Abstract
AIM: A hallmark of classical conditioning is that conditioned stimulus (CS) must be tightly coupled with unconditioned stimulus (US), often requiring temporal overlap between the two, or a short gap of several seconds. In this study, we investigate the temporal requirements for fear conditioning association between a strong artificial CS, high-frequency optogenetic activation of inputs into the lateral amygdala of rats, and a foot-shock to the animal with delays up to many minutes. METHODS: AAV-oChIEF-tdTomato viruses were injected into the auditory cortex and the medial geniculate nucleus of rats. An optical fiber was implanted just above the lateral amygdala of the animal. Optogenetic high-frequency stimuli (oHFS; containing five 1-s trains of 100 Hz laser pulses) were delivered to the lateral amygdala, before or after (with varying intervals) a foot-shock that elicits fear responses in the animal. Pre-trained lever-press behavior was used to assess the degree of fear recall by optogenetic test stimuli (OTS; 10 Hz for 2 min) 24 h after the association experiment. RESULTS: In contrast to the tight temporal requirement for classical conditioning with paired optogenetic moderate-frequency stimuli (oMFS; 10 Hz for 20 s) and foot-shock, oHFS followed by foot-shock with a 5-min or even 1-h (but not 3-h) interval could successfully establish an association to be recalled by OTS the next day. Meanwhile, foot-shock followed by oHFS with a 5-min (but not 1-h) interval could also establish the conditioning. Thus, distant association may be formed between temporally distant stimuli when the CS is strong.