PMC

Results from Experiment 1. Freezing behavior during the first two conditioning trials is graphed as a function of time, separately for rats in the predictable-shock/short-tone group (n = 10) and rats in the unpredictable-shock/short-tone group (n = 10). Each data point during the tone presentations represents the mean percentage of time that groups of rats displayed freezing behavior during a 2-s period; each data point during the intervals after the tones represents the mean percentage of time that groups of rats displayed freezing behavior during a 10-s period. Error bars indicate ±1 SEM. The asterisk indicates a significant difference between the behavior of the two groups (*p < .05).

Method and results from Experiment 4. Four groups of rats (n = 5–7 per group) received intrahippocampal infusions of muscimol or vehicle before auditory fear conditioning (10-s tone, 2-s foot shock; 70-s intertrial interval), as illustrated in (a). Conditioning was either partially reinforced (50% chance of a foot shock following a tone) or fully reinforced (100% chance of a foot shock following a tone). The next day, all the rats were returned to the conditioning context for 10 min. The day after that, all rats received 14 tone presentations, and auditory fear recall was measured as average freezing across the first two tone presentations for each group. The bar graphs in (b) show the mean percentage of time the rats displayed freezing behavior in each reinforcement-schedule group, separately for contextual fear-memory recall and auditory memory-fear recall. The small open circles represent the percentage of time that individual rats displayed freezing behavior. Error bars represent +1 SEM. Asterisks represent significant differences between groups (*p < .05, **p < .01).

Method and results for Experiment 1. Two of the groups of rats (n = 10 per group) were fear conditioned with five pairings of a 30-s tone with a 1-s foot shock, as illustrated in (a); there was a 210-s intertrial interval (ITI). For the predictable-shock/short-tone group (which received predictable training and a 30-s tone), each tone coterminated with a foot shock. For the unpredictable-shock/short-tone group (which received unpredictable training and a 30-s tone), each tone was paired with a foot shock that occurred pseudorandomly during the tone. Thus, interstimulus intervals (ISIs) were shorter for this group than for the predictable-shock/short-tone group. The other two groups of rats, the predictable-shock/long-tone and unpredictable-shock/long-tone groups, were fear conditioned the same way, except that the tones were 42 s instead of 30 s, as illustrated in (b). For the unpredictable-shock/long-tone group, each tone was paired with a foot shock that occurred pseudorandomly during the tone, so the average ISI was longer for this group than for the predictable-shock/long-tone group. The bar graphs show the mean percentage of time the rats displayed freezing behavior in each (c) short-tone group and (d) long-tone group, separately for contextual fear-memory recall and auditory fear-memory recall. The small open circles represent the percentage of time that individual rats displayed freezing behavior. Error bars represent +1 SEM. Asterisks indicate significant differences between groups (*p < .05, **p < .01).

Method and results from Experiment 2. Three groups of rats (n = 6–8 per group) were fear conditioned with pairings of a 30-s tone with a 1-s foot shock, as illustrated in (a); there was a 3-min interval after the tone. The three conditions had one, three, or six pairings of a tone and a foot shock. The average interval between tone onset and foot shock onset was held constant across the three groups. The day after fear conditioning, all the rats were returned to the conditioning context for 20 min. The day after that, the rats were placed in a novel context, and auditory fear recall was measured as average freezing across the first 2 of 15 tone presentations. In (b), results are shown separately for the two groups. The bars show the mean percentage of time that groups of rats displayed freezing behavior, and the small open circles represent the percentage of time that individual rats displayed freezing behavior. Error bars represent +1 SEM. Asterisks indicate significant differences between the group with one predictable trial and each of the other groups (**p < .01).

Results from Experiment 3. As illustrated in (a), the rats (n = 8–12 per group) received intrahippocampal infusions of muscimol or vehicle before auditory fear conditioning (30-s tone, 2-s foot shock, and 210-s intertrial interval) with foot shocks delivered at either predictable or unpredictable intervals after tone onset. The next day, all the rats were returned to the conditioning context for an 8-min contextual fear-memory test. The day after that, the rats were placed in a novel context, and eight tones were presented (auditory fear-memory test). A representative photomicrograph (b) shows one brain hemisphere after infusion with fluorescent muscimol. The pink fluorescent signal indicates the spread of the muscimol. The bar graphs (c) depict the mean percentage of time the rats displayed freezing behavior over the test sessions for contextual fear-memory recall (left) and auditory fear-memory recall (right). The bars show the mean for each group, and the small open circles represent the percentage of time that individual rats displayed freezing behavior. Asterisks represent significant differences between groups (*p < .05, **p < .01, ****p < .0001). Freezing behavior during the first two conditioning trials (d) is graphed as a function of time, separately for rats from each group. Each data point represents freezing during a 3-s period averaged over the first two trials of tone presentation during extinction. Error bars in (c) represent +1 SEM; error bars in (d) represent ±1 SEM.

Method and results for Experiment 5. Mice without implants (n = 6–7 per group) were fear conditioned with three pairings of a 30-s tone with a 2-s foot shock delivered with predictable or unpredictable timing, as illustrated in (a). Auditory fear recall was then tested in a novel context. The graph in (b) shows mean percentage of time the mice without implants displayed freezing behavior, separately for predictable- and unpredictable-conditioning groups. The small open circles represent the percentage of time that individual mice displayed freezing behavior. The mice in the ArchT groups received a bilateral infusion of an adenoassociated virus (AAV) expressing the silencing opsin ArchT fused to green fluorescent protein (GFP); the mice in the GFP groups received an AAV expressing GFP. Both viruses targeted cornu ammonis (CA) 1 of dorsal hippocampus. The mice that received brain implants were fear conditioned with three pairings of a 30-s tone with a 2-s foot shock, delivered along with 4-s applications of green laser light, under one of three conditions, as illustrated in (c). Mice in the unpredictable-conditioning/light-during-negative-prediction-error group (n = 10–12) received intrahippocampal delivery of green light (λ = 575 nm) during 4-s periods (the 2 s in which foot shock was actually delivered, and an additional second of light delivery before and after the time of the previous foot shock) surrounding the times at which foot shock had been administered on previous trials. Thus, on Trial 2, light was applied during the time at which the foot shock had occurred on Trial 1. On Trial 3, light was applied during the times at which foot shock had occurred on Trials 1 and 2. Mice in the predictable-conditioning/random-light group received intrahippocampal green light at the same times (relative to conditioned stimulus, or CS, onset) as mice in the unpredictable-conditioning/light-during-negative-prediction-error group, but foot shock had never occurred at these times for the predictable-conditioning/random-light groups. Mice in the predictable-conditioning/random-light group (n = 5–8) received intrahippocampal green light during 4-s periods in which foot shock had never been delivered. Expression of GFP after infection with ArchT is shown for a representative brain section in (d). The white overlay indicates the location of the fiber-optic tip and the estimated light spread. DG = dentate gyrus. Auditory fear-memory recall was measured across two tone presentations during a subsequent laser-free extinction session. The bar graphs show mean percentage of time that the mice displayed freezing behavior as a function of infusion type for (e) the unpredictable-conditioning/light-during-negative-prediction-error group, (f) the predictable-conditioning/random-light group, and (g) the unpredictable-conditioning/random-light group. The small open circles represent the percentage of time that individual mice displayed freezing behavior. Error bars indicate +1 SEM. Asterisks represent significant differences between infusion conditions (*p < .05, **p < .01).