"You're doing your surgery, but you don't know if the animal still feels it and you've just stolen its ability to respond," says biologist Robyn Crook of San Francisco State University (SFSU) in California. Until recently, researchers working with octopuses, squids, and other cephalopods routinely faced this dilemma, an ethical and, in some cases, legal challenge to studying these intelligent creatures in the laboratory. But Crook has now shown that both ordinary alcohol and magnesium chloride are effective anesthetics—crucial information for scientists pursuing cephalopod research.
Cephalopods might not seem to be ideal laboratory animals. They're exclusively marine, so a complex seawater system is needed to keep them alive, and they're disinclined to stay put—octopuses can escape through minuscule holes, while squids may jet right out of their tanks. But their unique biology and behavior have made them indispensable to researchers in many fields. Studies of the squid's giant axon helped spawn modern neuroscience decades ago, and the light organ of the bobtail squid (Euprymna scolopes) powered a revolution in the study of symbiotic host-microbe interactions. Today, some researchers are studying how the animals accomplish their striking feats of regeneration, while others use them in ecotoxicology studies; cephalopods even guide research into the origins of consciousness.
Because of their complex brains, cephalopods became the first invertebrates to be protected by laboratory animal laws. In 1991, the Canadian Council on Animal Care decided to extend the standards for vertebrate care to cephalopods, meaning, among other things, that researchers have to get ethical approval for their studies and must use anesthesia, when possible, for procedures that could cause pain. Since then, the United Kingdom, New Zealand, and some Australian states have passed similar regulations. The biggest expansion of cephalopod rights came in 2013, when an EU-wide directive gave them the same protections as vertebrates in scientific studies in 28 countries.
Although the new regulations create extra hurdles, EU researchers have generally been able to continue their work, says Graziano Fiorito, a cephalopod neuroscientist at the Anton Dohrn Zoological Station in Naples, Italy. "We can do whatever research is scientifically, logically justifiable," he says.
But a crucial question remained: Do the anesthetics widely used in cephalopods actually work? Most researchers use either ethanol or magnesium chloride because they immobilize cephalopods quickly and the animals soon recover without lasting effects. But paralysis isn't the same as anesthesia, and previous studies of cephalopod anesthesia all used behavioral indicators as a measure of efficacy. None addressed the question of whether the animals were unconscious or felt pain, Crook says.
At workshops, European researchers raised what Jennifer Mather, a cephalopod ethologist at the University of Lethbridge in Canada, calls "the horrible specter" that the drugs might be mere muscle relaxants, blocking motion but not sensation. In 2017, Crook, who was already studying the response of the cephalopod nervous system to pain and injury, returned home from one of these workshops determined to set the specter to rest.
Her team recorded nerve signals from three cephalopod species: a cuttlefish and two species of octopus. The experimental technique relied on the pallial nerve, which lies just inside the cephalopod mantle cavity, transmitting signals from the mantle to the brain and back. Exposed and accessible, the nerve can be hooked up to an electrode noninvasively. Even so, the procedure isn't always easy: "The octopuses like to pull out the electrode," says collaborator Samantha Brophy of SFSU, and cuttlefish research "is like working with a cat," says another SFSU collaborator, Hanna Butler-Struben. "You can't really force them to do anything."
Once the electrodes were hooked up, the team added anesthetics to the seawater in the animals' tanks, then pinched their skin lightly at regular intervals—not exactly a painful stimulus, but enough to produce a nerve signal in a conscious animal. As the anesthetized animals lost their physical responsiveness, the scientists wanted to see whether signals that mark the brain's ability to perceive sensations also disappeared.
Magnesium chloride and ethanol both passed the test in all three species—though each with its own caveat. Magnesium chloride cut off an animal's physical responses to stimuli 15 minutes before it deadened the signals for sensation, leaving the animal temporarily in a vulnerable state of appearing anesthetized when it wasn't yet. "That was a real eye-opener for us," Crook says. "That changed our practices in the lab." Ethanol shut down both movement and sensation simultaneously—although not before causing the animals to tense and rub their tentacles in typical cleaning behavior, a sign of irritation. That's not a big surprise, Fiorito says: "If I put ethanol in your eye, even in solution, you're not going to be in a good state."
The team also tested ether, which caused anesthesia but had a much longer recovery period. It's also hazardous for researchers, so Crook's team recommends against its use. Another drug candidate, MS-222, failed to cause anesthesia in a lower dose but was fatal in a higher one. A bath in chilled seawater, another proposed method, didn't cause anesthesia either. (On the other hand, they found that injections with lidocaine and magnesium chloride readily triggered local anesthesia, which might be useful in some experiments.)
The data, published 20 February in Frontiers in Physiology, come as a welcome relief to many scientists. "This is what we needed," Mather says. "I care about my animals and I want to see them properly cared for." Fiorito notes, however, that nerve signals don't give the full picture. Additional research "should show the real mechanism of the molecule used, and what receptors are silenced so that the animal can actually be in an anesthetized state."
Cephalopod research is not regulated in the United States, but most researchers, including Crook, already use magnesium chloride or ethanol as anesthetics when needed. Whether to implement official rules is under debate. In January, the Association for Assessment and Accreditation of Laboratory Animal Care International proposed altering its definition of "laboratory animal" to include cephalopods, but the Federation of American Societies for Experimental Biology opposed the change.
Brophy points out that the new data will make it easier for researchers to follow their consciences, regardless of what regulations require. "You can treat cephalopods humanely when you're doing research with them," she says. "And that should always be a standard, I believe."
*Correction 4 April, 6:25 p.m.: An earlier version of this story misspelled Hanna Butler-Struben's name.