Sarah Dalesman

Cue association and antipredator behaviour in a pulmonate snail, Lymnaea stagnalis

Associative learning may help to offset costs of unnecessary escape behaviour by providing accurate information about the current risk to potential prey. We investigated innate antipredator behaviour and cue association learning in naive gastropods. Juvenile laboratory-reared great pond snails, Lymnaea stagnalis (L.), were exposed to odour cues from a natural predator, tench, Tinca tinca (L.), and crushed conspecifics. The snails showed an innate antipredator behaviour to odour from T. tinca, by crawling above the water line (crawl-out response). This crawl-out response was significantly increased in the presence of alarm cues (crushed conspecifics). In a second experiment, juvenile L. stagnalis were exposed to tench odour and alarm cues in aquaria before being tested in behavioural assays. The behavioural responses to tench cue alone were similar to those seen in response to tench plus alarm cues presented together during the first experiment. Hence, L. stagnalis is apparently capable of relating potential predation risk to recent experience. In a final experiment snails were removed from pre-exposure cues for periods of 1, 4 and 8 days prior to behavioural assays. A raised level of avoidance persisted for at least 8 days, suggesting that this behaviour may be retained over timescales relevant to predation risk in the natural environment. The ability of organisms to modify antipredator behaviour based on recent experience, as found in L. stagnalis, would allow costs associated with unnecessary responses to be reduced while still allowing the organisms to avoid active predators.

Ecologically relevant stressors modify long-term memory formation in a model system

Stress can alter adaptive behaviours, and as well either enhance or diminish learning, memory formation and/or memory recall. We focus attention on how environmentally relevant stressors (e.g. predator detection, crowding, and low concentrations of environmental Ca(++)) alter memory formation in the pond snail, Lymnaea stagnalis. We specifically look at operant conditioning of aerial respiration and whether or not long-term memory forms following the acquisition of the learned event, not performing aerial respiration. We will also examine the strain differences in Lymnaea which allow or cause isolated populations to possess different heritable cognitive capabilities, as manifested by differing abilities to form long-term memory.

Low environmental calcium blocks long-term memory formation in a freshwater pulmonate snail.

The freshwater snail Lymnaea stagnalis (L.) is considered a calciphile and exhibits reduced growth and survival in environments containing less than 20 mg/l environmental calcium. Although it has no apparent effect on survival at 20 mg/l, reducing environmental calcium increases metabolic demand, and as such we consider that this level of calcium acts as a stressor on the snail. We exposed snails to acute periods of low environmental calcium and tested their ability to form intermediate-term memory (ITM) and long-term memory (LTM) following one trial operant conditioning (1TT) to reduce aerial respiratory activity in hypoxic conditions. We also assessed whether there were changes in the electrophysiological properties of a single neuron, right pedal dorsal 1 (RPeD1), which has been demonstrated to be necessary for LTM formation. Following training in high (80 mg/l) environmental calcium, L. stagnalis formed ITM and LTM lasting 24 h and demonstrated a significant reduction in all activity measured from RPeD1; however when snails were exposed to low (20 mg/l) environmental calcium they were able to form ITM but not LTM. Although no behavioral LTM was formed, a partial reduction in RPeD1 activtiy measured 24 h after training was observed, indicating a residual effect of training. The strong effect that environmental calcium concentration had on physiology and behavior in response to training to reduce aerial respiration in L. stagnalis suggests that it is an element of gastropod husbandry that needs to be carefully considered when studying other traits. This study also indicates that L. stagnalis found naturally in low calcium environments may be less able to adapt to novel stressors than populations found in harder waters.

Environmentally relevant stressors alter memory formation in the pond snail Lymnaea

Stress alters adaptive behaviours such as learning and memory. Stressors can either enhance or diminish learning, memory formation and/or memory recall. We focus attention here on how environmentally relevant stressors alter learning, memory and forgetting in the pond snail, Lymnaea stagnalis. Operant conditioning of aerial respiration causes associative learning that may lead to long-term memory (LTM) formation. However, individual ecologically relevant stressors, combinations of stressors, and bio-active substances can alter whether or not learning occurs or memory forms. While the behavioural memory phenotype may be similar as a result of exposure to different stressors, how each stressor alters memory formation may occur differently. In addition, when a combination of stressors are presented it is difficult to predict ahead of time what the outcome will be regarding memory formation. Thus, how combinations of stressors act is an emergent property of how the snail perceives the stressors.

