Untangling mysteries of the brain—with the remarkable biology of squid

“We’ve got tuuuuuubes!” fisherman Matt Rissell shouts as he leans over the gunwale of the Skipjack to check his line. After a salty April morning spent bobbing off the coast of Cape Cod, Massachusetts, the first squid of the season are coming in.

Rissell reels and a foot-long male Doryteuthis pealeii (though everyone on this boat refers to it by its older name Loligo) emerges from the waves spouting water and waggling its two tentacles and eight sucker-covered arms. The iridescent skin of its mantle—the “tube”—is flecked with pink, teal, and gold, until in a flash, as Rissell brings the squid over the side, it transforms to an angry maroon.

Other boats are out for the squid run, too. Poles bend across the small fleet of reel-and-rod outfits gathered here in shallow waters where the squid come to spawn each spring. The animals spend the rest of the year jetting around deep underwater canyons, ensnaring fish, crustaceans, and even fellow squid. Further out, commercial trawlers drag long nets, each year landing thousands of tons of squid, most of which are destined for the deep fat fryer.

The squid pulled into the Skipjack, however, have a more cerebral purpose. They will be taken to the Woods Hole Marine Biological Laboratory a few miles to the west, where for nearly a century, these squid have played a vital role in neuroscience research. The animals have helped scientists shed light on everything from the basics of nerve signaling to the evolution of complex brains. Study of the squid’s unique biology could eventually lead to improved therapies for neurological and genetic disorders in humans.

Last year, this research took a major step forward when a group of scientists at the laboratory successfully used the gene editing tool CRISPR-Cas9 to disable, or “knock out,” a gene in the Doryteuthis squid—a first for any member of the talented group of mollusks known as cephalopods. The work paves the way for scientists to investigate the genetics behind cephalopods’ near extraterrestrial abilities, from squid’s color-changing skin cells to cuttlefish’s duplicitous mating behavior to octopus’s capacity for memory and learning.

“How have they figured out different ways to make these complex behaviors?” wonders molecular biologist Josh Rosenthal as he reels a squid into the Skipjack, then flips it off his jig into a tank of water where it disappears in a bloom of black ink. “These things that basically came more from a clam than from a vertebrate.”

Historically, however, it was another feature of the squid that made them famous among neuroscientists. As the Skipjack heads back with at least 70 squid in the tank, Rosenthal, who led the CRISPR research at the Marine Biological Laboratory, yells over the engine: “It was their giant nerve cells!”

Big squid axons

A few hours later, I see what Rosenthal means as Pablo Miranda Fernandez, a neuroscientist from the National Institutes of Health, takes one of the squid from the Skipjack up to his dissection room and without ceremony lops off its head. He immediately goes to work at a table covered with cold seawater, slicing open the squid’s translucent body and gingerly removing its viscera with metal forceps. He peels back the squid’s hard inner shell, or “pen,” to reveal a pair of nerve fibers, called axons, extending from the severed end of the squid into its well-muscled mantle.

“Pretty good,” he says, measuring the width of the fiber, which is about a fourth as thick as a cooked strand of spaghetti. Tying off the ends of the axon, Fernandez plops it in a dish of calcium-free water, so as not to disrupt the ions inside, which enable the nerve to fire. Hundreds of times larger than the largest axon in humans, its girth allows electrical impulses to travel rapidly into the mantle, so the squid can quickly jet away from danger.  

Following the discovery of these giant fibers in 1936 (scientists initially thought they were blood vessels), researchers began using them for experiments on the chemical and electrical mechanisms of the nervous system and the brain. The squid axon was so big that scientists could attach electrodes to it and zap it, measuring changes in voltage. They could squeeze out the axoplasmic goo inside and study what it was made of.

Leonid Moroz, a neuroscientist at the University of Florida, calls the squid’s giant axon “nature’s gift to neuroscience.”

The study of squid nerves has resulted in hundreds of scientific papers and two Nobel Prizes. The first was awarded in 1963 for revealing how nerves transmit electrical impulses to communicate with other cells via a chain of biochemical reactions. This process, called an action potential, is a fundamental mechanism in all organisms with a nervous system. The second squid-inspired Nobel was awarded in 1970 for elucidating the role of neurotransmitters, such as adrenaline, in nerve signaling.

Today, precision tools that can measure and manipulate smaller nerve fibers have made the squid’s giant axon less essential for research, but the squid “still has a lot of mysteries and science we need to find out,” Fernandez says.

At the National Institutes of Health, for instance, Fernandez works with a team that studies whether certain proteins can be made within the squid’s axon, which extends from a cell body, rather than transported into the axon from the cell body. The work could eventually lead to improved therapies for damaged nerve cells in humans, Fernandez says, but if we don’t first understand how the basic process works in a squid cell, “we cannot even dream of doing that kind of stuff.”

Tunable genetics

Other squid nerves from the Skipjack catch will be used by Rosenthal to study the animal’s curious ability to alter genetic information in RNA molecules within their nerve cells at very high rates. This may allow the squid to “tune” how genes are expressed in different parts of its body—but no one knows for sure, Rosenthal says.

A better understanding of how RNA editing works in the squid could even lead to therapies for people. A biotech startup co-founded by Rosenthal is working to learn from the squid’s natural RNA editing skills to target diseases of the liver, eye, and central nervous system in humans, correcting harmful mutations without having to permanently alter anyone’s DNA.

But to investigate these and other mysteries of cephalopods, scientists need to be able to do genetic research on them. That requires three key pieces: an organism’s complete genetic code, the ability to manipulate that code, and the ability to grow the organism in a lab.

