A translational and multidisciplinary approach to studying the Garcia effect, a higher form of learning with deep evolutionary roots.

Animals, including humans, learn and remember to avoid a novel food when its ingestion is followed, hours later, by sickness - a phenomenon initially identified during World War II as a potential means of pest control. In the 1960s, John Garcia (for whom the effect is now named) demonstrated that this form of conditioned taste aversion had broader implications, showing that it is a rapid but long-lasting taste-specific food aversion with a fundamental role in the evolution of behaviour. From the mid-1970s onward, the principles of the Garcia effect were translated to humans, showing its role in different clinical conditions (e.g. side-effects linked to chemotherapy). However, in the last two decades, the number of studies on the Garcia effect has undergone a considerable decline. Since its discovery in rodents, this form of learning was thought to be exclusive to mammals; however, we recently provided the first demonstration that a Garcia effect can be formed in an invertebrate model organism, the pond snail Lymnaea stagnalis. Thus, in this Commentary, after reviewing the experiments that led to the first characterization of the Garcia effect in rodents, we describe the recent evidence for the Garcia effect in L. stagnalis, which may pave the way for future studies in other invertebrates and mammals. This article aims to inspire future translational and ecological studies that characterize the conserved mechanisms underlying this form of learning with deep evolutionary roots, which can be used to address a range of different biological questions.

Investigating the interactions between multiple memory stores in the pond snail Lymnaea stagnalis.

The pond snail Lymnaea stagnalis exhibits various forms of associative learning including (1) operant conditioning of aerial respiration where snails are trained not to open their pneumostome in a hypoxic pond water environment using a weak tactile stimulus to their pneumostome as they attempt to open it; and (2) a 24 h-lasting taste-specific learned avoidance known as the Garcia effect utilizing a lipopolysaccharide (LPS) injection just after snails eat a novel food substance (carrot). Typically, lab-inbred snails require two 0.5 h training sessions to form long-term memory (LTM) for operant conditioning of aerial respiration. However, some stressors (e.g., heat shock or predator scent) act as memory enhancers and thus a single 0.5 h training session is sufficient to enhance LTM formation lasting at least 24 h. Here, we found that snails forming a food-aversion LTM following Garcia-effect training exhibited enhanced LTM following operant condition of aerial respiration if trained in the presence of the food substance (carrot) they became averse to. Control experiments led us to conclude that carrot becomes a 'sickness' risk signal and acts as a stressor, sufficient to enhance LTM formation for another conditioning procedure.

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.

Snails go on a fast when acetylsalicylic acid comes along with heat stress: A possible effect of HSPs and serotonergic system on the feeding response.

A novel food followed by sickness, causes a taste-specific conditioned aversion, known as the 'Garcia effect'. We recently found that both a heat shock stressor (30 °C for 1 h - HS) and the bacterial lipopolysaccharide (LPS) can be used as 'sickness-inducing' stimuli to induce a Garcia effect in the pond snail Lymnaea stagnalis. Additionally, if snails are exposed to acetylsalicylic acid (ASA) present in aspirin tablets before the LPS injection, the formation of the Garcia effect is prevented. Here, we hypothesized that exposing snails to crushed aspirin before the HS (ASA-HS) would prevent the HS-induced 'sickness state' and - therefore -the Garcia effect. Unexpectantly, the ASA-HS procedure induced a generalized and long-lasting feeding suppression. We thus investigate the molecular effects underlying this phenomenon. While the exposure to the HS alone resulted in a significant upregulation of the mRNA levels of the Heat Shock Protein 70 (HSP 70) in snails' central ring ganglia, the ASA-HS procedure induced an even greater upregulation of HSP70, suggesting that the ASA-HS combination causes a severe stress response that inhibits feeding. Additionally, we found that the ASA-HS procedure induced a significant downregulation of the mRNA levels of genes involved with the serotoninergic system which regulates feeding in snails. Finally, the ASA-HS procedure prevented HS-induced upregulation of the mRNA levels of key neuroplasticity genes. Our study indicates that two sickness-inducing stimuli can have different physiological responses even if behavioral outcomes are similar under some learning contexts.

