Protein degradation through the ubiquitin-proteasome system [UPS] plays a critical role in some forms of synaptic plasticity. However, its role in memory formation in the amygdala, a site critical for the formation of fear memories, currently remains unknown. Here we provide the first evidence that protein degradation through the UPS is critically engaged at amygdala synapses during memory formation and retrieval. Fear conditioning results in NMDA-dependent increases in degradation-specific polyubiquitination in the amygdala, targeting proteins involved in translational control and synaptic structure and blocking the degradation of these proteins significantly impairs long-term memory. Furthermore, retrieval of fear memory results in a second wave of NMDA-dependent polyubiquitination that targets proteins involved in translational silencing and synaptic structure and is critical for memory updating following recall. These results indicate that UPS-mediated protein degradation is a major regulator of synaptic plasticity necessary for the formation and stability of long-term memories at amygdala synapses.

Previous functional magnetic resonance imaging (fMRI) studies with human subjects have explored the neural substrates involved in forming associations in Pavlovian fear conditioning. Most of these studies used delay procedures, in which the conditioned stimulus (CS) and unconditioned stimulus (UCS) coterminate. Less is known about brain regions that support trace conditioning, a procedure in which an interval of time (trace interval) elapses between CS termination and UCS onset. Previous work suggests significant overlap in the neural circuitry supporting delay and trace fear conditioning, although trace conditioning requires recruitment of additional brain regions. In the present event-related fMRI study, skin conductance and continuous measures of UCS expectancy were recorded concurrently with whole-brain blood oxygenation level-dependent (BOLD) imaging during direct comparison of delay and trace discrimination learning. Significant activation was observed within the visual cortex for all CSs. Anterior cingulate and medial thalamic activity reflected associative learning common to both delay and trace procedures. Activations within the supplementary motor area (SMA), frontal operculum, middle frontal gyri, and inferior parietal lobule were specifically associated with trace interval processing. The hippocampus displayed BOLD signal increases early in training during all conditions; however, differences were observed in hippocampal response magnitude related to the accuracy of predicting UCS presentations. These results demonstrate overlapping patterns of activation within the anterior cingulate, medial thalamus, and visual cortex during delay and trace procedures, with additional recruitment of the hippocampus, SMA, frontal operculum, middle frontal gyrus, and inferior parietal lobule during trace conditioning. These data suggest that the hippocampus codes temporal information during trace conditioning, whereas brain regions supporting working memory processes maintain the CS-UCS representation during the trace interval.

The mammalian target of rapamycin kinase (mTOR) regulates protein synthesis in neurons at the translational level through phosphorylation of several intracellular targets. Recent work in invertebrates indicates that mTOR-dependent translational control may be critical for the induction and maintenance of activity-dependent synaptic plasticity underlying memory formation. Here, we report that training rats in a simple fear conditioning procedure evokes a time-dependent increase in the phosphorylation of p70s6 kinase, a major direct downstream target of mTOR. When the activation of mTOR was prevented by posttraining injection of rapamycin into the amygdala, formation of the memory and the increase in p70s6 kinase phosphorylation was attenuated. Furthermore, when rapamycin was applied to the amygdala after the recall of a previously stored fear memory, subsequent retention was disrupted, indicating that local translational control at active synapses is required for the stability as well as the formation of long-term memory in this system.

The amygdala is known to be important for normal aversive Pavlovian learning in the rat. The relative contribution of the amygdala to the learning vs. performance of conditional fear with the GABAa agonist muscimol was assessed. Rats were prepared with cannulas aimed at the basolateral amygdala and trained in a contextual fear conditioning paradigm in which each subject received a series of footshocks in a distinctive observation chamber. Conditional responses evoked after exposure to the observation chamber were assessed 24 hr later. Rats that were pretreated with muscimol before performance showed a significantly attenuated fear response, and injections made before acquisition resulted in a much smaller decrement in conditional fear measured 24 hr after training. These results indicate that acquisition-related processes that may be occurring within the amygdala are more difficult to disrupt than those associated with performance.

The contribution of the medial prefrontal cortex (mPFC) to the formation of memory is a subject of considerable recent interest. Notably, the mechanisms supporting memory acquisition in this structure are poorly understood. The mPFC has been implicated in the acquisition of trace fear conditioning, a task that requires the association of a conditional stimulus (CS) and an aversive unconditional stimulus (UCS) across a temporal gap. In both rat and human subjects, frontal regions show increased activity during the trace interval separating the CS and UCS. We investigated the contribution of prefrontal neural activity in the rat to the acquisition of trace fear conditioning using microinfusions of the g-aminobutyric acid type A (GABA A ) receptor agonist muscimol. We also investigated the role of prefrontal N-methyl-D-aspartate (NMDA) receptormediated signaling in trace fear conditioning using the NMDA receptor antagonist 2-amino-5-phosphonovaleric acid (APV). Temporary inactivation of prefrontal activity with muscimol or blockade of NMDA receptor-dependent transmission in mPFC impaired the acquisition of trace, but not delay, conditional fear responses. Simultaneously acquired contextual fear responses were also impaired in drug-treated rats exposed to trace or delay, but not unpaired, training protocols. Our results support the idea that synaptic plasticity within the mPFC is critical for the long-term storage of memory in trace fear conditioning.