DAT isn’t all that: Cocaine also activates immune cells in the brain, increasing reward and addiction

You’ve probably heard of dopamine before. Whether you know every region of the brain by heart or are still learning how to tell gray matter from the spinal cord, dopamine is likely on your mental radar. As this fun (and informative) article from Slate describes, dopamine is involved in experiences of pleasure, reward, love, and addiction, among other processes in the brain. It’s kind of a big deal. Dopamine isn’t love or addiction itself: how dopamine shapes these experiences is complex and not yet fully understood.

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Strictly, dopamine is a neurotransmitter: a molecule that gets released from a neuron in the brain into the synapse, or the small space between neurons where they interact, and then binds to the surface of another neuron in order to cause a cellular response. In essence, neurotransmitters are little messengers that carry signals between neurons.

Despite the litany of complex functions it has in the brain, dopamine’s role is well established in a number of processes. Dopamine gets released in response to pleasurable stimuli, and is involved in the brain circuit that makes us take note and remember everyday rewards like tasty food or fun social interactions. It is also critical for the response to addictive drugs. For some drugs, like cocaine, the drug itself affects dopamine in the reward circuit in the brain.

Cocaine binds to and blocks dopamine transporters, or DATs, proteins that pump dopamine out of the synapse between neurons. But cocaine also causes changes in immune cells in the brain that have been more difficult to explain. Researchers in the Department of Psychology and Neuroscience at CU Boulder recently discovered that cocaine’s interaction with dopamine transporters does not fully explain addiction. Cocaine can also interact with pattern-recognition receptors, first activating immune cells in the brain and ultimately changing dopamine levels and behavior.

The brain’s immune system is made almost entirely of microglia, or brain macrophages, which are tightly tuned to gobble up any offending protein build-up or infectious agents without causing excessive damage. Like macrophages in other parts of the body, microglia are coated with pattern-recognition receptors that become activated when they bind to various danger signals. This activation sends a signal into the immune cell, ultimately causing the cell to release molecules that cry “danger” to other cells in the area. Overall, researchers refer to this process as inflammatory signaling, and it can have a huge range of effects, from the redness and swelling you see when you cut your finger to changes in gene expression within cells.

One such pattern receptor is Toll-like receptor 4, or TLR4, and its partner protein MD-2. This duo can bind to a huge range of danger signals produced by stressed cells, pathogens, or molecules introduced from outside the body. Alexis Northcutt, a graduate student researcher in Dr. Maier and Dr. Watkins’ lab, made a canny guess. Since TLR4 can bind to so many molecules, and because it is so important for turning on inflammation, it might play a role in cocaine’s effect on the brain.

Northcutt and her coworkers discovered that cocaine interacts with TLR4, triggering inflammatory signaling and altering behavior in experimental animals. Their findings were reported in the paper, “DAT isn’t all that: cocaine reward and reinforcement require Toll-like receptor 4 signaling”, recently published in Molecular Psychiatry.

First, Northcutt followed up that hunch by aligning the known structures of TLR4 and MD-2 with that of cocaine, showing that cocaine can bind to these proteins like other molecules that activate the immune system. They also observed that drugs used to treat overdose and addiction, like naloxone, fit into the same structure and may block cocaine from binding.

To confirm this observation, the researchers tested the interaction between cocaine and MD-2 using an in vitro, purified system. They found that cocaine binds to TLR4 and MD-2. Cocaine competes with a bacterial molecule that activates the immune system through TLR4, suggesting that cocaine might turn on immune cells like other danger signals. What happens to microglial cells, those brain immune cells expressing TLR4 and MD-2, when cocaine is present? Just like bacterial molecules, cocaine turns on the expression of genes involved in inflammatory signaling.

In order to really know how cocaine affects TLR4 and MD-2 pathways in the brain, Northcutt and coworkers had to move from the relatively pared-down experimental microglial cell culture system to the more complicated brains and behaviors of mice and rats.

