Immediate and Persistent Transcriptional Correlates of Long-Term Sensitization Training at Different CNS Loci in Aplysia californica

Editor: Bjorn Brembs, Universitaet Regensburg, Germany Received: September Immediate and Persistent Transcriptional Correlates of Long-Term Sensitization Training at Different CNS Loci in Aplysia californica Samantha Herdegen 0 Catherine Conte 0 Saman Kamal 0 Robert J. Calin-Jageman 0 Irina E. Calin-Jageman 0 0 Neuroscience Program, Dominican University , River Forest, Illinois , United States of America Repeated noxious stimulation produces long-term sensitization of defensive withdrawal reflexes in Aplysia californica, a form of long-term memory that requires changes in both transcription and translation. Previous work has identified 10 transcripts which are rapidly up-regulated after long-term sensitization training in the pleural ganglia. Here we use quantitative PCR to begin examining how these transcriptional changes are expressed in different CNS loci related to defensive withdrawal reflexes at 1 and 24 hours after long-term sensitization training. Specifically, we sample from a) the sensory wedge of the pleural ganglia, which exclusively contains the VC nociceptor cell bodies that help mediate input to defensive withdrawal circuits, b) the remaining pleural ganglia, which contain withdrawal interneurons, and c) the pedal ganglia, which contain many motor neurons. Results from the VC cluster show different temporal patterns of regulation: 1) rapid but transient up-regulation of Aplysia homologs of C/EBP, C/EBPc, and CREB1, 2) delayed but sustained up-regulation of BiP, Tolloid/BMP-1, and sensorin, 3) rapid and sustained up-regulation of Egr, GlyT2, VPS36, and an uncharacterized protein (LOC101862095), and 4) an unexpected lack of regulation of Aplysia homologs of calmodulin (CaM) and reductase-related protein (RRP). Changes in the remaining pleural ganglia mirror those found in the VC cluster at 1 hour but with an attenuated level of regulation. Because these samples had almost no expression of the VC-specific transcript sensorin, our data suggests that sensitization training likely induces transcriptional changes in either defensive withdrawal interneurons or neurons unrelated to defensive withdrawal. In the pedal ganglia, we observed only a rapid but transient increase in Egr expression, indicating that long-term sensitization training is likely to induce transcriptional - How are long-term memories maintained despite molecular turnover in the central nervous system? The answer to this question seems to depend, in part, on learning-induced changes in gene expression. In a wide range of species and learning paradigms, training that produces long-term memory also evokes changes in neuronal gene expression [13]. Moreover, blocking changes in gene expression has been repeatedly shown to impair the formation of long-term memory [48]. Thus, there is considerable interest in elucidating the specific transcriptional changes that accompany the encoding and maintenance of longterm memory. Sensitization in the marine mollusk Aplysia californica has proven a fruitful paradigm for studying the transcriptional mechanisms of long-term memory ( Fig. 1A). Sensitization is an increase in reflex responsiveness due to noxious stimulation [9]. This non-associative form of memory is observed across the entire animal kingdom [10]. Mechanistically, sensitization is thought to reflect the operation of basal plasticity mechanisms from which more complex forms of learning have evolved [11]. Moreover, sensitization in Aplysia shares many behavioral, physiological, and molecular characteristics with aspects of chronic pain in humans and other mammals [12, 13], and research in Aplysia has proven informative for helping to guide research into this important clinical problem [14]. A particular advantage of studying sensitization in Aplysia is that it can be induced and expressed unilaterally, by applying noxious shock to one side of the body (Fig. 1A). This produces a unilateral increase in the duration of defensive reflexes in Aplysia [15, 16] which can include changes in the siphon-withdrawal reflex (SWR) and the tail-withdrawal reflex (TWR). With repeated exposure to noxious stimulation, a long-term sensitization memory is induced on the trained side of the body which lasts more than 24 hours and requires changes in transcription [17]. Unilateral sensitization training allows powerful withinsubjects designs in which these transcriptional changes can be compared from the trained and untrained side of the same animal [18]. Although long-term sensitization training is simple, it produces complex changes in behavior. At the site of training, there is site-specific sensitization which can be observed as a change in the duration of SWRs elicited from the same location that was trained [16, 19]. In addition, sensitization generalizes beyond the region of training. For example, generalized sensitization can be observed in SWRs evoked by tail stimulation even when the tail is not included as a training site [20]. Finally, long-term sensitization training produces additional long-lasting changes Fig. 1. Long-term sensitization overview. A) Long-term sensitization is evoked in Aplysia by applying a strong shock (90mA) over most of one side of the body (training site). This produces a robust increase in the duration of defensive withdrawals evoked by innocuous stimulation to both the training site (site-specific sensitization) and to untrained sites on the same side of the body (generalized sensitization). In this paper, generalized sensitization was measured via the tail-elicited siphon withdrawal reflex (T-SWR). The T-SWR reflex is evoked by innocuous electrical shock (2 mA) to the left or right tail (tail test sites). This produces a defensive withdrawal of the siphon (grey) which is measured as the duration of contraction. B) CNS components related to defensive withdrawal reflexes. Defensive withdrawal reflexes are mediated by a number of cell types including: 1) VC nociceptors, which are located in the VC cluster within the pleural ganglia, 2) excitatory and inhibitory interneurons in the pleural ganglia, 3) motor neurons in the pedal ganglia, and 4) siphon, gill, and mantle motor neurons in the abdominal ganglia (not shown). C) Experimental protocol. Animals were given long-term sensitization training consisting of four rounds of noxious shock applied to one side of the body at 30 min intervals. CNS samples were then harvested either 1 hour or 24 hours after training. For animals harvested 24 hours after training, T-SWR behavior was also measured before and 24 hours after training. in behavior, including a persistent increase in heart rate [21] and a persistent decrease in feeding behavior [22]. The mechanisms mediating the behavioral effects of long-term sensitization training can be readily studied in Aplysia because the neural circuitry underlying defensive withdrawal of both the siphon and the tail (Fig. 1B) are relatively well defined [23]. Sensory input is mediated by the VC no (...truncated)