Electrophysiological and behavioral evidence demonstrating that predator detection alters adaptive behaviors in the snail Lymnaea

Abstract

Stress has been shown to both impair and enhance learning, long-term memory (LTM) formation, and/or its recall. The pond snail, Lymnaea stagnalis, both detects and responds to the scent of a crayfish predator with multiple stress-related behavioral responses. Using both behavioral and electrophysiological evidence, this investigation is a first attempt to characterize how an environmentally relevant stressor (scent of a predator) enhances LTM formation in Lymnaea. Using a training procedure that, in "standard" pond water (PW), results in an intermediate-term memory that persists for only 3 h, we found that training snails in "crayfish effluent" (CE) induces a memory that persists for 48 h (i.e., its now an LTM). In addition, if we use a training procedure that in PW produces an LTM that persists for 1 d, we find that snails trained in CE have an LTM that persists for at least 8 d. Furthermore, we describe how a single neuron (RPeD1), which has been shown to be a necessary site for LTM formation, reflects the behavioral changes in its firing properties that persist for the duration of the LTM. Finally, Lymnaea exhibit context-specific memory, that is, when a memory is formed in a specific context (food odorant), it is only recalled in that context. Here, we found that snails trained in CE demonstrate context generalization, that is, memory is recalled in multiple contexts. All data are consistent with the hypothesis that learning in a stressful, yet biologically relevant, environment enhances LTM and prolongs its retention.

Figure 1.

A, Operant conditioning of aerial respiratory behavior using the ITM-training procedure in PW 24 h after a 2 h CE exposure. Snails demonstrate ITM 3 h after operant conditioning of aerial respiratory behavior but do not demonstrate memory 24 h after. A significant reduction in the number of attempted pneumostome openings was observed after 3 h but not in the 24 h test for memory (24 h TM) or in the yoked controls compared with TS1 (3 h TM, n = 18, p < 0.01; 24 h TM, n = 22, p > 0.05; yoke, n = 20, p > 0.05). B, Representative electrophysiological recordings from RPeD1 in an untrained semi-intact preparation obtained 24 h after the intact snails were exposed to either PW (Naive, top) or CE (24 hrs after CE, bottom). No significant differences were found between naive and CE-exposed RPeD1s in any of the 10 measured electrophysiological parameters (n = 9; p > 0.05 in all analyses; data not shown).

Figure 2.

Behavioral data and representative electrophysiological recordings of intact Lymnaea and RPeD1 from semi-intact animals after the ITM-training procedure in either PW or CE. A, The ITM-training procedure in PW results in intermediate-term memory but does not result in LTM (i.e., a memory lasting 24 h; 3 h TM, n = 18, p < 0.05; 24 h TM, n = 44, p > 0.05; black bars). However, the ITM-training procedure in CE results in LTM. That is, the number of attempted pneumostome openings is significantly lower than TS1 and time-matched controls (i.e., memory at 24 and 48 h; white bars; 24 h TM, n = 35, p < 0.01; 48 h TM, n = 41, p < 0.01). Snails did not demonstrate memory formation in the 24 or 48 h yoked control groups (faded bars; n = 30, p > 0.05 and n = 20, p > 0.05 for 24 and 48 h yoked, respectively) or 72 h after training (white bar; n = 22; p > 0.05). B, Representative recordings from RPeD1 in the naive state (untrained and in PW), 24 h after PW–ITM-training, 48 h after ITM-training procedure in CE, 48 h CE–ITM yoke control, and 72 h after the ITM-training procedure in CE. C, Summary data for mean ± SEM spiking activity/600 s and number of spikes per burst (top and middle bar graphs, respectively; values log transformed). Results for the 48 h ITM-training procedure in CE are significantly lower than the naive state (n = 7; p < 0.01). Results for 24 h ITM-training procedure in PW-trained animals, 48 h ITM-training procedure in CE-yoked, and 72 h ITM-training procedure in CE- trained animals are not significantly different from the naive state (24 h PW–ITM, n = 8, p > 0.05; 48 h CE–ITM yoked, n = 8, p > 0.05; 72 h CE–ITM, n = 6, p > 0.05). Bottom bar graph demonstrates summary data for burst duration (mean ± SEM, values log transformed) of each treatment. ITM-training procedure in CE at 48 h are significantly lower than the naive state (n = 7; p < 0.05). ITM-training procedure in PW at 24 h, ITM-training procedure in CE at 48 h yoked, and ITM-training procedure in CE at 72 h are not significantly different from the naive state (24 h PW–ITM, n = 8, p > 0.05; 48 h CE–ITM yoked, n = 8, p > 0.05; 72 h CE–ITM, n = 6, p > 0.05). No significant differences were detected between treatments in other electrophysiological parameters measured (see Materials and Methods).

