Transcriptional changes before and after forgetting of a long-term sensitization memory in Aplysia californica

Abstract

Most long-term memories are forgotten, becoming progressively less likely to be recalled. Still, some memory fragments may persist, as savings memory (easier relearning) can be detected long after recall has become impossible. What happens to a memory trace during forgetting that makes it inaccessible for recall and yet still effective to spark easier re-learning? We are addressing this question by tracking the transcriptional changes that accompany learning and then forgetting of a long-term sensitization memory in the tail-elicited siphon withdrawal reflex of Aplysia californica. First, we tracked savings memory. We found that even though recall of sensitization fades completely within 1 week of training, savings memory is still detectable at 2 weeks post training. Next, we tracked the time-course of regulation of 11 transcripts we previously identified as potentially being regulated after recall has become impossible. Remarkably, 3 transcripts still show strong regulation 2 weeks after training and an additional 4 are regulated for at least 1 week. These long-lasting changes in gene expression always begin early in the memory process, within 1 day of training. We present a synthesis of our results tracking gene expression changes accompanying sensitization and provide a testable model of how sensitization memory is forgotten.

Registered Report: Transcriptional Analysis of Savings Memory Suggests Forgetting is Due to Retrieval Failure

“…Thus, defined in terms of overlapping regulation, the forgotten and new phases of memory showed no similarity ( Table 1 , column showing proportion of overlap). This was expected, as previous studies have also shown that the transcriptional response to sensitization training mostly fades over time and that the very few transcriptional changes that persist are difficult to detect with an array-wide screen ( Patel et al, 2018 ; Perez et al, 2018 ). Indeed, although we had confirmed upregulation of FMRFa via qPCR is these samples (see Materials and Methods), for the microarray analysis this transcript did not meet the threshold for being flagged as clearly regulated (LFC = 0.41 95% CI [0.19, 0.72], p = 0.02 before correction, but p = 1 after correction).…”

“…Quantitative PCR was conducted using Maxima SYBR Green/Fluorescein qPCR Master Mix (Thermo Scientific) and the MyIQ real time PCR system (Bio-Rad). Primers were validated for correct PCR efficiency; exact sequences are provided in Patel et al (2018) , their Supplemental Table 1. qPCR samples were analyzed in duplicate or triplicate and the relative amounts of each transcript were determined using the ddCT method and the Bio-rad IQ5 gene expression analysis. All qPCR expression levels were normalized to levels of histone H4, a transcript which is stable during LTS training.…”

“…Thus, we checked the savings-memory group for regulation of the transcript encoding the peptide neurotransmitter Phe-Met-Arg-Phe NH2 (FMRFa; GenBank: M11283.1; Schaefer et al, 1985 ). This transcript is strongly upregulated within 1 d of sensitization training and continues to be upregulated for more than one week ( Patel et al, 2018 ). As expected, we observed strong upregulation of FMRFa in this group (LFC = 1.28 95% CI [0.86, 1.69], n = 14).…”

“…We used these archival data to benchmark assessments of similarity. fades; 7 d after training only approximately seven transcripts remain regulated (Patel et al, 2018;Perez et al, 2018). Here, we asked whether savings memory reactivates the transcriptional changes observed with new memory formation (as predicted if forgetting is because of decay) or if savings has a distinct transcriptional profile (as predicted if forgetting is because of retrieval failure).…”

“…Long-term sensitization in Aplysia requires changes in transcription ( Sutton et al, 2001 ), and new memory storage is associated with regulation of over 1000 transcripts within 1 d of training ( Conte et al, 2017 ). As sensitization memory is forgotten most of this transcriptional response fades; 7 d after training only approximately seven transcripts remain regulated ( Patel et al, 2018 ; Perez et al, 2018 ). Here, we asked whether savings memory reactivates the transcriptional changes observed with new memory formation (as predicted if forgetting is because of decay) or if savings has a distinct transcriptional profile (as predicted if forgetting is because of retrieval failure).…”