As a student studying to complete my Bachelor of Science degree in biochemistry, molecular, and cellular biology, I have always been interested in anesthesia. I recently shadowed an anesthesiologist from Concord Hospital to gain a deeper understanding of the field. I wanted to apply my interest of anesthesia into research, so I reached out to my biology professor, Dr. Chen, because it was in his class where I became more interested in the cellular mechanisms of anesthesia. My contact with Dr. Chen led me to conduct research in a neuroscience lab for the first time.
I applied for a Summer Undergraduate Research Fellowship (SURF) to study the effect of a drug, ketamine, on short-term memory and learning. Ketamine is used commonly as an anesthetic for short-term sedation. We can study the effects of ketamine by imaging an area of the brain called the hippocampus, which plays a key role in learning and memory. I am studying ketamine because the effects of ketamine on learning and memory are not widely understood, so this research aims to understand this connection. To date, I have completed the 10-week program and gained many valuable skills during this time. I will continue the research with Dr. Chen throughout this academic year to incorporate into my Honors Senior Thesis.
For the SURF program, to complete the necessary tasks set for each week my hours related to that of a full-time job, Monday through Friday, at Rudman Hall on the Durham campus. The majority of my time in the lab consists of data configuration. The data comes from in vivo imaging that uses a fiber optic probe to image the hippocampus CA1 region of a mouse. We focus on the hippocampus because it is responsible for learning and memory formation. We label the regions of interest from the images taken throughout experimental cycles.
To conduct our experiment, we collect data by combining trace fear conditioning (TFC) with in-vivo imaging to record neuronal function during learning. TFC is a protocol to measure hippocampal dependent learning and memory formation in mice. It is different from classical conditioning, which is when the tone and the shock occur at the same time. Types of conditioning such as TFC and classical conditioning are used to learn more about how animals respond and learn about a stimulus. Trace fear conditioning is harder for mice to learn. For this reason, it works well for studying more complex memory and learning functions.
A tone is emitted 5 seconds before a small foot shock is administered in a behavior cage equipped with a speaker and camera, and this is repeated for 7 cycles. The repetitive cycles teach the mouse to associate the tone with the delayed shock. Neuronal firing is recorded with the fiber optic probe and mouse behavior is recorded using the video camera in the cage. Ketamine or saline(control) is administered after the first round of TFC, and the second round of TFC is recorded after three hours following administration to determine if there was a change in learning or memory. Data is recorded during TFC as well as resting periods in their home cage.
Some lab techniques that I have learned pertain to immunostaining. Immunostaining is completed after trace fear conditioning. Immunostaining is done by extraction of the brain from euthanized mice before slicing, staining, and fixing the slices to slides. I learned each step of the process like perfusion (delivering fixatives to the tissue), using the cryostat to slice the mice brains thin, and immunostaining with primary and secondary antibodies to target specific proteins. It is challenging to fix brain tissues on to slides without breakage. They are extremely delicate and must be manipulated with care.
After the immunostained slices are mounted on the glass slides, a confocal microscope is used for imaging and ImageJ is used to analyze the expression levels of the selected protein. This tool can also be used to confirm the accuracy of the placement of the imaging probe and virus expression level after in vivo imaging. When analysis is complete, we expect to see a disruption of neuronal firing after ketamine administration.
Many of the problems that I face involve data collection. Slow buffering and storage space have been problems due to large amounts of data. I am also working on a more efficient way to analyze data with Query in Excel.
The SURF experience allowed me to see first-hand how anesthesia drugs impact neurons in the brain, and how this relates to the specific cellular mechanisms occurring throughout drug administration. Following completion of my undergraduate degree, I am planning to attend medical school, where I will be able to apply important techniques I learned this summer, such as immunostaining. Studying anesthesia in the lab helped me understand its effect on the brain, which also applies to skills needed in medical school. I hope to continue to learn more skills as the weeks progress and I pursue more independent research this fall.
