Final Report Summary - FEAR MEMORY TRACE (Cellular mechanisms underlying formation of the fear memory trace in the mouse amygdala) Summary of project objectives: Memory allocation is a newly defined phase of the memory process which refers to the recruitment of specific neurons in a network that will encode a specific memory. The purpose of the project is to investigate the synaptic properties between amygdala neurons and determine the underlying biophysical mechanisms that allow neurons to compete with each other for encoding a specific memory, by combining state-of-the-art in vivo genetic manipulation, behavioural, electrophysiological as well as computational approaches. The objectives of the project include a) identifying the electrophysiological properties of neurons encoding the same memory, and b) to determine the contribution of specific biophysical mechanisms involved in manifesting the plasticity changes only to a specific subset of neurons. Description of the work performed during the project: 1) Training in and performing behavioural experiments. The fellow was extensively trained to perform auditory fear conditioning and conditioned taste aversion. 2) Training in intra-amygdala surgeries and patch-clamp recording from the transfected neurons. The fellow observed and participated in the design of intra-amygdala surgeries using viral vectors under the supervision of Dr. Balaji Jayaprakash, post-doctoral fellow in the Silva lab. 3) Performing of electrophysiological experiments with regards to biophysical property changes following behavioural training. The fellow performed several recordings from amygdala neurons following auditory fear conditioning in order to study changes in neuronal excitability following training. 4) Training in molecular and cellular techniques. Under the supervision of several members of the Silva lab, the fellow was trained in molecular and cellular techniques, such as western blot, genotyping, and viral DNA preparation. The fellow has constructed a large scale model network of amygdala and has run simulations that test the hypothesis that biophysical alterations following acquisition of one memory. 5) Construction and simulations in a model network of the prefrontal cortex. The fellow participated and supervised the construction of a model network of the prefrontal cortex that included the distinct interneuron cell types. 6) Initiation of electrophysiological studies at IMBB-FORTH. The fellow has initiated electrophysiological experiments in brain slices, performing either field recordings or current-clamp recordings from brain slices of the prefrontal cortex, hippocampus or amygdala. 7) Initiation of behavioural studies at IMBB-FORTH. The fellow has initiated behavioural experiments studying anxiety, with the open-field test and the elevated plus maze, working memory with the delayed alternation in the T-maze, emotional memory with context or auditory fear conditioning, reference memory with the left-right discrimination in the T-maze and recognition memory with three different types of object recognition tests.Description of the main results:• The fellow participated in a series of behavioural tests in order to test the short-term and long-term memory of mice, using sequential training in fear conditioning first and conditioned taste aversion after 3 or 5 hrs and found increased memory performance at the 3hr time point.• The fellow performed several electrophysiological recordings following behavioural training in auditory fear conditioning and conditioned taste aversion, and recorded measurements of intrinsic excitability, such as the input resistance, action potential properties and number of spikes elicited. She found that the intrinsic excitability of mice that received both the shock and the tone was increased3 hours, but not 5 hours, compared to mice that only received the tone. • The fellow conducted experiments on amygdala neurons transfected with channelrhodopsin and green fluorescent protein to determine the functionality of channelrhodopsin in the viral transfections used for behavioural experiments, and found that channelrhodopsin activation allowed the emergence of spikes.• The fellow conducted combined optical and electrophysiological experiments in cfos-GFP mice following auditory fear conditioning in order to distinguish between neurons activated during the short-term memory recall and determine whether their intrinsic excitability was similar. She recorded a few number of cells (n=4 for each of the four group), however, the technical difficulties along with time limitations did not allow for further experimentation. • The fellow constructed a large model network of the basolateral amygdala that incorporated plasticity mechanisms and simulated the learning-induced changes based on the data acquired by the fellow. The simulation results show that the observed physiological changes could explain the enhanced memory performance in mice, suggesting that physiological changes during a mneumonic episode could alter subsequent memories.• The fellow has initiated several transfer-of-knowledge activities since her return from UCLA. She has set up electrophysiological recordings in brain slices and is in the process of setting up recordings in combination with fluorescence imaging, as well. In addition, she has set up a behavioral facility that allows testing for several memory tasks and recording the anxiety status and locomotor activity of the animal.Expected final results and their potential impact and use: The final results of this project include (a) the identification of biophysical mechanisms involved in recruiting individual neurons for participation in the memory trace, (b) the development of computational models that will allow us to assess the role of these mechanisms in memory performance. Our results from both experimental and computational approaches show that acquisition of a specific memory alters the underlying neuronal networks in the brain in such a way that acquisition of a subsequent memory can be altered (in our case enhanced). These are very initial data to a field that is likely to advance in the next few years and could lead to developing new treatments/approaches for memory deficits by identifying new mechanisms for medical intervention. In addition, the fellow has contributed to extensive transfer-of-knowledge activities that will advance research in the region of Greece.