This method can help answer key questions in the field of neuroscience by providing a simpler, less error prone, and more quantitative method for verifying lesions and locating electrode locations. The main advantage of this technique is that it allows for the verification of lesions and electrode sites in the whole brain without the need for sectioning. The end product is a digital 3D volume of the brain and multiple brains can be processed in parallel.
Though this method will be demonstrated in the rat, it can also be applied to other organisms, such as mice and zebra finches. Generally, individuals new to this method will struggle because some steps require particular care to ensure best results. To begin, place an extracted brain in profusion solution in a 50 milliliter conical tube.
Store the sample for two to three days with gentle shaking at four degrees Celsius. Make sure that the osmium is diluted in water and not a buffer. This is because the water will act as a mild detergent, allowing the osmium to penetrate deep into the tissue.
Prepare 50 milliliters of 2%osmium tetroxide in double-distilled water. Then place the brain in a new 50 milliliter conical tube and add the osmium tetroxide solution. Close the tube and seal it with paraffin film to prevent leaks.
Store the sealed tube at four degrees Celsius with gentle shaking for two weeks. Make sure that the tube is placed horizontally. The brain is very thick and a long ways to travel by diffusion.
Placing the tube horizontally will allow the osmium to travel deeper into the tissue. After the sample incubates for two weeks, wash it with double-distilled water five times to remove all the unbound osmium tetroxide from the sample. Next, wash the sample with double-distilled water for 30 minutes at four degrees Celsius.
Replace the double-distilled water with four dilutions of ethanol to dehydrate the sample. To begin the resin infiltration process, move the sample through three acetone and three acetone resin dilutions, then immerse the sample in 100%resin at room temperature to continue the infiltration process. After this, transfer the sample to a disposable mold.
Infuse the sample with fresh 100%resin for four hours at room temperature. Degas the sample in a vacuum oven for 15 minutes at 45 degrees Celsius. Then cure the sample in an oven for 48 hours at 60 degrees Celsius.
Finally, when the sample has finished curing, peel off the disposable mold and scan it with the Micro-CT machine. In traditional sectioning, the orientation of the brain must be predetermined. Using this Micro-CT method, the digital volume of the sample brain can be manipulated in three dimensions and virtually sliced in any direction.
Scanning electron microscopy of the prepared rat brain confirm that the tissue was not significantly damaged for the purposes of Micro-CT imaging. A zebra finch brain was also prepared and scanned according to this protocol to test the utility of this method on other small animal brains. This technique can be used to find surface lesions in the brain, as well as lesions deep within the brain.
This technique can also be applied to the location of single tetrodes in situ, electrolytic lesions, electrode tracks, tetrode arrays in situ, and silicon probes in situ. While attempting this procedure, it's important to remember to allow the brain to incubate for long enough and with enough agitation. Following this procedure, other methods like optogenetics and two-photon imaging can be performed in order to answer additional questions about the role different brain areas play in different behaviors.
This technique could allow researchers in the field of neuroscience to better explore structure-function relationships between the brain and behavior in rats, mice, songbirds, and other small animals. Don't forget that working with osmium can be extremely hazardous and precautions, such as wearing gloves and working in a vacuum hood should always be taken while performing this procedure.
