Have you ever remembered an obscure fact and wondered how your brain held onto it?

Despite a nervous system very different from our own, one of the Marine Biological Laboratory’s (MBL) most famous research organisms can also form memories. Octopuses, known for their remarkable intelligence, have shown the ability to discriminate between new objects and familiar ones. But how are they doing it?

“Up to now, we have understood memory with a strong bias towards mammals,” said Jose Fabian Vergara-Ovalle, a Grass Fellow at MBL and professor at the National Autonomous University of Mexico. Learning more about memory formation in other animals could give new insights into how humans create memories, Vergara-Ovalle said, and broaden our understanding of conditions like Alzheimer’s disease.

“Because we know there is some neurodegeneration [in Alzheimer’s],” he said. “But how is neurodegeneration affecting the networks that actually [store] the memory?”

This summer in the Grass Lab, Vergara-Ovalle plans to use behavioral testing, imaging, and molecular techniques to investigate the genes and brain areas critical for memory formation in the California two-spot octopus (Octopus bimaculoides). Octopuses separated from the human lineage 550 million years ago, Vergara-Ovalle said.

“So they have their own nervous system that’s characteristic of them. It’s really different from ours,” he said. “But they can do things that look similar to us.”

Jose Fabian Vergara-Ovalle in the MBL Grass Lab.
Jose Fabian Vergara-Ovalle in the MBL Grass Lab. Credit: Alex Megerle

Out with the old, in with the new

To get the octopuses thinking, Vergara-Ovalle plans to use a test called novel object recognition, in which subjects are presented with and allowed to explore two identical objects. The next day, one of the objects is replaced with a new one. Researchers can then compare how a subject reacts—the idea being that an animal will explore a new object differently than a familiar one.

Novel object recognition allows for comparison between different animal models and is the primary test researchers use to study Alzheimer’s disease in rats or mice, Vergara-Ovalle said.

“So we already know so much about this particular test and which areas of the brain are important for memory formation in rats and mice, or mammals in general,” he said. “And now I want to figure out which areas are important in the octopus to create this recognition memory during the same test.”

The California two-spot octopus (Octopus bimaculoides).
A California two-spot octopus. Credit: Tom Kleindinst

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 MBL’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.”