The fear conditioned response under head fixation

In classic measures of fear induced freezing behavior, animals stop all movement except breathing. Many large-scale recording techniques in modern neuroscience such as wide-field and two-photon fluorescent imaging require animals be in a head-fixed preparation. Here we demonstrate that it is possible to measure freezing behavior in head-fixed mice based on video measurements of motion, the pupillary response, and electromyography of neck muscles. Animals were either conditioned to form an association between a tone (conditioned stimulus; CS) and a footshock (unconditioned stimulus; US) or were presented both tones and footshocks explicitly unpaired. Animals who were conditioned to form an association showed less movement, and a stronger pupil response when presented with the CS. We outline key considerations which are likely important in establishing a strong fear response in a head-fixed preparation.