Wrapping Up

In concluding our research, we didn’t see much of a difference in AChE staining between the two hemispheres in the three rat brains from Hampton-Sydney. We are going to try the stain one more time, because it was a bit faint, and just to make sure we’re not missing anything. We will use new tissue sections for the second round of AChE and Parvalbumin staining within the next couple of weeks. With so many steps in each staining procedure, one small miscalculation can throw off results. However, it could just be that the results are accurate, and that there is no difference in AChE staining with Anti-Chat saporin, which would refute our hypothesis for the pilot study.

Acetylcholinesterase Tissue Staining

The last staining step was targeting acetylcholinesterase, which is an enzyme that catalyzes the hydrolysis reaction of acetylcholine. We did this similarly to the parvalbumin staining, with a few tweaks to the protocol. First, the solution was prepared with a phosphate buffer and hydrogen peroxide. Then, a maleate buffer was used, which caused a slight peach color during incubation. TRIS Buffer rinses were followed by an incubation period , which was then followed by hydrogen peroxide and Tris Buffer rinses. The tissues from the three rat brains successfully reached a dark purple to black color, and were then mounted on gelatinous slides. These slides as well as the parvalbumin stained tissue slides will be subsequently analyzed by Dr. Burk to assess the effectiveness of  anti-ChAT saporin as a novel immunotoxin in the thalamic reticular nucleus. 

Parvalbumin Immunostaining

To effectively section the brains, we cryogenically froze and preserved them. After sectioning, we identified tissue sections around the target region based on hippocampal development from the hundreds of slices we made. We also selected tissues anterior and posterior to the target region, which we identified again based on hippocampal development, which was greater in posterior regions and less defined in anterior regions. When it came time to stain and plate the tissues, we repeatedly rinsed them in phosphate-buffered saline (PBS) buffer in order to remove any residue from the freezing, in which the brain was glued down.

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Unilateral Lesioning of Brain Tissues

So far in my research this summer, we have received and sectioned the rat brain tissues from Hampden-Sydney College. The brains received unilateral (in one hemisphere) infusions of anti-ChAT-saporin, the new immunotoxin we are testing. The other hemisphere was injected with saline solution as a control, which is known as a “sham lesion,” something similar to a placebo.

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Lesioning Thalamic Reticular Cholinergic Neurons

My name is Emily Masi and I am a rising sophomore studying neuroscience and public health here at the College. I have been working in Dr. Burk’s lab since the beginning of this semester, and its focus has been the role of orexin projections to the basal forebrain in attention, the effects of reward delay discounting on impulsivity and the role of the cholinergic system in this pathway, and the actions of acetylcholine at muscarinic and nicotinic receptors under cognitively challenging conditions.

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