The overall goal of this procedure is to visualize mitochondrial transport and cultured neurons over long durations using live cell fluorescence microscopy. This is accomplished by first labeling mitochondria in cultured neurons with an organ L targeted fluorescent protein. The second step of the procedure is to acquire images of mitochondrial transport under control conditions.
The third step of the procedure is to acquire time-lapse image series of mitochondrial transport after acute administration of neuromodulators such as serotonin. The final step of the procedure is to perform an analysis of the acquired time-lapse image series. Ultimately, results can be obtained that show that neurotransmitters modulate mitochondrial transport.
Such results are achieved only by imaging fluorescently labeled mitochondria in neurons over long durations. The main advantage of this method over existing techniques such as vital dye labeling or transient transfection of GFP, is that this method makes it feasible to perform long-term observations of mitochondrial transport in healthy cultured neurons. Demonstrating this procedure will be Chen, an associate fellow from my laboratory, In order to provide a stably heated and humidified environment.
The stage top incubator is connected to a standard tissue culture incubator via a closed circuit, A-P-F-T-E membrane lid for 35 millimeter glass Bottom culture dishes allows for gas exchange between the neuronal culture and the environment within the incubator. Together, these and other features allow for the long-term observation of cultured neurons under various experimental conditions. Transfer the culture dish containing cultured hippocampal neurons, expressing the mito turbo red protein to a tissue culture hood.
Replace the plastic lid with the PFTE membrane lid and move the culture dish to the microscope. Clip the base of the incubator to the microscope stage, and then carefully seat to the membrane covered culture dish. In the recessed opening, align the incubator enclosure with the steel posts on the incubator base and lower the enclosure onto the base.
Tighten the thumb screws until a good seal is achieved between enclosure and base. Turn on the microscope filter wheel controller, light source, and CCD camera prior to opening the imaging software. In this protocol slide book five is used for image acquisition.
Switch to a 63 x oil immersion objective and lower the objective turret. Carefully apply immersion oil to the surface of the objective. Click the focus window icon on the toolbar and turn on the brightfield lamp by moving the lamp slider.
To adjust the light intensity acquire focus on a field of cells using the oculars and find a cell of interest. Select the XI three filter under the scope tab using the oculars, find the plane of focus for mitochondria in the cells that have been labeled with the mito turbo red protein, and then switch to the CCD camera. Click on the image capture icon on the toolbar, starting with an initial exposure time of 100 milliseconds.
Find the optimum exposure by using the test and find best buttons in the capture window. Under capture type box. Check the time-lapse option and select the desired imaging interval and duration of imaging session under time-lapse capture options.
Start time-lapse image acquisition by clicking the start button at the bottom right side of the capture window. After capturing a control imaging session, administer neurotransmitters or other reagents through the injection ports on the top of the stage top incubator. Upon the addition of serotonin receptor agonist eight O-H-D-P-A-T-A previously stationary mitochondria indicated by the arrowhead moves along the axon of the hippocampal neuron.
The mean speed and directionality of individual mitochondria is summarized before the addition of serotonin. The majority of mitochondria are stationary, however, after the addition of serotonin, 42%of the mitochondria now exhibit directional movement and travel further distances than those exhibiting directional movement in the absence of serotonin. Conversely, upon the addition of dopamine, 92%of mitochondria are stationary by the last 15 minutes of an imaging session Following this procedure.
Other methods for monitoring more than one signal can be performed in order to answer additional questions. For example, how calcium activity and mitochondrial transport in neurons are related.
