Behavioral and Transcriptional Effects of Short or Prolonged Fasting on the Memory Performances of Lymnaea stagnalis

Introduction: The Garcia effect, a solid learning paradigm, was used to investigate the molecular and behavioral effects induced by different lengths of fasting on the cognitive functions in the pond snail Lymnaea stagnalis, a valid model systemMethods: Three experimental groups were used: Moderately hungry snails, food-deprived for 1 day (D1 snails), severely hungry snails (D5 snails), fasting for 5 days, and satiated snails with ad libitum access to food (AL snails). In the Garcia effect, a single pairing of an appetitive stimulus with a heat stressor results in a learned taste-specific negative hedonic shift. D5 snails were injected with bovine insulin and D1 snails with the insulin receptor antibody (Ab). As a control group, AL snails were injected with saline. Gene expression analyses were performed by Real-time PCR in snails' central nervous system (CNS).Results: AL snails are 'average learners', D1 snails are the best performers, whereas the D5 ones do not show the Garcia effect. Severely fasting snails injected with insulin 3h before the training procedure, show the Garcia effect, whereas injecting 1-day fasting snails with insulin receptor Ab blocks their ability to express memory. The differences in memory performances are associated with changes in the expression levels of selected targets involved in neuronal plasticity, energy homeostasis, and stress response.Discussion: Our results suggest that short-term fasting creates an optimal internal state in L. stagnalis' CNS, allowing a spike in insulin release and an upregulation of genes involved in neuroplasticity. Long-term fasting, instead, upregulates genes involved in energy homeostasis and animal survival

Lymnaea stagnalis as model for translational neuroscience research: from pond to bench

The purpose of this review is to illustrate how a reductionistic, but sophisticated, approach based on the use of a simple model system such as the pond snail Lymnaea stagnalis (L. stagnalis), might be useful to address fundamental questions in learning and memory. L. stagnalis, as a model, provides an interesting platform to investigate the dialog between the synapse and the nucleus and vice versa during memory and learning. More importantly, the "molecular actors" of the memory dialogue are well-conserved both across phylogenetic groups and learning paradigms, involving single- or multi-trials, aversion or reward, operant or classical conditioning. At the same time, this model could help to study how, where and when the memory dialog is impaired in stressful conditions and during aging and neurodegeneration in humans and thus offers new insights and targets in order to develop innovative therapies and technology for the treatment of a range of neurological and neurodegenerative disorders

Identification and characterization of the kynurenine pathway in the pond snail Lymnaea stagnalis

Dysregulation of the kynurenine pathway (KP) is implicated in many human diseases and disorders, from immunological, metabolic, neurodegenerative, and neuropsychiatric conditions to cancer, and represents an appealing target for new therapeutic approaches. In this intricate scenario, invertebrates, like Lymnaea stagnalis (LS), provide a flexible tool to unravel the complexity of the KP. Starting from the available LS genome and transcriptome, we identified putative transcripts of all KP enzymes containing an ORF; each predicted protein possessed a high degree of sequence conservation to known orthologues of other invertebrate and vertebrate model organisms. Sequences were confirmed by qualitative PCR and sequencing. At the same time, the qRT-PCR analysis revealed that Lym IDO-like, Lym TDO-like, Lym AFMID-like, Lym KMO-like, Lym AADAT-like, Lym KYAT I/III-like, Lym KYNU-like, Lym HAAO-like, and Lym ACMSD-like showed widespread tissue expression. Then, tryptophan, kynurenine, kynurenic acid, anthranilic acid, 3-hydroxy-kynurenine, xanthurenic acid, picolinic acid, and quinolinic acid were identified in the hemolymph of LS by UHPLC-Q exactive mass spectrometer. Our study provides the most thorough characterization to date of the KP in an invertebrate model, supporting the value of LS for future functional studies of this pathway at the cellular, synaptic, and behavioral levels

The Use of Psychotropic Medication in Pediatric Oncology for Acute Psychological and Psychiatric Problems: Balancing Risks and Benefits

Severe acute behavioral and emotional problems represent one of the most serious treatment-related adverse effects for children and adolescents who have cancer. The critical and severe nature of these symptoms often makes necessary the use of psychotropic drugs. A working group composed of experts in multiple disciplines had the task of creating an agreement regarding a management plan for severe acute behavioral and emotional problems (SABEPs) in children and adolescents treated for cancer. To obtain global information on the use of psychotropic drugs in pediatric oncology, the working group first developed and mailed a 15-item questionnaire to many Italian pediatric oncology centers. Overall, an evident lack of knowledge and education regarding the use of psychotropic medications for the treatment of SABEPs was found. Thus, by referring to an adapted version of the Delphi method of consensus and standard methods for the elaboration of clinical questions (PICOs), the working group elaborated evidence-based recommendations for psychotropic drugs in the pediatric oncology setting. Furthermore, based on a thorough multivariate analysis of needs and difficulties, a comprehensive management flow was developed to optimize therapeutic interventions, which allows more accurate and efficient matching of the acute needs of patients while guiding treatment options

