Characterization of the rapid transcriptional response to long-term sensitization training in Aplysia californica

Abstract

We used a custom-designed microarray and quantitative PCR to characterize the rapid transcriptional response to long-term sensitization training in the marine mollusk Aplysia californica. Aplysia were exposed to repeated noxious shocks to one side of the body, a procedure known to induce a longlasting, transcription-dependent increase in reflex responsiveness that is restricted to the side of training. One hour after training, pleural ganglia from the trained and untrained sides of the body were harvested; these ganglia contain the sensory nociceptors which help mediate the expression of longterm sensitization memory. Microarray analysis from 8 biological replicates suggests that long-term sensitization training rapidly regulates at least 81 transcripts. We used qPCR to test a subset of these transcripts and found that 83% were confirmed in the same samples, and 86% of these were again confirmed in an independent sample. Thus, our new microarray design shows strong convergent and predictive validity for analyzing the transcriptional correlates of memory in Aplysia. Fully validated transcripts include some previously identified as regulated in this paradigm (ApC/EBP and ApEgr) but also include novel findings. Specifically, we show that long-term sensitization training rapidly upregulates the expression of transcripts which may encode Aplysia homologs of a C/EBPγ transcription factor, a glycine transporter (GlyT2), and a vacuolar-protein-sorting-associated protein (VPS36).

Transcriptional analysis of a whole-body form of long-term habituation inAplysia californica

“…This is considerably less than the 1494 transcripts we found regulated immediately after long-term sensitization training using the same array platform, tissue sample, sample size, and statistical criteria (Herdegen et al 2014). The most likely explanation is a difference in statistical power due to different experimental designs.…”

“…We used the treat function from limma (McCarthy and Smyth 2009) to conduct two separate tests of significance: (1) against the standard null hypothesis of no regulation (H o : mean fold change ,1 or .1; note that a ratio of 1 is produced when both trained and untrained samples have the same levels of gene expression) and then (2) against a null of at least 1.1-fold change in either direction. We have found that transcripts regulated by this more stringent criterion are likely to generalize to an independent sample (Herdegen et al 2014).…”

“…Following the convention in the statistical software package we used (GraphPad Prism), P values are reported exactly except where they are smaller than 0.0001. Effect sizes are estimated using Microarray analysis was conducted exactly as in Herdegen et al (2014). Briefly, expression was normalized using the loess function (Smyth and Speed 2003).…”

“…ApCornichon was the only transcript among those regulated to pass an even more stringent test of regulation (.1.1-fold change in either direction, see Table 1). We have previously found with this microarray platform that transcripts meeting this stringent criterion are very likely to generalize to independent samples (Herdegen et al 2014). Indeed, in limited testing of 11 other transcripts which did not meet this more stringent criterion, none of them reached statistical significance in qPCR analysis (see Table 1).…”

“…Based on estimates from previous microarray designs (Moroz et al 2006), the ATA should cover .50%-60% of all neurally expressed transcripts. Complete details on the ATA design are in Herdegen et al (2014).…”