travel farther than the intact DNA. Comet Assay, an image analysis software, measures and compares the overall fluorescent intensity of the DNA in the nucleus with DNA that has migrated out of the nucleus. Fluorescent signal from the migrated DNA is proportional to DNA damage. Longer brighter DNA tail signifies increased DNA damage. Some of the parameters that are measured are tail moment which is a measure of both the amount of DNA and distribution of DNA in the tail, tail length and percentage of DNA in the tail. This assay allows to measure DNA repair as well since resolution of DNA damage signifies repair has taken place. The limit of sensitivity is approximately 50 strand breaks per diploid mammalian cell 1,2. Cells treated with any DNA damaging agents, such as etoposide, may be used as a positive control. Thus the comet assay is a quick and effective procedure to measure DNA damage.

Watch this Scientific Journal Video about Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay at JoVE.com

Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

The comet assay is an efficient way of detecting single- and double-strand breaks, including alkali-labile sites and DNA-DNA/DNA-protein cross-links on the DNA in all cells including hippocampal neurons. The method takes advantage of the differential migration of DNA in an electric field due to differences in amount of DNA damage.

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been ...

assaying-dna-damage-in-hippocampal-neurons-using-the-comet-assay

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JoVE Journal

Neuroscience

27.1K Views.  University of Alabama-Birmingham. The comet assay is an efficient way of detecting single- and double-strand breaks, including alkali-labile sites and DNA-DNA/DNA-protein cross-links on the DNA in all cells including hippocampal neurons. The method takes advantage of the differential migration of DNA in an electric field due to differences in amount of DNA damage.

Video: Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

Assaying &beta;-amyloid Toxicity using a Transgenic C. elegans Model

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant mechanism(s) of toxicity are still unclear. A&beta; is also deposited intramuscularly in Inclusion Body Myositis, a severe human myopathy. The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express human A&beta;. Depending on the tissue or timing of A&beta; expression, transgenic worms can have readily measurable phenotypes that serve as a read-out of A&beta; toxicity. For example, transgenic worms with pan-neuronal A&beta; expression have defects is associative learning (Dosanjh et al. 2009), while transgenic worms with constitutive muscle-specific expression show a progressive, age-dependent paralysis phenotype (Link, 1995; Cohen et al. 2006). One particularly useful C. elegans model employs a temperature-sensitive mutation in the mRNA surveillance system to engineer temperature-inducible muscle expression of an A&beta; transgene, resulting in a reproducible paralysis phenotype upon temperature upshift (Link et al. 2003). Treatments that counter A&beta; toxicity in this model [e.g., expression of a protective transgene (Hassan et al. 2009) or exposure to Ginkgo biloba extracts (Wu et al. 2006)] reproducibly alter the rate of paralysis induced by temperature upshift of these transgenic worms. Here we describe our protocol for measuring the rate of paralysis in this transgenic C. elegans model, with particular attention to experimental variables that can influence this measurement.

Assaying &amp;#946;-amyloid Toxicity using a Transgenic C. elegans Model

The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express the human &beta;-amyloid peptide (A&beta;). Induced expression of A&beta; in C. elegans muscle leads to a rapid, reproducible paralysis phenotype that can be used to monitor treatments that modulate A&beta; toxicity.

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant ...

Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured neurons for extended durations1-3. This is now possible through the use of vital dyes and fluorescent proteins with which cytoskeletal components, organelles, and other structures in living cells can be labeled and then visualized via dynamic fluorescence microscopy. For example, in embryonic chicken sympathetic neurons, mitochondrial movement was characterized using the vital dye rhodamine 1234. In another study, mitochondria were visualized in rat forebrain neurons by transfection of mitochondrially targeted eYFP5. However, imaging of primary neurons over minutes, hours, or even days presents a number of issues. Foremost among these are: 1) maintenance of culture conditions such as temperature, humidity, and pH during long imaging sessions; 2) a strong, stable fluorescent signal to assure both the quality of acquired images and accurate measurement of signal intensity during image analysis; and 3) limiting exposure times during image acquisition to minimize photobleaching and avoid phototoxicity.
Here, we describe a protocol that permits the observation, visualization, and analysis of mitochondrial movement in cultured hippocampal neurons with high temporal resolution and under optimal life support conditions. We have constructed an affordable stage-top incubator that provides good temperature regulation and atmospheric gas flow, and also limits the degree of media evaporation, assuring stable pH and osmolarity. This incubator is connected, via inlet and outlet hoses, to a standard tissue culture incubator, which provides constant humidity levels and an atmosphere of 5-10% CO2/air. This design offers a cost-effective alternative to significantly more expensive microscope incubators that don't necessarily assure the viability of cells over many hours or even days. To visualize mitochondria, we infect cells with a lentivirus encoding a red fluorescent protein that is targeted to the mitochondrion. This assures a strong and persistent signal, which, in conjunction with the use of a stable xenon light source, allows us to limit exposure times during image acquisition and all but precludes photobleaching and phototoxicity. Two injection ports on the top of the stage-top incubator allow the acute administration of neurotransmitters and other reagents intended to modulate mitochondrial movement. In sum, lentivirus-mediated expression of an organelle-targeted red fluorescent protein and the combination of our stage-top incubator, a conventional inverted fluorescence microscope, CCD camera, and xenon light source allow us to acquire time-lapse images of mitochondrial transport in living neurons over longer durations than those possible in studies deploying conventional vital dyes and off-the-shelf life support systems.

We describe a protocol that allows imaging of mitochondria in living neurons via fluorescence microscopy over long durations. Imaging over extended periods is accomplished through lentivirus-mediated expression of a mitochondrially targeted fluorescent protein and use of an inexpensive stage-top incubator that was designed and built in our laboratory.

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured ...

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual neurons, researchers are able to analyze properties related to cellular trafficking, cellular structure and individual protein localization using a variety of biochemical techniques. Results from such experiments are critical for testing theories addressing the neural basis of memory and learning. However, unambiguous results from these forms of experiments are predicated on the ability to grow neuronal cultures with minimum contamination by other brain cell types. In this protocol, we use specific media designed for neuron growth and careful dissection of embryonic hippocampal tissue to optimize growth of healthy neurons while minimizing contaminating cell types (i.e. astrocytes). Embryonic mouse hippocampal tissue can be more difficult to isolate than similar rodent tissue due to the size of the sample for dissection. We show detailed dissection techniques of hippocampus from embryonic day 19 (E19) mouse pups. Once hippocampal tissue is isolated, gentle dissociation of neuronal cells is achieved with a dilute concentration of trypsin and mechanical disruption designed to separate cells from connective tissue while providing minimum damage to individual cells. A detailed description of how to prepare pipettes to be used in the disruption is included. Optimal plating densities are provided for immuno-fluorescence protocols to maximize successful cell culture. The protocol provides a fast (approximately 2 hr) and efficient technique for the culture of neuronal cells from mouse hippocampal tissue.

We provide a protocol for the culture of highly purified hippocampal neurons from prenatal mouse brains without the use of a feeder glial cell layer.

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual ...

Calcium Phosphate Transfection of Primary Hippocampal Neurons

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this method on hard-to-transfect cells like primary neurons. Here we describe our detailed protocol for calcium phosphate transfection of hippocampal neurons cocultured with astroglial cells.

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this ...

Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. Technological advances have identified these structures as key elements in neuron connectivity and synaptic plasticity. The quantitative analysis of spine morphology using light microscopy remains an essential problem due to technical limitations associated with light's intrinsic refraction limit. Dendritic spines can be readily identified by confocal laser-scanning fluorescence microscopy. However, measuring subtle changes in the shape and size of spines is difficult because spine dimensions other than length are usually smaller than conventional optical resolution fixed by light microscopy's theoretical resolution limit of 200 nm.
Several recently developed super resolution techniques have been used to image cellular structures smaller than the 200 nm, including dendritic spines. These techniques are based on classical far-field operations and therefore allow the use of existing sample preparation methods and to image beyond the surface of a specimen. Described here is a working protocol to apply super resolution structured illumination microscopy (SIM) to the imaging of dendritic spines in primary hippocampal neuron cultures. Possible applications of SIM overlap with those of confocal microscopy. However, the two techniques present different applicability. SIM offers higher effective lateral resolution, while confocal microscopy, due to the usage of a physical pinhole, achieves resolution improvement at the expense of removal of out of focus light. In this protocol, primary neurons are cultured on glass coverslips using a standard protocol, transfected with DNA plasmids encoding fluorescent proteins and imaged using SIM. The whole protocol described herein takes approximately 2 weeks, because dendritic spines are imaged after 16-17 days in vitro, when dendritic development is optimal. After completion of the protocol, dendritic spines can be reconstructed in 3D from series of SIM image stacks using specialized software.