Lymnaea stagnalis come modello per la ricerca traslazionale nell'ambito delle Neuroscienze: dallo stagno al bancone di laboratorio

Gli alti costi in termini di gestione, tempo e lavoro legati all'uso dei mammiferi nella ricerca biomedica hanno reso sempre più urgente la necessità di individuare modelli alternativi più economici e semplici ma altrettanto efficaci. Lo scopo della mia tesi è stata la caratterizzazione della chiocciola di stagno Lymnaea stagnalis come modello per le Neuroscienze Traslazionali. Gli studi bioinformatici, molecolari e comportamentali condotti hanno dimostrato la validità e la versatilità di questo modello e hanno permesso di esaminare fenomeni finora osservati solo nei mammiferi. In particolare, è emerso che: (1) L. stagnalis consente di approfondire il dialogo tra il sistema nervoso e immunitario, inclusi gli effetti dell’infiammazione sulle funzioni cognitive. Il trattamento con uno stimolo infiammatorio (lipopolisaccaride - LPS) è stato in grado di modulare l’espressione degli enzimi della pathway delle chinurenine nel sistema nervoso centrale di L. stagnalis. Questa pathway è altamente conservata tra vertebrati e invertebrati e metabolizza il triptofano in diversi cataboliti neuroattivi, i quali possono essere sia neurotossici che neuroprotettivi. Lo stesso stimolo è stato in grado di alterare il comportamento e le performance cognitive delle chiocciole, come osservato in organismi più complessi, uomo incluso. Questi effetti possono essere eliminati trattando preventivamente gli animali con un composto antinfiammatorio come l’aspirina. (2) L. stagnalis è in grado di formare il Garcia effect, una forma di apprendimento complesso che consiste in un’avversione condizionata a un sapore nuovo a seguito della sua associazione a uno stimolo avverso che induce un malessere viscerale, incontrato fino a 48h ore dopo. Come precedentemente dimostrato nel modello di roditore e nell’uomo, è stata sufficiente una singola presentazione dei due stimoli per generare una duratura e specifica avversione per questo sapore. L. stagnalis rappresenta quindi un modello semplificato per lo studio dei meccanismi alla base del Garcia effect negli organismi più complessi. (3) L. stagnalis può essere utilizzata per studiare gli effetti del riscaldamento globale sulla resilienza, i comportamenti adattativi e le funzioni cognitive degli organismi. L'esposizione giornaliera a uno shock termico ha aumentato la sensibilità alle temperature delle chiocciole di laboratorio (mantenute a temperature costanti per generazioni), ma non è stato un fattore di stress per le chiocciole appena raccolte nei loro habitat naturali (dove sono state esposte a considerevoli escursioni termiche), né per la loro progenie nata e allevata in laboratorio. Questi risultati hanno suggerito un duplice ruolo della genetica e della plasticità fisiologica nella termo-tolleranza. (4) L. stagnalis permette di studiare gli effetti di composti naturali bioattivi su apprendimento e memoria. L'esposizione acuta al flavonoide quercetina si è dimostrata in grado di migliorare la formazione della memoria a lungo termine, mentre a livello molecolare ha indotto un’up-regolazione della via serotoninergica e di CREB1 (cAMP response element-binding protein 1), i quali svolgono un ruolo chiave e altamente conservato nei processi di plasticità sinaptica. Anche se i modelli animali non potranno mai riassumere l'intero fenotipo dei cervelli umani, i risultati presentati nella mia tesi hanno illustrato che, se spostata dallo stagno al banco di laboratorio, L. stagnalis rappresenta un modello valido per aprire nuove frontiere nelle Neuroscienze Traslazionali. L'obiettivo finale di questo progetto è quello di fornire un ulteriore strumento per promuovere lo spostamento della ricerca dal banco di laboratorio al letto dei pazienti, 'traducendo' i dati ottenuti nelle chiocciole ai mammiferi.The high costs in time and efforts associated with the use of mammals in biomedical research are creating a pressing demand for alternative models that are cheaper and simpler, but still effective. The aim of my thesis was the characterization of the pond snail Lymnaea stagnalis as a model for Translational Neuroscience. Different bioinformatics, molecular, and behavioural studies have been performed to show the validity and versatility of this model to study phenomena so far demonstrated only in mammals. In particular, it has been shown that (1) L. stagnalis can be used to elucidate the conserved dialogue between the immune and nervous systems and to study the effects of inflammation on cognitive functions. An immune challenge (i.e., injection of lipopolysaccharide – LPS) affected the transcriptional levels of the enzymes of the kynurenine pathway in L. stagnalis’ central nervous ganglia. This conserved pathway in vertebrates and invertebrates catabolizes the aminoacid tryptophan into several neuroactive metabolites which can exert both neuroprotective and neurotoxic effects. The same immune challenge was able to alter snails’ adaptive behaviours and to obstruct their ability to form or show long-term memory (LTM), as observed for more complex organisms. These behavioural effects were prevented by exposing L. stagnalis to an anti-inflammatory compound, like aspirin, before the LPS injection, suggesting the involvement of immune-related molecules in mediating LPS-induced sequelae. The results of these studies gave important translational contributions for elucidating the effects of inflammation on the central nervous system. (2) L. stagnalis is capable of the Garcia effect, a higher form of learning, consisting of a conditioned aversion to an appetitive food stimulus consumed hours before the exposure to an aversive, nausea-inducing stimulus. As previously demonstrated in rodents and humans, a single paired presentation of these stimuli was sufficient to create a long-lasting and taste-specific gustatory aversion. This study allowed to elucidate the causal underpinnings of the Garcia effect in higher animals. (3) L. stagnalis can be used to predict the effects of the current global warming on animal resilience, adaptive behaviours, and cognitive functions. Daily exposure to a thermal shock increased the thermal sensitivity of laboratory-reared snails, which have been maintained under constant laboratory temperatures for generations. However, this ‘habitat-related challenge’ did not appear to be a stressor in freshly collected snails, that had experienced severe thermal fluctuations in their natural environment, nor in their progeny born and raised in lab conditions. These results allowed a better understanding of the role of genetic changes and physiological plasticity on thermotolerance. (4) L. stagnalis is a versatile model to examine the effects of bioactive natural compounds on learning and memory. Exposure to the flavonoid quercetin upregulated the serotoninergic pathway and CREB1 (cAMP response element-binding protein 1), a key regulator of synaptic plasticity in several in vivo models, in L. stagnalis’ central ganglia. This molecular effect was accompanied by an enhancement of LTM acquisition, consolidation, recall, and reconsolidation. Although animal models can never summarize the full phenotype of human brains, findings presented in my thesis illustrated that, when moved from pond to bench, L. stagnalis represents a valid model to open new frontiers in Translational Neuroscience. The ultimate goal of this project is to provide an additional tool to promote and sustain the rational and move research from bench to bedside, ‘translating’ data from snails to mammals, and maybe to humans