Figure 3.

Behavioral data (intact snails) and representative electrophysiological recordings of RPeD1 from semi-intact preparations after the LTM-training procedure in either PW or CE. A, The LTM-training procedure in PW results in a memory that lasts 24 but not 48 h or 8 d (black bars; 24 h TM, n = 61, p < 0.01; 48 h TM, n = 26, p > 0.05; 8 d TM, n = 20, p > 0.05). However, the LTM-training procedure in CE results in a memory that persists for at least 8 d. That is, the number of attempted pneumostome openings is significantly lower than TS1 and not significantly greater than TS2 as well as time-matched controls (white bars; 8 d TM, n = 29, p < 0.01). Snails did not demonstrate LTM in the 8 d yoked control groups (faded bars; n = 21, p > 0.05) nor was LTM present 10 d after the LTM-training procedure in CE (white bar; n = 16, p > 0.05). B, Representative recordings from RPeD1 in a naive snail (untrained, PW), 24 h after the LTM-training procedure in PW, 48 h after the LTM-training procedure in PW, 8 d after the LTM-training procedure in CE, 8 d after the CE-yoke control, and 10 d after the LTM-training procedure in CE. C, Summary data for mean ± SEM spiking activity/600 s and number of spikes per burst (top and bottom bar graph, respectively, values log transformed). The data 24 h after the LTM-training procedure in PW are significantly lower than the naive state (n = 7, p < 0.01). However, the data 48 h after the LTM-training procedure in PW are not significantly different from the naive state (n = 7, p > 0.05). D, Summary data for mean ± SEM spiking activity/600 s, number of spikes per burst, and burst duration (top, middle, and bottom bar graphs, respectively, values log transformed). The data 8 d after the LTM-training procedure in CE-trained animals are significantly lower than the naive state (n = 8, p < 0.01) in all three measures. The data for the 8 d CE-yoked control and 10 d post-LTM-training procedure in animals are not significantly different from the naive state for all three measures (8 d CE–LTM yoked, n = 8, p > 0.05; 10 d CE–LTM, n = 8, p > 0.05). No significant differences were detected between treatments in other electrophysiological parameters measured (see Materials and Methods).

Figure 4.

Memory profile of Lymnaea given the ITM-training procedure with testing for memory also in CE and after 2 h direct exposure to hungry crayfish predators. A, Animals trained and tested in CE demonstrated memory after 24 h but not 72 h or in 24 h yoked controls (24 h TM, n = 20, p < 0.01; 72 h TM, n = 17, p > 0.05; 24 h yoked, n = 19, p > 0.05). B, Animals trained in CE immediately after 2 h in direct contact with hungry crayfish demonstrated memory after 24 h but not 72 h (24 h TM, n = 24, p < 0.01; 72 h TM, n = 20, p > 0.05). Yoked control group did not demonstrate memory at 24 h (24 h yoked, n = 19, p > 0.05).

Figure 5.

The effect of the LTM-training procedure in PW and CE on context-dependent LTM. Snails given the LTM-training procedure in PW demonstrate learning (TS2, n = 38, p < 0.01) and memory 24 h later (24 h TM, n = 38, p < 0.01; black bars); however, when challenged with a change of context test (i.e., carrot odor), they do not demonstrate memory (24 h carrot TM, n = 24, p > 0.05; hatched bar). Snails given the LTM-training procedure in CE demonstrate learning and memory (TS2, n = 36, p < 0.01; 24 h TM, n = 36, p < 0.01). However, when challenged with a change of context test, carrot scent, or PW, memory continued to be observed (carrot TM, hatched bar, n = 28, p < 0.01; PW, black bar, n = 34, p < 0.01). That is, context generalization was observed. Carrot-yoked controls did not demonstrate memory (carrot yoke, n = 20, p > 0.05, faded bar).