How Cuttlefish Think Outside of the Brain

Dwarf cuttlefish.

Source: Adam Dewan, via Flickr. Distributed under a CC BY-NC-SA 2.0 license.

Cuttlefish, like their fellow cephalopods squid and octopus, are known for their intelligent and complex behaviors. Scientists have shown that cuttlefish can count, solve mazes, and remember what, where, and when they last ate (a phenomenon known as episodic memory that was once thought to be unique to humans). A recent study even found cuttlefish can pass the “marshmallow test,” eating less food earlier if they know they will later be rewarded with an especially tasty treat if they wait.

Supporting these sophisticated behaviors is an impressive brain; the cuttlefish has one of the largest brain-to-body size ratios of any invertebrate. But their impressive nervous system doesn’t end there. More than 60 to 70 percent of cephalopod neurons are located outside the brain, with many of them found within the eight arms.

“A lot of the intelligence of cephalopods is demonstrated through the dexterity of their arms,” says Vinoth Sittaramane, a biologist at Georgia Southern University. “That got us to thinking: If they are able to use their arms for problem solving, and they possess so many neurons in their arms, it would only make sense that they are able to perform some of these thinking and memory tasks independent of the brain.”

Dwarf cuttlefish with arms posturing.

Source: Bill Abbott, via Wikimedia Commons. Distributed under a CC BY-SA 2.0 license.

In a new study, Sittaramane, with his students Jessica Bowers, Jack Wilson, and Tahirah Nimi, used a neural activity marker known as phosphorylated CREB (pCREB) to look at memory mechanisms in the unique cephalopod nervous system. pCREB has been shown to facilitate learning and memory formation in the neurons of many vertebrates and invertebrates. Sittaramame and colleagues decided to look for pCREB in cuttlefish arm sensory neurons after training on a learning and memory task.

Out on a Limb

For the new study, Sittaramane and colleagues developed a new automated, high-throughput system of testing and tracking cephalopod learning. They used dwarf cuttlefish, which are small but still capable of complex behaviors.

The first step was training the cuttlefish in what is known as an “inaccessible prey” experiment. Cuttlefish were shown tiny shrimp enclosed in a transparent tube. At first, the cuttlefish rapidly strike their tentacles at the tube to capture the prey, but these strikes decrease over time as the cuttlefish learn the prey is inaccessible.

The researchers evaluated memory across two timescales: short-term memory, which lasts minutes to hours, and long-term memory, which lasts for days or longer.

The cuttlefish showed behavioral evidence for short-term memory by reducing the tentacle strikes across five consecutive training sessions. To test long-term memory, the researchers tested the cuttlefish again four days after initial training. Compared to naïve, untrained cuttlefish, the trained cuttlefish showed reduced attacks on the tube, indicating they remembered their previous experience.

Dwarf cuttlefish.

Source: Elanna, via Flickr. Distributed under a CC BY-NC-SA 2.0 license.

During the inaccessible prey experiment, Sittaramane noticed that the oral, sensory surface of the arms repeatedly gripped the tube at the end of each tentacle strike. To assess learning through the arms, the researchers used anti-pCREB as a marker to label neural activity in the arms. They found that trained cuttlefish had significantly increased numbers of pCREB-positive neurons on the oral surface of their arms, notably in the suckers and surrounding folds of tissue.

Next, the researchers examined differences in pCREB expression in different parts of cuttlefish arms. In trained cuttlefish, they found more neural activity at the distal tips of the arms compared to the ends closer to the body, and in arm pairs 1 and 2 compared with arm pairs 3 and 4. These are the arm pairs and parts of the arms that the cuttlefish used to contact the tube surface during learning.

“This says that arms are potentially playing a role in some learning and memory processes,” says Sittaramane.

Future Questions

From an evolutionary perspective, Sittaramane says he can see the advantage of having localized memories in the peripheral nervous system.

“It might help an organism develop better abilities to survive,” he says. “Cephalopods use their arms a lot in many behaviors and they are able to sense chemical cues in the environment. It only makes sense to have localized memories.”

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Dwarf cuttlefish.

Source: Elanna, via Flickr. Distributed under a CC BY-NC-SA 2.0 license.

This experiment is a starting point for Sittaramane, who says we have a long way to go in understanding the details and mechanisms involved in learning and memory outside of the brain. He hopes that future experiments will be able to characterize the types of neurons involved and the neural circuits at play. Sittaramane and his colleagues also did some preliminary staining in certain brain regions to investigate how the arms might be communicating with the brain. He says that although they did not detect any increase in pCREB levels in the brain, the question of connections between neurons in the arms and neurons in the brain is still open.

Finally, Sittaramane is excited about the potential applications of this knowledge to human biomedicine. For instance, cephalopods, including cuttlefish, can regenerate lost arms. What does this mean for the localized memory processes within arms? Can they also be regenerated?

“Since they can regenerate arms, it raises questions of how these neurons can regenerate,” he says. “Cephalopods have a lot to teach us about regenerating neurons with complete functional ability. That is an important question for regenerative medicine.”