This article describes a working protocol to image dendritic spines from hippocampal neurons in vitro using Structured Illumination Microscopy (SIM). Super-resolution microscopy using SIM provides image resolution significantly beyond the light diffraction limit in all three spatial dimensions, allowing the imaging of individual dendritic spines with improved detail.

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. ...

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible are badly known. Excitotoxicity, a process believed to contribute to neuron damage induced by both insults, is mediated by activation of glutamate receptors that promotes Ca2+ influx and mitochondrial Ca2+ overload. A substantial change in intracellular Ca2+ homeostasis or remodeling of intracellular Ca2+ homeostasis may favor neuron damage in old neurons. For investigating Ca2+ remodeling in aging we have used live cell imaging in long-term cultures of rat hippocampal neurons that resemble in some aspects aged neurons in vivo. For this end, hippocampal cells are, in first place, freshly dispersed from new born rat hippocampi and plated on poli-D-lysine coated, glass coverslips. Then cultures are kept in controlled media for several days or several weeks for investigating young and old neurons, respectively. Second, cultured neurons are loaded with fura2 and subjected to measurements of cytosolic Ca2+ concentration using digital fluorescence ratio imaging. Third, cultured neurons are transfected with plasmids expressing a tandem of low-affinity aequorin and GFP targeted to mitochondria. After 24 hr, aequorin inside cells is reconstituted with coelenterazine and neurons are subjected to bioluminescence imaging for monitoring of mitochondrial Ca2+ concentration. This three-step procedure allows the monitoring of cytosolic and mitochondrial Ca2+ responses to relevant stimuli as for example the glutamate receptor agonist NMDA and compare whether these and other responses are influenced by aging. This procedure may yield new insights as to how aging influence cytosolic and mitochondrial Ca2+ responses to selected stimuli as well as the testing of selected drugs aimed at preventing neuron cell death in age-related diseases.

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Intracellular Ca2+ remodeling in aging may contribute to excitotoxicity and neuron damage, processes mediated by Ca2+ overload. We aimed at investigating Ca2+ remodeling in the aging brain using fluorescence and bioluminescence imaging of cytosolic and mitochondrial Ca2+ in long-term cultures of rat hippocampal neurons, a model of neuronal aging.

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible ...

Stripe Assay to Study the Attractive or Repulsive Activity of a Protein Substrate Using Dissociated Hippocampal Neurons

Growing axons develop a highly motile structure at their tip, termed the growth cone. The growth cone contacts extracellular environmental cues to navigate axonal growth. Netrin, slit, semaphorin, and ephrins are known guidance molecules that can attract or repel axons upon binding to receptors and co-receptors on the axon. The activated receptors initiate various signaling molecules in the growth cone that alter the structure and movement of the neuron. Here, we describe the detailed protocol for a stripe assay to assess the ability of a guidance molecule to attract or repel neurons. In this method, dissociated hippocampal neurons from E15.5 mice are cultured on laminin-coated dishes processed with alternating stripes of ectodomain of fibronectin and leucine-rich transmembrane protein-2 (FLRT2) and control immunoglobulin G (IgG) fragment crystallizable region (Fc) protein. Both axons and cell bodies were strongly repelled from the FLRT2-coated stripe regions after 24 h of culture. Immunostaining with tau1 showed that ~90% of the neurons were distributed on the Fc-coated stripes compared to the FLRT2-Fc-coated stripes (~10%). This result indicates that FLRT2 has a strong repulsive effect on these neurons. This powerful method is applicable not only for primary cultured neurons but also for a variety of other cells, such as neuroblasts.

Axon guidance molecules regulate neuronal migration and targeted growth-cone navigation. We present a powerful method, the stripe assay, to assess the ability of guidance molecules to attract or repulse neurons. In this protocol, we demonstrate the stripe assay by showing FLRT2's ability to repel cultured hippocampal neurons.

Growing axons develop a highly motile structure at their tip, termed the growth cone. The growth cone contacts extracellular environmental cues to ...

