The overall goal of the following experiment is to visualize neuronal receptors as they're inserted into the plasma membrane in cultured primary mouse neurons. To do this mouse GL are obtained from the cerebral cortex of embryonic or neonatal mice and grown on semi-permeable collagen coated culture inserts. The glial cultures are suspended over primary embryonic hippocampal neurons on cover slips.
Then following seven days of conditioning, the neurons are transfected with a super ecliptic fluorine tagged dopamine D two receptor. The tagged receptor localizes to intracellular vesicles, but remains non fluorescent within the vesicles acidic environment. When the vesicle merges with the plasma membrane, however, the Florin is exposed to the neutral pH of the extracellular environment and fluoresces brightly enabling detection by total internal reflection fluorescence microscopy.
This method can help answer key questions in the membrane protein trafficking field, enabling us to visualize trafficking of any membrane proteins that can be labeled with fluorine, such as glutamate receptors and gpro coupled receptors. Demonstrating the procedure together with me today will be breeding dry and, and we one, two other members of our research group The day before the dissection procedure, T 75 flasks with collagen solution, set the flasks upright and allow them to dry overnight. On the day of glial culture, warm the glial medium for at least 30 minutes in a 37 degrees Celsius water bath following euthanization, dissect the cerebral cortices of embryonic or neonatal mice under a dissection microscope.
Place the removed cortices in a 15 milliliter conical tube filled with ice, cold, artificial cerebral spinal fluid, or A CSF. Next, remove all but two milliliters of the A CSF and add 100 microliters of pepane solution and 20 microliters of 1%DNAs.One. Place the tube in a 37 degrees Celsius water bath and allow the tissue to digest for 20 minutes.
Following the digestion carefully wash the brain tissues by carefully removing the propane solution and adding 10 milliliters of prewarm glial medium. Then taking care to avoid disturbing the tissue. Remove as much of the glial medium as possible.
Add two milliliters of fresh glial medium, and using a fire polished glass past pipette tri rate until the tissue is no longer visible. Avoid forming bubbles once the tissue is completely dissociated. Add an additional eight milliliters of fresh glial medium and pipette the suspension through a 70 micron cell strainer.
Collect the flow through in a 50 milliliter conical tube. Transfer the dissociated cells from two to three embryos into a collagen coated T 75 flask and culture at 37 degrees Celsius in a tissue culture incubator overnight the next day aspirate the medium. Rinse the cells once with 10 milliliters of PBS.
Then add 10 to 15 milliliters of fresh glial medium to each flask. Incubate the cultures for 10 to 12 days, changing the medium every three to four days. Once confluent, rinse the glial cells with PBS.
Then add five millimeters of 0.05%trips into the flask and allow the cells to detach for five minutes at 37 degrees Celsius. Following the short incubation, add glial medium and pipette to detach the GL from the bottom of the flask. Split the cells into two new collagen coated T 75 flasks.
Once these reach confluence tryps anize as before and seed the contents of each flask into six well plates containing collagen coated culture inserts. Placed over two milliliters of glial medium culture, the cells for one to seven days. These glial culture inserts will be used to condition the neuronal cultures.
Add four milliliters of plating medium to each well of a six well plate containing cleaned polylysine coated cover slips and place the plate in a 37 degrees Celsius incubator overnight. This step increases the likelihood of neuronal survival, most likely by removing residual polylysine the following day. Remove the plating medium and replace it with two milliliters of fresh plating.
Medium containing 2%of the medium. Supplement B 27 for culture of hippocampal neurons Under a dissecting microscope, dissect the hippo campi from the brain tissue of four E 15 to 18 embryonic mice. Place the hippo campi in a 15 milliliter conical tube with ice cold dissection buffer.
Next, dissect the striatum from the brain tissue of three E 15 to 18 embryonic mice and place them in a separate 15 milliliter conical tube with ice cold dissection buffer. Digest the tissue with pepane as before. Then using a hemo cytometer.
