This method can be used to facilitate the visualization of very fine astrocyte processes for the evaluation of astrocyte neuronal interactions at the synapse during disease and steady states. This technique can also be applied in any mouse model, cell population, or brain region. To prepare a sharp electrode with an appropriate resistance, pull a borosilicate glass single barrel electrode with a filament on a micropipette puller.
Store the pulled electrodes in a closed container to prevent dust from entering the tips and keep the electrodes elevated from the bottom of the box to prevent the tips from breaking. To fill an electrode with lucifer yellow dye solution, place an electrode in the vertical position with the tip facing downward, and pipette one to two microliters of lucifer yellow solution into the back of the electrode. Wait five to 10 minutes for the solution to move to the tip via capillary action before gently securing the filled electrode in an electrode holder connected to a manipulator with the silver wire of the electrode holder in contact with the lucifer yellow inside the electrode.
For the dye ejection test, gently place a lightly fixed brain slice into a glass bottom dish filled with 0.1 molar PBS at room temperature and hold the slice in place with a platinum harp with nylon strings. Connect the electrode to a voltage source and place the ground electrode into the bath containing the brain slice. Move the objective of a confocal microscope to the brain region of interest and lower the electrode into the solution using the 10X water immersion lens, moving the electrode to the center of the field of view.
Under bright-field illumination, examine the electrode carefully with the 40X water immersion lens to ensure that the electrode appears clear and without debris or bubbles. Switch to confocal laser scanning microscopy with a 488-nanometer laser. Turn on the stimulator at 12 volts to test the dye ejection.
A large fluorescent dye cloud should be visible around the tip of the electrode. Then, under bright-field slowly lower the electrode toward the slice, stopping just above the surface. To fill an astrocyte of interest with iontophoresis, use the infrared differential interference contrast to identify astrocytes with elongated oval-shaped somata about 10 micrometers in diameter, 40 to 50 micrometers below the slice surface.
This is the stratum radiatum of the hippocampus directly below the pyramidal cell layer. As we move through the tissue, we can see blood vessels, neurons, astrocytes, and other cell types. Once an astrocyte has been selected, move the cell to the center of the field of view and slowly lower the electrode tip into the slice, navigating through the tissue until the electrode is on the same plain as the cell body.
Once the cell body of the astrocyte is clearly visible and outlined, slowly and gently advance the electrode forward, moving the electrode until the tip impales the soma of the cell. Move the focus of the objective slowly up and down to note if the electrode is inside the soma. Once the electrode tip is inside the cell, turn on the stimulator at 0.5 to one volts to continuously eject current into the cell.
Using the confocal microscope, watch the cell film, increasing the digital zoom to visualize the details of the cell and to make sure that the tip of the electrode is visible inside the cell as necessary. Wait for about 15 minutes until the finer branches and processes appear defined before turning off the voltage and gently withdrawing the electrode tip from the cell. To image the filled cell, wait 15 to 20 minutes for the cell to return to its original form before adjusting the setting on the confocal microscope to make sure that the finer branches and processes appear defined under the 40X objective.
Set up a Z stack with a step size of 0.3 micrometers, moving the objective while imaging until there is no signal from the cell and set that step as the top. Then move the objective down, focusing through the cell until there is no signal and set that step as the bottom. After the imaging is complete, check the electrode for dye ejection.
If a large dye cloud appears, the electrode can be used for the next slice. Otherwise, replace the electrode. By generating a maximum intensity projection, a detailed view of the structure of an astrocyte in its domain can be observed.
A four X magnitude zoom into the astrocyte reveals a maximum intensity projection of one major branch, several secondary branches, and the distribution of the surrounding branchlets and leaflets. Here, the original image of a CA1 astrocyte is shown. The cell body, major branches, processes, and territory volume enclosed by the astrocyte are reconstructed in these subsequent images.
After the reconstructions were created, the volumes of the soma, entire cell, and territory were quantified. And the number of major branches were counted. As cytoskeletal protein that labels intermediate astrocyte filaments is expressed in the cell soma, major branches, and some secondary astrocyte branches, but not in the finer branches and processes.
In this representative analysis, no significant differences were found in the number of primary branches labeled by glial fibrillary acidic protein and those visualize by lucifer yellow. Further, the cell area and volume of the astrocyte labeled by glial fibrillary acidic protein were significantly smaller than the area and volume visualized with lucifer yellow, demonstrating that glial fibrillary acidic protein is a reliable marker for labeling major branches but is not useful for determining the overall area or volume of the cell. The amount of dye released from the electrode tip is dependent on the electrode resistance which must be high enough to allow a steady stream of dye to be ejected.
Future studies can examine the different components of astrocyte structure by super resolution light microscopy or by immunohistochemical analysis of the expression of specific proteins within the astrocyte structure.
