Five-minute exposure to a novel appetitive food substance is sufficient time for a microRNA-dependent long-term memory to form.

The Garcia effect is a unique form of conditioned taste aversion which requires that a novel food stimulus be followed sometime later by a sickness state associated with the novel food stimulus. The long-lasting associative memory resulting from the Garcia effect ensures that organisms avoid toxic foods in their environment. Considering its ecological relevance, we sought to investigate whether a brief encounter (5 min) with a novel, appetitive food stimulus can cause a persisting long-term memory (LTM) to form that would in turn block the Garcia effect in Lymnaea stagnalis. Furthermore, we wanted to explore whether that persisting LTM could be modified by the alteration of microRNAs via an injection of poly-L-lysine (PLL), an inhibitor of Dicer-mediated microRNA biogenesis. The Garcia effect procedure involved two observations of feeding behavior in carrot separated by a heat stress (30 °C for 1 h). Exposing snails to carrot for 5 min caused a LTM to form and persist for 1 week, effectively preventing the Garcia effect in snails. In contrast, PLL injection following the 5-min carrot exposure impaired LTM formation, allowing the Garcia effect to occur. These results provide more insight into LTM formation and the Garcia effect, an important survival mechanism.

Better together: isolation impedes memory formation for configural learning in Lymnaea stagnalis.

Social interactions play an important role in learning and memory. There is great variability in the literature regarding the effects of social isolation on cognition. Here, we investigated how memory formation was affected when Lymnaea stagnalis, our model system, were socially isolated at three different time periods: before, during or after the configural learning training procedure. Each group of snails underwent configural learning where we recorded and compared their feeding behaviour before and after the pairing of an appetitive food stimulus with predator kairomones (i.e. the training procedure). We found that isolating snails before the training procedure had no effect on their learning and memory. However, when snails were isolated either during the training procedure or immediately after the training procedure, they no longer formed memory. These data provide further insight into how isolation impacts cognitive functioning in the context of higher-order learning.

No food for thought: an intermediate level of food deprivation enhances memory in Lymnaea stagnalis.

Nutritional status plays an important role in cognitive functioning, but there is disagreement on the role that food deprivation plays in learning and memory. In this study, we investigated the behavioral and transcriptional effects induced by different lengths of food deprivation: 1 day, which is a short time period of food deprivation, and 3 days, which is an 'intermediate' level of food deprivation. Snails were subjected to different feeding regimens and then trained for operant conditioning of aerial respiration, where they received a single 0.5 h training session followed by a long-term memory (LTM) test 24 h later. Immediately after the memory test, snails were killed and the expression levels of key genes for neuroplasticity, energy balance and stress response were measured in the central ring ganglia. We found that 1 day of food deprivation was not sufficient to enhance snails' LTM formation and subsequently did not result in any significant transcriptional effects. However, 3 days of food deprivation resulted in enhanced LTM formation and caused the upregulation of neuroplasticity and stress-related genes and the downregulation of serotonin-related genes. These data provide further insight into how nutritional status and related molecular mechanisms impact cognitive function.

Remember the poke: microRNAs are required for long-term memory formation following operant conditioning in Lymnaea.

MicroRNAs (miRNAs) play an important role in learning and memory formation by controlling the expression of genes through epigenetic processes. Although miRNAs unquestionably play a role in memory, past literature focusing on whether miRNAs play key roles in the consolidation of associative long-term memory in Lymnaea contained confounding variables. Using operant conditioning of aerial respiratory behaviour, we investigated long-term memory (LTM) formation after injection of poly-L-lysine (PLL), an inhibitor of Dicer-mediated miRNA biogenesis, in Lymnaea stagnalis. Homeostatic breathing experiments were also performed to test whether PLL affects breathing. Homeostatic breathing was significantly suppressed 45 min but not 24 h after PLL injection. The operant conditioning procedure involved two 30-min training sessions separated by 1 h to cause LTM. Using this operant conditioning procedure, LTM formation was significantly impaired when snails were injected with PLL 15 min after the second training session. In contrast, when snails were injected with PLL 24 h before the first training session, LTM formation was not impaired. These results are consistent with past literature and highlight an important role for miRNAs in LTM formation.

A change in taste: the role of microRNAs in altering hedonic value.

The mechanisms associated with neophobia and anhedonia remain largely unknown. Neuropsychological disorders such as depression and schizophrenia are associated with excessive fear and anhedonia, and have been linked to microRNA 137. We hypothesized that microRNAs (miRNAs) in the snail Lymnaea stagnalis are important for regulating feeding behaviour through either preventing neophobia or establishing hedonic value. To test these hypotheses, we used an injection of poly-l-lysine (PLL) to inhibit miRNA biogenesis and observed its effects on feeding behaviour. We repeated these experiments with pre-exposure to novel stimuli capable of eliciting neophobia to disentangle the processes predicted to regulate feeding behaviour. Next, we exposed snails to food stimuli of high hedonic value after PLL injection to reset their hedonic value for that food. Finally, we consolidated our results with previous research by examining the effect of PLL injection on a one-trial appetitive classical conditioning procedure (1TT) to induce long-term memory (LTM). We found that miRNAs are likely not required for preventing neophobia. Moreover, we discovered that snails experienced anhedonia in response to inhibition of miRNA biogenesis, resulting in diminished feeding behaviour for food stimuli with a previously high hedonic value. Snails showed diminished feeding behaviour for multiple food stimuli of high hedonic value post-1TT with PLL injection. This finding suggests that PLL causes anhedonia rather than an impairment of LTM formation following the 1TT procedure. This is the first evidence suggesting that inhibiting the biogenesis of miRNAs contributes to anhedonia in L. stagnalis.

Configural learning in freshly collected, smart, wild Lymnaea.

An inbred laboratory strain (W-strain) of Lymnaeastagnalis is capable of configural learning. Configural learning, a higher form of learning, is an association between two stimuli experienced together that is different from the simple sum of their components. In our configural learning procedure, a food odour (carrot odour, CO) is experienced together with crayfish effluent (CE) (i.e. CO+CE). Following configural learning, CO now elicits a fear state rather than increased feeding. We hypothesized that freshly collected wild strains of predator-experienced L. stagnalis also possess the ability to form configural learning, even though they experience crayfish daily in their environment. We therefore subjected freshly collected wild strain L. stagnalis to the configural learning procedure. Following the configural learning procedure, CO became a risk signal and evoked anti-predator behaviours. Thus, configural learning was demonstrated in wild, freshly collected snails. We believe that configural learning occurs in the snail's natural environment and is important for their survival.