Ion channel chameleons: Switching ion selectivity by alternative splicing.

Voltage-gated sodium and calcium channels are distinct, evolutionarily related ion channels that achieve remarkable ion selectivity despite sharing an overall similar structure. Classical studies have shown that ion selectivity is determined by specific binding of ions to the channel pore, enabled by signature amino acid sequences within the selectivity filter (SF). By studying ancestral channels in the pond snail (Lymnaea stagnalis), Guan et al. showed in a recent JBC article that this well-established mechanism can be tuned by alternative splicing, allowing a single CaV3 gene to encode both a Ca2+-permeable and an Na+-permeable channel depending on the cellular context. These findings shed light on mechanisms that tune ion selectivity in physiology and on the evolutionary basis of ion selectivity.

Retinoic acid reduces the formation of, and acutely modulates, invertebrate electrical synapses.

Communication between cells in the nervous system is dependent on both chemical and electrical synapses. Factors that can affect chemical synapses have been well studied, but less is known about factors that influence electrical synapses. Retinoic acid, the vitamin A metabolite, is a known regulator of chemical synapses, but few studies have examined its capacity to regulate electrical synapses. In this study, we determine that retinoic acid is capable of rapidly altering the strength of electrical synapses in an isomer- and cell-dependent manner. Furthermore, we provide evidence that this acute effect might be independent of either the retinoid receptors or the activation of a protein kinase. In addition to the rapid modulatory effects of retinoic acid, we provide data to suggest that retinoic acid is also capable of regulating the formation of electrical synapses. Long-term exposure to both all-trans-retinoic acid or 9-cis-retinoic acid reduced the proportion of cell pairs forming electrical synapses, as well as reduced the strength of electrical synapses that did form. In summary, this study provides insights into the role that retinoids might play in both the formation and modulation of electrical synapses in the central nervous system.NEW & NOTEWORTHY Retinoids are known modulators of chemical synapses and mediate synaptic plasticity in the nervous system, but little is known of their effects on electrical synapses. Here, we show that retinoids selectively reduce electrical synapses in a cell- and isomer-dependent manner. This modulatory action on existing electrical synapses was rapid and nongenomic in nature. We also showed for the first time that longer retinoid exposures inhibit the formation of electrical synapses.

Same same, but different: exploring the enigmatic role of the pituitary adenylate cyclase-activating polypeptide (PACAP) in invertebrate physiology.

Evidence has been accumulating that elements of the vertebrate pituitary adenylate cyclase-activating polypeptide (PACAP) system are missing in non-chordate genomes, which is at odds with the partial sequence-, immunohistochemical-, and physiological data in the literature. Multilevel experiments were performed on the great pond snail (Lymnaea stagnalis) to explore the role of PACAP in invertebrates. Screening of neuronal transcriptome and genome data did not reveal homologs to the elements of vertebrate PACAP system. Despite this, immunohistochemical investigations with an anti-human PAC1 receptor antibody yielded a positive signal in the neuronal elements in the heart. Although Western blotting of proteins extracted from the nervous system found a relevant band for PACAP-38, immunoprecipitation and mass spectrometric analyses revealed no corresponding peptide fragments. Similarly to the effects reported in vertebrates, PACAP-38 significantly increased cAMP synthesis in the heart and had a positive ionotropic effect on heart preparations. Moreover, it significantly modulated the effects of serotonin and acetylcholine. Homologs to members of Cluster B receptors, which have shared common evolutionary origin with the vertebrate PACAP receptors, PTHRs, and GCGRs, were identified and shown not to be expressed in the heart, which does not support a potential role in the mediation of PACAP-induced effects. Our findings support the notion that the PACAP system emerged after the protostome-deuterostome divergence. Using antibodies against vertebrate proteins is again highlighted to have little/no value in invertebrate studies. The physiological effects of vertebrate PACAP peptides in protostomes, no matter how similar they are to those in vertebrates, should be considered non-specific.

Large-scale spatial drivers of avian schistosomes in Northern Michigan inland lakes.

