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We characterized the transcriptional response accompanying maintenance of long-term sensitization (LTS) memory in the pleural ganglia of "Aplysia californica" using microarray (N = 8) and qPCR (N = 11 additional samples). We found that 24 h after memory induction there is strong regulation of 1198 transcripts (748 up and 450 down) in a pattern that is almost completely distinct from what is observed during memory encoding (1 h after training). There is widespread upregulation of transcripts related to all levels of protein production, from transcription (e.g., subunits of transcription initiation factors) to translation (e.g., subunits of eIF1, eIF2, eIF3, eIF4, eIF5, and eIF2B) to activation of components of the unfolded protein response (e.g., CREB3/Luman, BiP, AATF). In addition, there are widespread changes in transcripts related to cytoskeleton function, synaptic targeting, synaptic function, neurotransmitter regulation, and neuronal signaling. Many of the transcripts identified have previously been linked to memory and plasticity (e.g., Egr, menin, TOB1, IGF2 mRNA binding protein 1/ZBP-1), though the majority are novel and/or uncharacterized. Interestingly, there is regulation that could contribute to metaplasticity potentially opposing or even eroding LTS memory (down-regulation of adenylate cyclase and a putative serotonin receptor, up-regulation of FMRFa and a FMRFa receptor). This study reveals that maintenance of a "simple" nonassociative memory is accompanied by an astonishingly complex transcriptional response.

Habituation is the simplest form of learning, but we know little about the transcriptional mechanisms that encode long-term habituation memory. A key obstacle is that habituation is relatively stimulus-specific and is thus encoded in small sets of neurons, providing poor signal/noise ratios for transcriptional analysis. To overcome this obstacle, we have developed a protocol for producing whole-body long-term habituation of the siphon-withdrawal reflex (SWR) of "Aplysia californica." Specifically, we constructed a computer-controlled brushing apparatus to apply low-intensity tactile stimulation over the entire dorsal surface of "Aplysia" at regular intervals. We found that 3 d of training (10 rounds of stimulation/day; each round = 15 min brushing at a 10-sec ISI; 15-min rest between rounds) produces habituation with several characteristics favorable for mechanistic investigation. First, habituation is widespread, with SWR durations reduced whether the reflex is evoked by tactile stimulation to the head, tail, or the siphon. Second, long-term habituation is sensitive to the pattern of training, occurring only when brushing sessions are spaced out over 3 d rather than massed into a single session. Using a custom-designed microarray and quantitative PCR, we show that long-term habituation produces long-term up-regulation of an apparent "Aplysia" homolog of cornichon, a protein important for glutamate receptor trafficking. Our training paradigm provides a promising starting point for characterizing the transcriptional mechanisms of long-term habituation memory.