This protocol will allow researchers to answer important questions about neuronal patterns of connectivity and spatial organization that underlies function within a circuit. The main advantage of this technique is that it enables visualization of neuronal morphologies located deep within intact three-dimensional tissue. This method is straightforward and thus easily reproducible.
First-time experimenters should be comfortable performing this technique. It is imperative to have a clear tissue sample in order to obtain high-quality data. The visual demonstration of appropriate tissue clarity and clearing techniques will enable easily reproducible results.
To begin, place the hydrogel-submerged brain tissue in a vacuum incubator for three hours at 37 degrees Celsius with minus 90 kilopascal vacuum. Leave the tube top unscrewed to allow the vacuum to form properly. Wash the tissue with PBS for 10 minutes at room temperature with gentle shaking.
Place the polymerized tissue sample in the electrophoresis chamber, keeping track of the orientation of the tissue within the chamber. Fill the chamber and reservoir with the supplied electrophoresis SDS buffer. Run the sample at 70 volts, one ampere, and 35 degrees Celsius with constant current for about two hours per milliliter of tissue.
Check the sample periodically for sufficient clearing of brain tissue by removing it from the chamber and replacing it in the same orientation. After the sample has finished clearing, wash it in PBS overnight at room temperature, replacing the PBS with fresh PBS as often as possible. Following the final wash in PBS, wash the tissue for five minutes in deionized water at room temperature three times.
The tissue will become opaque and may expand. Incubate the tissue in the refractive index matching solution for at least four hours at room temperature. During the incubation, construct a suitable housing chamber to image the sample.
Start by using a glass slide as a base for mounting, then lay down either rubber or plastic spacers and secure them with super glue, making sure that there aren't any holes. Place the clear tissue into the mounting chamber pre-filled with refractive index matching solution. Securely mount the tissue by placing a glass coverslip on top and sealing it with nail polish.
Image this tissue by adding a drop of refractive index matching mounting solution directly on top of the glass. To construct a large tissue imaging chamber for tissues with more than a five millimeter thickness, use a 10 centimeter glass dish with a high wall. Place a 50 milliliter conical tube in the center, making sure that the diameter of the tube is large enough to accept the barrel of the objective lens used.
Make 3%agarose in water and pour it in the space between the glass dish and the conical tube, then allow it to cool for one hour. This will form a ring of solid agarose. Securely adhere the tissue to the bottom of the chamber using super glue and fill the chamber with refractive index matching solution, which may start to polymerize unless preserved from air and stored at four degrees Celsius.
Acquire the image using a confocal microscope. Turn on all the relevant imaging equipment. Place the sample on the stage.
Carefully approach the immersion media with the objective and form a continuous column of media. Using epifluorescence, find an appropriate imaging field. Start by setting the resolution and scan speed settings.
If imaging using a confocal microscope, fully close the pinhole to obtain the smallest optical section and thus the best Z resolution. Gradually increase the laser power or sensor gain until a suitable image is obtained with a high signal-to-noise ratio. If utilizing standard EGFP or tdTomato two-color imaging, set the light collection settings.
Set the Z-stack parameters based on the observed start and end points of the tissue. Set the step size based on the desired Z resolution. Smaller step sizes will yield a greater Z resolution, but may lead to sample bleaching.
When satisfied with the image acquisition settings, acquire the image. Ensure that the image has a high signal-to-noise ratio and shows distinct boundaries of structures. After image acquisition, the representative cell morphology was analyzed using embedded statistics and classifying scripts within the analysis software.
The collected data reflects that Neuron 2 has larger than dendritic structure with a higher density of spines compared to Neuron 1. To substantiate this result, standard Scholl analysis was performed, which affirms that Neuron 2 is more dendritically complex than Neuron 1 as denoted by the increased number of Scholl intersections at 50 to 100 micrometers from the soma. Neuron 1 contains a larger proportion of spines with more filopodia-like shapes and is an immature spine subtype.
Spines with defined heads, called mushroom spines, likely contain more developed and mature synapses. Thus, Neuron 2 is more mature with a higher density of mature spines. After clearing, the tissue chunks can be stained using traditional immunohistochemistry.