Avian schistosomes are snail-borne trematode parasites (Trichobilharzia spp.) that can cause a nasty skin rash in humans when their cercariae mistake us for their normal bird hosts. We sought to investigate drivers of the spatial distribution of Trichobilharzia cercaria abundance throughout Northern Michigan lakes. For 38 sites on 16 lakes, we assessed several dozen potential environmental predictors that we hypothesized might have direct or indirect effects on overall cercaria abundance, based on known relationships between abiotic and biotic factors in wetland ecosystems. We included variables quantifying local densities of intermediate hosts, temperature, periphyton growth rates, human land use and hydrology. We also measured daily abundance of schistosome cercariae in the water over a 5-week period, supported by community scientists who collected and preserved filtered water samples for qPCR. The strongest predictor of cercaria abundance was Lymnaea host snail density. Lymnaea density was higher in deeper lakes and at sites with more deciduous tree cover, consistent with their association with cool temperature habitats. Contrary to past studies of human schistosomes, we also found a significant negative relationship between cercaria abundance and submerged aquatic vegetation, possibly due to vegetation blocking cercaria movement from offshore snail beds. If future work shows that these effects are indeed causal, then these results suggest possible new approaches to managing swimmer's itch risk in northern MI lakes, such as modifying tree cover and shallow-water vegetation at local sites.

Activity-dependent modulation of neuronal KV channels by retinoic acid enhances CaV channel activity.

The metabolite of vitamin A, retinoic acid (RA), is known to affect synaptic plasticity in the nervous system and to play an important role in learning and memory. A ubiquitous mechanism by which neuronal plasticity develops in the nervous system is through modulation of voltage-gated Ca2+ (CaV) and voltage-gated K+ channels. However, how retinoids might regulate the activity of these channels has not been determined. Here, we show that RA modulates neuronal firing by inducing spike broadening and complex spiking in a dose-dependent manner in peptidergic and dopaminergic cell types. Using patch-clamp electrophysiology, we show that RA-induced complex spiking is activity dependent and involves enhanced inactivation of delayed rectifier voltage-gated K+ channels. The prolonged depolarizations observed during RA-modulated spiking lead to an increase in Ca2+ influx through CaV channels, though we also show an opposing effect of RA on the same neurons to inhibit Ca2+ influx. At physiological levels of Ca2+, this inhibition is specific to CaV2 (not CaV1) channels. Examining the interaction between the spike-modulating effects of RA and its inhibition of CaV channels, we found that inhibition of CaV2 channels limits the Ca2+ influx resulting from spike modulation. Our data thus provide novel evidence to suggest that retinoid signaling affects both delayed rectifier K+ channels and CaV channels to fine-tune Ca2+ influx through CaV2 channels. As these channels play important roles in synaptic function, we propose that these modulatory effects of retinoids likely contribute to synaptic plasticity in the nervous system.

Disruptions in network plasticity precede deficits in memory following inhibition of retinoid signaling.

Retinoic acid, the active metabolite of vitamin A, is important for vertebrate cognition and hippocampal plasticity, but few studies have examined its role in invertebrate learning and memory, and its actions in the invertebrate central nervous system are currently unknown. Using the mollusc Lymnaea stagnalis, we examined operant conditioning of the respiratory behavior, controlled by a well-defined central pattern generator (CPG), and used citral to inhibit retinoic acid signaling. Both citral- and vehicle-treated animals showed normal learning, but citral-treated animals failed to exhibit long-term memory at 24 h. Cohorts of citral- or vehicle-treated animals were dissected into semi-intact preparations, either 1 h after training, or after the memory test 24 h later. Simultaneous electrophysiological recordings from the CPG pacemaker cell (right pedal dorsal 1; RPeD1) and an identified motorneuron (VI) were made while monitoring respiratory activity (pneumostome opening). Activity of the CPG pneumostome opener interneuron (input 3 interneuron; IP3) was also monitored indirectly. Vehicle-treated conditioned preparations showed significant changes in network parameters immediately after learning, such as reduced motorneuron bursting activity (from IP3 input), delayed pneumostome opening, and decoupling of coincident IP3 input within the network. However, citral-treated preparations failed to exhibit these network changes and more closely resembled naïve preparations. Importantly, these citral-induced differences were manifested immediately after training and before any overt changes in the behavioral response (memory impairment). These studies shed light on where and when retinoid signaling might affect a central pattern-generating network to promote memory formation during conditioning of a homeostatic behavior.NEW & NOTEWORTHY We provide novel evidence for how conditioning-induced changes in a CPG network are disrupted when retinoid signaling is inhibited. Inhibition of retinoic acid signaling prevents long-term memory formation following operant conditioning, but has no effect on learning. Simultaneous electrophysiological and behavioral analyses indicate network changes immediately following learning, but these changes are prevented with inhibition of retinoid signaling, before any overt changes in behavior. These data suggest sites for retinoid actions during memory formation.

