Here we demonstrate a high content receptor trafficking assay that is compatible with primary neuronal culture preparation. This method separately labels the surface an inter cellar receptor pools of fixed neurons. Enabling presentation of data as a ratio of normalized surface or internalized receptor density to the overall density of that receptor.
The high content and receptor trafficking assay provides an effected means for measuring bulk changes in receptors trafficking profiles within a neural network in response to various factors. Consuming much less time and materials than alternative methods. Disruptions and in preceptors trafficking have been linked to multiple neurological disorders, and are considered an attractive targets for drug therapy.
For examples studies have shown one of the earliest signs of Alzheimer's Disease is synaptic loss and diminished synaptic and preceptor pools. This technique requires many different steps of varying difficulty. Particularly because handling the 96 well plate and manipulating the wells can prove difficult for someone without experience.
And because the results of the experiment are highly sensitive to improper handling, it is useful to view the various steps being performed. Instructor To begin, feed the neurons in the 96 well plate by removing 100 microliters of the pre-existing media from each well. And replacing it with 100 microliters of media pre-warmed to 37 degrees Celsius.
Every three to four days. A day in vitro 14, use a multi-pipet to remove 100 microliters of media from each well and pool the media. Add two microliters of TTX Stock solution to one milliliter of the pooled conditioned media to create a four micromolar solution of TTX.
Treat the neurons in each well with 100 microliters of four micromolar TTX solution. If there is not enough pooled condition media add some of the previously stored media. Incubate in a 5%CO2 incubator at 37 degrees Celsius for four hours.
After this remove the existing TTX containing media and add 200 microliters of pre-warmed neuronal media, and incubate the micro plate at room temperature for fifteen minutes. First remove the neuronal media from the micro plate. Add fifty microliters of the anti gluA1 or anti gluA2 antibody solution to the corresponding wells of the micro plate.
Add fifty microliters of neuronal media to the secondary antibody only control wells. Incubate at room temperature for twenty minutes to allow for antibody binding. After this remove the existing media from wells.
Wash the micro plate three times with 100 microliters of room temperature neuronal media per well to remove any unbound antibodies. Then add 100 microliters 100 micromolar of DHPG stock solution to each well. Incubate in a 5%carbon dioxide incubator at 37 degrees Celsius for ten minutes.
Next remove the DHPG solution and add 100 microliters of neuronal media to each well. Repeat this process a second time. Then incubate in the 5%carbon dioxide incubator at 37 degrees Celsius for five minutes.
On the day of the experiment prepare a solution of 4%paraformaldehyde and 4%sucrose NPBS. Remove the media from each well and replace it with 100 microliters of the paraformaldehyde and sucrose solution. Repeat this process for each addition time point.
Incubate the micro plate at 4 degrees Celsius for twenty minutes. Then remove the fixative and add 100 microliters of DPBS to each well. Remove the DPBS and add 150 microliters of blocking buffer to each well.
Incubate at room temperature for ninety minutes. Remove the blocking buffer and add 50 microliters of the secondary antibody solution to each well. Incubate at room temperature for sixty minutes while keeping the plate protected from light.
Remove the antibody solution and add 100 microliters of TBS to each well. Incubate at room temperature for five minutes. Repeat this process, removing the media from the wells adding TBS, and incubating at room temperature four additional times.
After this, remove the TBS from the micro plate. Add 100 microliters of a solution of 4%paraformaldehyde and 4%sucrose in PBS to each well. Incubate at room temperature for fifteen minutes.
Remove the paraformaldehyde solution and add 100 microliters of TBS in each well, and incubate at room temperature for five minutes. Repeat this step two additional times. First prepare a solution of TBS containing 2%saponin and vortex the solution briefly to fully dissolve the saponin powder.
Using a 2 micrometer filter, filter the solution to remove any particles that could cause autofluorescence. Add 150 microliters of this 2%saponin solution to each well, and incubate at room temperature for fifteen minutes. Then remove the saponin solution, and add 150 microliters of blocking buffer to each well.
Incubate at room temperature for ninety minutes. After this remove the blocking buffer and add fifty microliters of the secondary antibody solution to each well. Incubate at room temperature for sixty minutes.
Then add 100 microliters of TBS to each well and incubate at room temperature for five minutes. Repeat this process adding TBS and incubating at room temperature four additional times. Using an infrared laser imaging system image the 96 well micro plate according the manufacturers instructions.
Set the scan resolution to 84 micrometers. The scan quality to medium and the focus offset according the base height of the 96 well micro plate used. Click on the Image Studio menu button, export image for digital media, and export the images with a resolution of 300 dpi in TIFF format.
Open the image in ImageJ Fiji. Split the color channels by clicking on the image menu and then color, split channels. Then open the ROI manager from analyze, tools.
Check the box for labels in the ROI manager, in order to classify the circles with numbers. In the 6 80, or red channel, select the region of interest by selecting the circle tool and drawing a circle that accurately fits the first well. Then press Ctrl plus T, dragging the circle to the next well.
Repeat this process until all the wells are circled. From the ROI manager click measure. Select the values that appear and copy them to a spread sheet.
Next click on the green channel and transpose the selected ROIs to the image. From the ROI manager click measure. Select the values that appear and copy them to a spread sheet.
After this calculate the changes the surface receptor expression as outlined in the text protocol. In this procedure the persistence of arc in regulating ampa receptor trafficking is examined using the high content ampa receptor trafficking and assay. Neurons are treated with the sodium-ion channel blocker TTX which inhibits action potentials and reduces arc levels.
Followed by DHPG which induces arc translation and ubiquitination. Both surface and internalize pools of gluA1 and gluA2 containing apa receptor subunits were measured at five and fifteen minutes after DHPG washout. ArcKR neurons show increased gluA1 endositosis when treated with DHPG, compared to while type neurons.
This effect is not seen when neurons are treated with TTX only. Surface expression of the gluA2 subunit is increased significantly at short time points compared to wile type neurons. Indicating a potential subunit replacement.
Insure that the cells are illiquid fixed paraformaldehyde should be prepared fresh on the day of the experiment. Cells must be re fixed after labeling for the surface receptors. Other methods such as confocal microscopy will be able to provide single celled resolution to further characterize related changed in neuronal structure and receptor localization.
Disrupting the temporal dynamics of arc alters ampa receptors trafficking in response to MgluR mediated LTD. Future directions will be to measure ampa receptors trafficking in response to other stimuli. This assay maybe adaptable to other cell types, treatments and receptors, provided that the procedure is carefully adjusted and appropriately validated.
Care must be taken to ensure that cell densities, treatment times, fixations steps, permeabilization steps, and reagent selections are optimized your system of interest. For successful and efficient completion of the assay it's important to prepare the DHPG solution and the 4%paraformaldehyde, 4%sucrose solution on the day of the experiment. Control well treated with vakeel or TTX are required to ensure that the effects observed are specific to treatment.
It's also critical to include wells treated with only the secondary antibodies to control for background fluorescence produced by non specific binding.
