The anesthetic ketamine could be a game changer for treating severe depression, but there are still many questions about how the drug works, including how it affects the brain’s cells and circuits. Newly reported research has taken advantage of the ability of zebrafish to exhibit “giving up” behavior. Using techniques including brain imaging, and a unique virtual reality system, a team of researchers from HHMI’s Janelia Research Campus, Harvard, and Johns Hopkins identified where ketamine acts in the zebrafish brain. Their study found that exposure to ketamine causes a lasting suppression of this giving up behavior by overstimulating astroglia.
The findings, which also show that astrocytes are similarly activated in mice, indicate that astrocyte activation may be a critical component of ketamine’s effects cross species, and could help researchers get a clearer picture of how antidepressants work in the brain, potentially leading to the development of safer and more effective drugs to treat depression.
“Our paper suggests that these astroglia, this non-neuronal cell population, are playing a very important role, and that some of the key effects of these antidepressant compounds go through changes in astroglial physiology,” said Alex Chen, a joint PhD student in lab of Misha Ahrens, PhD, Janelia senior group leader, and in the Engert Lab at Harvard. “I think our research suggests that targeting these astrocytes to find new treatments could be an interesting way to go,” added Marc Duque Ramírez, a PhD student in the Engert Lab.
Chen and Duque are co-lead authors of the team’s published paper in Neuron, titled “Ketamine induces plasticity in a norepinephrine astroglial circuit to promote behavioral perseverance.” In their paper the team wrote, “Understanding how fast-acting antidepressants act on non-neuronal cell types to alter their interaction with key behavioral circuits could lead to new insight into the cellular and circuit pathways that go awry during major depressive disorder and provide the means to design new fast-acting, effective therapeutics.”
The newly reported project started when the team, led by Duque and Chen, wanted to see if they could use zebrafish to test antidepressants that were known to work in humans and had previously been tested in rodents. Because zebrafish are small and translucent, researchers can image each animal’s entire brain to better track the drug’s effects. Understanding how antidepressants work on a molecular level has confounded scientists for decades, with much of the work focused on the drugs’ effects on neurons.
The millimeters-long zebrafish may not get depressed exactly like humans do, but the Ahrens Lab had shown in earlier work that these animals exhibit a trait known as futility-induced passivity, or “giving up”—a behavior that has also been seen in rodents, and which is used to study depression. Using a virtual reality setup, the researchers fixed the fish in place and showed them different visual patterns. When the fish saw a pattern simulating backward motion, they wiggled their tails as though swimming forward. When the pattern changed to one simulating being stuck in place, the animals would struggle at first, then give up, become passive, and stop swimming. “Previously, we showed that when swims become futile (i.e., stop moving the fish forward through virtual reality), fish at first produce higher-vigor swims but eventually stop swimming altogether, akin to ‘‘giving up” the team explained.
Example video of a larval zebrafish in the assay for futility-induced passivity or “giving up” behavior. Using a virtual reality setup, the team fixed a larval zebrafish fish in place and showed it different visual patterns. The first 10 seconds show the fish at rest. When the fish sees a pattern simulating backward motion (closed loop), it wiggles its tail as though swimming forward and the pattern responds to the fish’s actions. When the pattern changes to one simulating being stuck in place (open loop), the fish struggles at first, then gives up, becomes passive, and stops swimming. [Duque, Chen, Hsu, et al.]
Previous research by the Ahrens Lab found the giving up action is associated with a type of glial cell called radial astrocytes. As the fish registers that it isn’t getting anywhere, it swims harder, and astroglia activity ramps up. When astroglia activity reaches a threshold, the cells signal to neurons for the fish to stop swimming. “Futile swims enhance firing of hindbrain noradrenergic neurons, and this noradrenergic signal is integrated over time through intracellular calcium signaling in a population of hindbrain astrocytes to suppress swimming,” they continued. Prior findings, they said, “… indicate that there is a noradrenergic-astroglial circuit that integrates information about behavioral futility and induces switching from an active to a passive behavioral state.”