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.

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.

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.

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.

Diversity of trematode cercariae among naturally infected lymnaeid snails from Phayao, Thailand.

Lymnaeids are aquatic snails playing an important role in the transmission of many parasitic trematode species of veterinary and medical importance. In this study, we assessed the presence of cercarial flukes in naturally infected lymnaeid snails from Phayao province, Thailand, and determined the species diversity of both the intermediate snail hosts and parasite larvae. A total of 3,185 lymnaeid snails were collected from paddy fields at 31 sites in eight districts of Phayao province between October 2021 and December 2022. Larval fluke infection was assessed using the cercarial shedding method. The collected snails as well as emerging cercariae were identified at the species level via morphological and molecular methods. The sequences of snail internal transcribed spacer region 2 (ITS2) and cercarial 28S ribosomal RNA gene (28S rDNA) and cytochrome C oxidase1 (Cox1) were determined by PCR amplification and sequencing. Three species of lymnaeid snails were detected in this study, including Radix (Lymnaea) rubiginosa (Michelin, 1831), Radix (Lymnaea) swinhoei (Adams, 1866) and Austropeplea viridis (Quoy & Gaimard, 1832), of which R. rubiginosa was the most abundant, followed by A. viridis and R. swinhoei. The overall rate of trematode cercarial infection in the lymnaeid snails was 2.8% (90/3,185); the cercarial infection rate in R. rubiginosa and A. viridis was 3.5% (60/1,735) and 3.1% (30/981), respectively. No larval fluke infection was observed in the studied R. swinhoei (0/469). Nine morphotypes of cercariae were detected at 15 sites from four districts. The emerging cercariae were molecularly identified as Clinostomum sp., Aporocotylidae sp., Apharyngostrigea sp., Trichobilharzia sp., Apatemon sp., Pegosomum sp., Petasiger sp., Echinostoma revolutum and Plagiorchis sp. These findings emphasize the occurrence and diversity of trematode cercariae among naturally infected lymnaeid snails in Phayao province and could contribute to broadening our understanding of the host-parasite relationships between trematodes and their first intermediate hosts as well as developing effective interventions to control trematode parasitic diseases.

Morphological and molecular identification of lymnaeid snail and trematodes cercariae in different water bodies in Perak, Malaysia.

Lymnaeid snails play a crucial role in the transmission of trematode cercariae as an intermediate host that can infect humans, ruminants like buffalo, and other animals, resulting in serious economic losses. The purpose of the study was to identify the morphological and molecular characteristics of snails and cercariae collected from water bodies near buffalo farms that were integrated with palm oil in Perak, Malaysia. The presence or absence of snails in 35 water bodies was examined via cross-sectional study. From three marsh wetlands, 836 lymnaeid snails were gathered in total. Each snail's shell was morphologically identified to determine its family and species. The cercarial stage inside each snail's body was observed using the crushing method and trematode cercariae types were determined. In addition, the target gene Cytochrome c oxidase subunit 1 (Cox1) and the ribosomal internal transcribed spacer 2 (ITS2) region were used to identify the snail species and cercarial types according to the species level. The findings indicated that the collected snails belong to the family lymnaeidae and Radix rubiginosa species. In snails, the cercarial emergence infection rate was 8.7%. Echinostome, xiphidiocercariae, gymnocephalous, brevifurcate-apharyngeate distome cercariae (BADC), and longifurcate-pharyngeal monostome cercariae (LPMC) are the five morphological cercarial types that were observed. The cercariae were identified using morphological and molecular techniques, and they are members of the four families which are Echinostomatidae, Plagiorchiidae, Fasciolidae, and Schistosomatidae. Interestingly, this is the first study on R. rubiginosa and several trematode cercariae in Perak water bodies near buffalo farms that are integrated with palm oil. In conclusion, our research shown that a variety of parasitic trematodes in Perak use R. rubiginosa as an intermediate host.

