Investigating c-Fos as a Marker of Neural Activity in Planarians

Matthew Dodge, Verrazzano Class of 2025, completed major in Biology (7-12) and minor in ASL

My research investigated a common model organism known as planaria, a freshwater flatworm known for its regenerative ability and unique structure. It is similar to vertebrates, or organisms with backbones, which is evident through the conserved neurotransmitters, like serotonin and dopamine that is found in humans and planaria. This makes it a viable model organism for researchers to use in their laboratories.

However, little research has been done on the expression of a specific protein in planaria, known as c-Fos. It is produced by an Immediate Early Gene (IEG), meaning it has a rapid and transient production. In science, it is a known neural marker, meaning once the neuron is activated, the c-Fos protein is expressed and is visible under a confocal microscope for specific period of time. This information could be useful for a variety of reasons, including learning and memory research, mental health research, biomedicine, and specifically epilepsy, where neurons are activated and firing uncontrollably. By verifying the presence of c-Fos in planaria, we open the door for laboratories to expand epilepsy research and overcome financial burdens that come with vertebrate research.

Our research began by causing seizures in 20 worms using kainic acid, which activates neurons and causes synchronous, high-frequency firing. At 0 minutes, we killed 5 worms, then fixed them on a microscope slide. At 30, 120, and 240 minutes we repeated these steps. Once all the worms were fixed, we then observed the worms under a confocal microscope. It works similar to an MRI. It takes photos on individual planes of axis to create a high-resolution image that is unblurred by light coming from other angles. For instance, instead of taking a photo of an uncut cucumber, the confocal microscope would cut a thin slice out from the middle, then lay the slice flat and take a photo of it. The microscope will not only show us a 3D visual of the entire Planaria inside out, it will show us the fluorescence of the c-Fos protein. It shows up bright red in photos when present, and darker where it is not as present.

Each photo was analyzed on a software called ImageJ, which allowed me to calculate the amount of fluorescence specifically in the head and body of the flatworm. Once this data was collected, I created two line graphs to visualize the data over the course of 240 minutes. The data showed a temporal expression similar to that of vertebrates in the head. While not perfect, the repetition of this procedure with guidelines and protocols for confocal imaging will help us in the future. However, our data is promising and provides a foundation for the potential use of planaria as a model organism in neuroscience.

I expected this research to be trickier than it was, due to the first attempt being a little less than pleasant. We ran this experiment before measuring c-Fos expression using a Western Blot. This did not turn out as nicely as intended, leading to insufficient data to support our hypothesis. Having repeated the experiment again using a different approach, I am satisfied with the results, and happy with the work I put in.

This research can definitely be improved and implemented to build a strong foundation for the use of planaria as a model organism for neuroscience research. The validation of the presence of c-Fos would eliminate financial burdens caused by vertebrate research and allow for more accessible data. Further research can be done to evaluate the nervous system of planaria and map the areas of the brain firing under different stressors including seizures, chemical exposures, memorization, etc.

Our hypothesis was supported by the presence of a c-Fos-like temporal expression over the course of 240 minutes, specifically peaking at 30 minutes in the head of the planaria. While more research can be done to validate the presence of c-Fos, this lays the groundwork for further neuroscience research using a cheaper, small, and easier to handle model organism. Further research can be done to validate the presence of c-Fos and produce a nicer temporal expression.

This research experience granted me one of a kind access to equipment many students are unable to access. My favorite experience was using the confocal microscope, a one of a kind piece of equipment that takes beautiful images of planaria samples. Working in these settings invigorated my passion for learning. What also motivated me was the history of our school grounds and the Willowbrook State School. As a future New York City school teacher, being aware of the different learning disabilities that may impact your students and responding appropriately is necessary to maintain a safe and positive learning environment.