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.

A Novel Behavioral Display in Lymnaea Induced by Quercetin and Hypoxia.

AbstractThe pond snail Lymnaea stagnalis employs aerial respiration under hypoxia and can be operantly conditioned to reduce this behavior. When applied individually, a heat shock (30 °C for 1 h) and the flavonoid quercetin enhance long-term memory formation for the operant conditioning of aerial respiration. However, when snails are exposed to quercetin before the heat shock, long-term memory is no longer enhanced. This is because quercetin prevents the heat-induced upregulation of heat-shock proteins 70 and 40. When we tested the memory outcome of operant conditioning due to the simultaneous exposure to quercetin and 30 °C, we found that Lymnaea entered a quiescent survival state. The same behavioral response occurred when snails were simultaneously exposed to quercetin and pond water made hypoxic by bubbling nitrogen through it. Thus, in this study, we performed six experiments to propose a physiological explanation for that curious behavioral response. Our results suggest that bubbling nitrogen in pond water, heating pond water to 30 °C, and bubbling nitrogen in 30 °C pond water create a hypoxic environment, to which organisms may respond by upregulating the heat-shock protein system. On the other hand, when snails experience quercetin together with these hypoxic conditions, they can no longer express the physiological stress response evoked by heat or hypoxia. Thus, the quiescent survival state could be an emergency response to survive the hypoxic condition when the heat-shock proteins cannot be activated.

LPS-Induced Garcia Effect and Its Pharmacological Regulation Mediated by Acetylsalicylic Acid: Behavioral and Transcriptional Evidence.

Lymnaea stagnalis learns and remembers to avoid certain foods when their ingestion is followed by sickness. This rapid, taste-specific, and long-lasting aversion-known as the Garcia effect-can be formed by exposing snails to a novel taste and 1 h later injecting them with lipopolysaccharide (LPS). However, the exposure of snails to acetylsalicylic acid (ASA) for 1 h before the LPS injection, prevents both the LPS-induced sickness state and the Garcia effect. Here, we investigated novel aspects of this unique form of conditioned taste aversion and its pharmacological regulation. We first explored the transcriptional effects in the snails' central nervous system induced by the injection with LPS (25 mg), the exposure to ASA (900 nM), as well as their combined presentation in untrained snails. Then, we investigated the behavioral and molecular mechanisms underlying the LPS-induced Garcia effect and its pharmacological regulation by ASA. LPS injection, both alone and during the Garcia effect procedure, upregulated the expression levels of immune- and stress-related targets. This upregulation was prevented by pre-exposure to ASA. While LPS alone did not affect the expression levels of neuroplasticity genes, its combination with the conditioning procedure resulted in their significant upregulation and memory formation for the Garcia effect.

Agricultural Use of Insecticides Alters Homeostatic Behaviors and Cognitive Ability in Lymnaea stagnalis.

Lymnaea stagnalis is an ecologically important, stress-sensitive, freshwater mollusk that is at risk for exposure to insecticides via agricultural practices. We provide insight into the impact insecticides have on L. stagnalis by comparing specific behaviors including feeding, locomotion, shell regeneration, and cognition between snails collected at two different sites: one contaminated by insecticides and one not. We hypothesized that each of the behaviors would be altered in the insecticide-exposed snails and that similar alterations would be induced when control snails were exposed to the contaminated environment. We found no significant differences in locomotion, feeding, and shell regeneration of insecticide-exposed L. stagnalis compared with nonexposed individuals. Significant changes in feeding and shell repair were observed in nonexposed snails inhabiting insecticide-contaminated pond water. Most importantly, snails maintained and trained in insecticide-contaminated pond water did not form configural learning, but this cognitive deficit was reversed when these snails were maintained in insecticide-free pond water. Our findings conclude that insecticides have a primarily negative impact on this higher form of cognition in L. stagnalis. Environ Toxicol Chem 2023;42:2466-2477. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Prey populations with different predation histories show differences in behavioral and transcriptional effects under acute predation threat.

