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The overall goal of this procedure is to image intracellular calcium signals in stri AAL astrocytes from adult mice using genetically encoded calcium indicators such as cyto G CAMP three. This is accomplished by first injecting the virus into the mouse striatum. Then the mouse is allowed to recover for two to three weeks following surgery.

Next, the mouse is sacrificed and acute brain slices are prepared. The final step is to perform confocal calcium imaging of the brain slices. Ultimately, immunohisto chemistry is used to show that the Cyto G Camp three is expressed only in astrocytes but not neurons, and that the virus injection did not cause overt astrocyte reactivity.

The main advantage of this technique over existing methods like transgenic or nochi M, is that it allows robust expression of target protein and the rapid and flexible experimental implementation. This method can help answer key questions in the as osteocyte calcium signaling field, such as how to reliably measure as osteocyte calcium signals in the branches and branchlets of stratas osteocytes. Prepare for the virus injection by autoclaving the micro pipettes and surgical instruments just before surgery.

Retrieve the prepared A A V 5G camp three viral aliquots from the negative 80 degree Celsius freezer and store them on ice. The next step is to fill the micro pipette with virus. Begin by tightly connecting one end of a piece of tubing through an appropriately sized tubing compression fitting to a glass syringe with a reinforced plunger.

Then use an appropriately sized removable needle compression fitting to attach the autoclave micro pipette to the other end of the piece of tubing. Next, using a one milliliter syringe with a 27 gauge needle, fill the syringe with mineral oil colored with Sudan red four. Then inject the mineral oil into the syringe tubing, micro pipette system to remove air bubbles.

After placing a clean piece of parfum under the glass micro pipette, dispense 10 microliters of the viral vector onto the param. Now, suck the viral vector into the micro pipette by moving the plunger of the syringe backward at 0.5 microliters per minute. When finished, mark the boundary between the vector and the oil on the glass pipette.

After anesthetizing a mouse, shave the hair on its head. After fitting the mouse in the stereotaxic frame, place the mouse's nose in an anesthesia and ventilation system and maintain continuous two to 3%iso fluorine. Also apply artificial tears ointment to both eyes.

Next, administer 0.05 milliliters of buprenorphine by subcutaneous injection for pain relief wiping from the center and working outward In a circular motion, clean the surgical area with 10%povidone iodine, followed by 70%alcohol, three times alternating between the two solutions. Next, make a skin incision on top of the head, which starts between the eyes and ends between the ears. After removing the periosteum by swiping with a cotton swab, dry the surface of the skull with dental cotton balls and make a mark around the desired injection site using a high speed drill and a small steel burr, drill around the edge of the mark on the skull.

Use saline as needed to remove the bone chips and to clean the surface of the skull. The next step is to lower the pipette containing the viral vector into the brain. Using the micro manipulator, advance the pipette to the following coordinates as measured from the pile surface positive 0.8 millimeters anterior posterior positive, 2.0 millimeters medial lateral and negative 2.4 millimeters dorsal ventral.

Note that these coordinates mean that the tip of the microinjection needle sits just above the left dorsal lateral striatum, meaning that it only slightly penetrates within this nucleus. Start the injection with the injection rate set at 0.2 microliters per minute and inject approximately one to 1.5 microliters of the virus. Leave the pipette in place after the injection for 10 minutes and then withdraw the pipette Slowly finish by closing the surgical wound with continuous external nylon sutures.

After the mouse recovers from anesthesia, place it in a clean cage on a heating pad for 24 hours and monitor its general health at least once per day. Two to weeks after the viral injection, prepare the cutting solution and recording solution as specified in the text protocol. Fill the brain slice holding beaker with recording solution and keep it at 34 degrees Celsius.

After terminally, anesthetizing and decapitating the mouse, extract the brain and use a blade to remove the unin injected right hemisphere. Next, use super glue to mount the left hemisphere onto the vibram tray. Then fill the vibram tray with ice cold cutting buffer, keeping it saturated with 95%oxygen and 5%carbon dioxide.

Now cut four to five coronal slices or six to seven sagittal sal brain slices at 300 micrometer thickness. Transfer the slices to the slice holding beaker warmed at 34 degrees Celsius for 30 minutes. Then incubate the brain slices in oxygenated recording buffer for at least 30 minutes at room temperature with the slice holding beaker covered with aluminum foil for keeping the slices away from ambient light before imaging the intracellular calcium transient between one and five hours.

After slicing, place a slice in the recording chamber under a confocal microscope super. Fuse it with oxygenated recording buffer at room temperature with a flow rate of one to two milliliters per minute. Place a platinum harp with nylon strings on top of the slice to minimize movement.

Next, locate the dorsal striatum with bright field luminescence under a 10 x water immersion objective. Switch to the 40 x water immersion objective lens and turn on the 488 nanometer line of the argon laser by changing the focal plane. Find cells located about 20 micrometers below the slice surface, displaying basal fluorescence in soma branches and branchlets.

Avoid any cells that display fluorescence level above average as they tend to be compromised cells. Use frame scan mode to start scanning. Usually the laser intensity adjusted to 0.5 to 5%of the maximum output is sufficient to image the intracellular calcium concentration with cyto G Camp three for monitoring the intracellular calcium dynamics in the whole territory of astrocytes.

Use a frame scan with a scan rate of one second per frame or faster if necessary. To image intracellular calcium in processes. Use digital magnification of two to threefold to monitor one to two astrocytes in the whole field of view.

To avoid saturated pixels during recording, use high low lookup mode to pseudo color. The cells always start with a pilot recording for about five minutes to test if a red color appears as an indication of saturation. If so, lower the laser output power or lower the PMT gain offset values, and perform another pilot recording to test the pixel values in the unsaturated range.

Typically, the combined parameters used for imaging astrocyte intracellular calcium in the satu are as follows, laser output power 0.5 to 5%of 10 milliwatts PMT 550 to 650 volts gain 2.0 to 3.5 x and offset zero to 5%localization of cyto G CAMP three expression and new NA marker for neurons show that cyto G CAMP three does not co localize with neurons. However, cyto G CAMP three expression does co localize with S 100 beta A marker for astrocytes. A summary of these COLOCALIZATION experiments shows that cyto G Camp three expression was specific to stri AAL astrocytes, a comparison of G Camp three levels and glutamine, synthetase or GS levels in virus injected mice and wild type mice are shown here.

These studies showed that there were no significant changes in glutamine synthetase expression in Sal astrocytes following virus injection indicating no overt astrocyte reactivity using glutamine synthesis levels as a metric shown here is a flattened Zack image showing 10 astrocytes. The traces show intracellular calcium signals recorded over five minutes from these 10 cells. This shows a flattened and zoomed in Z stack image for a single stri AAL astrocyte.

The traces show intracellular calcium signals recorded over five minutes from the indicated branches of the cell Once mastered, the microinjection can be done in one to two hours if it is performed properly after its development. This technique paved the way for the researchers in the field of as osteocyte calcium signaling to explore bidirectional communications between as osteocytes and neurons in Therial micro circuit.