| BackgroudIn classical Pavlovian fear conditioning as a model system, an initially innocuous stimulus (the to-be conditioned stimulus-CS; for example, a light, tone, or distinctive place) is paired with an innately aversive unconditioned stimulus (US; e.g., a footshock) and the subject (typically a rat or mouse) comes to exhibit a conditioned fear response (CR) to the CS. When repeated exposed to the fear-eliciting CS in the absence of the aversive US, the animals will show a decline in conditioned fear responses. That is attributed to a process called fear extinction. However, fear extinction is not permanent because extinction does not directly modify the existing memory but instead leads to the formation of a new memory that suppresses activation of the initial trace. The efficacy of this inhibition, however, is strongly contingent on spatial, sensory, and temporal variables. Specifically, in rodents and humans alike, the extinguished CR may be returned due to uncovering phenomena, which usually include three general conditions:(i) renewal, when the CS is presented outside of the extinction context;(ii) reinstatement, when the original US is given unexpectedly; or (iii) spontaneous recovery, when a substantial amount of time has passed. In clinical settings, where extinction-based exposure therapy is widely used as treatment for a number of anxiety-related disorders, including phobias and post-traumatic stress, exposure treatments are effective in some cases; however, they do not benefit everyone, and of those who do benefit, many show a return of fear due to spontaneous recovery, reinstatement, or renewal. Previous studies have showed a retrieved memory (i.e. reconsolidation) transiently returns to a liable state that can persists for several hours. More recently, several studies have reported that during this liable state, the memory is amenable to disruption and can erase the initial fear memory, and suppress the CR return. Renewal, reinstatement, and spontaneous recovery of fear responding are prevented by acute exposure to cues previously paired with footshock (a retrieval manipulation) if that exposure is followed10min or1hour later (but not6hours later) by repeated exposure to the same cues in extinction sessions. The "memory retrieval-extinction" behavioral procedure to interfere with reconsolidation of fear cues has been verified in rats and humans. However, the mechanism of memory erasure is still unclear.This neural plasticity is largely mediated by morphological and functional modification of synapses, a process that depends on both synthesis and degradation of proteins. A number of studies have highlighted the role of protein synthesis in the processes of learning and memory. However, the role of protein degradation has not been fully understood. Eukaryotic protein degradation by the proteasome and the lysosome is a dynamic and complex process in which ubiquitin-proteasome system (UPS) has a key regulatory role. Previous study has showed microinjection of proteasome inhibitor into the nucleus accumbens (NAc) could disrupt cocaine reward memory extinction, but it remains unclear whether the UPS activity involves in auditory fear conditioning (AFC) extinction. More recently, it is reported that in contextual fear conditioning model, the polyubiquitylation levels of hippocampus CA1increased after memory retrieval. Put all together, we proposed a hypothesis that protein degradation induced by retrieved memory mediates fear memory erasure. In the present study, using AFC paradigm, in the basolateral amygdala (BLA), the involvement of UPS in AFC fear memory erasure is investigated. ObjectiveFinding out the role of protein degradation in memory erasure andinvestigating the possible mechanism.Results1. Microinjection of proteasome inhibitor PLac into the BLA impairs AFC extinction.Rats received bilateral microinfusion of vehicle or PLac before AFC extinction training, after24hours, the LTM test was performed. Compared with the vehicle group, the Freezing value of βLac group has significantly reduced.2. The βLac block the effect of retrieval erases AFC memory spontaneous recovery.All of the animals were fear conditioned using three tone-shock pairing, and were then divided into three experimental groups:retrieval+extinction group (Ret), retrieval+βLac+extinction group (Ret+βLac) and no retrieval+extinction group (No Ret). Rats received bilateral microinfusion of vehicle or βLac0.5hr before the retrieval or no retrieval training. After retrieval or no retrieval training1hour, the extinction training was performed. Twenty-four hours after extinction, all groups were tested for a long-term memory (LTM), and two weeks later for spontaneous recovery. During the last four trials of extinction and LTM test, the groups did not differ from one another (repeated-measures ANOVA, P>0.1). For spontaneous recovery test, Compared with the retrieval+extinction group, the freezing value of the other two groups have significantly increase. This result suggests that the UPS-dependent protein degradation induced by retrieval is necessary to spontaneous recovery erasure.3. βLac block the effect of retrieval erases AFC memory renewal.For renenwal, rats of three groups (Ret, Ret+pLac and No Ret) were fear conditioned in context A. twenty-four hours later, no retrieval or retrieval training were performed in context B. Rats received bilateral microinfusion of vehicle or3Lac30min before the retrieval or no retrieval training, and1hr after no retrieval or retrieval, extinction training occurred in context B. Twenty-four hours after extinction, all groups were tested for LTM in context B, and twenty-four later for renewal in context A. We found that compared with the retrieval+extinction group, the freezing value of the other two groups have significantly increase when tested for renewal.4. βLac block the effect of retrieval erases AFC memory reinstatement.In reinstatement experiment, all procedures (described above) were conducted in context A. The microinjection as well as the no retrieval or retrieval training were performed as above. Rats of three groups exhibited similar levels of freezing (a measure of fear expression) during fear conditioning (repeated-measures ANOVA, P>0.05), across the last four trials of extinction (repeated-measures ANOVA, P>0.1). Twenty-four hours after extinction, rats received five unsignaled footshocks and were tested for reinstatement the next day. Compared with the retrieval+extinction group, the freezing value of the other two groups have significantly increase when tested for reinstatement.5. increase of poly-ubiquitination in the BLA induced by fear memory retrieval is the consequence of AFC memory erasure.According the different sacrificed time (rats were sacrificed1or2hr after retrieval/no retrieval or immediately after extinction training), animals are divided into five groups:no retrieval1hr group (No Ret); no retrieval1hr+extinction group (No Ret+Ext); retrieval1hr group (Ret1hr); retrieval1hr+extinction group (Ret+Ext); retrieval2hr group (Ret2hr). GST-S5a pulldown analysis shows the poly-ubiquitination levels. There is a significantly difference between the Ret and No Ret grop; and no significantly difference between the No Ret and No Ret+Ext group. Compared with the Ret2hr group or No Ret+Ext group, the poly-ubiquitination levels of the Ret+Ext group have significantly increase. ConclusionsFor the first time, we found the remarkable role of UPS in the BLA in memory erasure. We demonstrate that in the BLA, protein degradation involves in retrieval+extinction rather than extinction only. |
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