Predator detection induces both behavioral and physiological responses in prey organisms. Our model organism, the pond snail Lymnaea stagnalis, shows multiple defensive behaviors in response to predator cues. In this study, we investigated and compared the transcriptional effects induced by the exposure to a predator scent (i.e., crayfish effluent - CE) in a strain of lab-inbred snails (i.e., W snails), which have been raised and maintained under standardized laboratory conditions for generations and a strain of freshly collected snails (i.e., Margo snails), which live in a crayfish-free pond. Neither the W- strain nor the Margo Lake snails used in this study have actually experienced crayfish. However, the W strain innately recognizes crayfish as a threat. We found that, following the exposure to CE, both strains showed significantly higher mRNA levels of serotonin-related genes. This is important, as the serotonergic system modulates predator detection and vigilance behaviors in pond snails. However, the expression levels of CREB1 and HSP70 were only upregulated in CE-exposed W snails but not in Margo ones. As CREB1 plays a key role in learning and memory formation, whereas HSP70 is involved in stress response, we investigated whether these differences in CREB1 and HSP70 mRNA levels would reflect differences in predator-induced learning (e.g., configural learning). We found that only W snails formed configural learning memory, whereas Margo snails did not. Thus, while both the strains molecularly respond to the CE by upregulating the serotoninergic system, only W snails behaviorally recognize CE as a threat and, therefore, form configural 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.

Novel taste, sickness, and memory: Lipopolysaccharide to induce a Garcia-like effect in inbred and wild strains of Lymnaea stagnalis.

Food is not only necessary for our survival but also elicits pleasure. However, when a novel food is followed sometime later by nausea or sickness animals form a long-lasting association to avoid that food. This phenomenon is called the 'Garcia effect'. We hypothesized that lipopolysaccharide (LPS) could be used as the sickness-inducing stimulus to produce a Garcia-like effect in inbred and wild populations of Lymnaea stagnalis. We first demonstrated that the injection of 25 µg (6.25 µg/mL) of Escherichia coli-derived LPS serotype O127:B8 did not by itself alter snails' feeding behavior. Then we showed that the presentation of a novel appetitive stimulus (i.e., carrot slurry) and LPS resulted in a taste-specific and long-lasting feeding suppression (i.e., the Garcia-like effect). We also found strain-specific variations in the duration of the long-term memory (LTM). That is, while the LTM for the Garcia-like effect in W-strain snails persisted for 24h, LTM persisted for 48h in freshly collected Margo snails and their F1 offspring. Finally, we demonstrated that the exposure to a non-steroidal anti-inflammatory drug, aspirin (acetylsalicylic acid) before the LPS injection prevented both the LPS-induced sickness state and the Garcia-like effect from occurring. The results of this study may pave the way for new research that aims at (1) uncovering the conserved molecular mechanisms underlying the Garcia-like effect, (2) understanding how cognitive traits vary within and between species, and (3) creating a holistic picture of the complex dialogue between the immune and central nervous systems.

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.

CNS serotonin content mediating food deprivation-enhanced learning is regulated by hemolymph tryptophan concentration and autophagic flux in the pond snail.

Nutritional status affects cognitive function in many types of organisms. In the pond snail Lymnaea stagnalis, 1 day of food deprivation enhances taste aversion learning ability by decreasing the serotonin (5-hydroxytryptamin; 5-HT) content in the central nervous system (CNS). On the other hand, after 5 days of food deprivation, learning ability and the CNS 5-HT concentration return to basal levels. How food deprivation leads to alterations of 5-HT levels in the CNS, however, is unknown. Here, we measured the concentration of the 5-HT precursor tryptophan in the hemolymph and CNS, and demonstrated that the CNS tryptophan concentration was higher in 5-day food-deprived snails than in non-food-deprived or 1-day food-deprived snails, whereas the hemolymph tryptophan concentration was not affected by the duration of food deprivation. This finding suggests the existence of a mediator of the CNS tryptophan concentration independent of food deprivation. To identify the mediator, we investigated autophagic flux in the CNS under different food deprivation conditions. We found that autophagic flux was significantly upregulated by inhibition of the tropomyosin receptor kinase (Trk)-Akt-mechanistic target of rapamycin complex 1 (MTORC1) pathway in the CNS of 5-day food-deprived snails. Moreover, when autophagy was inhibited, the CNS 5-HT content was significantly downregulated in 5-day food-deprived snails. Our results suggest that the hemolymph tryptophan concentration and autophagic flux in the CNS cooperatively regulate learning ability affected by different durations of food deprivation. This mechanism may underlie the selection of behaviors appropriate for animal survival depending on the degree of nutrition.

