Researchers from California Institute of Technology have recently described how two different neural subtypes act as a gate that controls the outflow of "fear" from the amygdala region of our brains.

David J. Anderson and colleagues discovered that this fear microcircuit contains two, antagonistic neurons - having opposing functions - which in effect 'see-saw' back and forth to control the 'fear flow'.

"Imagine that one end of a seesaw is weighted and normally sits on a garden hose, preventing water—in this analogy, the fear impulse—from flowing through it," says Anderson. "When a signal that triggers a fear response arrives, it presses down on the opposite end of the seesaw, lifting the first end off the hose and allowing fear, like water, to flow." Once the flow of fear has begun, that impulse is transmitted to other regions of the brain that regulate fearful behavior like your stomach clinching, heart pounding or throat tightening.

"Now that we know about this 'seesaw' mechanism," he adds, "it may someday provide a new target for developing more specific drugs for treating fear-based psychiatric illnesses like post-traumatic stress disorder, phobias, or anxiety disorders."

The research is published this week in the journal Nature.

To unlock this mystery Anderson and his team had to uncover markers in the genes that would identify, and allow them to tell the difference between the two neuronal cell types in the amygdala. Led by postdoctoral fellow Wulf Haubensak, the group found its marker in a gene that encodes for an enzyme known as protein kinase C-delta (PKCδ). PKC-delta is expressed in about half the neurons within a subdivision of the amygdala's central nucleus, the part of the amygdala that controls fear output.

With fellow postdocs Prabhat Kunwar and Haijiang Cai, Haubensak was able to fluorescently tag neurons in which the protein kinase is expressed. This allowed the researchers to map the connections of these neurons, as well as to monitor and manipulate their electrical activity.

The studies "revealed that PKC-delta + neurons form one end of a seesaw, by making connections with another population of neurons in the central nucleus that do not express the enzyme, which are called PKC-delta - neurons," said Anderson. The team also demonstrated that the kinase-positive neurons inhibit outflow from the amygdala - proving that they act like the end of the seesaw resting on the garden hose.

The researchers still didn't have the complete story: What happens to the seesaw during exposure to a fear-cliciting signal? They had hypothesized that the fear-signal would push down on the opposite end of the see-saw - opening the gate, and allowing the fear-signal to flow - but, how could they test this theory?

Enter neurophysiologist Andreas Lüthi and his student Stephane Ciocchi, from the Friedrich Miescher Institute in Basel, Switzerland. In work done independently from that of the Anderson lab, Lüthi and Ciocchi had managed to record electrical signals from the amygdala during exposure to fear-inducing stimuli. Interestingly enough, they had found two types of neurons that responded in opposite ways to the fear-inducing stimulus: one type increased its activity, while the other type decreased its activity. Like Anderson, they had begun to think that these neurons formed a seesaw that controls fear output from the amygdala.

And so the two teams joined forces to determine whether the cells Lüthi had been studying corresponded to the PKC-delta positive and PKC-delta negative cells Anderson's lab had isolated. In what Anderson refers to as a "sophisticated experiment," the two teams performed electrophysiological recordings while simultaneously turning the PKC-delta positive neurons on or off using a genetic method developed by Henry Lester, Caltech's Bren Professor of Biology.

The results of the experiment were "gratifyingly clear," says Anderson. The cells that decreased their activity in the face of fear-inducing stimuli clearly corresponded to the PKC-delta positive neurons Anderson's lab had isolated, while those that increased their activity corresponded to the PKC-delta negative neurons.

"These results supported the hypothesis that PKC-delta positive neurons were indeed at the opposite end of the seesaw from the one that the fear signal 'presses down' on, consistent with the finding that PKC-delta positive neurons crimp the 'fear hose,'" says Anderson.

The marriage of molecular biology and electrophysiology created by the collaboration between Anderson's and Lüthi's laboratories has revealed properties of the fear circuit that could not have been discovered in any other way, Anderson says. "The functional geography of the brain is organized like that of the world," he notes. "It's divided into continents, countries, states, towns and cities, neighborhoods and houses; the houses are analogous to the different types of neurons. Previously, it had only been possible to dissect the amygdala at the level of different towns, or of neighborhoods at best. Now, using these new genetic techniques, we are finally down to the level of the houses."

And that, he adds, is what will make it possible for us to fully understand the networks of communication that exist between neurons within a subdivision of the brain, as well as between subdivisions and different areas. "While these studies shed light on only a small part of the picture, they are an important step in that direction," Anderson says.

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