The overall aim of this procedure is to quantify changes in fluorescently Tag nicotinic receptors in mouse brain with chronic nicotine application. This is accomplished by first implanting alpha four YFP knockin mice with saline or nicotine filled mini osmotic pumps. Following treatment, the Misa intra Cardi fixed and the brains are cryo sectioned.
Next, the cover slipped brains are imaged using spectral and focal microscopy. The final step of the procedure is linear spectral on mixing and analysis of alpha four YFP expression levels. Ultimately, the results show the capability of spectral confocal microscopy and linear on mixing to accurately quantify upregulation of alpha four YFP nicotinic asto choline receptor subunits at submicron resolution in mouse brain neurons exposed to chronic nicotine.
The main advantage of this technique over existing methods such as antibody or radioligand bodying, is that the Y FPS genetically encoded to be attached to the receptor, thus allowing direct and more precise imaging of the tag receptor. This facilitates high resolution subcellular imaging of receptor localization in CNS neurons and obviates. Any potential issues of probe specificity, uneven staining, or probe access to antigens in deep layers of the tissue.
Demonstrating the procedure will be Anthony rda, a graduate student in my lab After removing the brain that has been fixed by intracecal perfusion, cut off the cerebellum with a razor blade. Place a small drop of mounting compound in the bottom of the mold under the brain and place the brain in a plastic embedding mold rostral side up. Then place the mold in the dry ice.
Once the brain is frozen in the proper orientation, fill the rest of the mold with mounting compound. Next, use a cryostat to collect 30 micron thick brain sections. Transfer the sections to coated slides using two fine paintbrushes.
Remember to protect the brain sections from light by always covering with a slide tray lid. Store the brain sections in slide boxes containing one anhydrous calcium sulfate stone. The slide box should be in a Ziploc sealed bag to avoid moisture and subsequent freezer burn When stored at minus 20 degrees Celsius.
Ensure that the slides have minimal exposure to any light source to minimize photo bleaching prior to imaging. By keeping them covered with the slide tray lid, the pipette tip can be cut to help lessen bubble formation When cover slipping. Use a mounting medium that does not inhibit fluorescent protein fluorescence, such as MO Wild 4 88.
Make sure the mo equilibrates to room temperature before cover slipping. In order to avoid air bubbles cover slip, the brain section slides one at a time. Do not use nail polishing when cover slipping as it will quench.
YFP fluorescence. Collect images of brain regions using a spectral confocal microscope system, which allows for very accurate quantification of YFP fluorescence even in tissue with significant levels of autofluorescence. To perform linear spectral unmixing on a sample image taken with a spectral confocal microscope, one must first acquire reference spectra for both YFP and tissue autofluorescence.
First, obtain a high signal to noise fluorescence reference spectrum by imaging a soluble YFP transfected cell line using the 4 88 nanometer laser line. Next, using the same laser line, obtain an autofluorescence reference spectrum in the desired brain region from a wild type mouse brain section. After obtaining a spectral confocal image from a brain region from the alpha four YFP mouse, devolve the image into its YFP and auto fluorescence signals by applying a linear spectral unmixing algorithm that uses the reference spectrums of both YFP and wild type mouse autofluorescence from the same brain region.
Open the unmixed alpha four YFP image in image analysis software such as Image J, and then calculate the mean pixel intensity for the region of interest. Repeat the same linear spectral and mixing and analysis to a spectral and focal image from the same brain region in a wild type mouse brain section to obtain a background residual value. Now the corrected alpha four YFP intensity can be obtained by subtracting the mean background residual value averaged over three to five wild type mice from the uncorrected alpha four YFP value shown here is a true color projection of a Lambda stack of images of the medial ular from an alpha four YFP mouse taken with a Nikon C one si spectral confocal microscope below are shown plots of spectra from a region of interest, which includes alpha four YFP containing neurons in green with a distinct YFP peak at 5 27 nanometers, and a region of interest outside the medial ULA in red, lacking the YFP peak separation of the YFP and autofluorescence signals is possible through linear spectral unmixing using the YFP and Wildtype brain Autofluorescence reference spectra yielding an alpha four YFP unmixed image and an autofluorescent unmixed image shown plotted below are the significantly overlapping YFP and Autofluorescence reference spectra used for unmixing in green and yellow respectively using spectral confocal microscopy upregulation of alpha four nicotinic acetylcholine receptors in the hippocampus of alpha four YFP knockin mice exposed to chronic nicotine can be seen here in a tiled montage of alpha four YFP fluorescence from the hippocampus.
The two selection areas are the approximate locations on the inferior limb of the Perent path where analyses were performed for each mouse. Alpha four YFP fluorescence was significantly higher in the perent path of mice exposed to chronic nicotine rather than chronic saline. After watching this video, you should have a clear understanding of how to image changes in alpha four YFP nicotinic receptors in knock in mouse brains with chronic nicotine administration by knowing how to one fix and cryo section, the mouse brain and two image using spectral confocal microscopy and linear spectrally and mixed images from the brain section.
These basic concepts can also be applied and extended to the study of expression of other ion channels and receptors.
