To find these elusive patterns of activity, the Stanford team employed a strategy rooted in evolutionary biology. Rather than trying to decode every signal in the incredibly complex human brain, they looked for patterns that were conserved across species. If a brain-activity pattern is present in both humans and mice—species that diverged from a common ancestor 70 million years ago—it is likely essential for survival.
"This approach allowed us to focus our study on the key principles that were shared between mice and humans," said lead co-author Isaac Kauvar.
The researchers sought activity patterns that met a strict set of criteria: they had to be induced by the same stimulus, measurable in the same way, synchronized with the same behaviors, and blocked by the same pharmacological interventions in both species.
The "Eye Puff" Experiment
To trigger a measurable emotional response, the researchers needed a stimulus that was safe, reproducible, and effective for both mice and humans. They found the answer in a common ophthalmological tool: the "eye puff" device used by eye doctors to check for glaucoma.
While a puff of air to the eye is not painful, it is inherently unpleasant. For the study, the team administered series of precisely timed puffs to participants. When asked to describe the experience, human subjects labeled the puffs as "annoying," "unpleasant," and "uncomfortable." Critically, repeated puffs led to a cumulative feeling of annoyance that lasted even after the stimulus stopped—a hallmark of an emotional state.
This state, while irritating, is adaptive. Deisseroth noted that any repeated negative event is information the brain must use to guide future behavior.
To record the brain activity during these events, the researchers worked with a unique cohort of patients at Stanford Hospital. These individuals suffered from medication-resistant epilepsy and had already undergone surgery to have electrodes implanted deep within their brains. These electrodes allow neurologists to locate the "focus" of a patient’s seizures, but they also provide a rare opportunity for scientists to observe human brain activity at high resolution.
"These patients typically spend about a week in a hospital bed with limited mobility… while the treatment team waits for spontaneous seizures to occur," said lead co-author Tony Liu. During these waiting periods, the patients volunteered to participate in the eye-puff study.
The Two-Phase Pattern
As the participants reacted to the air puffs, the researchers tracked their brain activity and physical responses. They observed a distinct two-phase neural pattern.
The first phase occurred within roughly 200 milliseconds of the puff. This was a sharp, short-lived spike of activity that functioned like a "news broadcast," alerting the entire brain to the sensory event. Immediately following this, the subjects would reflexively blink.
The second phase was more complex. Lasting for about 700 milliseconds, this longer phase was localized to specific emotion-related circuits across the brain. During this period, subjects exhibited "post-puff" eye squinting or rapid additional blinks—behaviors that were not reflexive but rather a self-protective, emotional response to an unpleasant situation.
When the team ran the same experiment on mice, they found the exact same two-phase pattern. Just like the humans, the mice showed a cumulative negative emotional state after repeated puffs, evidenced by a decreased interest in seeking rewards—a classic sign of a "bummed-out" state.
