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As depicted in Figure 2A, on postnatal day 70 animals were habituated to the conditioning context for 10 min. And 24 h after habituation, animals were given a cued fear conditioning session. Fear conditioning started with a 2-min habituation period, followed by five instances of a 30-s 85 dB 2.8 kHz auditory stimulus co-terminating with a 1-s 0.6 mA foot shock, followed by a 1-min inter-trial interval. And 24 and 48 h after conditioning, fear extinction and extinction recall were assessed. During extinction recall, rats were exposed to a 2-min habituation period, after which 20 presentations of 20 seconds of the auditory stimulus were given, with an inter-trial interval of 5 s. The freezing was automatically assessed by the Ethovision 9.0 behavioral software package (Noldus Information Technology B.V., Wageningen, the Netherlands) and was determined using the Activity Monitor feature of the software package, blind to the genotype of the animal. The apparatus was cleaned before and after each animal using a tissue slightly dampened with 70% EtOH.
Fear conditioning behavioral results during the extinction recall sessions. (A) Schematic representation of the experimental paradigm. (B,C) The data are expressed as the mean of the time spent freezing during conditioned stimulus presentations SEM of 10 independent determinations. *p
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To analyze fear extinction learning, extinction sessions were divided into five blocks representing the average freezing responses to four auditory cue presentations each. Average freezing to all auditory cue presentations during the recall sessions was used as an index for fear extinction recall.