Assessment of Hippocampal Dendritic Complexity in Aged Mice Using the Golgi-Cox Method

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching variations of the neuronal dendrites within the hippocampus are implicated in cognition and memory formation. There are several approaches to Golgi staining, all of which have been useful for determining the morphological characteristics of dendritic arbors and produce a clear background. The present Golgi-Cox method, (a slight variation of the protocol that is provided with a commercial Golgi staining kit), was designed to assess how a relatively low dose of the chemotherapeutic drug 5-flurouracil (5-Fu) would affect dendritic morphology, the number of spines, and the complexity of arborization within the hippocampus. The 5-Fu significantly modulated the dendritic complexity and decreased the spine density throughout the hippocampus in a region-specific manner. The data presented show that the Golgi staining method effectively stained the mature neurons in the CA1, the CA3, and the dentate gyrus (DG) of the hippocampus. This protocol reports the details for each step so that other researchers can reliably stain tissue throughout the brain with high quality results and minimal troubleshooting.

Here we present a Golgi-Cox protocol in extensive detail. This reliable tissue stain method allows for a high-quality assessment of the cytoarchitecture in the hippocampus, and throughout the entire brain, with minimal troubleshooting.

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching ...

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic transmission during repetitive nerve firing. Impaired endocytosis in pathological conditions leads to decreases in synaptic strength and brain functions. Here, we describe methods used to measure synaptic vesicle endocytosis at the mammalian hippocampal synapse in neuronal culture. We monitored synaptic vesicle protein endocytosis by fusing a synaptic vesicular membrane protein, including synaptophysin and VAMP2/synaptobrevin, at the vesicular lumenal side, with pHluorin, a pH-sensitive green fluorescent protein that increases its fluorescence intensity as the pH increases. During exocytosis, vesicular lumen pH increases, whereas during endocytosis vesicular lumen pH is re-acidified. Thus, an increase of pHluorin fluorescence intensity indicates fusion, whereas a decrease indicates endocytosis of the labelled synaptic vesicle protein. In addition to using the pHluorin imaging method to record endocytosis, we monitored vesicular membrane endocytosis by electron microscopy (EM) measurements of Horseradish peroxidase (HRP) uptake by vesicles. Finally, we monitored the formation of nerve terminal membrane pits at various times after high potassium-induced depolarization. The time course of HRP uptake and membrane pit formation indicates the time course of endocytosis.

Synaptic vesicle endocytosis is detected by light microscopy of pHluorin fused with synaptic vesicle protein and by electron microscopy of vesicle uptake.

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic ...

Assaying Circuit Specific Regulation of Adult Hippocampal Neural Precursor Cells

Adult neurogenesis is a dynamic process by which newly activated neural stem cells (NSCs) in the subgranular zone (SGZ) of the dentate gyrus (DG) generate new neurons, which integrate into an existing neural circuit and contribute to specific hippocampal functions. Importantly, adult neurogenesis is highly susceptible to environmental stimuli, which allows for activity-dependent regulation of various cognitive functions. A vast range of neural circuits from various brain regions orchestrates these complex cognitive functions. It is therefore important to understand how specific neural circuits regulate adult neurogenesis. Here, we describe a protocol to manipulate neural circuit activity using designer receptor exclusively activated by designer drugs (DREADDs) technology that regulates NSCs and newborn progeny in rodents. This comprehensive protocol includes stereotaxic injection of viral particles, chemogenetic stimulation of specific neural circuits, thymidine analog administration, tissue processing, immunofluorescence labeling, confocal imaging, and imaging analysis of various stages of neural precursor cells. This protocol provides detailed instructions on antigen retrieval techniques used to visualize NSCs and their progeny and describes a simple, yet effective way to modulate brain circuits using clozapine N-oxide (CNO) or CNO-containing drinking water and DREADDs-expressing viruses. The strength of this protocol lies in its adaptability to study a diverse range of neural circuits that influence adult neurogenesis derived from NSCs.

The goal of this protocol is to describe an approach for analyzing behavior of adult neural stem/progenitor cells in response to chemogenetic manipulation of a specific local neural circuit.

Adult neurogenesis is a dynamic process by which newly activated neural stem cells (NSCs) in the subgranular zone (SGZ) of the dentate gyrus (DG) generate ...