Anesthetics have been used in surgery since the mid-1800s, but their exact mechanisms of action continue to be the subject of intense investigation. Past studies have suggested they block neurotransmitter receptors postsynaptically. Now a new study, published January 9 in Cell Reports, uncovers a novel presynaptic mechanism by which general anesthetics directly interfere with neurotransmitter release to induce an apparent state of anesthesia.

“Anesthesia is at least a two-step process,” explains study author Bruno van Swinderen, associate professor of neuroscience at the University of Queensland Brain Institute in Australia. “A better way of looking at it is that there are two categories: the presynaptic effects and the postsynaptic effects.”

Early general anesthetics, namely chloroform, ether, and nitrous oxide, first gained popularity as recreational drugs. But ever since their first documented and highly successful use in surgery in the 1840s, scientists have been trying to figure out exactly how they work.

The first major clue as to their mechanism of action came in the 1990s when researchers, as reported in this 1994 paper and others, found that general anesthetics bind to the brain’s abundant GABA A receptors, located in the plasma membrane of postsynaptic neurons. There, they inhibit synaptic activity and induce a state of anesthesia. But that was only the start of the story. “GABA A is clearly important, but it’s not everything,” says Roderic Eckenhoff, professor of anesthesiology at the University of Pennsylvania Perelman School of Medicine in Philadelphia, who was not involved with the current study. “You can still produce anesthesia in animals with malfunctioning or deleted GABA A receptors.”

The recent paper attempted to elucidate the presynaptic contributors. Two of the most commonly used modern general anesthetics, propofol and etomidate, are promiscuous, interacting with a long list of neuronal receptors as well as presynaptic proteins. The latter interested van Swinderen. Previous studies had shown that presynaptic SNARE proteins bind anesthetics. SNAREs are located in the presynaptic neuron and are essential for neurotransmitter release. The studies also showed that a mutation in at least one SNARE protein, syntaxin 1A, confers resistance to anesthetics. These results prompted van Swinderen and colleagues to investigate how anesthetics might interfere with the SNARE machinery.

Because neurotransmitter release involves so many different proteins, the researchers had to find a way to study the SNARE proteins in isolation. To do so, they used photo-activated localization microscopy (PALM), a technique that allows observation and tracking of individual molecules in living cells. When they exposed cultured rat neurons and live Drosophila larvae to clinical doses of propofol and etomidate, PALM revealed that the drugs increased clustering of syntaxin 1A at the presynaptic neuronal membrane. This traffic jam impeded the ability of syntaxin 1A to interact with another critical SNARE protein, SNAP-25, resulting in partial inhibition of neurotransmitter release.

“This is a major new piece of information since it shows an interaction between anesthetics and SNARE proteins in living cells,” says Zheng Xie, an associate professor of anesthesia and critical care at the University of Chicago, who was not involved with the study.

When the researchers conducted similar experiments using two propofol analogs that are nearly identical but do not induce anesthesia, these compounds decreased syntaxin 1A clustering and improved its mobility. This further suggested that propofol exerts its anesthetic effects by interfering with SNARE protein machinery, something the analogs failed to do.

“It’s never been accepted that a potential target of anesthetics could be the presynaptic machinery,” van Swinderen says. “If we could learn how to interfere with a step in SNARE formation safely, it could potentially be a new class of drugs.” The authors also plan to investigate whether other categories of anesthetics function similarly and what other cellular mechanisms may be involved.

Xie suggests that much work remains before researchers can unequivocally show that propofol produces anesthesia by inhibiting the neurotransmitter release machinery directly. “The authors show, convincingly, that propofol directly interacts with release machinery proteins,” Xie says. “Whether this plays a role in the ‘anesthetic state’ must wait for studies in animals, as proposed by the authors themselves.”