Fear really resides in a different area of the brain than its inhibitory mechanisms

Extinguishing the fear response does not consist of erasing the memory of the fear provoking stimuli, but creating new, competitive memory traces. It has been suspected for some time that neuronal brain circuits responsible for extinguishing fear differ from circuits involved in reoccurrence of the fear response. This assumption has finally been experimentally confirmed. Novel experiments, described in the Proceedings of the National Academy of Sciences (PNAS), have been conducted by scientists from the Nencki Institute of Experimental Biology of the Polish Academy of Sciences and the International Institute of Molecular and Cell Biology in Warsaw. This research team was headed by Dr Ewelina Knapska, Dr Jacek Jaworski and Prof. Leszek Kaczmarek.

"Research has been carried out using a special, genetically modified strain of rats developed in the Nencki Institute. As a result we were able to observe the connections between neurons activated in the brains of animals experiencing fear," explains Dr Ewelina Knapska, head of the Laboratory of Emotions Neurobiology in the Nencki Institute.

Fear, a strong, spontaneous reaction of the organism to a given stimulus, is significant in evolutionary terms. An animal experiencing fear has a better chance of survival in unfriendly environment. However, excessive fear causes anxiety disorders, which can significantly hinder functioning of the organism.

Anxiety disorders in humans and animals can be treated by behavioural therapy. It involves exposure to fear triggering stimulus or stimuli in a safe environment. Multiple exposures to such stimuli cause the individual not to react with fear in response to them.

In practice, extinguishing fear is neither permanent nor complete. People with the disorder may well stop reacting to the stimulus in the therapist's office, but any change in the environment, for example entering the street, may cause fear to recur. In as many as 70-80% of people with the disorder fear re-emerges within a few years from treatment completion.

"Earlier studies have suggested that the memory trace of fear is established within the structures of the amygdala, which are controlled by the prefrontal cortex and the hippocampus," says Dr Knapska.

Brain structures responsible for learning and extinguishing fear have developed early in the evolution process and therefore can be studied experimentally in animals, such as rats. For the experiments to be possible at all, a method had to be developed for tracing the reaction of individual neurons to fear stimulus or the lack of it. To achieve this, the rat genome had to be modified. This genetic modification was developed by Dr Jacek Jaworski from the International Institute of Molecular and Cell Biology and Dr Morgan Sheng from the Massachusetts Institute of Technology (MIT).

"In the brains of modified rats, a certain fluorescent protein accumulates in the synaptic endings of active neurons," explains Dr Jaworski. It is known that the accumulated protein remains in activated cells for several dozen hours. That makes it easy to later identify the cells which reacted to a given stress stimulus in microscopic preparations. Illuminated with light of a given range, they glow in green.

Genetically modified rats exhibiting the desired reaction to the stress stimulus were exposed to situations of high or low level of fear. The procedure was designed in such a way as to ensure that the fluorescence marker would accumulate only in the cells which react to the fear triggering stimulus. Analysis of microscope images of the animal brain tissue identified within the lateral nucleus of the amygdala two subpopulations of neurons, partially mixed spatially, but functionally independent.

"We have studied the nervous connections from the prefrontal cortex and the hippocampus to the amygdala. The first two brain structures tell the animal, where and when it has previously encountered the present situation, while the amygdala processes emotions. It turns out that the connections activated at the time of the reoccurrence of the fear reaction are different from those activated when fear is extinguished," explains Dr Knapska.

Research methods developed by the scientists from Warsaw will in the future guide the search for pharmacological agents, which would precisely target individual neuronal networks responsible for fear and its inhibition. That would constitute an important progress in phobia treatment. For compounds developed to date impact not the individual neuronal circuits but entire brain structures. This means that once applied they could erase memory traces in an uncontrolled manner.

"Our method of marking active neurons is flexible. In the future we plan to use it to gain knowledge about neuronal connections typical for behavioural contexts other than fear," stresses Dr Knapska.

Studies have been financed from grants awarded by the Foundation for Polish Science, National Science Centre, 7 Framework Programme of the European Commission and ERA-NET NEURON program co-financed by the National Centre for Research and Development.

The Nencki Institute of Experimental Biology of the Polish Academy of Sciences has been established in 1918 and is the largest non-university centre for biological research in Poland. Priority fields for the Institute include neurobiology, neurophysiology, cellular biology and biochemistry and molecular biology -- at the level of complexity from tissue organisms through cellular organelles to proteins and genes. There are 31 labs at the Institute, among them modern Laboratory of Confocal Microscopy, Laboratory of Cytometry, Laboratory of Electron Microscopy, Behavioural and Electrophysiological Tests. The Institute is equipped with state-of-the-art research equipment and modernized animal house, where lab animals are bred, also transgenic animals, in accordance with the highest standards. Quality of experiments, publications and close ties with the international science community, place the Institute among the leading biological research centres in Europe.