Genes Upregulated by Operant Conditioning of Escape Behavior in the Pond Snail Lymnaea stagnalis.

The pond snail Lymnaea stagnalis is capable of learning by both classical conditioning and operant conditioning. Although operant conditioning related to escape behavior with punishment has been examined by some research groups, the molecular mechanisms are not known. In the present study, we examined changes in the expression levels of cAMP-response element binding protein 1 (CREB1), CREB2, CREB-binding protein (CBP), and monoamine oxidase (MAO) in the Lymnaea central nervous system (CNS) using real-time PCR following operant conditioning of escape behavior. CREB1 and CREB2 are transcription factors involved in long-term memory in Lymnaea; CBP is a coactivator with CREB1; and MAO is a degrading enzyme for monoamines (e.g., serotonin) with important roles in learning and memory in Lymnaea. In operant conditioning, the punishment cohort, in which snails escaping from the container encountered aversive KCl, exhibited significantly fewer escape attempts than the control cohort, in which snails escaping from the container encountered distilled water, during both the training and memory test periods. After the operant conditioning, CREB1 and CREB2 were upregulated, and the ratio of CREB1/CREB2 was also increased, suggesting that the operant conditioning of escape behavior involves these factors. MAO was also upregulated, suggesting that the content of monoamines such as serotonin in the CNS decreased. The upregulated genes identified in the present study will help to further elucidate learning and memory mechanisms in Lymnaea.

Expression Level Changes in Serotonin Transporter are Associated with Food Deprivation in the Pond Snail Lymnaea stagnalis.

In the pond snail Lymnaea stagnalis, serotonin (5-HT) plays an important role in feeding behavior and its associated learning (e.g., conditioned taste aversion: CTA). The 5-HT content in the central nervous system (CNS) fluctuates with changes in the nutritional status, but it is also expected to be influenced by changes in the serotonin transporter (SERT) expression level. In the present study, we identified SERT in Lymnaea and observed its localization in 5-HTergic neurons, including the cerebral giant cells (CGCs) in the cerebral ganglia and the pedal A cluster neurons and right and left pedal dorsal 1 neurons in the pedal ganglia by in situ hybridization. Real-time PCR revealed that the SERT mRNA expression level was lower under severe food deprivation than under mild food deprivation in the whole CNS as well as in a single CGC. These results inversely correlated with previous data that the 5-HT content in the CNS was higher in the severely food-deprived state than in the mildly food-deprived state. Furthermore, in single CGCs, we observed that the 5-HT level was significantly increased in the severely food-deprived state compared with the mildly food-deprived state. Our present findings suggest that changes in the SERT expression level associated with food deprivation may affect 5-HT signaling, probably contributing to learning and memory mechanisms in Lymnaea.

FOXO in Lymnaea: Its Probable Involvement in Memory Consolidation.

Food deprivation activates forkhead box O (FOXO), a transcription factor downstream of insulin receptors. In the pond snail Lymnaea stagnalis, insulin signaling and food deprivation improve memory consolidation following conditioned taste aversion (CTA) learning. We investigated the subcellular localization of FOXO in Lymnaea and changes in its expression levels following food deprivation, CTA learning, and insulin administration. Immunohistochemistry revealed that Lymnaea FOXO (LymFOXO) was located in the central nervous system (CNS) neuronal cytoplasm in food-satiated snails but was mainly in neuronal nuclei in food-deprived snails. Following CTA acquisition, LymFOXO translocated to the nuclei in food-satiated snails and remained in the nuclei in food-deprived snails. Contrary to our expectations, insulin administered to the CNS did not induce LymFOXO translocation into the nuclei in food-satiated snails. Real-time PCR was used to quantify LymFOXO mRNA levels, its target genes, and insulin signaling pathway genes and revealed that LymFOXO mRNA was upregulated in food-deprived snails compared to food-satiated snails. Insulin applied to isolated CNSs from food-satiated snails increased LymFOXO compared to vehicle-treated samples. Food deprivation prepares FOXO to function in the nucleus and enhances CTA learning in snails. Insulin application did not directly affect LymFOXO protein localization. Thus, insulin administration may stimulate pathways other than the LymFOXO cascade.

