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

Abstract

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.

Figure 1.

Whole-body habituation training apparatus. (A) Photo of the training apparatus. A windshield-wiper motor (1) rotates a long brush (2) attached to a control arm (3) suspended via a frame (4) directly over a rack (5) holding up to four colanders just at the surface of the tank. Each forward-then-back rotation (arrow) drags the bristles on the brush through the colanders, applying tactile stimulation over much of the dorsal surface of the body of each animal. (B) Top-down view of brush completing a back-stroke through a colander, with bristles spragging along the dorsal surface of an animal (head is oriented down and to the left). (C) Side-view as the brush completes a back-stroke through a colander as the animal crawls in the opposite direction. Photos were taken with a digital camera, converted to grayscale, and then processed with an HDR-like filter to enhance contrast.

Figure 2.

Examples of the response of VC sensory neurons to repetitive brushing stimulation in reduced preparations. Brush stimuli (arrows) were delivered at ∼10-sec ISI. Most VC neurons tested exhibited hyperpolarization, a common response to off-field stimulation (A), 25% fired 1–2 spikes/stimulus (B), and 25% showed no response (data not shown). Note that A and B are not simultaneous recordings and that stimulus markers are approximate.

Figure 3.

Short- and long-term effects of whole-body habituation training. (A) Experimental design. (Open arrows) SWR measures obtained by stimulating the siphon, head, or tail before (baseline) and 24 h after training. (Closed arrows) S-SWR measures obtained 1 h after each round of training to monitor short-term retention (ST1-4) or 1 h before the next round of training to measure long-term retention (LT1-3, ∼14 h after the end of prior training). Alternating bars represent the daily habituation sessions given to trained animals. (B) Changes in S-SWR responses during and after training (n = 12/group). The dotted line at 100% indicates no change in behavior. Group means are shown with error bars representing 95% confidence intervals. Means labeled with (*) indicate a P < 0.05 for comparison between trained and control group with Holm–Sidak correction for multiple comparison. (C) Long-term habituation by site of stimulation. Shown are changes in S-SWR (siphon), H-SWR (head), and T-SWR (tail) responses 24 h after training by condition (same animals as in B). Label for significance same as in B.

Figure 4.

Differential effectiveness of massed and spaced protocols for whole-body habituation training. (A) Experimental design. Same as in Figure 2, but with a massed group receiving a single block of whole-body habituation training encompassing the same total number of stimuli as in massed training (2700) condensed into a single training session. (B) Changes in S-SWR responses during and after training (n = 14/group). Means labeled with (*) indicate a P < 0.05 for comparison between trained and control group with Holm–Sidak correction for multiple comparison. Contrasts from control to massed were not significant. (C) Long-term habituation by site of stimulation (same animals as in B). Shown are changes in S-SWR (siphon), H-SWR (head), and T-SWR (tail) responses 24 h after training by condition. Label for significance same as in B.

Figure 5.

Dynamics of short-term habituation. (A) Experimental design. S-SWR durations were measured before and just after 1 d of standard spaced training or standard massed training. (B) Short-term changes in S-SWR responses (n = 8/group). Means marked (*) indicate a P < 0.05 for comparison between trained and massed group with Holm–Sidak correction for multiple comparison. (C) Test for delayed effects after a single day of training. Shown are changes in S-SWR responses 24, 48, and 72 h after training by condition. Label for significance same as in B.

Figure 6.

Dishabituation to moderate shock. (A) Experimental design. Same as in Figure 2, but with a moderate tail shock (untrained + shock, trained + shock) or sham shock (trained + shock) administered after 24-h tests but just before a set of dishabituation measures. Also, both baseline and 24-h measures consisted of 4 T-SWR measures followed by 4 S-SWR measures. (B) Changes in S-SWR responses during and after training (n = 8/group). Means labeled (*) or (°) indicate a P < 0.05 for comparison with the untrained + shock condition for the trained + shock and trained + sham conditions, respectively, with Holm–Sidak correction for multiple comparison. There was a significant change in “both” trained + shock and trained + sham conditions after the dishabituating/sham shock, but in opposite directions, leading to a significant difference between these groups in the dishabituation measures. (C) Long-term habituation by site of stimulation (same animals as in B). Shown are changes in S-SWR (siphon), and T-SWR (tail) responses 24 h after training by condition. Label for significance same as in B.

Figure 7.

Long-term habituation for microarray experiment. (A) Experimental design. Same as in Figure 4, but only ST1 and 24-h measures were made after training. Immediately after 24-h tests, each animal was sacrificed and pleural ganglia were harvested for transcriptional analysis (test tubes). (B) Changes in S-SWR responses during and after training (n = 12/group). Means labeled (*) indicate a P < 0.05 for comparison between trained and control group with Holm–Sidak correction for multiple comparison. Contrasts from control to massed were not significant. (C) Long-term habituation by site of stimulation (same animals as in B). Shown are changes in S-SWR (siphon), H-SWR (head), and T-SWR (tail) responses 24 h after training by condition. Label for significance same as in B.

Figure 8.

Regulation of cornichon after long-term habituation training. Mean fold change in cornichon expression in standard spaced-trained animals relative to controls (black) and massed-trained animals (gray) relative to controls (n = 14/group). Each bar represents a group mean with 95% confidence interval. A log₂ scale is used to give equal weight to up- and down-regulated transcripts. The dotted line at 1 represents no regulation (equal expression relative to controls). (*) indicates expression is significantly different from control at P < 0.05. Significance of paired comparisons from spaced to trained animals is marked with brackets.