At the Heller Lab of Neuroepigenetics, I have been working on a core project: What role does the cyclic dependent kinase 5 (CDK5) play in stress and depression?
My endeavors in the project hinge on two aims. First, I need to identify the most effective time point at which the mice display a stressed phenotype. Second, I wish to investigate the behavioral and biochemical effects of stress to different brain regions in the mice.
As of now, separate cohorts of mice have undergone the CUS paradigm at stressing intervals of 2, 3, and 4 weeks. The stressing procedure entails the mice undergoing tube restraint, overcrowding and white noise treatment, and cage rotations set at 100 rpm. In the evenings, a series of stressors that include wet bedding and titled cage setups are utilized. A separate batch of controls will be in the same room under the same environmental conditions but will be spatially separated from the other CUS group. At the conclusion of the stress protocol, the mice will undergo a series of behavioral tests, such as the forced swim test (FST), marble burying test (MBT), and sucrose preference test (SPT). All of these tests are methods of discerning whether the mice display a significant stressed phenotype. The mice utilized in this experiment total around 100.
The second part of this experiment involves determining gene expression levels between the stressed and control groups through biochemical tissue analysis. In order to investigate gene expression changes from this paradigm, I extract RNA directly from brain tissue, convert it into complementary DNA (cDNA) via the reverse transcriptase enzyme, and then run a quantitative polymerase chain reaction (qPCR). The qPCR is able to ascertain the copies of CDK5 mRNA transcripts present after designed primers are able to cut at specific sites on the DNA. GAPDH is used as the baseline control measurement, because of its ubiquity in cellular respiration processes. All qPCR values are normalized to the GAPDH counts.
For the future, I plan on checking protein expression for CDK5 using a Western Blot method and eventually checking for specific histone modifications after stress treatment as well.
Research at the Nestler Lab done by Elizabeth Heller at Mt. Sinai previously confirmed that CDK5 in vivo epigenetic manipulation affected drug behavior for mice. The stress paradigm will add another level to the overall narrative of CDK5 expression and can provide the best path forward into future in vivo experiments.
My involvement in this experiment over several years now has really provided an understanding of the patience that is integral in the domain of science. Many of my experiments encountered complications that proved to be frustrating. Much of the stress paradigm involved working seven days a week in making sure that the protocol was followed rigorously. The whole experience, however, has truly made me more appreciative of putting in maximum effort. Being able to fail and have support and resources to try again is something invaluable I learned as a young scientist, student, and young adult.