How are memories made?
To form long-term memories, the neurons that will store those memories need to strengthen their connections to each other, Vergara-Ovalle explained. In order to make the protein machinery to strengthen those connections, an organism needs to overexpress certain genes—in other words, ramp up the process of switching on those genes and producing the specified proteins. The genes that get overexpressed after learning are called immediate early genes (IEGs), and their expression spikes over the five to 60 minutes after you learn something, Vergara-Ovalle said.
He wants to find out if the genes for two proteins in particular—abbreviated CREB and C/EBP—are overexpressed after learning in the octopus. Both genes are IEGs in the California sea hare (Aplysia californica), which is a mollusk, like the octopus. When CREB and C/EBP are overexpressed, he said, it leads to a chain-reaction of overexpression in other genes that eventually produces the proteins that strengthen neuronal connections and form memories.
Vergara-Ovalle plans to use a technique called in situ hybridization to visualize if CREB and C/EBP are overexpressed after learning, and in which parts of the brain. If one of the genes is overexpressed in a particular lobe, it follows that the lobe is important for forming that memory.
Vergara-Ovalle will use immunohistochemistry—a type of imaging—to see where CREB and C/EBP proteins are present. In other animals like mammals and snails, both proteins are activated by phosphorylation—a chemical tweak that adds a phosphate group—so he will also use a technique called Western blot to separate proteins by molecular weight and see how much of CREB and C/EBP are phosphorylated.
Vergara-Ovalle has been working with staff in the ’s Marine Resources Center to prepare for his experiments. He plans to investigate if overexpression is occurring in other genes besides CREB and C/EBP, as well; he would love to see those two overexpressed, he said, but is not completely convinced it will happen.
“Especially with these new emerging models, you have so many questions,” he said. “Most of the time you think that you will know the answer, and that’s not the case.”