Sensory mediation of memory blocking stressors in the pond snail Lymnaea stagnalis.

SUMMARY The great pond snail, Lymnaea stagnalis, is commonly used as a model species to study how stress affects the ability to form long-term memory (LTM); however, we still have little information about how the snail senses stressful stimuli. The osphradium is an external sensory organ that demonstrates electrophysiological responses to a variety of external chemical stimuli. We examined the role, if any, played by the osphradium in sensing two environmental stressors, crowding and low environmental calcium, both known to block LTM in intact animals. We severed the osphradial nerve, blocking external sensory input from this organ to the central nervous system, and then exposed the snails to low environmental calcium or crowding stress to assess whether these stressors continued to block LTM formation. When exposed to low environmental calcium, snails with their osphradial nerve severed responded as if they were maintained in our standard calcium environment. That is, they did not respond to low calcium as a stressor blocking LTM; therefore, the osphradium plays a crucial role in mediating how snails respond to this stressor. However, following crowding, LTM formation was blocked in both control groups and snails that had the osphradial nerve severed, indicating that sensory information from the osphradium is not required to sense crowded conditions. Together these data show that two stressors that result in the same behavioural phenotype, blocking LTM formation, do so via two distinct sensory pathways.

Low external environmental calcium levels prevent forgetting in Lymnaea.

SUMMARY Forgetting may allow an animal to react more appropriately to current conditions, rather than continuing to exhibit a previously learned, possibly maladaptive behaviour based on previous experience. One theory is that forgetting is an active process, whereby the previously learnt response is replaced by new learning that interferes with the older memory. Hence, we hypothesized that an appropriately timed environmental stressor that blocks long-term memory (LTM) formation would also block forgetting. Lymnaea stagnalis (L.) is a freshwater snail, which requires environmental calcium of at least 20 mg l–1 to meet its requirements. Low environmental Ca2+ (i.e. 20 mg l–1) in their environment acts as a stressor, and prevents LTM formation. Here, we asked whether a low Ca2+ environment would also prevent forgetting, concordant with the retrograde interference model of Jenkins and Dallenbach. Snails were operantly conditioned to reduce aerial respiration in hypoxia. When maintained in standard conditions (80 mg l–1 Ca2+), snails demonstrated LTM following training lasting 24 h, but not 72 h; however, when trained in standard conditions then exposed to a low Ca2+ environment (20 mg l–1) immediately following training, they retained memory for at least 96 h, indicating that forgetting had been blocked. Thus, when exposed to low environmental Ca2+, Lymnaea will fail to form new memories, but will also continue to retain information previously learned and remembered as the low calcium blocks forgetting.

What's hot: the enhancing effects of thermal stress on long-term memory formation in Lymnaea stagnalis

SUMMARY The pond snail, Lymnaea stagnalis, naturally inhabits slow flowing, shallow and stagnant environments in the northern temperate zone. Consequently, it will experience wide temperature fluctuations dependent on prevailing weather conditions. We hypothesize that periods of warming act as a thermal stressor to alter memory formation. Snails were exposed to an acute 1 h period of 30°C pond water and we determined how memory formation following operant conditioning of aerial respiration was affected. In the snails used here (Dutch strain), a single 0.5 h training session (TS) results in intermediate-term (3 h) but not long-term memory (LTM). Applying the thermal stressor during training caused memory enhancement (i.e. LTM lasting 24 h). However, the breathing rate also increased in warm water, which might explain the enhanced memory. Therefore, we applied the thermal stressor (1 h at 30°C) up to 4 h before or 1 h after training. This did not alter baseline breathing rate during the period when snails would experience training. However, the thermal stressor whether experienced prior to or following the single TS, resulted in an enhanced memory that persisted up to 48h (i.e. LTM). We conclude that memory enhancement is due to the stress associated with the thermal stimulus.