The newly reported research found that a brief exposure to ketamine suppressed the giving up behavior by overstimulating astroglia. This overstimulation, which occurs through ketamine’s stimulation of noradrenergic neurons that activate astrocytes, appears to subsequently reduce the astroglia counter’s sensitivity, causing the fish to continue swimming normally, even when it isn’t getting anywhere.
The researchers demonstrated that ketamine suppressed this giving up behavior from zebrafish for more than a day. Although the animals still struggled when their swimming was not effective, they did not give up as easily and were less passive. “Here we showed that a single dose of ketamine causes a persistent increase in behavioral perseverance primarily by inducing plasticity within a hindbrain neuromodulatory circuit consisting of astroglia and neurons,” they wrote.
The authors also tested other fast-acting antidepressants, such as psychedelic compounds, and found a reduction in passivity similar to that observed with ketamine. Conversely, stress-inducing treatments, such as chronic glucocorticoids, increased the giving up behavior.
Whole-brain imaging revealed that ketamine increased the amount of calcium at the astrocytes, showing that the drug activated these cells for many minutes after administration. The researchers think that although short or fast increases in astroglia calcium might drive giving up behavior, the after-effects of this ketamine-induced flood of calcium reduce the astroglia’s response to the futility signal that drives giving up behavior, making the fish more robust in these behavioral situations—or less likely to give up—in the future.
“It’s desensitized because during ketamine it was so hyperactivated,” said Ahrens, a senior author on the paper. “It’s like when you take a cold shower—afterwards you are a little less sensitive to the cold—but at a cellular and molecular level.”
Example video of a ketamine-treated larval zebrafish in the assay for futility-induced passivity or “giving up” behavior following recovery from the acute effects of ketamine treatment. Using a virtual reality setup, the team fixed the ketamine-treated larval zebrafish fish in place and showed it different visual patterns. The first 10 seconds show the fish at rest. When the fish sees a pattern simulating backward motion (closed loop), it wiggles its tail as though swimming forward and the pattern responds to the fish’s actions. When the pattern changes to one simulating being stuck in place (open loop), the ketamine-treated fish struggles at first, but does not give up as easily and is less passive than an untreated fish. [Duque, Chen, Hsu, et al.]
The researchers also found that this same mechanism was at work in mammals. Co-lead author Eric Hsu, a graduate student at Johns Hopkins, found that astrocytes were similarly activated in mice, both when they exhibit “giving up” behavior and when they are exposed to ketamine. “Results from their studies in both species, they noted, “… indicate that the dynamics of astrocytic activity in mice are remarkably similar to those in zebrafish. And while the behaviors and brain regions are not identical, they pointed out, “… these results suggest that astrocyte engagement during intense arousal states is evolutionarily conserved, opening the possibility for cross-species comparisons of the effects of drugs on the neural pathways that control futility-related state-switching behavior.”
Co-senior author Dwight Bergles, PhD, a professor of neuroscience at Johns Hopkins, further suggested, “This evidence of cross-species conservation increases the likelihood that comparable mechanisms exist in humans.”
The team’s study demonstrated that ketamine is acting on the astrocytes by increasing norepinephrine levels, though how it does that and how that changes neuronal and astroglia physiology as a result is still unknown. But the findings do point to a potential role for astroglia in depression and could help inform future research. “We need to be careful in taking these results too literally, but this could be a model for pieces of the mammalian brain,” Ahrens said.
“The primary implication of these studies is that the long-lasting behavioral effects of ketamine, including its rapid and persistent antidepressant effects, should be re-evaluated in the context of its rapid, profound effects on endogenous monoaminergic neuromodulation and neuron-astroglial communication,” the authors stated. “Our work suggests potential links between astroglial modulation of brain state with previous work on the role of astroglia in both depression and the efficacy of antidepressants.”