A thermal stressor, propranolol and long-term memory formation in freshly collected Lymnaea.

A heat stressor (1 h at 30°C) in Lymnaea stagnalis before operant conditioning training of aerial respiration is sufficient to enhance long-term memory (LTM) formation in 'average' cognitive ability, laboratory-reared, inbred snails. However, in freshly collected outbred snails, the same heat stressor blocks LTM formation in 'smart' cognitive phenotype but not in average cognitive phenotype strains. Here, we hypothesize that (1) preventing the stress associated with the heat stressor before training allows LTM to form in the smart phenotype strains; and (2) alleviating the stress before a memory recall session allows a formed LTM to be recalled in the smart phenotype strains. We found that an injection of propranolol, which mitigates the stressor, before snails experience the heat stressor enabled two strains of the smart phenotype snails to form LTM, consistent with our first hypothesis. However, the injection of propranolol before a memory test session did not alleviate a memory recall block in the smart phenotype snails. Thus, our second hypothesis was not supported. Therefore, smart cognitive phenotype snails encountering a heat stressor have an inability to form LTM, but this inability can be overcome by the pre-injection of propranolol.

Stress before training alters memory retrieval of a non-declarative memory in Lymnaea.

Stress alters both memory formation and its retrieval. Here, we show that a combination of stressors before an associative learning event alters memory retrieval of a non-declarative memory in an invertebrate model system. Previously, two combinations of stressors were purported to prevent long-term memory (LTM) formation in 'smart' Lymnaea and this inability to form LTM was considered to be a cost of being smart. Here, we show that is not the case. The specific combinations of stressors used here cause emotional memory formation. Previously, it was shown that propranolol, a synthetic beta-blocker, altered emotional memory in Lymnaea. We show here that when propranolol but not saline is injected into smart snails before they perceive the combination of stressors, these snails form LTM. We then show that the injection of propranolol but not saline before a memory activation session allowed the memory to be recalled. That is, LTM formed but was not retrievable unless propranolol was injected pre-retrieval. Thus, the smart snails formed LTM in the face of the stressors but could not retrieve it.

Shell damage leads to enhanced memory formation in Lymnaea.

Ecologically relevant stressors alter the ability of the pond snail, Lymnaea stagnalis, to form long-term memory (LTM). Here, we show that an environmentally relevant stressor, shell damage, has a dramatic effect on the enhancement of LTM formation. Damage in the form of a shell clip 24 h before operant conditioning training resulted in long-term memory (LTM) formation following a single 0.5 h training session (TS). Typically, in these snails, two 0.5 h TSs with a 1 h interval between the sessions are required to cause LTM formation. We show here that even with a 72 h interval between shell clip and training, memory enhancement still occurred. The stress associated with shell clip could be mitigated by an ongoing high-Ca2+ pond water environment, an injection of propranolol and a DNA methylation blocker. However, use of an anaesthetic (MgCl2) during the clip or intermittent exposure to the high-Ca2+ pond water environment did not mitigate the stress associated with the shell clip. Shell clip was also sufficient to cause juvenile snails, which neither learn nor form memory, to gain the capacity to form LTM. Together, the experiments demonstrate that shell clipping is an environmentally relevant stressor that can cause enhancement of LTM formation.

Configural learning: a higher form of learning in Lymnaea.

Events typically occur in a specific context and the ability to assign importance to this occurrence plays a significant role in memory formation and recall. When the scent of a crayfish predator (CE) is encountered in Lymnaea stagnalis strains known to be predator experienced (e.g. the W-strain), enhancement of memory formation and depression of feeding occur, which are part of a suite of anti-predator behaviours. We hypothesized that Lymnaea possess a form of higher-order conditioning, namely configural learning. We tested this by simultaneously exposing W-strain Lymnaea to a carrot food odour (CO) and predator scent (CE). Two hours later, we operantly conditioned these snails with a single 0.5 h training session in CO to determine whether training in CO results in long-term memory (LTM) formation. A series of control experiments followed and demonstrated that only the CO+CE snails trained in CO had acquired enhanced memory-forming ability. Additionally, following CE+CO pairing, CO no longer elicited an increased feeding response. Hence, snails have the ability to undergo configural learning. Following configural learning, CO becomes a risk signal and evokes behavioural responses phenotypically similar to those elicited by exposure to CE.

Green tea and cocoa enhance cognition in Lymnaea.

