A metaplasticity-like mechanism supports the selection of fear memories: role of protein kinase a in the amygdala

Abstract

How the brain determines which memories are selected for long-term storage is critical for a full understanding of memory. One possibility is that memories are selected based on the history of activity and current state of neurons within a given memory circuit. Many in vitro studies have demonstrated metaplasticity-like effects whereby prior neuronal activity can affect the ability of cells to express synaptic plasticity in the future; however, the significance of these findings to memory is less clear. Here we show in rats that a single pairing of a light with shock, insufficient to support either short- or long-term fear memory, primes future learning such that another trial delivered within a circumscribed time window lasting from ∼60 min to 3 d results in the formation of a long-lasting and robust fear memory. Two adequately spaced training trials support long-term fear memory only if the two trials are signaled by the same cue. Furthermore, although a single training trial does not support formation of an observable fear memory, it does result in the phosphorylation of several targets of protein kinase A (PKA) in the amygdala. Accordingly, blocking PKA signaling in the amygdala before the first training trial completely prevents the ability of that trial to facilitate the formation of long-term fear memory when a second trial is delivered 24 h later. These findings may provide insight into how memories are selected for long-term storage.

Figure 1.

Optimal trial-spacing permits long-term memory formation using two training trials. A, Rats received a single pairing of light that predicted shock (n = 16) or two trials separated by intertrial intervals ranging from 4 min to 30 d (n's = 8–30/group) and memory was tested 2 d following the last trial. B, Spacing the trials by 60 min to 3 d resulted in a very robust memory. C, D, Superior memory in rats trained with a 60 min interval versus those trained with a single trial or two trials spaced by 4 min is still observed when time in the training chamber is equated. E, Levels of contextual fear did not differ between groups in rats left in during the ITI. F, Rats trained with a 60 min ITI and removed during the ITI showed higher levels of contextual fear compared with the group trained with a single trial. G, Rats were trained with a single trial and tested for short term memory 1 h later and long-term memory 2 d later. Another group was only tested for long term memory. H, Rats showed no evidence of short or long term memory. I, Rats pre-exposed to the light showed similar levels of fear as those not pre-exposed to the light cue (*p < 0.05 vs 1 trial; #p < 0.05 vs 4 min). In all graphs, error bars indicate ±SEM.

Figure 2.

Fear memory in rats trained with a 60 min or 24 h intertrial interval is long lasting. A, Rats were trained with a 4 min (n = 9), 60 min (n = 8) or 24 h (n = 8) training interval and tested 14 d after the second training trial. B, Rats with a 60 min or 24 h intertrial interval showed robust retention. C, Another group of rats (n = 8) trained with a 60 min intertrial interval and tested 30 d later showed very robust fear memory. D, Rats were given a single pairing of light and shock followed by a pairing of odor with shock. Rats were tested 2 d later in the presence of the light, odor, or light and odor in compound. E, There was no evidence of summation between the light and odor, as the rats showed negligible amounts of fear when the cues were presented in compound (*p < 0.05 vs 4 min).

Figure 3.

Fear memory in rats trained with two trials is cue specific. A, Separate groups of rats (n's = 6–13/group) were trained with one of the listed sets of stimuli. B, Only rats trained with two trials at a 60 min intertrial interval using the same cue show a long-term memory (#p values <0.05 vs all groups).

Figure 4.

Phosphorylation of PKA targets in the amygdala following training with a single trial or two trials. A, Rats were given a single training trial and killed 4 min (n = 6), 60 min (n = 13), or 24 h (n = 6) later or (B) given two trials separated by 60 min (n = 8) and killed 1 h later. “Box” controls (n = 8) were exposed to the training apparatus twice separated by 60 min but did not experience the light or shock and were killed 1 h later. Home cage rats (n = 13) were transported on the day of training and killed from their home cage. Sixty minutes after a single trial there was a significant increase in the phosphorylation of MAPK (C), GLUR1 at SER845 (D) and CREB at SER 133 (E). One hour after two trials spaced by 60 min there was a significant increase in phosphorylation of GLUR1 (D) and CREB (E) in the amygdala, but no change in MAPK (C). Representative images from the Western blots are shown below each graph (*p < 0.05 vs HC; **p < 0.05 vs 4 min; #p < 0.05 vs 24 HR; ##p < 0.05 vs BOX).

Figure 5.

Blocking PKA activity in the amygdala before trial 1 prevents the priming of future learning. A, ACSF (n = 12) or Rp-cAMPS (n = 14) was infused into the amygdala (18 μg/0.5 μl/hemisphere) 30 min before the first light shock trial. Rats were administered another trial 24 h later. B, Rats infused with Rp-cAMPS showed no evidence of memory when tested 2 d after the final trial. C, Reactivity to the footshock was not affected by the PKA inhibitor. D, Both groups show intact fear memory when retrained with an odor that signaled shock (*p < 0.05). E, Representative Nissl-stained images in rats infused with ACSF or Rp-cAMPS into the amygdala. F, Cannulae placements for all rats in this experiment.