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Computation of innate threats and defensive behaviour in the mouse

Periodic Reporting for period 1 - defence_SC (Computation of innate threats and defensive behaviour in the mouse)

Reporting period: 2017-09-01 to 2019-08-31

When faced with imminent threats, choosing the correct behaviour at the right time is crucial for survival. Animals have to constantly process information from the environment, and correctly identify not only potentially harmful situations, but also the defensive response most likely to succeed. For example, is there anywhere to hide? Is there enough time to escape? In this project we study how the brain implements defensive behavioural choices. Neurons use electrical signals to collect information from the environment, communicate with other neurons, and form behavioural responses. The aim of the study is to measure the electrical signals in single neurons when mice face threatening cues and have to decide whether to escape or freeze, and to understand the activity patterns that control the choice of each behaviour.
Recent developments in recording and behavioural techniques, which are used throughout this project, provide a unique opportunity for understanding the basic biology of how the brain makes decisions, at the level of individual neurons. This is important for understanding the mechanisms by which the brain generates behaviour, and is also the level of resolution necessary for devising therapeutic strategies for mental health problems, such as anxiety. Anxious individuals often perceive the environment as over-threatening, and engage in defensive actions too often in detriment of other behaviours. Thus, understanding how neuronal activity causes defensive behaviour in the mouse has the potential to have a positive impact in the treatment of anxiety disorders in humans.
In this project we investigated how the decision between escape and freezing is computed at the neuronal and synaptic level in two midbrain circuits that are critical for organizing defensive responses in the mouse. We studied defensive behaviour using a paradigm that was established in the host laboratory, in which mice are faced with either threatening ultrasonic sweep or visual looming stimuli that mimick an approaching predator or object from above. When a shelter is placed in the arena, mice rapidly escape to the shelter when faced with either threatening cue. However, when the shelter is removed, in response to the same threatening cues, mice freeze instead, in order to avoid detection.
We developed a novel cutting edge electrophysiological and behavioural setup to translate the freely-moving innate behviours into head-fixed preparation for the purpose of intracellular recordings of neuronal activity. We built a state-of-the-art set up (Figure 1), in which awake mice are head-fixed and placed on a light-weight platform which contains a shelter, and is floating on an air table (Neurotar Mobile Home Cage). Mice are able to move the platform with their paws, and therefore fictively walk and explore the environment. During exploration, the threatening stimuli are presented, which elicit defensive responses comparable to those observed in freely behaving mice. Two electrophysiological recording techniques were used: high-density Neuropixels silicon probes, to record simultaneously extracellular action potentials from hundreds of neurons, across all layers of the relevant midbrain structures; and whole-cell patch-clamp, to record sub- and supra-threshold events in single neurons during behaviour.
Using this set up we found neurons in the superior colliculus and in the periaqueductal gray that respond to threatening auditory stimuli, visual stimuli, or to both (comprising about 40% of the neurons). We could then divide them further by the nature of the response, which could either be a phasic response at the beginning and at the end of the stimulus, or a persistent response lasting longer then the stimulus presentation. Other neurons were found to respond exclusively during the defensive behavior and not during stimulation that did not cause a behavioural response, indicating that they are behavior neurons. Current efforts are focused on collecting more data to understand the mechanisms underlying the neuronal activity patterns during escape and freezing episodes.
These findings were presented to the scientific community both at the institutional level, and at international workshops and conferences. The researcher will keep on disseminating the results to the wider public through lectures and various outreach activities.
For the purpose of the project we have developed a unique state-of -the-art setup which allows us to record with two innovative techniques from neurons in the mouse brain while they are awake, and engaged in an innate behaviour, choosing between escape and freezing when faced with imminent threatening stimuli. The system allows recording of spiking activity from hundreds of neurons simultaneously and intracellular recordings from single neurons, with synaptic resolution. During the project period, the researcher advised and guided other researchers in the field on different aspects of the system, and participated as a teaching assistant in the international high-density Neuropixels probe course. Once published, this new technique and the scientific results from the project will be available for the research community to use. Moreover, the results will advance our understanding of one of the most evolutionary conserved behaviours, observed in species ranging from flies to deer and humans and will contribute to our knowledge of how brain circuits compute decisions. In addition, they may also provide knowledge that important for devising therapeutic strategies for mental health disorders in humans.

Figure 1: electrophysiological recordings during innate bahaviour in the Neurotar arena. A. The recording setup consists of an air table, a bridge onto which the mouse is head-fixed, and a light-weight carbon cage with a shelter. Two high speed cameras used from above and from the side of the animal, and looming spots or ultrasonic sweeps are played using a monitor and an ultrasonic speaker. B. Left, example of mouse track over 6 minutes of exploration in the arena. Right, example of speed trace when the mouse is escaping from a looming stimulus. C. Example of single unit recordings in the PAG with the Neuropixels probe during presentation of an ultrasonic sweep (grey). Top, peristimulus time histogram. Bottom, raster plots of 5 trials. D. Example of whole cell-recording from a PAG neuron during movement in the arena.