Stagnalis Central Nervous System Research Articles

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

BackgroundThe 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.ResultsUsing 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.ConclusionsTogether, 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.

Ion channel profiling of the Lymnaea stagnalis ganglia via transcriptome analysis

BackgroundThe 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.ResultsIn 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.ConclusionsOur 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.

Gap Junction Coding Innexin in Lymnaea stagnalis: Sequence Analysis and Characterization in Tissues and the Central Nervous System.

Connections between neurons called synapses are the key components underlying all nervous system functions of animals and humans. However, important genetic information on the formation and plasticity of one type, the electrical (gap junction-mediated) synapse, is understudied in many invertebrates. In the present study, we set forth to identify and characterize the gap junction-encoding gene innexin in the central nervous system (CNS) of the mollusk pond snail Lymnaea stagnalis. With PCR, 3′ and 5′ RACE, and BLAST searches, we identified eight innexin genes in the L. stagnalis genome, named Lst Inx1–Lst Inx8. Phylogenetic analysis revealed that the L. stagnalis innexin genes originated from a single copy in the common ancestor of molluskan species by multiple gene duplication events and have been maintained in L. stagnalis since they were generated. The paralogous innexin genes demonstrate distinct expression patterns among tissues. In addition, one paralog, Lst Inx1, exhibits heterogeneity in cells and ganglia, suggesting the occurrence of functional diversification after gene duplication. These results introduce possibilities to study an intriguing potential relationship between innexin paralog expression and cell-specific functional outputs such as heterogenic ability to form channels and exhibit synapse plasticity. The L. stagnalis CNS contains large neurons and functionally defined networks for behaviors; with the introduction of L. stagnalis in the gap junction gene field, we are providing novel opportunities to combine genetic research with direct investigations of functional outcomes at the cellular, synaptic, and behavioral levels.

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 WIN55,212-2 (WIN55) into the snail before operant conditioning obstructed learning and memory formation. This effect of WIN55 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 AM251 enhanced long-term memory formation in snails and reduced the duration of the effects of the severe traumatic stressor on learning and memory. Neither WIN55 nor AM251 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.

Cross-platform metabolic profiling: application to the aquatic model organism Lymnaea stagnalis.

The freshwater pond snail Lymnaea stagnalis is used in several studies on molecular and behavioral neurobiology and ecotoxicology showing its successful application as a model organism. In the present study, a cross-platform metabolomic approach has been evaluated to characterize the organ molecular phenotypes of L. stagnalis central nervous system (CNS), digestive gland (DG), and albumen gland (AG). Two types of tissue disruption methods were evaluated of which beads beating was the preferred method. To obtain a broad picture of the hydrophilic and lipophilic metabolome, two complementary analytical platforms have been employed: liquid chromatography (LC) and gas chromatography (GC) coupled to high-resolution mass spectrometry. Furthermore, to increase the power to separate small polar metabolites, hydrophilic interaction liquid chromatography (HILIC) was applied. The analytical platform performances have been evaluated based on the metabolome coverage, number of molecular features, reproducibility, and multivariate data analysis (MVDA) clustering. This multiplatform approach is a starting point for future global metabolic profiling applications on L. stagnalis.

Transcriptome Analysis of the Octopus vulgaris Central Nervous System

BackgroundCephalopoda are a class of Mollusca species found in all the world's oceans. They are an important model organism in neurobiology. Unfortunately, the lack of neuronal molecular sequences, such as ESTs, transcriptomic or genomic information, has limited the development of molecular neurobiology research in this unique model organism.ResultsWith high-throughput Illumina Solexa sequencing technology, we have generated 59,859 high quality sequences from 12,918,391 paired-end reads. Using BLASTx/BLASTn, 12,227 contigs have blast hits in the Swissprot, NR protein database and NT nucleotide database with E-value cutoff 1e−5. The comparison between the Octopus vulgaris central nervous system (CNS) library and the Aplysia californica/Lymnaea stagnalis CNS ESTs library yielded 5.93%/13.45% of O. vulgaris sequences with significant matches (1e−5) using BLASTn/tBLASTx. Meanwhile the hit percentage of the recently published Schistocerca gregaria, Tilapia or Hirudo medicinalis CNS library to the O. vulgaris CNS library is 21.03%–46.19%. We constructed the Phylogenetic tree using two genes related to CNS function, Synaptotagmin-7 and Synaptophysin. Lastly, we demonstrated that O. vulgaris may have a vertebrate-like Blood-Brain Barrier based on bioinformatic analysis.ConclusionThis study provides a mass of molecular information that will contribute to further molecular biology research on O. vulgaris. In our presentation of the first CNS transcriptome analysis of O. vulgaris, we hope to accelerate the study of functional molecular neurobiology and comparative evolutionary biology.