Context-Dependent Role of miR-124 in Retinoic Acid-Induced Growth Cone Attraction of Regenerating Motorneurons.

During development and regeneration, growth cones at the tips of extending axons navigate through a complex environment to establish accurate connections with appropriate targets. Growth cones can respond rapidly to classical and non-classical guidance cues in their environment, often requiring local protein synthesis. In vertebrate growth cones, local protein synthesis in response to classical cues can require regulation by microRNAs (miRNAs), a class of small, conserved, non-coding RNAs that post-transcriptionally regulate gene expression. However, less is known of how miRNAs mediate growth cone responses to non-classical cues (such as retinoic acid (RA)), specifically in invertebrates. Here, we utilized adult regenerating invertebrate motorneurons to study miRNA regulation of growth cone attraction to RA, shown to require local protein synthesis. In situ hybridization revealed the presence of miR-124 in growth cones of regenerating ciliary motorneurons of the mollusc Lymnaea stagnalis. Changes in the spatiotemporal distribution of miR-124 occurred following application of RA, and dysregulation of miR-124 (with mimic injection), disrupted RA-induced growth cone turning in a time-dependent manner. This behavioural regulation by miR-124 was altered when the neurite was transected, and the growth cone completely separated from the soma. miR-124 did not, however, appear to be involved in growth cone attraction to serotonin, a response independent of local protein synthesis. Finally, we provide evidence that a downstream effector of RhoGTPases, ROCK, is a potential target of miR-124 during RA-induced growth cone responses. These data advance our current understanding of how microRNAs might mediate cue- and context-dependent behaviours during axon guidance.

A combined bioinformatics and LC-MS-based approach for the development and benchmarking of a comprehensive database of Lymnaea CNS proteins.

Applications of key technologies in biomedical research, such as qRT-PCR or LC-MS-based proteomics, are generating large biological (-omics) datasets which are useful for the identification and quantification of biomarkers in any research area of interest. Genome, transcriptome and proteome databases are already available for a number of model organisms including vertebrates and invertebrates. However, there is insufficient information available for protein sequences of certain invertebrates, such as the great pond snail Lymnaea stagnalis, a model organism that has been used highly successfully in elucidating evolutionarily conserved mechanisms of memory function and dysfunction. Here, we used a bioinformatics approach to designing and benchmarking a comprehensive central nervous system (CNS) proteomics database (LymCNS-PDB) for the identification of proteins from the CNS of Lymnaea by LC-MS-based proteomics. LymCNS-PDB was created by using the Trinity TransDecoder bioinformatics tool to translate amino acid sequences from mRNA transcript assemblies obtained from a published Lymnaea transcriptomics database. The blast-style MMSeq2 software was used to match all translated sequences to UniProtKB sequences for molluscan proteins, including those from Lymnaea and other molluscs. LymCNS-PDB contains 9628 identified matched proteins that were benchmarked by performing LC-MS-based proteomics analysis with proteins isolated from the Lymnaea CNS. MS/MS analysis using the LymCNS-PDB database led to the identification of 3810 proteins. Only 982 proteins were identified by using a non-specific molluscan database. LymCNS-PDB provides a valuable tool that will enable us to perform quantitative proteomics analysis of protein interactomes involved in several CNS functions in Lymnaea, including learning and memory and age-related memory decline.

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.

Ion channel profiling of the Lymnaea stagnalis ganglia via transcriptome analysis.

BACKGROUND: The pond snail Lymnaea stagnalis (L. stagnalis) has been widely used as a model organism in neurobiology, ecotoxicology, and parasitology due to the relative simplicity of its central nervous system (CNS). However, its usefulness is restricted by a limited availability of transcriptome data. While sequence information for the L. stagnalis CNS transcripts has been obtained from EST libraries and a de novo RNA-seq assembly, the quality of these assemblies is limited by a combination of low coverage of EST libraries, the fragmented nature of de novo assemblies, and lack of reference genome. RESULTS: In this study, taking advantage of the recent availability of a preliminary L. stagnalis genome, we generated an RNA-seq library from the adult L. stagnalis CNS, using a combination of genome-guided and de novo assembly programs to identify 17,832 protein-coding L. stagnalis transcripts. We combined our library with existing resources to produce a transcript set with greater sequence length, completeness, and diversity than previously available ones. Using our assembly and functional domain analysis, we profiled L. stagnalis CNS transcripts encoding ion channels and ionotropic receptors, which are key proteins for CNS function, and compared their sequences to other vertebrate and invertebrate model organisms. Interestingly, L. stagnalis transcripts encoding numerous putative Ca2+ channels showed the most sequence similarity to those of Mus musculus, Danio rerio, Xenopus tropicalis, Drosophila melanogaster, and Caenorhabditis elegans, suggesting that many calcium channel-related signaling pathways may be evolutionarily conserved. CONCLUSIONS: Our study provides the most thorough characterization to date of the L. stagnalis transcriptome and provides insights into differences between vertebrates and invertebrates in CNS transcript diversity, according to function and protein class. Furthermore, this study provides a complete characterization of the ion channels of Lymnaea stagnalis, opening new avenues for future research on fundamental neurobiological processes in this model system.