Identification of Putative Molecules for Adiponectin and Adiponectin Receptor and Their Roles in Learning and Memory in Lymnaea stagnalis.

Adiponectin enhances insulin sensitivity, which improves cognition in mammals. How adiponectin affects the mechanism's underlying cognition, however, remains unknown. We hypothesized that experiments using the pond snail Lymnaea stagnalis, which has long been used in learning and memory studies and in which the function of insulin-like peptides affect learning and memory, could clarify the basic mechanisms by which adiponectin affects cognition. We first identified putative molecules of adiponectin and its receptor in Lymnaea. We then examined their distribution in the central nervous system and changes in their expression levels when hemolymph glucose concentrations were intentionally decreased by food deprivation. We also applied an operant conditioning protocol of escape behavior to Lymnaea and examined how the expression levels of adiponectin and its receptor changed after the conditioned behavior was established. The results demonstrate that adiponectin and adiponectin's receptor expression levels were increased in association with a reduced concentration of hemolymph glucose and that expression levels of both adiponectin and insulin-like peptide receptors were increased after the conditioning behavior was established. Thus, the involvement of the adiponectin-signaling cascade in learning and memory in Lymnaea was suggested to occur via changes in the glucose concentrations and the activation of insulin.

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.

Comparison between relative and absolute quantitative real-time PCR applied to single-cell analyses: Transcriptional levels in a key neuron for long-term memory in the pond snail.

Quantitative real-time PCR (qPCR) is a powerful method for measuring nucleic acid levels and quantifying mRNA levels, even in single cells. In the present study, we compared the results of single-cell qPCR obtained by different quantification methods (relative and absolute) and different reverse transcription methods. In the experiments, we focused on the cerebral giant cell (CGC), a key neuron required for the acquisition of conditioned taste aversion in the pond snail Lymnaea stagnalis, and examined changes in the mRNA levels of 3 memory-related genes, cAMP-response element binding proteins (LymCREB1 and LymCREB2) and CREB-binding protein (LymCBP), during memory formation. The results obtained by relative quantification showed similar patterns for the 3 genes. For absolute quantification, reverse transcription was performed using 2 different methods: a mixture of oligo d(T) primers and random primers (RT method 1); and gene-specific primers (RT method 2). These methods yielded different results and did not show consistent changes related to conditioning. The mRNA levels in the samples prepared by RT method 2 were up to 3.3 times higher than those in samples prepared by RT method 1. These results suggest that for qPCR of single neurons, the efficacy and validity do not differ between relative and absolute quantification methods, but the reverse transcription step critically influences the results of mRNA quantification.

Molecular and functional characterization of an evolutionarily conserved CREB-binding protein in the Lymnaea CNS.

In eukaryotes, CREB-binding protein (CBP), a coactivator of CREB, functions both as a platform for recruiting other components of the transcriptional machinery and as a histone acetyltransferase (HAT) that alters chromatin structure. We previously showed that the transcriptional activity of cAMP-responsive element binding protein (CREB) plays a crucial role in neuronal plasticity in the pond snail Lymnaea stagnalis. However, there is no information on the molecular structure and HAT activity of CBP in the Lymnaea central nervous system (CNS), hindering an investigation of its postulated role in long-term memory (LTM). Here, we characterize the Lymnaea CBP (LymCBP) gene and identify a conserved domain of LymCBP as a functional HAT. Like CBPs of other species, LymCBP possesses functional domains, such as the KIX domain, which is essential for interaction with CREB and was shown to regulate LTM. In-situ hybridization showed that the staining patterns of LymCBP mRNA in CNS are very similar to those of Lymnaea CREB1. A particularly strong LymCBP mRNA signal was observed in the cerebral giant cell (CGC), an identified extrinsic modulatory interneuron of the feeding circuit, the key to both appetitive and aversive LTM for taste. Biochemical experiments using the recombinant protein of the LymCBP HAT domain showed that its enzymatic activity was blocked by classical HAT inhibitors. Preincubation of the CNS with such inhibitors blocked cAMP-induced synaptic facilitation between the CGC and an identified follower motoneuron of the feeding system. Taken together, our findings suggest a role for the HAT activity of LymCBP in synaptic plasticity in the feeding circuitry.

