Giving zebrafish ketamine can stop them from “giving up” during tasks where trying to swim is futile. A new study led by researchers from the Engert Lab and Fishman Lab at Harvard and the Ahrens Lab at Janelia Research Campus, which appears in the journal Neuron (PDF) this week, found that the drug causes a huge surge in calcium signalling in non-neuronal astrocyte cells. After the ketamine wears off, the neural circuit remains less sensitive to futility, requiring a bigger struggle before it can be activated.
Program in Neuroscience (PiN) graduate students and study first authors Marc Duque Ramirez and Alex Chen of the Engert Lab at Harvard and Ahrens Lab at Janelia and graduate student Eric Hsu of the Bergles Lab at Johns Hopkins wanted to investigate a circuit that appears to govern the “giving up” behavior. “When I joined the lab, we were interested in studying this ‘giving up’ behavior that had been previously established by the team, where we have the fish in a virtual reality environment,” says Duque Ramirez. “We can make the fish think it’s swimming against a current by showing it a visual stimuli that makes it think it’s moving even though it’s actually stuck in place, and the fish will move its tail to fight the current…In that previous work, they showed that if you switch from that to making the fish think its swims don’t work anymore, it goes from swimming happily to getting stressed and struggling and then giving up.”
In the assays Duque Ramirez and his colleagues performed, the larval zebrafish are partially immobilized with their heads held in place by an agarose gel but with their tails free to move. This setup allowed them to perform whole brain imaging on the translucent fish while also using their tail movement as a measure of whether the fish were giving up. They paired this assay with whole brain imaging to see where calcium showed up in the brain while the fish does different things.
“The cool thing about this circuit that originally made me interested is it adds astrocytes, this non-neuronal cell as an active computational element,” says Duque Ramirez. “And so the general idea is that these astrocytes in the fish hindbrain, they integrate noradrenergic futility signals over time.If there had been a lot of futile swims in a short period of time, norepinephrine would trigger increases in intracellular calcium in glia, and once glial calcium reaches a certain threshold, it activates an inhibitory population that suppresses swimming. If ketamine is changing the giving up behavior by tapping into this circuit, we could learn something new about how ketamine acts in the brain.”
They treated the fish by adding ketamine to the water. The drug initially has a strong dissociative effect that prevents normal swimming, so the researchers would wait an hour before placing the zebrafish in the futility assay. They found that ketamine-treated fish took much longer to give up compared to controls.
“We saw in the normal swimming periods, they swim the same, so it’s not that ketamine is messing up all the swimming,” says Duque Ramirez. “But when we go into these open-loop futile states, where their swims don’t work, they still seem to still struggle. They still sense that something is wrong, but they don’t give up. They keep trying a lot longer.”
Whole brain imaging revealed that the ketamine-treated fish were experiencing huge surges of calcium in the astrocytes in the hindbrain. Duque Ramirez and his colleagues believe the calcium surge is essentially causing adaptation in the circuit that dampens its excitability, so that going forward, it delays or even eliminates the giving up behavior.
Duque Ramirez and his colleagues also performed an experiment where they treated the fish with corticosteroids that made the fish more likely to give up sooner than controls. Following the corticosteroids with ketamine allowed them to rescue the phenotype in zebrafish.
The team also performed a calcium imaging experiment in the adult mouse cortex and found that the astrocytes in mice are also activated during a futility test.
The study’s co-authors say these results emphasize the importance of considering the roles of non-neuronal cells. Most studies focus exclusively on neurons, but this study’s findings indicate that astrocytes could have an important role in driving ketamine’s antidepressant effects. Duque Ramirez adds that since whole brain imaging and behavioral assays are much easier in translucent larval zebrafish than in mice, zebrafish assays could be used as a first pass for testing and screening novel antidepressants.