Transcriptome analysis provides genome annotation and expression profiles in the central nervous system of Lymnaea stagnalis at different ages.

BACKGROUND: The pond snail, Lymnaea stagnalis (L. stagnalis), has served as a valuable model organism for neurobiology studies due to its simple and easily accessible central nervous system (CNS). L. stagnalis has been widely used to study neuronal networks and recently gained popularity for study of aging and neurodegenerative diseases. However, previous transcriptome studies of L. stagnalis CNS have been exclusively carried out on adult L. stagnalis only. As part of our ongoing effort studying L. stagnalis neuronal growth and connectivity at various developmental stages, we provide the first age-specific transcriptome analysis and gene annotation of young (3 months), adult (6 months), and old (18 months) L. stagnalis CNS. RESULTS: Using the above three age cohorts, our study generated 55-69 millions of 150 bp paired-end RNA sequencing reads using the Illumina NovaSeq 6000 platform. Of these reads, ~ 74% were successfully mapped to the reference genome of L. stagnalis. Our reference-based transcriptome assembly predicted 42,478 gene loci, of which 37,661 genes encode coding sequences (CDS) of at least 100 codons. In addition, we provide gene annotations using Blast2GO and functional annotations using Pfam for ~ 95% of these sequences, contributing to the largest number of annotated genes in L. stagnalis CNS so far. Moreover, among 242 previously cloned L. stagnalis genes, we were able to match ~ 87% of them in our transcriptome assembly, indicating a high percentage of gene coverage. The expressional differences for innexins, FMRFamide, and molluscan insulin peptide genes were validated by real-time qPCR. Lastly, our transcriptomic analyses revealed distinct, age-specific gene clusters, differentially expressed genes, and enriched pathways in young, adult, and old CNS. More specifically, our data show significant changes in expression of critical genes involved in transcription factors, metabolisms (e.g. cytochrome P450), extracellular matrix constituent, and signaling receptor and transduction (e.g. receptors for acetylcholine, N-Methyl-D-aspartic acid, and serotonin), as well as stress- and disease-related genes in young compared to either adult or old snails. CONCLUSIONS: Together, these datasets are the largest and most updated L. stagnalis CNS transcriptomes, which will serve as a resource for future molecular studies and functional annotation of transcripts and genes in L. stagnalis.

Decreased 14-3-3 expression correlates with age-related regional reductions in CNS dopamine and motor function in the pond snail, Lymnaea.

Ageing is associated in many organisms with a reduction in motor movements. We have previously shown that the rate of feeding movements of the pond snail, Lymnaea, decreased with age but the underlying cause is not fully understood. Here, we show that dopamine in the cerebro-buccal complex is an important signalling molecule regulating feeding frequency in Lymnaea and that ageing is associated with a decrease in CNS dopamine. A proteomic screen of young and old CNSs highlighted a group of proteins that regulate stress responses. One of the proteins identified was 14-3-3, which can enhance the synthesis of dopamine. We show that the Lymnaea 14-3-3 family exists as three distinct isoforms. The expression of the 29 kDa isoform (14-3-3Lym3) in the cerebro-buccal complex decreased with age and correlated with feeding rate. Using a 14-3-3 antagonist (R18) we were able to reduce the synthesis of L-DOPA and dopamine in ex vivo cerebro-buccal complexes. Together these data suggest that an age-related reduction in 14-3-3 can decrease CNS dopamine leading to a consequential reduction in feeding rate.

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.