Insulin and Memory in Invertebrates.

Insulin and insulin-like peptides (ILP) help to maintain glucose homeostasis, whereas insulin-like growth factor (IGF) promotes the growth and differentiation of cells in both vertebrates and invertebrates. It is sometimes difficult to distinguish between ILP and IGF in invertebrates, however, because in some cases ILP has the same function as IGF. In the present review, therefore, we refer to these peptides as ILP/IGF signaling (IIS) in invertebrates, and discuss the role of IIS in memory formation after classical conditioning in invertebrates. In the arthropod Drosophila melanogaster, IIS is involved in aversive olfactory memory, and in the nematode Caenorhabditis elegans, IIS controls appetitive/aversive response to NaCl depending on the duration of starvation. In the mollusk Lymnaea stagnalis, IIS has a critical role in conditioned taste aversion. Insulin in mammals is also known to play an important role in cognitive function, and many studies in humans have focused on insulin as a potential treatment for Alzheimer's disease. Although analyses of tissue and cellular levels have progressed in mammals, the molecular mechanisms, such as transcriptional and translational levels, of IIS function in cognition have been far advanced in studies using invertebrates. We anticipate that the present review will help to pave the way for studying the effects of insulin, ILPs, and IGFs in cognitive function across phyla.

Profile of dorsal root ganglion neurons: study of oxytocin expression.

Although dorsal root ganglion (DRG) neurons have been so far classified according to the difference in their fibers (Aß, Aδ, and C), this classification should be further subdivided according to gene expression patterns. We focused on oxytocin (OXT) and its related receptors, because OXT plays a local role in DRG neurons. We measured the mRNA levels of OXT, OXT receptor (OXTR), vasopressin V1a receptor (V1aR), transient receptor potential cation channel subfamily V member 1 (TRPV1), and piezo-type mechanosensitive ion channel component 2 (Piezo2) in single DRG neurons by using real-time PCR, and then performed a cluster analysis. According to the gene expression patterns, DRG neurons were classified into 4 clusters: Cluster 1 was characterized mainly by Piezo2, Cluster 2 by TRPV1, Cluster 4 by OXTR, and neurons in Cluster 3 did not express any of the target genes. The cell body diameter of OXT-expressing neurons was significantly larger in Cluster 1 than in Cluster 2. These results suggest that OXT-expressing DRG neurons with small cell bodies (Cluster 2) and large cell bodies (Cluster 1) probably correspond to C-fiber neurons and Aß-fiber neurons, respectively. Furthermore, the OXT-expressing neurons contained not only TRPV1 but also Piezo2, suggesting that OXT may be released by mechanical stimulation regardless of nociception. Thus, mechanoreception and nociception themselves may induce the autocrine/paracrine function of OXT in the DRG, contributing to alleviation of pain.

Acetylation of the influenza A virus polymerase subunit PA in the N-terminal domain positively regulates its endonuclease activity.

The post-translational acetylation of lysine residues is found in many nonhistone proteins and is involved in a wide range of biological processes. Recently, we showed that the nucleoprotein of the influenza A virus is acetylated by histone acetyltransferases (HATs), a phenomenon that affects viral transcription. Here, we report that the PA subunit of influenza A virus RNA-dependent RNA polymerase is acetylated by the HATs, P300/CREB-binding protein-associated factor (PCAF), and general control nonderepressible 5 (GCN5), resulting in accelerated endonuclease activity. Specifically, the full-length PA subunit expressed in cultured 293T cells was found to be strongly acetylated. Moreover, the partial recombinant protein of the PA N-terminal region containing the endonuclease domain was also acetylated by PCAF and GCN5 in vitro, which facilitated its endonuclease activity. Mass spectrometry analyses identified K19 as a candidate acetylation target in the PA N-terminal region. Notably, the substitution of the lysine residue at position 19 with glutamine, a mimic of the acetyl-lysine residue, enhanced its endonuclease activity in vitro; this point mutation also accelerated influenza A virus RNA-dependent RNA polymerase activity in the cell. Our findings suggest that PA acetylation is important for the regulation of the endonuclease and RNA polymerase activities of the influenza A virus.