Sensory input from the osphradium modulates the response to memory-enhancing stressors in Lymnaea stagnalis

SUMMARY In the freshwater environment species often rely on chemosensory information to modulate behavior. The pond snail, Lymnaea stagnalis, is a model species used to characterize the causal mechanisms of long-term memory (LTM) formation. Chemical stressors including crayfish kairomones and KCl enhance LTM formation (≥24 h) in Lymnaea; however, how these stressors are sensed and the mechanism by which they affect the electrophysiological properties of neurons necessary for memory formation are poorly understood. Here, we assessed whether the osphradium, a primary chemosensory organ in Lymnaea, modulates LTM enhancement. To test this we severed the osphradial nerve proximal to the osphradium, using sham-operated animals as controls, and assessed the behavioral and electrophysiological response to crayfish kairomones and KCl. We operantly conditioned aerial respiratory behavior in intact, sham and osphradially cut animals, and tested for enhanced memory formation after exposure to the chemical stressors. Sham-operated animals displayed the same memory enhancement as intact animals but snails with a severed osphradial nerve did not show LTM enhancement. Extracellular recordings made from the osphradial nerve demonstrate that these stressors evoked afferent sensory activity. Intracellular recordings from right pedal dorsal 1 (RPeD1), a neuron necessary for LTM formation, demonstrate that its electrophysiological activity is altered by input from the osphradium following exposure to crayfish kairomones or KCl in sham and intact animals but no response is seen in RPeD1 in osphradially cut animals. Therefore, sensory input from the osphradium is necessary for LTM enhancement following exposure to these chemical stressors.

How Stress Alters Memory in ‘Smart’ Snails

Cognitive ability varies within species, but whether this variation alters the manner in which memory formation is affected by environmental stress is unclear. The great pond snail, Lymnaea stagnalis, is commonly used as model species in studies of learning and memory. The majority of those studies used a single laboratory strain (i.e. the Dutch strain) originating from a wild population in the Netherlands. However, our recent work has identified natural populations that demonstrate significantly enhanced long-term memory (LTM) formation relative to the Dutch strain following operant conditioning of aerial respiratory behaviour. Here we assess how two populations with enhanced memory formation (i.e. ‘smart’ snails), one from Canada (Trans Canada 1: TC1) and one from the U.K. (Chilton Moor: CM) respond to ecologically relevant stressors. In control conditions the Dutch strain forms memory lasting 1–3 h following a single 0.5 h training session in our standard calcium pond water (80 mg/l [Ca2+]), whereas the TC1 and CM populations formed LTM lasting 5+ days following this training regime. Exposure to low environmental calcium pond water (20 mg/l [Ca2+]), which blocks LTM in the Dutch strain, reduced LTM retention to 24 h in the TC1 and CM populations. Crowding (20 snails in 100 ml) immediately prior to training blocks LTM in the Dutch strain, and also did so in TC1 and CM populations. Therefore, snails with enhanced cognitive ability respond to these ecologically relevant stressors in a similar manner to the Dutch strain, but are more robust at forming LTM in a low calcium environment. Despite the two populations (CM and TC1) originating from different continents, LTM formation was indistinguishable in both control and stressed conditions. This indicates that the underlying mechanisms controlling cognitive differences among populations may be highly conserved in L. stagnalis.

A flavonol present in cocoa [(−)epicatechin] enhances snail memory

SUMMARY Dietary consumption of flavonoids (plant phytochemicals) may improve memory and neuro-cognitive performance, though the mechanism is poorly understood. Previous work has assessed cognitive effects in vertebrates; here we assess the suitability of Lymnaea stagnalis as an invertebrate model to elucidate the effects of flavonoids on cognition. (−)Epicatechin (epi) is a flavonoid present in cocoa, green tea and red wine. We studied its effects on basic snail behaviours (aerial respiration and locomotion), long-term memory (LTM) formation and memory extinction of operantly conditioned aerial respiratory behaviour. We found no significant effect of epi exposure (15 mg l−1) on either locomotion or aerial respiration. However, when snails were operantly conditioned in epi for a single 0.5 h training session, which typically results in memory lasting ~3 h, they formed LTM lasting at least 24 h. Snails exposed to epi also showed significantly increased resistance to extinction, consistent with the hypothesis that epi induces a more persistent LTM. Thus training in epi facilitates LTM formation and results in a more persistent and stronger memory. Previous work has indicated that memory-enhancing stressors (predator kairomones and KCl) act via sensory input from the osphradium and are dependent on a serotonergic (5-HT) signalling pathway. Here we found that the effects of epi on LTM were independent of osphradial input and 5-HT, demonstrating that an alternative mechanism of memory enhancement exists in L. stagnalis. Our data are consistent with the notion that dietary sources of epi can improve cognitive abilities, and that L. stagnalis is a suitable model with which to elucidate neuronal mechanisms.