The overall goal of this procedure is to induce long-term increase or decrease in astrocyte GQG protein coupled receptor signaling in response to changes in neuronal synaptic transmission. This is accomplished by first preparing acute hippocampal slices from wild type mice. The second step is to apply sulur Rumine 1 0 1 to the slices to label astrocytes.
Next, the acute hippocampal slices are incubated for four to six hours tetrodatoxin to block neuronal action potentials or in regular artificial cerebral spinal fluid. The final step is to load the astrocytes in the stratum radi atom of the slice with calcium indicator using a back pressure loading protocol. Ultimately, confocal microscopy is used to show changes in astrocyte GQ GPCR signaling activity by recording changes in spontaneous and evoked astrocyte calcium transient.
This method provide insight into astrocytes receptor plasticity and associated changes in TQG PCR signaling. It can also be used to measure homeostatic plasticity of metabotropic receptors in neurons. To begin this procedure, turn on the vibrato and make sure the drainage is closed.
Then secure the cutting chamber in the vibrato and pack ice around the cutting chamber. Next, remove factory grease from the double edge razor blade by soaking it in 70%ethanol for five minutes, and then rinsing with double distilled water. Cut it into halves carefully and mount one half onto the cutting block for brain slice preparation.
After obtaining a mouse brain bisected with the chilled razor blade in a Petri dish to allow more surface area for cooling and oxygenation. Let the bisected hemispheres sit in the ice cold slicing buffer for two to three minutes and become completely cool and more solid. After that, apply a thin layer of crazy glue on the platform of the vibrato glue, both hemispheres to the platform.
With the cut sides down and the lateral sides up and the olfactory bulb facing forward, then secure the platform in the cutting chamber. Fill the cutting chamber with ice cold well oxygenated slicing buffer. Continue oxygenating the cutting chamber while preparing 300 micron thick para sagittal slices using the vibrato After slicing, dissect the hippocampus and the adjacent endrin cortex out of each para sagittal slice in the ice cold well oxygenated slicing buffer.
To make a transfer pipette break off the long tip of a glass pasture pipette and top the broken part with a pipette bulb. Transfer the hippocampal slices to the incubation chamber in the 35 degree Celsius water bath one at a time. Please note that for visibility purposes, these steps are being shown with the incubation chambers outside the 35 degrees Celsius water bath.
Incubate the hippocampal slices for 20 minutes in one micromolar SR 1 0 1 diluted in low calcium A CSF in the 35 degree water bath. Then transfer them to the low calcium A CSF without SR 1 0 1 for an additional 10 minutes. Warm recovery.
Subsequently transfer the slices to the control or experimental A CSF for the remaining 15 minutes of the warm incubation. After the 45 minute recovery, carefully move the incubation chambers from the 35 degrees Celsius water bath to the benchtop and allow the slices to continue to incubate at room temperature for three hours in total before beginning the bolus loading protocol. In this procedure, prepare a pipette from a boro silicic glass capillary pulled to a resistance of approximately 1.3 mega ohm when filled with dye solution.
Next place a hippocampal slice into a recording chamber and continuously perfused with oxygenated A CSF at 1.5 milliliters per minute only use a hippocampal slice that has a high percentage of healthy CA one parametal cells and smooth appearing surface and discard if it looks unhealthy. Using differential interference contrast optics, locate a suitable field of astrocytes in the stratum radium 40 to 70 microns below the slice surface. Then place the glass pipette preloaded with D solution into a standard patch clamp micro electrode holder.
Lower it to the surface of the slice above the field with the pipette at the surface of the slice. Apply positive pressure to the pipette to begin ejecting dye. Slowly lower the pipette to approximately 40 microns below the slice surface using a micro manipulator and allow the dye to eject for approximately 45 to 60 seconds.
Then lower the pipette an additional 35 microns and eject dye for approximately 45 to 60 seconds. Afterward, slowly retract the pipette tip from the slice to ensure that a large number of astrocytes take up the dye. It is usually helpful to inject a second dye bolus a short distance away.
To do so, raise the pipette back to the surface of the slice and make sure that the pipette is not clogged. Then move the pipette approximately 80 to 100 microns away from the first injection site along the stratum radium repeat bolus injection at this site and allow 30 to 45 minutes before imaging the astrocytes to set up the confocal microscope for imaging. Limiting slice Exposure to laser light is of the utmost importance as high exposure can lead to dye bleaching and or phototoxicity.
Set the default values for each laser to a high photo multiplier setting, one x gain and 0.5%laser output power hour. Next, apply a 1.5 x zoom for better visualization of the astrocyte processes. Then set the field resolution to 512 pixels by 512 pixels.
After that, set the scan speed to the fastest possible, which is about 1.2 seconds per scan. Using the one-way scan mode, collect emission spectra using band pass filters of 503 to 548 nanometers for the 488 nanometer laser and 624 to 724 nanometers for the 559 nanometer laser. Then confirm the identity of cells loaded with calcium dye as astrocytes by visualizing the SR 1 0 1 co labeling.
Using the 559 nanometer laser to record astrocyte calcium activity. First, draw the boxes over the regions of interest within the cell using the image acquisition software in this case over astrocyte cell bodies. Then draw one box over the background as reference.
Obtain 10 minutes of baseline recording of spontaneous calcium activity from the ROIs. Afterward, apply an agonist of interest at sequentially increasing concentrations and leave a minimum of five minutes between applications to reduce possible receptor desensitization. At the end of the recording, apply a cocktail of agonists for other astrocytic GQ, GPCR as a positive control for the Inpact astrocytic GQ GPCR signaling pathways.
At the completion of the calcium recording, take still images with the 488 nanometer and 559 nanometer lasers for later confirmation of astrocyte identity and ROI placement. Here are the representative images of cells in the recording field, incubated in control conditions or in TTX that have taken up Oregon Green BAFTA 1:00 AM calcium indicator dye and SR 1 0 1. Overlay of both signals indicates the astrocytes loaded with calcium indicator boxes are drawn over individual astrocytes somas to record fluorescence intensity over time in the green channel to monitor calcium activity in astrocytes.
Here are the sample traces from the recording boxes of calcium activity in the astrocytes astrocytes incubated in TTX show increased spontaneous activity and more robust evoked group one MGL R calcium responses as evidenced by the changes in the pattern of response. Examples of single peak, multi peak and plateau calcium transient are shown and here are the representative traces of astrocyte calcium recordings from slices incubated in 5.0 millimolar potassium A CSF to depolarize the neurons and increase their basal firing rate compared to 2.5 millimolar potassium control A CSF astrocytes incubated with 5.0 millimolar potassium A CSF exhibit, fewer spontaneous somatic calcium transients and weaker DHPG evoked responses compared to astrocytes incubated in control. A CSF Astrocyte calcium imaging provides a nice functional readout of changes in G-protein coupled receptor signaling.
However, it's unable to pinpoint where in the signaling pathway these changes are taking place. Therefore, a complimentary approach would be to use fluorescence activated cell sorting or flow cytometry on astrocytes expressing green fluorescent protein, then apply an an antibody to the G-protein coupled receptor of interest, and then run western blots to look for changes in receptor expression levels. After watching this video, you should have a good understanding of how to measure scaling of astrocyte tqt PCR activity, following changes in neuronal synaptic transmission by recording spontaneous and Agnes evoked the calcium events in astrocytes.