In-vitro acetylation of SARS-CoV and SARS-CoV-2 nucleocapsid proteins by human PCAF and GCN5.

Recently, the novel coronavirus (SARS-CoV-2), which has spread from China to the world, was declared a global public health emergency, which causes lethal respiratory infections. Acetylation of several proteins plays essential roles in various biological processes, such as viral infections. We reported that the nucleoproteins of influenza virus and Zaire Ebolavirus were acetylated, suggesting that these modifications contributed to the molecular events involved in viral replication. Similar to influenza virus and Ebolavirus, the coronavirus also contains single-stranded RNA, as its viral genome interacts with the nucleocapsid (N) proteins. In this study, we report that SARS-CoV and SARS-CoV-2 N proteins are strongly acetylated by human histone acetyltransferases, P300/CBP-associated factor (PCAF), and general control nonderepressible 5 (GCN5), but not by CREB-binding protein (CBP) in vitro. Liquid chromatography-mass spectrometry analyses identified 2 and 12 acetyl-lysine residues from SARS-CoV and SARS-CoV-2 N proteins, respectively. Particularly in the SARS-CoV-2 N proteins, the acetyl-lysine residues were localized in or close to several functional sites, such as the RNA interaction domains and the M-protein interacting site. These results suggest that acetylation of SARS-CoV-2 N proteins plays crucial roles in their functions.

Another Example of Conditioned Taste Aversion: Case of Snails.

Conditioned taste aversion (CTA) in mammals has several specific characteristics: (1) emergence of a negative symptom in subjects due to selective association with a taste-related stimulus, (2) robust long-term memory that is resistant to extinction induced by repeated presentation of the conditioned stimulus (CS), (3) a very-long-delay presentation of the unconditioned stimulus (US), and (4) single-trial learning. The pond snail, Lymnaea stagnalis, can also form a CTA. Although the negative symptoms, like nausea, in humans cannot be easily observed in invertebrate animal models of CTA, all the other characteristics of CTA seem to be present in snails. Selective associability was confirmed using a sweet sucrose solution and a bitter KCl solution. Once snails form a CTA, repeated presentation of the CS does not extinguish the CTA. A long interstimulus interval between the CS and US, like in trace conditioning, still results in the formation of a CTA in snails. Lastly, even single-trial learning has been demonstrated with a certain probability. In the present review, we compare, in detail, CTA in mammals and snails, and discuss the possible molecular events in CTA.

Monoamines, Insulin and the Roles They Play in Associative Learning in Pond Snails.

Molluscan gastropods have long been used for studying the cellular and molecular mechanisms underlying learning and memory. One such gastropod, the pond snail Lymnaea stagnalis, exhibits long-term memory (LTM) following both classical and operant conditioning. Using Lymnaea, we have successfully elucidated cellular mechanisms of learning and memory utilizing an aversive classical conditioning procedure, conditioned taste aversion (CTA). Here, we present the behavioral changes following CTA training and show that the memory score depends on the duration of food deprivation. Then, we describe the relationship between the memory scores and the monoamine contents of the central nervous system (CNS). A comparison of learning capability in two different strains of Lymnaea, as well as the filial 1 (F1) cross from the two strains, presents how the memory scores are correlated in these populations with monoamine contents. Overall, when the memory scores are better, the monoamine contents of the CNS are lower. We also found that as the insulin content of the CNS decreases so does the monoamine contents which are correlated with higher memory scores. The present review deepens the relationship between monoamine and insulin contents with the memory score.

Acetylation of lysine residues in the recombinant nucleoprotein and VP40 matrix protein of Zaire Ebolavirus by eukaryotic histone acetyltransferases.

