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

Highlights

• We developed a new Aplysia microarray with high convergent and predictive validity.

• Long-term sensitization training rapidly regulates 81 transcripts in pleural ganglia.

• Regulated transcripts include putative homologs of C/EBPgamma, GLYT2, and VPS36.

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 long-lasting, 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 long-term 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 up-regulates 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).

Introduction

The encoding of new memories triggers rapid changes in neuronal gene expression (Bailey et al., 1996, Tischmeyer and Grimm, 1999). This early wave of transcriptional change seems to set the stage for long-term memory maintenance, as blocking transcription during encoding impairs retention across a wide range of species and learning contexts (e.g. Chew et al., 1995, Esdin et al., 2010, Hermitte et al., 1999, Sangha, 2003, Watanabe et al., 2005). Thus, characterizing the immediate transcriptional response to memory encoding is an important step in understanding the long-term cellular and network changes that underlie long-term memory.

Here we use microarray and qPCR to characterize the rapid transcriptional response to long-term sensitization in the marine mollusk Aplysia californica. Aplysia have long served as an attractive model organism for studying the molecular mechanisms of memory. One particular focus has been long-term sensitization (Pinsker, Hening, Carew, & Kandel, 1973), a learning paradigm in which repeated exposure to a noxious stimulus produces a long-lasting, transcription-dependent increase in reflex responsiveness (Castellucci, Blumenfeld, Goelet, & Kandel, 1989). Sensitization of the tail-elicited siphon withdrawal reflex provides an especially attractive system for transcriptional analysis because (1) sensitization can be applied and expressed unilaterally (Scholz & Byrne, 1987), allowing for powerful within-subjects comparisons, (2) sensitization memory is known to depend at least in part on physiological changes in the VC nociceptors of the pleural ganglia (Cleary, Lee, & Byrne, 1998), providing a behaviorally-relevant target for transcriptional analysis, and (3) transcriptional and behavioral changes can be correlated at the level of individual animals, allowing exploration of individual differences in retention (Bonnick et al., 2012).

Previous research has shown that long-term sensitization training produces a rapid (within 1 h) increase in the expression of six different transcripts in the pleural ganglia: ApC/EBP (GenBank: U00994; Alberini, Ghirardi, Metz, & Kandel, 1994), ApCREB1 (GenBank: NM_001256437; Bartsch, Casadio, Karl, Serodio, & Kandel, 1998), ApEgr (GenBank: KC608221; Cyriac et al., 2013), ApTBL-1 (GenBank: U57369; Liu et al., 1997), ApCalmodulin (GenBank: NM_001204580; Zwartjes et al., 1998), and an reductase-related protein (GenBank: NM_001204605; Zwartjes et al., 1998). To date, however, there have been no comprehensive efforts to characterize the rapid transcriptional response to long-term sensitization training (though see Castellucci et al., 1988, Liu et al., 1997). Considerable effort, however, has gone into characterizing the transcriptional response of Aplysia neurons to exposure to serotonin (5-hydroxytryptamine, 5-HT), a neuromodulator released during sensitization training that is critical to its induction (Glanzman et al., 1989). For example, a microarray study in Aplysia kurodai has characterized the rapid transcriptional response to soaking whole animals in 200 μM 5-HT for 2 h (Lee et al., 2008). Many others have examined changes in transcription or protein expression at different time points after 5-HT exposure in whole animals, ganglia, or cultured neurons (e.g. Barzilai et al., 1989, Monje et al., 2011). In a previous analysis of a small set of transcripts, however, we have shown that 5-HT and behavioral training do not always produce similar patterns of transcriptional change in A. californica (Bonnick et al., 2012).

To facilitate characterization of learning-related transcriptional changes, we developed a new microarray design that leverages the entire known EST pool for Aplysia in a more compact format than previously available arrays. We find that this new platform has high convergent and predictive validity, and present 4 new transcripts which are strongly, consistently, and rapidly up-regulated by long-term sensitization training.