Freezeframe software

Contextual fear conditioning test was adapted from previously described protocols (Kimura et al., 2010 (link); Montagne et al., 2018 (link)). The experiments were performed using a standard conditioning chamber equipped with a stainless-steel grid floor attached to a shock source that was controlled by FreezeFrame software (Coulbourn Instruments), and a digital camera was mounted on the ceiling to monitor behavior. During training, mice were placed in the conditioning chamber for 5 min and received four footshocks (0.4 mA, duration 1 s) 1 min apart starting after 2 min. Contextual memory was tested in the same chamber the next morning without footshock. The automated FreezeFrame system was used to score the percentage of total freezing time with a threshold set at 10% and minimal bout duration of 0.25 s.

Contextual fear conditioning tests were conducted in the same manner as previously described [36] (link). Briefly, mice were first habituated for 30 minutes in the behavioral suite and then placed in fear conditioning chambers. A training protocol was administered using Freeze Frame software (Coulbourn Instruments), which consists of two instances of a low tone paired with a mild electric foot shock (0.7 mA) in a 3-minute time frame. The amount of time that the mice were immobile, designated as “freezing” in this protocol was recorded by the software. For short-term recall, mice were placed back in the same chamber without a tone or foot shock, to measure contextual fear conditioning 2 hours after testing. For long-term recall, mice were again placed in the same chamber 24 hours after testing. Both outputs were measured using Freeze View software (Coulbourn Instruments) after individually adjusting a baseline freezing threshold for each mouse that was tested.

We conducted fear conditioning and testing following previous methods.8 (link)
For fear conditioning, mice underwent 5 trials of a conditioned stimulus (tone; 80 dB, 6000 Hz) co-terminated with a foot shock (0.8 mA, 2 s). We used an intensity of 1 mA in the chemogenetic experiments. Each tone lasted for 30 s, with a 90 s intertrial interval.
For recall tests (1, 2 and 3), mice were placed in a different cage with a different shape (35 × 20 × 20 cm) from the fear conditioning context, and 3 tones were presented with a 90 s intertrial interval. Recall 1 took place 24 hours after fear conditioning to determine the formation of fear memory. To test fear memory expression recalls 2 and 3 took place 24 hours and 10 days after fear extinction, respectively.
For fear extinction training, 15 tones were presented. Each tone lasted for 30 s, with a 60 s intertrial interval. Mice were placed in the same context as for recall tests.
The primary outcome measured was freezing time. Freezing was defined as the complete absence of movement except for normal respiration. Freezing time was defined as the percentage of total time in a state of freezing during tone presentation and was calculated automatically using FreezeFrame software (Coulbourn Instruments).8 (link)