Expression of freezing and fear-potentiated startle during sustained fear in mice.

Fear-potentiated acoustic startle paradigms have been used to investigate phasic and sustained components of conditioned fear in rats and humans. This study describes a novel training protocol to assess phasic and sustained fear in freely behaving C57BL/6J mice, using freezing and/or fear-potentiated startle as measures of fear, thereby, if needed, allowing in vivo application of various techniques, such as optogenetics, electrophysiology and pharmacological intervention, in freely behaving animals. An auditory Pavlovian fear conditioning paradigm, with pseudo-randomized conditioned-unconditioned stimulus presentations at various durations, is combined with repetitive brief auditory white noise burst presentations during fear memory retrieval 24 h after fear conditioning. Major findings are that (1) a motion sensitive platform built on mechano-electrical transducers enables measurement of startle responses in freely behaving mice, (2) absence or presence of startle stimuli during retrieval as well as unpredictability of a given threat determine phasic and sustained fear response profiles and (3) both freezing and startle responses indicate phasic and sustained components of behavioral fear, with sustained freezing reflecting unpredictability of conditioned stimulus (CS)/unconditioned stimulus (US) pairings. This paradigm and available genetically modified mouse lines will pave the way for investigation of the molecular and neural mechanisms relating to the transition from phasic to sustained fear.

Standardizing the analysis of conditioned fear in rodents: a multidimensional software approach.

Data comparability between different laboratories strongly depends on the individually applied analysis method. This factor is often a critical source of variation in rodent phenotyping and has never been systematically investigated in Pavlovian fear conditioning paradigms. In rodents, fear is typically quantified in terms of freezing duration via manual observation or automated systems. While manual analysis includes biases such as tiredness or inter-personal scoring variability, computer-assisted systems are unable to distinguish between freezing and immobility. Consequently, the novel software called MOVE follows a semi-automatized approach that prefilters video sequences of interest for the final human judgment. Furthermore, MOVE allows integrating additional data sources (e.g. force-sensitive platform, EEG) to reach the most accurate and precise results. MOVE directly supports multi-angle video recordings with webcams or standard laboratory equipment. The integrated manual key logger and internal video player complement this all-in-one software solution. Calculating the interlaboratory variability of manual freezing evaluation revealed significantly different freezing scores in two out of six laboratories. This difference was minimized when all experiments were analyzed with MOVE. Applied to a genetically modified mouse model, MOVE revealed higher fear responses of CB1 deficient mice compared to their wild-type littermates after foreground context fear conditioning. Multi-angle video analysis compared to the single-camera approach reached up to 15% higher accuracy and two fold higher precision. Multidimensional analysis provided by integration of additional data sources further improved the overall result. We conclude that the widespread usage of MOVE could substantially improve the comparability of results from different laboratories.