The temperature sensitivity of memory formation and persistence is altered by cold acclimation in a pond snail

There are reports on the inability of inbred, laboratory-reared Lymnaea stagnalis to perform feeding and aerial respiration in the cold. It has also been suggested that laboratory-bred snails have an inability to perform aerial respiration in winter months in the laboratory. Here, we used an inbred, laboratory-reared strain of Lymnaea (the S-strain) to demonstrate that the snails are capable of performing those behaviours in a cold (4 degrees C) environment after a 2 day acclimation period. In addition, the inbred snails were able to perform aerial respiration during winter months at room temperature (20 degrees C) in the laboratory. The persistence of long-term memory (LTM) was extended for at least 4 weeks by placing S-strain snails into a 4 degrees C environment following training. Typically, the cold block (CB) procedure (1 h at 4 degrees C) immediately after a training session blocks LTM formation in the S-strain but not in a freshly collected strain. Four weeks at 4 degrees C transformed the S-strain phenotype into one resisting the CB procedure. Thus, with a 4 week cold spell snails gain a resistance to the CB procedure, and that would explain why freshly collected snails are resistant to the procedure. However, we found that F1 progeny of a freshly collected strain reared in the laboratory were resistant to the CB procedure. This suggests that an unknown selection resulted in the Sstrain being susceptible to the CB procedure

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

What can we teach Lymnaea and what can Lymnaea teach us?

This review describes the advantages of adopting a molluscan complementary model, the freshwater snail Lymnaea stagnalis, to study the neural basis of learning and memory in appetitive and avoidance classical conditioning; as well as operant conditioning of its aerial respiratory and escape behaviour. We firstly explored ‘what we can teach Lymnaea’ by discussing a variety of sensitive, solid, easily reproducible and simple behavioural tests that have been used to uncover the memory abilities of this model system. Answering this question will allow us to open new frontiers in neuroscience and behavioural research to enhance our understanding of how the nervous system mediates learning and memory. In fact, from a translational perspective, Lymnaea and its nervous system can help to understand the neural transformation pathways from behavioural output to sensory coding in more complex systems like the mammalian brain. Moving on to the second question: ‘what can Lymnaea teach us?’, it is now known that Lymnaea shares important associative learning characteristics with vertebrates, including stimulus generalization, generalization of extinction and discriminative learning, opening the possibility to use snails as animal models for neuroscience translational research