New, Forgotten, and Savings Memory for Long-Term Sensitization in Aplysia Pleural Ganglia

We used a custom-designed microarray to characterize the transcriptional response that accompanies longt-term sensitization training in the marine mollusk Aplysia californica. We characterized transcription for a new memory (1 day after training), a forgotten memory (8 days after training) and for a long-term savings memory (8 days after training but 1 day after a reminder that re-instates the sensitization memory). All animals received a 1-day unilateral LTS training protocol (4 rounds of noxious shock at 30 min intervals, each shock 10s of 1/2s off 1/2on 90mA 60-hz biphasic square-wave shock). This produced robust sensitization in all animals (measured as an increase in T-SWR response from baseline to 24h after training). In the forgetting condition, animals received training on day 1, and post-tests on days 7 and 8 showed little-to-no evidence of recall (T-SWR responses on the trained side returned to close to baseline). In the savings condition, animals received training on day 1 that produced strong sensitization, showed forgetting on day 7, and then received a weak bilateral shock as a reminder (day 7), producing a long-term savings memory on day 8 (a re-instatement of the behavioral expression of sensitization on the previously trained side). In the new memory condition, animals received sham training on day 1, then real training on day 7, producing robust sensitization measured on day 8. Pleural ganglia were harvested on day 8. Samples from a left-trained and right-trained animal were combined to smooth out lateralized gene expression. The pleural ganglia contains the VC nociceptors, which are thought to play a primary role in expressing LTS memory. This is a 3 (Phase of Memory: New, Forgotten, Savings) x 2 (Sensitization: Trained vs. Untrained) mixed design. Each array is a two-color approach hybridized to a matched trained and untrained set of samples (with each sample containing tissue from a left- and right-trained animal). There are 8 biological replicates for each of the 3 phases of memory (New, Forgotten, Savings), yielding 24 arrays run on 3 plates. Dye swaps were used for 1/2 the samples in each condition, and both condition and dye swap was balanced across plates.