Identification of the role of C/EBP in neurite regeneration following microarray analysis of a L. stagnalisCNS injury model

BackgroundNeuronal regeneration in the adult mammalian central nervous system (CNS) is severely compromised due to the presence of extrinsic inhibitory signals and a reduced intrinsic regenerative capacity. In contrast, the CNS of adult Lymnaea stagnalis (L. stagnalis), a freshwater pond snail, is capable of spontaneous regeneration following neuronal injury. Thus, L. stagnalis has served as an animal model to study the cellular mechanisms underlying neuronal regeneration. However, the usage of this model has been limited due to insufficient molecular tools. We have recently conducted a partial neuronal transcriptome sequencing project and reported over 10,000 EST sequences which allowed us to develop and perform a large-scale high throughput microarray analysis.ResultsTo identify genes that are involved in the robust regenerative capacity observed in L. stagnalis, we designed the first gene chip covering ~15, 000 L. stagnalis CNS EST sequences. We conducted microarray analysis to compare the gene expression profiles of sham-operated (control) and crush-operated (regenerative model) central ganglia of adult L. stagnalis. The expression levels of 348 genes were found to be significantly altered (p < 0.05) following nerve injury. From this pool, 67 sequences showed a greater than 2-fold change: 42 of which were up-regulated and 25 down-regulated. Our qPCR analysis confirmed that CCAAT enhancer binding protein (C/EBP) was up-regulated following nerve injury in a time-dependent manner. In order to test the role of C/EBP in regeneration, C/EBP siRNA was applied following axotomy of cultured Lymnaea PeA neurons. Knockdown of C/EBP following axotomy prevented extension of the distal, proximal and intact neurites. In vivo knockdown of C/EBP postponed recovery of locomotory activity following nerve crush. Taken together, our data suggest both somatic and local effects of C/EBP are involved in neuronal regeneration.ConclusionsThis is the first high-throughput microarray study in L. stagnalis, a model of axonal regeneration following CNS injury. We reported that 348 genes were regulated following central nerve injury in adult L. stagnalis and provided the first evidence for the involvement of local C/EBP in neuronal regeneration. Our study demonstrates the usefulness of the large-scale gene profiling approach in this invertebrate model to study the molecular mechanisms underlying the intrinsic regenerative capacity of adult CNS neurons.

Transcriptome analysis of the central nervous system of the mollusc Lymnaea stagnalis

BackgroundThe freshwater snail Lymnaea stagnalis (L. stagnalis) has served as a successful model for studies in the field of Neuroscience. However, a serious drawback in the molecular analysis of the nervous system of L. stagnalis has been the lack of large-scale genomic or neuronal transcriptome information, thereby limiting the use of this unique model.ResultsIn this study, we report 7,712 distinct EST sequences (median length: 847 nucleotides) of a normalized L. stagnalis central nervous system (CNS) cDNA library, resulting in the largest collection of L. stagnalis neuronal transcriptome data currently available. Approximately 42% of the cDNAs can be translated into more than 100 consecutive amino acids, indicating the high quality of the library. The annotated sequences contribute 12% of the predicted transcriptome size of 20,000. Surprisingly, approximately 37% of the L. stagnalis sequences only have a tBLASTx hit in the EST library of another snail species Aplysia californica (A. californica) even using a low stringency e-value cutoff at 0.01. Using the same cutoff, approximately 67% of the cDNAs have a BLAST hit in the NCBI non-redundant protein and nucleotide sequence databases (nr and nt), suggesting that one third of the sequences may be unique to L. stagnalis. Finally, using the same cutoff (0.01), more than half of the cDNA sequences (54%) do not have a hit in nematode, fruitfly or human genome data, suggesting that the L. stagnalis transcriptome is significantly different from these species as well. The cDNA sequences are enriched in the following gene ontology functional categories: protein binding, hydrolase, transferase, and catalytic enzymes.ConclusionThis study provides novel molecular insights into the transcriptome of an important molluscan model organism. Our findings will contribute to functional analyses in neurobiology, and comparative evolutionary biology. The L. stagnalis CNS EST database is available at .

An expressed sequence tag survey of gene expression in the pond snail Lymnaea stagnalis, an intermediate vector of Fasciola hepatica

The pond snail Lymnaea stagnalis is an intermediate vector for the liver fluke Fasciola hepatica, a common parasite of ruminants and humans. Yet, despite being a disease of medical and economic importance, as well as a potentially useful comparative tool, the genetics of the relationship between Lymnaea and Fasciola has barely been investigated. As a complement to forthcoming F. hepatica expressed sequence tags (ESTs), we generated 1320 ESTs from L. stagnalis central nervous system (CNS) libraries. We estimate that these sequences derive from 771 different genes, of which 374 showed significant similarity to proteins in public databases, and 169 were similar to ESTs from the snail vector Biomphalaria glabrata. These L. stagnalis ESTs will provide insight into the function of the snail CNS, as well as the molecular components of behaviour and response to parasitism. In the future, the comparative analysis of Lymnaea/Fasciola with Biomphalaria/Schistosoma will help to understand both conserved and divergent aspects of the host-parasite relationship. The L. stagnalis ESTs will also assist gene prediction in the forthcoming B. glabrata genome sequence. The dataset is available for searching on the world-wide web at http://zeldia.cap.ed.ac.uk/mollusca.html.

