This summer, I had the privilege of working with Dr. Ethan Goldberg and Dr. Joanna Mattis in the Department of Neurology at the Children's Hospital of Philadelphia. My project focused on Dravet Syndrome, an epileptic disorder caused by a mutation of the Scn1a gene. The pathology of Dravet Syndrome is thought to be due to interneuron dysfunction. One circuit containing interneurons is located in the hippocampus and involves the perforant path (the projection from the entorhinal cortex to granule cells in the dentate gyrus) and parvalbumin interneurons. Normally, parvalbumin interneurons project inhibitory input onto granule cells and prevent them from being hyperexcited by the perforant path. However, in Dravet Syndrome, it is theorized that these parvalbumin interneurons are intrinsically dysfunctional and cannot be excited and therefore cannot project their inhibitory input onto granule cells, leading them to become "overexcited". Therefore, it can be hypothesized that activation of parvalbumin interneurons will decrease granule cell response to perforant path input in Scn1a mice (an attempt to rescue hyperactivity). During the summer, I wanted to test this hypothesis by optogenetically stimulating the parvalbumin cells while simultaneously using calcium imaging to see the granule cells' response. In order to do this, I had to get Chrimson (a red-shifted opsin that would allow for ontogenetic stimulation of the parvalbumin cells) into the parvalbumin cells and GCaMP (a genetically encoded calcium indicator for calcium imaging) in the granule cells.
I was able to create a viral mix that included both of these molecules. The two viruses I mixed were AAV9.syn.FLEX.ChrimsonR.tdT and AAV9-syn-jGCaMP7s, both relying on a synapsin promoter that could be used to target all neurons. I imaged dentate gyrus injected with this viral mix and saw great GCaMP expression and Chrimson expression throughout the dentate gyrus, ensuring I could use this mixture in future experiments. This would allow for both opsin and GCaMP to land in the appropriate places in the mouse's brain.
The next part of my project involved figuring out the best way to target the dentate gyrus. I tried a variety of strategies. The first strategy was a subdural viral injection in a newborn mouse, simply sticking a needle under the dura and injecting virus on either side of the brain. When this was unsuccessful, I then used an intraparenchymal approach with a stereotactic method. I had to modify this strategy a number of times to include larger volumes of virus injected, longer waiting times to leave the needle in the brain, as well as increasing the number of injection sites. I also varied the age of the mice I injected from newborn mice to "young adult mice" (12 days old) to adult mice.
This summer was a great hands-on experience. I was able to learn so much about physiology, anatomy, cell biology, and a variety of valuable laboratory techniques through this project. I was even able to explore surgical methods when working with the mice, giving me insight into an important aspect of future medical education.