Too Hot to Eat: Wild and Lab-Bred Lymnaea stagnalis Differ in Feeding Response Following Repeated Heat Exposure.

AbstractAcute extreme heat events are increasing in frequency and intensity. Understanding their effects on ectothermic organisms' homeostasis is both important and urgent. In this study we found that the exposure to an acute heat shock (30 °C for 1 hour) repeated for a seven-day period severely suppressed the feeding behavior of laboratory-inbred (W-strain) Lymnaea stagnalis, whereas the first-generation offspring of freshly collected wild (F1 D-strain) snails raised and maintained under similar laboratory conditions did not show any alterations. The W-strain snails might have inadvertently been selected against heat tolerance since they were first brought into the laboratory many (∼70) years ago. We also posit that the F1 D-strain snails do not perceive the heat shock as a sufficient stressor to alter their feeding response because their parental populations in wild environments have repeatedly experienced temperature fluctuations, thus becoming more tolerant and resilient to heat. The different responses exhibited by two strains of the same species highlight the importance of selecting the most appropriate strain for addressing questions about the impacts of global warming on organisms' physiology and behavior.

Aspirin reverts lipopolysaccharide-induced learning and memory impairment: first evidence from an invertebrate model system.

By employing a reductionistic (but not simplistic) approach using an established invertebrate model system, the pond snail Lymnaea stagnalis, we investigated whether (1) lipopolysaccharide (LPS)-induced inflammation would cause a sickness state and impair cognitive function, and-if so-(2) would aspirin (acetylsalicylic acid-ASA) restore the impaired cognition. To test our hypotheses, we first determined if the injection of 25 mg (6.25 µg/mL) of Escherichia coli-derived LPS serotype O127:B8 altered homeostatic behavior, aerial respiration, and then determined if LPS altered memory formation when this behavior was operantly conditioned. Next, we determined if ASA altered the LPS-induced changes in both aerial respiration and cognitive functions. LPS induced a sickness state that increased aerial respiration and altered the ability of snails to form or recall long-term memory. ASA reverted the LPS-induced sickness state and thus allowed long-term memory both to be formed and recalled. We confirmed our hypotheses and provided the first evidence in an invertebrate model system that an injection of LPS results in a sickness state that obstructs learning and memory, and this impairment can be prevented by a non-steroidal anti-inflammatory.

Epicatechin Alters the Activity of a Neuron Necessary for Long-Term Memory of Aerial Respiratory Behavior in Lymnaea stagnalis.

Epicatechin (EpiC) enhances long-term memory (LTM) formation in the pond snail Lymnaea stagnalis. Here we investigated at the level of a single neuron, RPeD1, which is a necessary site for LTM formation of operant conditioning of aerial respiration, how EpiC may bring about its enhancing effect on LTM formation. When snails were operantly conditioned in EpiC (15 mg/l) by a single 0.5 h training session, which typically only results in memory lasting ∼3 h, they now formed LTM lasting at least 24 h. We recorded from RPeD1 in semi-intact preparations made from snails 24 h after a single 0.5 h training session in EpiC or pond water (PW) and found that the firing and bursting rate of RPeD1 decreased significantly in the EpiC preparations compared to the PW preparations. However, the excitability (i.e., number of spikes evoked by injected depolarizing current) of RPeD1 was not different between the two preparations. We next performed "in vitro" operant training in semi-intact preparations made from naïve snails. In the training, we applied a gentle tactile stimulus to the pneumostome area every time the semi-intact preparation began to open. The preparations exposed to EpiC-saline (15 mg/l) exhibited significantly increased RPeD1 excitability compared with saline only preparations. These results suggest that EpiC can alter some electrophysiological properties of a neuron that is a necessary site for learning and memory formation.