Determine the number of cells in each neuronal suspension and seed. Each cover slip with 0.4 times 10 to the sixth cells and culture them in a 37 degrees Celsius incubator overnight. To prepare the glial culture inserts for conditioning the neuronal cultures, aspirate the medium from the glial culture inserts and plate, and replace each with two milliliters of fresh plating.
Medium the next day place the cover slips with the neurons in each well of the dish underneath the glial culture inserts. Continue incubating at 37 degrees Celsius every three to four days. Replace half of the medium with fresh pre warmed feeding, medium containing B 27 until they're ready for transfection and imaging experiments.
Neurons will be transfected at day seven and imaged at day eight of culture. Consistent neuronal culture is the key to successful live imaging experiments. This figure shows mouse hippocampal neurons cultured using this protocol from 11 days in vitro to 18 days in vitro.
It is clear from the images that the neurons have developed extensive processes and that the dendritic processes are covered densely with dendritic spines. Indications that these neurons are healthy in culture for each neuronal culture. Combined two microliters of lipectomy in 2001 microgram of DNA and coding the dopamine D two receptor tagged with super ecliptic Florin.
Mix and incubate for 20 minutes at room temperature. During the incubation, remove and transfer one milliliter of the medium from underneath and one milliliter from inside each glial culture insert to the same conical tube. Then add the same volume of feeding medium with B 27 to the saved medium and incubate at 37 degrees Celsius.
This medium will be used for neuronal culture following transfection after the 20 minute lip effect mean DNA incubation momentarily. Remove the glial culture inserts from the six well plates. Add the 200 microliter lipectomy DNA mixture to each cover slip with neurons.
Avoid generating bubbles under the membrane of the culture. Replace the glial culture inserts and incubate at 37 degrees Celsius for two to four hours. After the incubation, remove the glial inserts again and aspirate the transfection solution from each well then add the two milliliters of medium that was previously saved to the neurons and replace the glial culture inserts.
Finally, remove the glial medium from each insert and add two milliliters of the saved medium to each culture. The cells for an additional 24 to 72 hours to allow expression of the transfected CD NA.The TIRF imaging system used in this video is a custom set up with a 100 milliwatt cyan laser for excitation and an electron multiplying charge coupled device camera for a detector 30 minutes before the start of imaging, warm A CSF to 37 degrees Celsius in a water bath. Also, turn on the laser and camera and heating insert and place the heating insert on the microscope stage.
Place a cover slip with transfected neurons in a live imaging chamber and quickly add pre warmed A CSF. Place the chamber on the heating insert on the microscope stage under epi fluorescent imaging mode, identify the transfected neurons. Then under TIRF imaging mode, photobleach the neurons for one minute to eliminate any preexisting fluorine fluorescence on the plasma membrane.
Next, set the gain setting of the electron multiplier on the E-M-C-C-D camera to maximum and to begin taking images for one minute at 10 hertz. Each recording will contain 600 images once all of the imaging is complete. Analyze the data using Image J to determine the membrane insertion of dopamine D two receptor.
A mouse sal medium spiny neuron was transfected with super ecliptic. Florin tagged dopamine D two receptor PDR D two live TIRF Imaging was then performed in which 600 images were taken at a rate of one image every 100 milliseconds. This image shows the maximum intensity projection of the time-lapse recording white arrowhead indicate where individual vesicles containing pH DD two have fused with the plasma membrane.
This image shows the YT maximum intensity projection in which the XYT stack is rotated 90 degrees around the Y axis, and the maximum pixel on each X axis line is projected on a single y axis pixel. To give information about the timeline of insertion, these data demonstrate that this method can be successfully employed to examine the regulation of plasma membrane insertion of receptors in cultured mouse neurons After its development. This technique paved the way for researchers in the field of neuronal receptor trafficking to explore regulations of other neurono plasma membrane receptors, channels, and transporters.