Acetylation of histones and other proteins plays crucial roles in transcriptional regulation, chromatin organization, and other biological processes. It has been recently reported that the nucleoprotein (NP) of influenza virus is acetylated in infected cells, and this modification contributes to the RNA polymerization activity of the virus. As the influenza virus, the Ebolavirus contains single-stranded negative-sense RNA as its viral genome, which interacts with NP and other viral proteins. In this study, we performed a series of biochemical experiments and revealed that the recombinant Ebolavirus NP and the viral matrix protein VP40, which binds with NP, were acetylated by eukaryotic histone acetyltransferases, such as P300/CREB-binding protein (P300/CBP) and P300/CBP-associated factor (PCAF), in vitro. Mass spectrometry was used to identify the lysine residues that were potential acetylation targets in NP and VP40. The identified lysine residues in NP were located in the RNA-binding cleft and the VP35-binding domain. Potentially acetylated lysine targets in VP40 were identified in the basic patch, which is necessary for constructing oligomers. These results suggest that the acetylation of these lysine residues is involved in the interactions between viral proteins.

Influenza A virus nucleoprotein is acetylated by histone acetyltransferases PCAF and GCN5.

Histone acetylation plays crucial roles in transcriptional regulation and chromatin organization. Viral RNA of the influenza virus interacts with its nucleoprotein (NP), whose function corresponds to that of eukaryotic histones. NP regulates viral replication and has been shown to undergo acetylation by the cAMP-response element (CRE)-binding protein (CBP) from the host. However, whether NP is the target of other host acetyltransferases is unknown. Here, we show that influenza virus NP undergoes acetylation by the two host acetyltransferases GCN5 and P300/CBP-associated factor (PCAF) and that this modification affects viral polymerase activities. Western blot analysis with anti-acetyl-lysine antibody on cultured A549 human lung adenocarcinoma epithelial cells infected with different influenza virus strains indicated acetylation of the viral NP. A series of biochemical analyses disclosed that the host lysine acetyltransferases GCN5 and PCAF acetylate NP in vitro MS experiments identified three lysine residues as acetylation targets in the host cells and suggested that Lys-31 and Lys-90 are acetylated by PCAF and GCN5, respectively. RNAi-mediated silencing of GCN5 and PCAF did not change acetylation levels of NP. However, interestingly, viral polymerase activities were increased by the PCAF silencing and were decreased by the GCN5 silencing, suggesting that acetylation of the Lys-31 and Lys-90 residues has opposing effects on viral replication. Our findings suggest that epigenetic control of NP via acetylation by host acetyltransferases contributes to regulation of polymerase activity in the influenza A virus.

Effects of 5-HT and insulin on learning and memory formation in food-deprived snails.

The pond snail Lymnaea stagnalis learns conditioned taste aversion (CTA) and consolidates it into long-term memory (LTM). How well they learn and form memory depends on the degree of food deprivation. Serotonin (5-HT) plays an important role in mediating feeding, and insulin enhances the memory consolidation process following CTA training. However, the relationship between these two signaling pathways has not been addressed. We measured the 5-HT content in the central nervous system (CNS) of snails subjected to different durations of food deprivation. One-day food-deprived snails, which exhibit the best learning and memory, had the lowest 5-HT content in the CNS, whereas 5-day food-deprived snails, which do not learn, had a high 5-HT content. Immersing 1-day food-deprived snails in 5-HT impaired learning and memory by causing an increase in 5-HT content, and that the injection of insulin into these snails reversed this impairment. We conclude that insulin rescues the CTA deficit and this may be due to a decrease in the 5-HT content in the CNS of Lymnaea.

Pharmacological effects of cannabinoids on learning and memory in Lymnaea.

Cannabinoids are hypothesized to play an important role in modulating learning and memory formation. Here, we identified mRNAs expressed in Lymnaeastagnalis central nervous system that encode two G-protein-coupled receptors (Lymnaea CBr-like 1 and 2) that structurally resemble mammalian cannabinoid receptors (CBrs). We found that injection of a mammalian CBr agonist WIN 55,212-2 (WIN 55) into the snail before operant conditioning obstructed learning and memory formation. This effect of WIN 55 injection persisted for at least 4 days following its injection. A similar obstruction of learning and memory occurred when a severe traumatic stimulus was delivered to L. stagnalis In contrast, injection of a mammalian CBr antagonist AM 251 enhanced long-term memory formation in snails and reduced the duration of the effects of the severe traumatic stressor on learning and memory. Neither WIN 55 nor AM 251 altered normal homeostatic aerial respiratory behaviour elicited in hypoxic conditions. Our results suggest that putative cannabinoid receptors mediate stressful stimuli that alter learning and memory formation in Lymnaea This is also the first demonstration that putative CBrs are present in Lymnaea and play a key role in learning and memory formation.