Fluoride alters feeding in lab-bred pond snails but not in wild snails or their progeny.

Increasing concentrations of fluoride in natural bodies of water due to anthropogenic activities can lead to potentially detrimental effects on residing species. Here we investigated the differences in fluoride exposure on feeding behaviour between freshly collected (i.e., wild) and lab-bred Lymnaea stagnalis and if developmental exposure plays a key role in fluoride tolerance. We show that wild snails that reside in naturally fluoridated waters and their fluoride naïve lab-reared progeny have a fluoride tolerance that does not suppress feeding when introduced to a fluoridated food stimulus. These results were also seen in our lab-bred snails who were exposed to artificially fluoridated pond water (at similar concentrations to natural levels) throughout development. However, lab-bred snails that have never been exposed to fluoride, or only exposed during the egg stage demonstrated suppression of feeding in the fluoridated food stimulus condition compared to an unfluoridated food stimulus. Genetic diversity and phenotypic plasticity are suspected to be two key underlying mechanisms for fluoride ion tolerance. These results are critical in understanding how parental and developmental exposure can influence a phenotypic tolerance to a potential chemical pollutant.

Fluoride affects memory by altering the transcriptional activity in the central nervous system of Lymnaea stagnalis.

Fluoride (F-), has been found to affect learning and memory in several species. In this study, we exposed an F--naïve, inbred strain of Lymnaea stagnalis to a concentration of F- similar to that naturally occurring in wild ponds. We found that the exposure to F- before the configural learning procedure obstructs the memory formation and blocks the configural learning-induced upregulation of CREB1, GRIN1, and HSP70 in snails' central ring ganglia. Along with altering the mRNA levels of these key genes for memory formation, a single acute F- exposure also upregulates Cytochrome c Oxidase, a major regulatory enzyme of the electron transport chain, which plays direct or indirect roles in reactive oxygen species production. As the central nervous system is sensitive to oxidative stress and consistent with previous studies from mammals, our results suggest a potential role of oxidative stress in memory impairment. To our knowledge, this is the first study investigating the neuronal mechanism of memory impairment in an invertebrate species that is exposed to natural F- levels.

Nature versus nurture in heat stress induced learning between inbred and outbred populations of Lymnaea stagnalis.

Changing environmental conditions often lead to microevolution of traits that are adaptive under the current selection pressure. Currently, one of the major selection pressures is the rise in temperatures globally that has a severe impact on the behavioral ecology of animals. However, the role of thermal stress on neuronal plasticity and memory formation is not well understood. Thermal tolerance and sensitivity to heat stress show variation across populations of the same species experiencing different thermal regimes. We used two populations of the pond snail Lymnaea stagnalis: one lab-bred W-snails and the other wild Delta snails to test heat shock induced learning and memory formation for the Garcia effect learning paradigm. In Garcia effect, a single pairing of a heat stressor (30 °C for 1h) with a novel taste results in a taste-specific negative hedonic shift lasting 24h as long-term memory (LTM) in lab bred W-snails. In this study we used a repeated heat stress procedure to test for increased or decreased sensitivity to the heat before testing for the Garcia effect. We found that lab-bred W-snails show increased sensitivity to heat stress after repeated heat exposure for 7days, leading to enhanced LTM for Garcia effect with only 15min of heat exposure instead of standard 1h. Surprisingly, the freshly collected wild snails do not show Garcia effect. Additionally, F1 generation of wild snails raised and maintained under laboratory conditions still retain their heat stress tolerance similar to their parents and do not show a Garcia effect under standard learning paradigm or even after repeated heat stressor. Thus, we found a differential effect of heat stress on memory formation in wild and lab bred snails. Most interestingly we also show that local environmental (temperature) conditions for one generation is not enough to alter thermal sensitivity in a wild population of L. stagnalis.

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.