In a what could be a major step forward in depression research, a team led by BBRF grantees has reported the results of experiments that they say reveals the mechanism through which the rapid anti-depressant effects of ketamine are initiated, within minutes of the drug’s arrival in the brain.

With this cellular mechanism in mind, the same investigators sought to identify novel targets in a specific subset of neurons in the brain’s prefrontal cortex that might be engaged to generate similarly rapid and dramatic antidepressant effects, possibly through “cocktails” composed of multiple drugs. The use of multiple alternate, synergistically acting agents, in theory, increases the likelihood of antidepressant effectiveness while at the same time offers an opportunity to reduce some of the serious side effects that limit the clinical utility of powerful psychoactive drugs like ketamine.

The search for ketamine’s mechanism of action has been a focal point of research for years, since the previously unexpected effectiveness of the drug, originally used in much higher doses as an anesthetic, was discovered in the 1990s. BBRF Scientific Council vice president John H. Krystal, M.D., and Scientific Council member emeritus Dennis S. Charney, M.D., were involved in that pathbreaking research. Dr. Krystal, chair of psychiatry at Yale, is a past BBRF Colvin Prize winner and 3-time BBRF grantee; Dr. Charney is also a Colvin Prize winner.

The new research was led by Conor Liston, M.D., Ph.D., a BBRF Scientific Council member and 2013 BBRF Young Investigator, and Joshua Levitz, Ph.D., at Weill Cornell Medicine. Francis S. Lee, M.D., Ph.D., chair of psychiatry at the same institution and a BBRF Scientific Council member and 3-time BBRF grantee, was another senior member of team. First author of the team’s paper, appearing in the journal Cell, was Hermany Munguba, Ph.D., a 2022 BBRF Young Investigator.

Various theories have been put forward about ketamine’s mechanism of action. The drug is known to block a key receptor called the NMDA receptor in excitatory neurons of the cortex; when injected, it is known to break down into a variety of compounds called metabolites. Yet other drugs specifically inhibiting the NMDA receptor, on their own, have not demonstrated powerful antidepressant effects; nor have various ketamine metabolites, tested individually. It has been suggested, also, that the ketamine molecule in some way interacts with the body’s naturally occurring, or “endogenous” opioid system, perhaps helping to explain its uniquely rapid antidepressant impact.

In a variety of sophisticated experiments involving mouse models of depression (as induced by chronic stress) and a variety of molecular biology techniques, Dr. Liston and colleagues focused their work, most broadly, on mechanisms in the prefrontal cortex (PFC), which is well known to be central in mediating the effects of depression. Within the PFC, the medial portion, or mPFC, was central in the research. Building upon what past research has established about various mechanisms relevant both to depression and ketamine’s impact on the brain, the team further honed their attention on a class of cells called interneurons (INs)—cells comparatively sparse in number that punctuate the physical space in the cortex dominated by much more numerous excitatory (“pyramidal”) neurons. Interneurons act as brakes on neural excitation, the absence of which sends the brain into a seizure state like that seen in epilepsy.

Within the population of interneurons within the mPFC, the team focused still further on those INs that are distinguished from others by their expression of a protein called somatostatin (Sst). This subset of INs is known to regulate the integration of synapses into neural circuits. Synapses are the tiny gaps across which neurons connect with other neurons. There are trillions of them in the human brain.

This makes synapses particularly interesting in the context of depression treatments. Past work in the Liston lab has shown ketamine’s dramatic effect upon the formation of new synapses in the brains of depressed mice. Some are brand new; others are “restored” synapses, lost when the animals undergo a period of chronic stress. Yet Dr. Liston and colleagues found that ketamine’s ability to promote the formation of synapses, while it may be important in its antidepressant action, does not initiate the drug’s impact on behavior—it is only essential in the maintenance of ketamine’s initial impact, which is virtually immediate.

The new research indicates to the team the answer to the mystery of what initiates ketamine ‘s antidepressant impact. The driver of the behavioral effect, they find, is the drug’s impact on Sst-expressing interneurons in the mPFC. Even more specifically, it is in the engagement of a type of opioid receptor called the mu-opioid receptor (MOR) in these particular neurons.

MORs—to make the story a bit more complex—are of a class called G protein-coupled receptors, or GPCRs. This superfamily of receptors is found on cells everywhere in the body and throughout the brain. An estimated 20%-30% of FDA-approved drugs interact with cells by docking at GPCRs, making them intensely interesting to science. When an opioid molecule binds to a mu-opioid receptor, it activates a complex of “G proteins” inside the cell, triggering a series of inhibitory events. This results, ultimately, in an opioid’s ability to reduce pain or induce behavioral effects like euphoria or sedation.

Dr. Liston and colleagues found that when the ketamine molecule binds at a specific subtype of G protein (called G_i/o), it sets off a distinct kind of signaling in the cell—an action they determined to be both necessary and sufficient in itself to induce ketamine’s dramatic antidepressant effect within minutes, in mice that displayed depressed behavior after being subjected to chronic stress.

It is not certain, but probable, that the same mechanism, assuming it is validated in forthcoming research, is also engaged in the human brain, since most important pathways and structures in the mammalian brain have been “conserved” across eons of time by evolution.

This major finding about ketamine was only part of the research reported in the new paper. Other aspects make it broader in application, regarding potential development of new and more effective depression therapies.

Based on their finding of the centrality of GPCR-mediated signaling within Sst-expressing interneurons in the mPFC, both in driving the antidepressant effects of ketamine and the opposing depressive effects caused by chronic stress, the team used molecular biology tools to identify an ensemble of different GPCRs in the same subtype of neurons. They characterized the expression of these cells before and after chronic stress, in so doing revealing many potential targets for antidepressant drugs.

The researchers then tested two such potential targets in Sst-expressing cortical interneurons—experimentally stimulating one receptor, the relaxin-3 receptor, and inhibiting another one, called prokineticin receptor 2, to test their impact on depression symptoms in stressed mice.

Then they made a “cocktail” consisting of three drugs to—they hoped—synergistically target several highly specific cellular targets of this type, based on their model of ketamine’s mechanism of action. Their aim was to reproduce ketamine-like antidepressant effects, but with fewer side effects. Ketamine, in people, can cause dissociation (an out-of-body feeling), blood pressure instability, anxiety, and carries the risk of abuse.

These experiments were preliminary, and intended as a proof of principle for combining subtherapeutic doses of multiple compounds guided by GPCR expression patterns in neuronal subtypes central in initiating rapid antidepressant effects, to selectively engage specific circuit elements while minimizing side effects. Among other things, much more work needs to be done on the cocktail concept, the team acknowledged, studying drug-drug interactions, the ability of components to penetrate the protective blood-brain barrier, as well as the efficacy and durability of any antidepressant effects.

The team also included Joseph M. Stujenske, M.D., Ph.D., 2024 BBRF Young Investigator.