First, they found that in one region at the center of the brain’s reward circuit, cocaine treatment in rats increased expression of the inflammatory cytokine IL1β – which is turned on by signals from TLR4. Treatment with naloxone, which can block cocaine from interacting with TLR4, prevented IL1β expression in that brain region after cocaine treatment.

When the researchers treated rats with cocaine, dopamine levels spiked in the brain – a result they expected based on previous research. Adding the TLR4-blocking drug, naloxone, brought dopamine levels down. They also found that blocking inflammatory signaling by blocking the cytokine IL1β from signaling to other cells also reduced dopamine levels.

This suggests a preliminary pathway: cocaine binds to TLR4 and MD-2, turning on expression of the inflammatory cytokine IL1β. IL1β gets released from microglia, interacts with other cells, and triggers them to release more dopamine. But that’s not the whole story: cocaine leads to more dopamine release than other molecules that bind to and activate TLR4 and MD-2. This is likely due to its previously known role in blocking dopamine from getting sucked back into neurons through dopamine transporters (DATs).

Once the researchers determined that cocaine causes changes to inflammatory signaling in the brain, they looked at how these changes altered behavior in experimental animals.

Northcutt used conditioned place preference experiments to determine if treatment with cocaine, or a mix of cocaine and inhibitors, would change rats’ preference for a particular compartment in an experimental box. Rats were treated with cocaine or cocaine plus inhibitors in one of two box compartments for a few days in a row. Then they were allowed to explore the two compartments on their own without drug treatment. Rats conditioned with cocaine strongly preferred the treatment compartment where they were injected with the drug. However, when treated with naloxone first, which blocks cocaine from binding with TLR4 and MD-2, the rats no longer preferred the treatment compartment. They were no longer conditioned – suggesting that TLR4 might be important for drug-seeking behavior.

How does this relate to human behavior where cocaine is self-administered? To address this, the researchers trained rats to press a lever to serve themselves food. Then, the rats were moved to a setup where pressing a lever would both turn on a light and trigger an injection: with a control, with cocaine, or with cocaine and the TLR4 inhibitor naloxone. With the inhibitor present, the rats pressed the lever at a slower rate, triggering cocaine injections less often.

In a similar experiment, mice were trained to poke their noses into small portals that would dispense sugar solution they could lick up. Like before, some mice were injected with cocaine when they nose-poked for sugar. In order to determine how important cocaine binding and signaling through TLR4 was for the cocaine-seeking response in this experiment, the researchers compared mice with a functional TLR4 signaling pathway to those with a defective TLR4 pathway. In these mice, cocaine could bind to TLR4, but that binding event wouldn’t turn on IL1β expression or other aspects of inflammatory signaling. Mice with a non-functional TLR4 signaling pathway triggered fewer cocaine injections than those mice where cocaine could activate TLR4.

Taken together, these experiments show that cocaine binding to TLR4 is one way that cocaine changes behavior in mammals, suggesting that this inflammatory effect could be important for drug addiction in humans.

In general, inflammatory signaling in the brain excites neurons, leading to greater release of neurotransmitters like dopamine. And since dopamine is so important for reward circuits in the brain, drugs like cocaine that turn on inflammation can have compounded effects on experiences of reward, pleasure, and addiction.

Northcutt found that cocaine’s effect on dopamine levels and behavior isn’t just because it interacts with dopamine transporters on neurons, but because it alters the overall state of the immune cells in the brain. Others have found similar synergistic effects on the brain for opioid drugs like morphine. These discoveries might change how researchers think about addiction and study how drugs interact with the brain. They may also help us find new ways to help treat addiction.

As we’ve learned over the past few decades, drug addiction isn’t a personal or moral weakness. It can’t be overcome with speeches, harsh laws, or wanting it badly enough. To cure addiction, people need concrete psychological and medical treatments. By figuring out exactly what happens in the brain during drug use, researchers step closer to developing medication that can treat drug abuse. The inflammatory signaling pathways flipped on by cocaine binding to TLR4 might be useful targets for future drugs to treat overdose or addiction.

By Bridget Menasche

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