Weak involvement of octopamine in aversive taste learning in a snail.

The pond snail Lymnaea stagnalis is capable of learning taste aversion by pairing presentations of a sucrose solution and an electric shock and consolidating it into long-term memory (LTM), which is referred to as conditioned taste aversion (CTA). We asked here if the neurotransmitter octopamine is involved in CTA. We first determined the levels of octopamine and its catabolites in the central nervous system (CNS) of snails with varying degrees of food deprivation, because CTA grades are correlated with degrees of food deprivation. We next manipulated the octopamine signaling using both an agonist and an antagonist of octopamine receptors and correlated their respective effects with CTA grades. We found that snails with the least amount of food-deprivation obtained the best CTA grade and had low levels of octopamine; whereas the most severely food-deprived snails did not form CTA and had the highest CNS octopamine levels. In modestly food-deprived snails, octopamine application increased the basal level of feeding response to a sucrose solution, and it did not obstruct CTA formation. Application of phentolamine, an octopamine receptor antagonist, to the most severely food-deprived snails decreased the basal level of feeding elicited by sucrose, but it did not enhance CTA formation. We conclude that octopamine involvement in CTA formation in Lymnaea is at best weak, and that the changes in CNS octopamine content are an epiphenomenon.

Structural and Biochemical Analyses on the RNA-dependent RNA Polymerase of Influenza Virus for Development of Novel Anti-influenza Agents.

The PA, PB1, and PB2 subunits, components of the RNA-dependent RNA polymerase of influenza A virus, and the nucleoprotein (NP) interact with the genomic RNA of influenza viruses and form ribonucleoproteins. Especially, the PB2 subunit binds to the host RNA cap [7-methylguanosine triphosphate (m7GTP)] and supports the endonuclease activity of PA to "snatch" the cap from host pre-mRNAs. In this study, we describe a novel Val/Arg/Gly (VRG) site in the PB2 cap-binding domain, which is necessary for interaction with acetyl-CoA found in eukaryotic histone acetyltransferases (HATs). In vitro experiments revealed that the recombinant PB2 cap-binding domain that includes the VRG site interacts with acetyl-CoA; moreover, it was found that this interaction could be blocked by CoA and various HAT inhibitors. Interestingly, m7GTP also inhibited this interaction, suggesting that the same active pocket is capable of interacting with acetyl-CoA and m7GTP. To elucidate the importance of the VRG site on PB2 function and viral replication, we constructed a PB2 recombinant protein and recombinant viruses including several patterns of amino acid mutations in the VRG site. Substitutions of 2 or 3 amino acid residues of the VRG site to alanine significantly reduced PB2's binding ability to acetyl-CoA and its RNA polymerase activity. Recombinant viruses containing the same mutations could not be replicated in cultured cells. These results indicate that the PB2 VRG sequence is a functional site that is essential for acetyl-CoA interaction, RNA polymerase activity, and viral replication. I will also discuss some novel functions of NP in this review.

Relationship between the grades of a learned aversive-feeding response and the dopamine contents in Lymnaea.

The pond snail Lymnaea learns conditioned taste aversion (CTA) and remembers not to respond to food substances that initially cause a feeding response. The possible relationship between how well snails learn to follow taste-aversion training and brain dopamine contents is not known. We examined this relationship and found the following: first, snails in the act of eating just before the commencement of CTA training were poor learners and had the highest dopamine contents in the brain; second, snails which had an ad libitum access to food, but were not eating just before training, were average learners and had lower dopamine contents; third, snails food-deprived for one day before training were the best learners and had significantly lower contents of dopamine compared to the previous two cohorts. There was a negative correlation between the CTA grades and the brain dopamine contents in these three cohorts. Fourth, snails food-deprived for five days before training were poor learners and had higher dopamine contents. Thus, severe hunger increased the dopamine content in the brain. Because dopamine functions as a reward transmitter, CTA in the severely deprived snails (i.e. the fourth cohort) was thought to be mitigated by a high dopamine content.