A flavonoid, (-)-epicatechi (Epi), enhances long-term memory (LTM) formation in Lymnaea and reverses memory obstruction caused by stress. Many foods contain substantial amounts of Epi, (e.g. green tea and cocoa). In humans eating such foods may directly or indirectly enhance cognition. We directly test whether operant conditioning training Lymnaea in these natural foods result in the same effects as training snails in pure Epi. We found that exposure to products containing high concentrations of Epi (e.g. green tea and cocoa) during training enhanced memory formation and could even reverse a learning and memory deficit brought about by stress. Epi can be photo-inactivated by exposure to ultraviolet light. We found that following photo-inactivation of Epi, memory enhancement did not occur. Photo-inactivation of foods containing Epi (e,g. green tea) blocked their ability to enhance LTM. Our data are thus consistent with the hypothesis that dietary sources of Epi can have positive benefits on cognitive ability and be able to reverse memory aversive states.

Strain-specific differences of the effects of stress on memory in Lymnaea.

Stress alters the ability to form, recall and maintain memory according to the Yerkes-Dodson/Hebb (YDH) law. The effects of environmentally relevant stressors, such as low environmental calcium and crowding, on learning and memory have previously been described in a laboratory-reared 'average' strain of Lymnaea stagnalis (i.e. the Dutch strain) as well as two strains of freshly collected L. stagnalis with enhanced memory formation abilities (i.e. 'smart' snails). Here, we use L. stagnalis to study the effects of other environmentally relevant stressors on memory formation in two other strains of freshly collected snails, one 'smart' and one 'average'. The stressors we examined are thermal, resource restriction combined with food odour, predator detection and, for the first time, tissue injury (shell damage). We show that the same stressor has significantly different effects on memory formation depending on whether snails are 'smart' or 'average'. Specifically, our data suggest that a stressor or a combination of stressors act to enhance memory in 'average' snails but obstruct memory formation in 'smart' snails. These results are consistent with the YDH law and our hypothesis that 'smart' snails are more easily stressed than 'average' snails.

Juveniles of Lymnaea 'smart' snails do not perseverate and have the capacity to form LTM.

Previously, it was concluded that the nervous systems of juvenile snails were not capable of mediating long-term memory (LTM). However, exposure and training of those juvenile snails in the presence of a predator cue significantly altered their ability to learn and form LTM. In addition, there are some strains of Lymnaea which have been identified as 'smart'. These snails form LTM significantly better than the lab-bred strain. Here, we show that juveniles of two smart snail strains not only are capable of associative learning but also have the capacity to form LTM following a single 0.5 h training session. We also show that freshly collected 'wild' 'average' juveniles are also not able to form LTM. Thus, the smart snail phenotype in these strains is expressed in juveniles.

Epicatechin, a component of dark chocolate, enhances memory formation if applied during the memory consolidation period.

Epicatechin (Epi), a flavanol found in foods such as dark chocolate has previously been shown to enhance memory formation in our model system, operant conditioning of aerial respiration in Lymnaea. In those experiments snails were trained in Epi. Here we ask whether snails exposed to Epi before training, during the consolidation period immediately following training, or 1 h after training would enhance memory formation. We report here that Epi is only able to enhance memory if snails are placed in Epi-containing pond water immediately after training. That is, Epi enhances memory formation if it is applied during the memory consolidation period as well as if snails are trained in Epi-containing pond water.

Heat stress enhances LTM formation in Lymnaea: role of HSPs and DNA methylation.

Environmentally relevant stressors alter the memory-forming process in Lymnaea following operant conditioning of aerial respiration. One such stressor is heat. Previously, we found that following a 1 h heat shock, long-term memory (LTM) formation was enhanced. We also had shown that the heat stressor activates at least two heat shock proteins (HSPs): HSP40 and HSP70. Here, we tested two hypotheses: (1) the production of HSPs is necessary for enhanced LTM formation; and (2) blocking DNA methylation prevents the heat stressor-induced enhancement of LTM formation. We show here that the enhancing effect of the heat stressor on LTM formation occurs even if snails experienced the stressor 3 days previously. We further show that a flavonoid, quercetin, which inhibits HSP activation, blocks the enhancing effect of the heat stressor on LTM formation. Finally, we show that injection of a DNA methylation blocker, 5-AZA, before snails experience the heat stressor prevents enhancement of memory formation.