Researchers map brain circuits that signal imminent danger and create memories that help avoid threats

An article in the magazine Current Biology describes experiments to map a brain circuit responsible for the immediate detection of threats and the formation of fear memories.

“We wanted to locate a brain region associated with fear signaling and understand how this region can identify environments previously associated with physical or predatory threats, such as a location where the individual was exposed to an aversive physical stimulus,” said Newton Sabino Canteras, full professor in the Department of Anatomy at the Institute of Biomedical Sciences of the University of São Paulo (ICB-USP) in Brazil and last author of the article.

The hippocampus is known to be involved in spatial navigation and orientation. This brain region also detects threats from the environment. Anything dangerous leaves a trace there, making it possible to calculate the exact location of the threat, Canteras explained. A central structure that is closely connected to the hippocampus is the subiculum.

“The subiculum is basically the star of the show. It transmits information related to environmental threats to the hypothalamus. We wanted to study how it behaves when the animal is exposed to an environment that was previously associated with an aversive stimulus,” he said.

The researchers used fiber photometry to observe activity in the subiculum. “We inserted a virus that expresses a calcium-sensitive protein that is able to register cellular activity. It emits fluorescence in response to cellular activity,” Canteras said.

The behavioral paradigm they used consisted of habituating a mouse to an apparatus with two boxes connected by a corridor. First, the mouse was confined to one box and received aversive physical stimuli (electric shocks to the paws). The next day, it was placed in the apparatus so that it could avoid the aversive box.

“In this type of test, the animal stretches and sniffs, moves in one direction and the other, but does not go back into the box where its paws were shocked. It shows what we call avoidance behavior,” he said.

A photometric analysis focusing on the dorsal premammillary nucleus (PMd), a critical interface of the neuronal circuit under study, showed that the PMd becomes particularly active when the animal approaches and avoids the threatening source.

“It is a very clear threat detector that interacts dynamically with the source. If the mouse turns its back to the source, the PMd is not activated, but if it looks at the source or approaches it, the PMd sounds the alarm. This is one of the most important findings of the study,” he said.

The researchers then inactivated the PMd using a widely used chemogenetic technique called DREADDs, short for designer receptors that are activated exclusively by designer drugs.

“We injected a virus that expressed a receptor (the protein hM4Di) that silenced the PMd in the presence of a certain drug. The drug prevented the cells from firing. As soon as the drug was stopped, they resumed their normal function.

“When the PMd was silenced in this way, the animal drastically reduced its defensive response. Instead of avoiding the threatening box, it went back in as if it were a harmless environment – as if nothing had happened,” he said.

They concluded that PMd inactivation affected both contextual fear responses and fear memory reconsolidation. Therefore, after PMd inactivation, the animal considers the environment safe and moves in it the next day as if there were no risks.

Next, they investigated the functional roles of the main targets of PMd in the nervous system: the periaqueductal gray area or PAG (in the brainstem) and the ventral anteromedial thalamus (in the thalamus).

“There is a technique that allows me to specifically deactivate the projection that goes to the brain stem or the thalamus. I insert a virus into the PMd that expresses a protein that acts as a light-sensitive chlorine channel. When I illuminate the terminal fields, those terminals are silenced and I can manipulate a specific projection pathway,” Canteras said.

The researchers observed that although inactivation of the PAG projection pathway reduced the animal’s defensive behavior, it responded well to fear the following day, showing that fear memory was not impaired.

“Behavioral expression is impaired, but fear memory recovery is not. On the other hand, inactivation of the thalamic pathway has virtually no immediate effect but significantly impairs fear memory recovery,” he said, adding that both events are mainly mediated by different projection sites in the nucleus.

The co-first authors of the article are Juliette Viellard (ICB-USP and Institute of Neurodegenerative Diseases, University of Bordeaux, France) and Fernando Melleu (ICB-USP).

The other co-authors are Alicia Tamais, Alisson de Almeida, Carolina Zerbini, Juliane Ikebara, Karolina Domingues, Miguel de Lima, Simone Motta (all ICB-USP); and Fernando A. Oliveira (Cellular and Molecular Laboratory, Center for Mathematics, Computation and Cognition, Federal University of the ABC).