The overall goal of this video is to describe the methodology for the construction and testing of Cell-based Neurotransmitter Fluorescent Reporters, or CNiFERs, which can be used to optically monitor the release of specific neurotransmitters in vivo. This method can help answer key questions in neuroscience concerning the timing and the release of neuromodulators in the brain. The main advantage of this technique is that it can be adapted for any type of neurotransmitter or neuromodulator, that signals through GPCS.
Regional demonstration of this method is helpful. It is the in vivo injection, and subsequent imaging of CNiFERs are technically challenging. To begin this procedure, see the HEK293 cells which contain the front based calcium detector and chimeric g protein in a T-25 flask.
Then, grow the cells in a humidified incubator, at 37 degrees Celsius, with five percent CO2, until about 50%confluent. Afterward, aspirate the media from the T-25 flask. Add two milliliters of the lenta virus media mixture, and incubate the T-25 flask at 37 degrees Celsius, with 5%CO2.
The next day, harvest the infected HEK293 cells by adding one milliliter of trypsin. Next, resuspend the cell pellet in five milliliters of HEK293 growth media. Seed one milliliter of cells in a T-75 flask for freezing and storage, and 1.5 milliliters of cells in a T-25 flask for FACS analysis.
For the 10 point agonist curve, to test the infected cells, seed the first two rows of a fibronectin coated 96 well plate with 100 microliters of the cell suspension per well. Then, incubate the HEK293 cells growing in a T-25 flask, a T-75 flask, and a 96 well plate until about 90%confluent at 37 degrees Celsius with 5%CO2 for one to two days. Before FACS analysis, determine the functional expression of the GPCR by preparing a drug plate with 10 different agonist concentrations that bracket the predicted EC 50.
Prepare different concentrations of agonist using a serial dilution method, and create a template, to keep track of the drug concentrations. Next, set the plate reader temperature to 37 degrees Celsius. Then, program the 96 well fluorometric plate reader for measuring fret, and performing solution transfers.
For measuring fret with a genetically encoded fret based Calcium sensor, TNXXL, set the excitation wavelength at 436 plus or minus 4.5 nanometers. Then, set the emission filters, and the cut off filters, for ECFP and citrine. Next, program the plate reader to measure the emissions every four seconds, for a total of 180 seconds.
Choose the options for 100 microleader plate volume, 150 microliter pipette height, and delivery of 50 micro liters of drug from the threefold compound plate. Set the time point for drug delivery at 30 seconds. After that, aspirate the media from rows A and B, and add 100 microliters of ACSF to the 96 well sniffer plate, that is about 90%confluent.
Then, load the threefold drug plate and 96 well sniffer plate into the plate reader. Allow 30 minutes to equilibrate the plates at 37 degrees Celsius, before starting the program. After the plate reader run, export the fluorescence reader values to a text file.
Then, import this file to a previously made spreadsheet template that normalizes the florescence intensities to the pre-stimulus base lines, calculates the peak fret ratio for each agonist concentration, and generates a dose response curve. Prepare the CNiFER injection pipette by putting a glass capillary on a vertical electrode puller. Use a pair of forceps to break the tip of the electrode to a diameter of approximately 40 micrometers.
Place an ACSF soaked sponge on a previously formed two by three millimeter thin skull window of an anesthetized mouse, to keep moist while preparing the cells for injection. Next, harvest the CNiFER clone that was grown in a T-75 flask to about 80%confluency. Aspirate the media, and wash the cells with sterile PBS.
Remove PBS, and use 10 milliliters of ACFS to dislodge the cells from the bottom of the flask. Then, tritcherate the cells to dissociate the cell clumps. Centrifuge for two minutes in a cell culture centrifuge.
Remove the supernatant, and resuspend the pellet in 100 microliters of ACSF. Subsequently, centrifuge for 30 seconds at 1400 times G, and remove the supernatant, leaving a pellet, covered in ACSF. Afterward, back fill the injection pipette with mineral oil.
Load the pipette onto a nano injector. Put five microliters of CNiFER cell suspension onto a strip of plastic paraffin film near the mouse preparation. Draw up the CNiFER cells into the pulled pipette.
Now, move the pipette to the target x and y coordinates. Lower the pipettes, and pierce the thinned skull, prepared previously. Continue to about 200 to 400 micrometers below the skull's surface, in layers two and three of the cortex.
Inject 4.6 nanoliters of CNiFER cells at the deepest site with the nano injector. Note movement in the oil and cell interface. And then, wait for five minutes for the cells to dispense.
Withdraw the pipette approximately 100 micrometers and inject another 4.6 nanoliters of CNiFER cells and again, wait five minutes. Afterward, withdraw the pipette slowly and gently to prevent backflow of the CNiFERs. In this procedure, place the imaging platform with the head restrained mouse under a 10x water immersion objective in a two photon imaging microscope.
Insert the filter cube for fret imaging that has a dichroic mirror at 505 nanometers and band pass filters that span 460 nanometers to 500 nanometers for measuring ECFP and 520 nanometers to 560 nanometers for measuring citrine. Then, add ACSF to the well, containing the thinned skull window and lower the 10x water immersion objective into the ACSF. Use the eye piece in conjunction with mercury lamp, and GFP filter cube to locate the CNiFERs.
Now, switch to the 40x water immersion objective. Next, select the appropriate light path for two photon imaging. Turn on the near infrared femtosecond pulse laser.
Select the wavelength of 820 nanometers, and a power setting of five to 15%Set the pmt one and pmt two voltage to a submaximal value, typically 500-1000 volts, depending on the pmt. Then, set the gain to one for each channel, and zero the z position for the objective. Lower the objective approximately 100 to 200 micrometers from the cortical surface, and start the xy scan.
Adjust the laser power, gain, and pmt voltage for each channel to optimize the signal to noise ratio of the CNiFER's florescence. Next, use the software to restrict the imaging to a region that contains the CNiFER cells, as well as a background region. Select the cowman line, averaging for two, for a suitable signal to noise ratio, and use a scan rate of 3 to one hertz at four microseconds per pixel.
After that, draw an ROI around the CNiFER cells. Surrounding about three to four cells per plane. Set up real time analysis of ROI average intensities.
Then, start acquisition to monitor the CNiFER florescence over time and begin electrical stimulation or behavioral experiment, while monitoring fret. In this example, D2R CNiFER fret's response was measured on a plate reader with a solution delivery system. This graph shows the fret response during the application of dopamine to D2 CNiFERs.
Note that ECFP emission decreases while citrine emission increases with dopamine. And here is a plot of the corresponding fret ratio. Shown here are the dose response curves for the response of D2 CNiFERs to dopamine and to norepinepherine.
In addition, the responsive control CNiFERs lacking the D2R is presented. This bar graph shows the fret ratio response for other neurotransmitters and modulators at 50 nano molar and one micro molar. Here, electrical stimulation of DA neurons in the substantia nigra, elicits a large change in the fret ratio for D2 CNiFERs.
Note how the amplitude of the response increases with an increasing intensity of electrical stimulation. Coupling stimulation with cocaine injection augments the CNiFER response. After watching this video, you should have a good understanding of how to create, test, and use CNiFERs for in vivo imaging.
Throughout the development of CNiFERs, it is essential to maintain sterile technique, because these cells will be ultimately implanted in living mice. The realization of CNiFERs provides an important tool for researches in the field of neuroscience to optically measure the release of any neurotransmitter, or neuromodulator, that signals through a G protein coupled receptor. Thank you for watching, and good luck with your experiments.
