The overall goal of this Quantification of Filamentous Actin Punta in cultured neurons, is to introduce a useful method for studying the integrity and cognitivity of synaptic structures. This method can help address key questions in the neuroscience field about spinogenisis, synaptic plasticity, and synaptic stability. The main advantage of this technique is that it provides a detailed protocol for primary neuronal culture, following F-actin puncta identification and quantification.
When the target of achievement is unknown, it's necessary to estimate the general integrity of ratia fundantric structures. We first had the idea for this method when were studying the neuron damage induced by drugs of abuse like cocaine, and HIV toxic proteins like, tat and JP 120 Two days before setting up the culture coat 12, 35 millimeter glass bottom dishes, with two milliliters of Poly-L lysine working solution. And store the dishes in the hood overnight to maintain their sterility.
The next day, discard the coating agent, and rinse the dishes with double-distilled water. Allow the dishes to dry for one hour under the hood. Then transfer two milliliters of plating medium to each dish.
And incubate the dishes overnight at 37 degrees Celsius and five percent carbon dioxide. One the day of the culture, place the harvested brain in a petri dish containing cold HBSS. Then, securing a brain with tweezers, use the curved forceps to separate the hemispheres, and to remove the meninges.
Isolate the frontal cortices, and transfer the pieces into a sterile 15 milliliter centrifuge tube. Add two milliliters of fresh HBSS, and 20 microliters of trypsin EDTA to the tube, and incubate the neural tissues for 10 to 15 minutes at room temperature. With gentle swirling every few minutes.
At the end of the incubation, use a glass pipette to aspirate all of the old HBSS. And rinse the cortices two times with fresh HBSS. After the second wash, immerse the tissue in trypsin inhibitor.
Mix well, and let the sample sit for five minutes at room temperature. Then wash the tissues two times with fresh HBSS. Now use a glass pipette with a rubber bulb to very slowly triturate the brain pieces 10 to 15 times.
Then, using a calibrated pipette equipped with a sterile tip with a reduced diameter, very slowly triturate the tissue slurry again. When the solution is homogeneous, plate the cells at the appropriate density in the pre-coated culture dishes for an overnight incubation in the cell culture incubator. After 24 hours, replace the plating medium with freshly prepared complete growth serum-free narrow basal medium.
And return the cells to the cell culture incubator for at least 12 to 14 days. Low density plating is important for visualizing the individual neurons. And the neuron basal medium is critical in minimizing the perforation of astrocise within the cell cultures.
To label the cells for immunocytochemistry, first wash the glass bottom cell culture dish two times with PBS. Then fix the cells with four percent paraformaldehyde for 15 minutes at room temperature. Followed by two PBS washes.
Next, permeablize the cells with 0.1 percent Triton X 100 in PBS for five minutes, and label them with an F-actin specific stain at room temperature. After 20 minutes, rinse the cells two time with PBS and block any non-specific cell surface staining with 10%normal goat serum. At the end of the incubation, label the overnight cells at four degrees celcius with chicken polyclonal anti-map two antibody.
The next morning, rinse the cells two times with PBS and label them with appropriate secondary antibody. After two hours at room temperature, rinse the cells with PBS again, and label them with 10 microliters of huxs for three minutes at room temperature. Then wash the cells two final times with PBS an preserve them with 100 microliters of anti-fade re-agent.
Within three days of this staining, turn on the fluorescent microscope and select the 20 X objective. Next, in the microscope software, set the pixel image size to 1600 by 1200 and the 0.17 micron per pixel image resolution to 1 X zoom. Transfer the dish of cells onto the microscope stage.
And acquire images of the co labeled F-actin map 2 neurons under the green and red fluorescent channels. When all of the images have been obtained choose five images of individual neurons in the green, red and blue channels with clearly defined dendritic arbors. And use continuous map two immunofluorescence to identify the F-actin rich structures in the second ordered dendritic segments.
Rotate the selected region of images horizontally. And copy and paste the region as a new image. Then subtract the image background fluorescence and use the software to count the number of bright green F-actin puncta.
Use trained independent observers to measure the length of the selected dendritic segments. And then export the puncta numbers, and dendritic segment length data to a spreadsheet file. Be sure that observers include F-actin posture puncta with a pink fluorescence intensity of at least 50%above average intensity of staining in the dendritic shaft in each selected segment.
Finally, calculate the density according to the formula, expressing the data as the number of F-actin puncta per ten microns of dendrite. Here, differential interference contrast images demonstrate the morphological changes observed in developing fetal rat cortical neurons at days 4 to 27 en vitro. Note the increase in length and number of the dendrites as the cultured rat primary neurons mature.
Since phalloidin labeling of F-actin is very rapid it is possible to visually estimate the integrity of the synapto-dendritic network before proceeding with the immunocytochemical anti-body labeling. Map2 stains the intact dendrites, confirming that the F-actin puncta are located on the neuronal dendrites, and not localized to other cells, such as astrocytes. In high resolution images, F-actin rich structures containing phi filopodia, spine protrusions, and F-actin patches can be observed.
In this experiment for assessing HIV 1 tat induce synapto-dendritic injury, Map2 staining revealed fewer dendritic branches and diminished F-actin in rat cortical neurons following the HIV 1 tat treatment. Indeed, F-actin puncta increase or decrease in response to the experimental treatment. For example, in this experiment when the cultured neurons were treated uncompetitive NMDA receptor antagonist memantine, the F-actin positive puncta experienced a significant increase in density.
In contrast, treatment with combination of methamphetamine plus HIV1 tat resulted in a significant loss of the F-actin puncta. After acquiring high-resolution images of the coal labeled F-actin Map2 neurons, remember to have two trained observers select segments of the second ordered dendrites for analysis of the puncta densities. After its development this technique paved the way for researchers in the field of neuroscience research.
Not only for the monitoring of acutes synaptopathy, but also for the study of synaptic recovery and experimental neural restoration processes. After watching this video you should have a good understanding of how to develop a reliable method measuring the synaptic integrity of the neural dendritic network.