HPLC and electrospray ionization mass spectrometry as tools for the identification of APGWamide-related peptides in gastropod and bivalve mollusks: comparative activities on Mytilus muscles

The APGWamide-related neuropeptides, predicted by the cDNA of the APGWamide precursor of Mytilus edulis, have been sought by means of HPLC and electrospray mass ionization. The three predicted peptides KPGWamide, RPGWamide and TPGWamide were detected in the three main muscles and surprisingly an ion at m/z 429 corresponding to the gastropod peptide APGWamide was also demonstrated. Similar investigations performed in Lymnaea stagnalis central nervous system (CNS) revealed the occurrence of mussel APGWamide-related peptides (APGWamide-RPs) demonstrating for the first time the presence and the expression of the two precursors in both gastropod and bivalve mollusks. The absence of homologous domain in the Mytilus precursor [P. Favrel, M. Mathieu, Molecular cloning of a cDNA encoding the precursor of Ala-Pro-Gly-Trp-amide related neuropeptides from the bivalve Mytilus edulis. Neurosci. Lett. 1996;205:210–214] and the Lymnaea precursor [A.B. Smit, C.R. Jiménez, R.W. Dirks, R.P. Croll, W.P.M. Geraerts, Characterization of cDNA clone encoding multiple copies of the neuropeptide APGWamide in the molluscs Lymnaea stagnalis. J. Neurosci. 1992;12:1709–1715] eliminates the hypothesis of an alternative splicing of a single gene and suggests the likelihood of two genes probably resulting from duplication of an ancestral gene before the divergence between gastropods and bivalves. The similar potency observed on contraction assay and the differential distribution of the various peptides suggest that they may exert distinct activities on multiple targets.

Leech egg-laying-like hormone: structure, neuronal distribution and phylogeny

Cells immunoreactive to antisera specifically directed against Lymnaea stagnalis caudo dorsal cells egg-laying hormone (CDCH) or against α- and β-peptides (CDCP), encoded on the egg-laying hormone precursor, were detected in central nervous system (CNS) of the rhynchobdellid leech Theromyzon tessulatum. A co-localization of the CDC-like hormone and CDC-like peptides was found in T. tessulatum as in L. stagnalis CNS. ≈45 immunoreactive cells to the anti-CDCH were detected in leech brain but this number varies according to the stage of the animal life cycle, i.e. it reaches a maximum just before egg-laying while after it decreases to 2–3 cells. CDCH and α-CDCP epitopes recognized by anti-CDCH and anti- α-CDCP were contained in neurosecretory granules. Following an extensive purification, including HPGPC and reverse-phase HPLC, the CDC-like hormone contained in the T. tessulatum CNA was isolated. The sequence (GSGVSNGGTEMIQLSHIRERQRYWAQDNLRRRFLEK-amide) was established by a combination of automated Edman degradation, arginyl-endopeptidase digestion, electrospray mass spectrometry measurement and carboxypeptidase A treatment. The results demonstrate that the peptide recognized by the anti-CDCH in the leech CNS possesses 27.8, 37.2 and 47.2% sequence identity with Aplysia parvula, Lymnaea stagnalis and Aplysia californica ELH, respectively. This molecule was named the leech egg-laying-like hormone (L-ELH). The secondary structure prediction of the L-ELH and all mollusks ELH, revealed the existence of a conserved segment (segment 29–34) in a strong helicoidal bend that might be important for receptor recognition and/or activation. This finding constitutes the first biochemical characterization of an egg-laying hormone in other invertebrates than mollusks.

The effect of some Frescon analogs on the aquatic snail, Lymnaea stagnalis: II. Correlation of neurotoxicity to Molluscicidal effectiveness

Although the molluscicide Frescon is a strong neurotoxin to the Lymnaea stagnalis central nervous system in vitro, it is probable that the exposure of the whole animal to this molluscicide fails to result in central nervous system abnormalities: Frescon does not appear to reach the brain in sufficient quantity to disrupt its normal activity. However, only those Frescon analogs found to be neurotoxic were molluscicidal, suggesting some related mode, if not site, of action. Frescon and its analogs may act by affecting excitable tissues other than the nervous system (e.g., the snail musculature) by altering certain functional and/or structural membrane properties.