The overall goal of this procedure is to evaluate glucose sensing in ventral medial hypothalamic or VMH neurons from older mice, using membrane potential sensitive dye as an index of neuronal activity. This is accomplished by first performing cardiac perfusion to flush blood out of the brain of an anesthetized mouse prior to removal of the brain. The second step is to create coronal brain slices and dissect the VMH.
Next, the VMH neurons are dissociated and cultured on cover slips. The final step is to measure the fluorescence of the neurons as glucose is decreased in the presence of membrane potential dye. Ultimately, each neurons change in fluorescence is used to quantify the response of that glucose inhibited neuron to glucose changes.
The main advantage of this technique over existing methods, such as electrophysiology, is the ability to study neurons from a adult mice. This allows us to study disease states in adulthood. Begin this procedure by placing the dissected brain on the chuck in the Vibram chamber with the Vibram set to slow speed at level two and high amplitude at level nine, cut slices at 300 to 500 microns until reaching the hypothalamic area.
Then cut thin slices at 100 microns from the posterior to the anterior to isolate the precise VMH region. Pay close attention to anatomical changes at bgma minus 2.30 millimeters. The third ventricle is separated into dorsal and ventral sections.Corona.
Once the cross sections of the third ventricle fuse cut two 500 micron slices, these slices will contain the correct region of VMH. Transfer these slices to a Petri dish on ice containing culture media. Then dissect the VMH.
Use a pipette to gently transfer the VMH pieces into milliliters of culture media on ice. In this step, remove the culture media containing the VMH from ice and allow acclimation to room temperature. Add 20 units per milliliter of pape to four milliliters of culture media, then invert to mix and place in a 34 degree Celsius water bath Check and mix by inversion every minute until the digestion media is no longer cloudy.
After that, filter it into a sterile flask and transfer the tissue to the digestion media. Next, digest the tissue by shaking at 100 RPM at 34 degrees Celsius for 30 minutes. During the digestion, prepare one milliliter of media containing 8%bovine serum albumin by dissolving 80 milligrams of BSA in one milliliter of culture media, and filtering it into a conical tube.
Wash the tissue by transferring it to five milliliters of culture media in a conical tube and slowly inverting it. Once afterward, add 30 microliters of DNA's enzyme to six milliliters of culture, media, and mix to make tation media. Then aspirate the wash media and add three milliliters of the tation media.
Use the glass pipettes to tri in the order of largest to smallest gently tritrate 10 times, and then wait four minutes for larger pieces to settle. Next, use the second pipette to transfer the top two milliliters containing the dissociated cell suspension to a new conical tube. Add two milliliters of tri media tri 10 times and wait three minutes.
Use the third pipette to transfer the top two milliliters to the cell suspension. After that, add one milliliter of tri media tri rate five times and wait two minutes. Use the fourth pipette to transfer the top two milliliters to the cell suspension.
Now layer the dissociated cell suspension on top of the 8%BSA, being careful not to mix them. Then centrifuged 1000 RPM for five minutes. Next place the autoclave six by eight millimeter cloning cylinders in the center of each completely dry cover slip.
Then aspirate and resuspend the pellet in 440 microliters of warm growth media. Subsequently, fill the cloning cylinders with the neuronal suspension and place it in the incubator for 20 minutes. Remove the cloning cylinders and wash away the debris very gently.
Then fill each dish with two milliliters of growth media and allow the cells to recover for at least one hour before any assays are performed In this procedure, make the recording solution and adjust the pH to 7.4. Now add four milliliters of room temperature buffer to a blue MPD vial mix thoroughly and add five microliters of dye per milliliter of recording solution to it. After that, incubate the cells in two milliliters of 2.5 millimolar glucose recording solution with dye at 34 degrees Celsius for 30 minutes protected from light.
Then fill the syringe pumps with either 2.5 or 0.1 millimolar glucose recording solution and the profusion system with 2.5 millimolar glucose recording solution. Connect the tubing from 60 milliliter syringes to a manifold. The manifold output tubing should be connected to an inline heater, and then polyethylene tubing, which is then connected to a closed chamber.
Clear the bubbles by running the perfusion system and tapping with a hard object if necessary. Then set the perfusion rate to 0.5 milliliters per minute and use minimal light for the least time possible to set up the system. Next, transfer a 25 millimeter cover slip with adherent neurons to the closed chamber.
Being careful not to allow the cover slip to dry and not to introduce air bubbles. Align the slip in the grooves of the bottom piece. Place a couple drops of recording solution along the sides and gently fit the top piece to hold the cover slip in place.
Then use a dropper to fill the chamber with recording solution and place an 18 millimeter cover slip on top. Gently fit the white ring into the chamber to hold the smaller cover slip in place. After that, press the white ring down very slowly and lightly to prevent air bubbles from being introduced into the closed chamber.
Start the syringe pump for the 2.5 millimolar glucose recording solution. Then press down on the white ring and connect to the closed chamber. Slowly release pressure from the white ring and connect to the tubing leading to a waste container.
For MPD Imaging Center, the neurons using low bright field light. Try to minimize the amount of time the cells are being exposed to light and allow the perfusion system to run for 10 minutes for stabilization of temperature, focus and die equilibrium while priming. Open the METAMORPH program and take a bright field image of the neurons at 10 times magnification.
Use the autofocus function to take three stacks of fluorescent images and determine the focus within five microns. At the end of the 10 minute priming period, check the focus once more, then begin to record fluorescent images every 30 seconds for 40 minutes. Establish a baseline at 2.5 millimolar glucose for 10 minutes.
Then decrease glucose for 15 minutes, and finally return to 2.5 millimolar glucose for 15 minutes. After recording all fluorescent images, take a brightfield image of the neurons. This figure shows a brightfield image of the healthy VMH neurons from an adult mouse.
This image was taken 24 hours after dissociation and has been used for MPD imaging. A few examples of neurons that would or would not be used for analysis are marked shown. Here are the representative MPD fluorescence traces of a GI neuron indicated by green and a non-GI neuron indicated by orange cells were perfused with 2.5 millimolar glucose recording solution for 10 minutes, followed by a decrease to 0.1 millimolar G for 15 minutes and a return to 2.5 millimolar G for 15 minutes.
This figure shows the percentage of the adult VMH neurons, which reversibly depolarize in response to decreased glucose detected using fluorescent MPD imaging. The magnitude of the glucose change is positively correlated with the percentage of depolarizing neurons Following this procedure. Other methods, such as single cell R-T-P-C-R can be performed to identify specific proteins expressed by glucose sensing neurons.