For decades, this has been possible in mice and other classic model organisms, such as fruit flies and nematode worms, enabling innumerable advances in biology and medicine. But cephalopods—with their treasure trove of evolutionary oddities—have proved less amenable to genetic research (and not just because of the octopus’s notorious ability to escape from tanks).

The difficulties encountered by Rosenthal’s team editing just one gene in one squid species illustrate the challenges involved.

Cephalopod Operation

The first hurdle was sequencing Doryteuthis pealeii’s genome, which the team needed in order to know where to make the cut, says Marine Biological Laboratory neurobiologist Carrie Albertin, who led the genome sequencing work on the squid. “Cephalopod genomes are large and complicated,” she says. 

Where the human genome consists of about 3.2 billion letters, or bases, the squid’s genome has about 4.5 billion letters, more than half of which are made up of repetitive sequences. Sequencing those letters, Albertin says, is like piecing together an enormous jigsaw puzzle that depicts an empty blue sky. “Whenever you're developing something new,” she says, “you’ve got to figure out how to overcome whatever weird challenge biology decides to throw at you.”

After an expensive effort to sequence those billions of fragments of squid DNA and fit them together, biology threw the team another curve ball. Unlike other squid, Doryteuthis eggs have a thick rubbery outer layer, or chorion, which can’t easily be punctured by the fragile needles used to inject the molecular editing tool CRISPR-Cas9 into the egg. It’s a game of embryonic Operation: If the needle doesn’t puncture far enough, the CRISPR-Cas9 won’t reach its target, but if the needle punctures too far, the egg won’t develop.

“I failed miserably at it for years,” says St. Mary’s College embryologist and squid editing team member Karen Crawford.

After much trial and error, enabled by the steady supply of squid eggs from the Atlantic catch, Crawford found a way to use micro-scissors to make a slit in the chorion big enough for the needle to pass through, yet small enough to reseal behind the needle and leave the egg intact. “I got very good at making holes,” Crawford says.

For the first knockout, the team chose a gene responsible for pigmentation in the squid. They selected the pigmentation gene because it would be easy to see whether the edit had worked. And it did. In September 2020, the group reported in the journal Current Biology that the gene had been disrupted in 90 percent of cells in the edited squid, representing a key advance toward making the squid and other cephalopods amenable to genetic research. While unaltered squid were speckled with colorful chromatophores, the knockout squid were clear as glass.

Since then, the group has experimented with knocking out other genes, Rosenthal says, such as the two genes that enable RNA editing. Though the function of this genetic trick is still unclear, it seems to be essential for the squid: Larvae that lack the RNA editing genes die soon after hatching.

This summer, the group is focused on adding, or “knocking in,” a gene to the squid to produce a protein that fluoresces green when it binds to calcium, which flows into the axon when a nerve fires. Combined with the pigmentation knockout, this would allow researchers to literally watch as nerves develop and begin working in transparent squid.

Squid culture

Despite these advances in Dorytheuthis pealeii research, and the distinguished career of the species in service of science, the squid has a serious drawback as a genetic research organism: It can’t easily be grown in a lab. “It’s such a large adult,” Crawford says. “And it likes deep, cold ocean water.”

Wild caught Dorytheuthis can be kept in tanks, but they don’t survive more than a few days. And while eggs sourced from wild squid can be fertilized in the lab, the hatchlings have a complex diet and can’t be kept alive long enough to reproduce, which is necessary for scientists to establish different genetic lines.

Across the Marine Biological Laboratory campus from the dissection room, however, an alternative to Dorytheuthis floats peacefully in a plastic tub of water treated to mimic the ocean off the coast of Japan. Shining a light into the tub, Taylor Sackmar, a cephalopod culture specialist, reveals a pebble-sized hummingbird bobtail squid, Euprymna barryi, transparent but for its reflective red eyes.

The month-old hatchling is the first-generation offspring of two genetically edited parents, which float in nearby tanks like dumplings with tentacles. Sackmar moves the light to the mother squid to point out stripes of missing color on her skin, indicating that she lacks two genes responsible for pigmentation. Her eggs, when fertilized by a male that also lacks those genes, hatch albino, see-through children.

“Offspring from this female are the leading edge of the CRISPR research,” whispers Sackmar, as though not to disturb the squid.

Unlike Dorytheuthis, the bobtail squid can hatch, grow to adulthood, and reproduce within the lab. Though the work is still in early stages, Sackmar says the vision is for the lab to one day supply scientists around the world with squid eggs and adults for genetic investigations. The lab is also working to establish full life-cycle cultures of other cephalopods, including flamboyant cuttlefish, striped pyjama squid, and the golf-ball-size California two-spot octopus. “If this does take off as we project, other labs are going to want more of these,” Sackmar says.

A number of challenges remain, though, to scaling up the use of these cephalopods as research organisms. For instance, it’s still not possible to fertilize bobtail squid eggs in vitro, so any gene editing can’t begin until the mother squid decides to reproduce. Squid also take a relatively long time to mature after hatching, slowing research.

“People who love squid say, Oh, this is the best thing ever. But the reality is the route is not going to be easy,” says Miguel Holmgren, another National Institutes of Health neuroscientist who uses the squid’s giant axon for research.

Moroz, the Florida neuroscientist, thinks the bobtail squid is too simple to answer many questions out there about cephalopod neurobiology, though he calls the research an “extremely important step.” With their combination of neural complexity and evolutionary distinctiveness, he says any basic research on cephalopods “really will speed up our understanding of the brain, the same way the squid giant axon did.”

“These organisms have been around since the end of the Paleozoic” 250 million years ago, Crawford says. “They’ve got a lot of stories to tell.”