Recombinant Lymnaea stagnalis Lymna-DF-amide 2

Basic Research Questions

• What is Lymna-DF-amide 2 and how does it relate to other neuropeptides?

Lymna-DF-amide 2 is one of five tridecapeptides identified from the central nervous system of the pond snail Lymnaea stagnalis. These peptides follow the general sequence Pro-Xaa-Asp-Arg-Ile-Ser-Yaa-Ser-Ala-Phe-Ser-Asp-Phe.NH2, where Xaa is either Tyr or Phe and Yaa is either Asn, Ser, or Gly . Lymna-DF-amides are named for their C-terminal Asp-Phe-amide sequence, which shares structural similarity with the C-terminal region of mammalian cholecystokinin (CCK) and gastrin, suggesting they belong to an evolutionary conserved Asp-Phe-amide superfamily .

• What neuroanatomical regions express Lymna-DF-amide 2 in Lymnaea stagnalis?

Lymna-DF-amides are expressed within specific neurons of the central nervous system of Lymnaea stagnalis. Detection is typically performed using antisera that recognize the biologically active C-termini shared with cholecystokinin and gastrin. Research indicates differential expression patterns across various ganglia of the snail's central nervous system . Immunohistochemical mapping can reveal the precise neuroanatomical distribution, which is essential for understanding the peptide's physiological functions.

• What are the fundamental experimental approaches for studying Lymna-DF-amide 2?

The fundamental approaches for studying Lymna-DF-amide 2 include:

• Molecular identification through PCR-based cloning and sequencing of the precursor gene

• Peptide isolation using HPLC and mass spectrometry

• Immunohistochemical localization with specific antibodies

• Expression analysis using in situ hybridization to detect mRNA distribution

• Functional bioassays to determine physiological effects

• Recombinant expression systems to produce sufficient quantities for research

Researchers typically begin with transcript identification before proceeding to peptide characterization and functional studies .

Advanced Research Questions

• What expression systems are most effective for producing recombinant Lymna-DF-amide 2?

The optimal expression system for recombinant Lymna-DF-amide 2 production depends on research objectives:

The critical consideration is ensuring correct C-terminal amidation, which is essential for biological activity of Lymna-DF-amides. Many researchers employ enzymatic methods post-expression to achieve proper amidation when using bacterial systems.

• How can neuronal co-cultures be designed to investigate Lymna-DF-amide 2 signaling mechanisms?

Neuronal co-culture systems for investigating Lymna-DF-amide 2 signaling can be established by:

• Isolating identified neurons from Lymnaea central nervous system through enzymatic digestion of ganglia

• Plating neurons expressing Lymna-DF-amide 2 together with potential target neurons

• Maintaining cultures in hemolymph-supplemented medium to preserve neuronal health

• Employing electrophysiological recordings to measure synaptic communication

• Using calcium imaging with fluorescent indicators to visualize cell-specific responses

• Applying recombinant Lymna-DF-amide 2 at varying concentrations to observe dose-dependent effects

• Combining with CREB pathway inhibitors to assess involvement in synaptic plasticity

This approach allows for direct observation of peptide-mediated signaling between specific neurons and can reveal whether Lymna-DF-amide 2 modulates synaptic properties related to learning and memory formation.

• What methodologies can distinguish between the five Lymna-DF-amide variants in tissue samples?

Distinguishing between the five Lymna-DF-amide variants requires sophisticated analytical approaches:

• High-resolution liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) using multiple reaction monitoring (MRM) to detect specific fragmentation patterns

• Isoelectric focusing combined with western blotting using variant-specific antibodies

• Custom peptide arrays with variant-specific antibodies for immunological differentiation

• MALDI-imaging mass spectrometry for spatial distribution analysis in tissue sections

• Targeted proteomics approaches using isotopically labeled internal standards

These techniques can quantify the relative abundance of each variant in different neuronal populations, such as the type I and type II neurons described in Lymnaea .

• How do Lymna-DF-amides interact with neural circuits governing reproductive behavior in Lymnaea stagnalis?

The interaction between Lymna-DF-amides and reproductive neural circuits can be investigated through:

• Systematic mapping of Lymna-DF-amide expression in relation to egg-laying hormone (ELH) producing neurons

• Comparing peptide processing and sorting in type I versus type II ELH-producing neurons

• Electrophysiological recordings from identified neurons in the reproductive circuit before and after Lymna-DF-amide application

• Behavioral assays measuring egg-laying responses following peptide microinjection

• Co-localization studies with other reproductive neuropeptides using multi-label immunohistochemistry

• Calcium imaging to visualize network-level responses to Lymna-DF-amide application

Research on ELH-producing neurons in Lymnaea has revealed cell type-specific sorting of neuropeptides, suggesting differential processing mechanisms that may also affect Lymna-DF-amide trafficking and release .

• What approaches can determine if Lymna-DF-amide 2 influences CREB-dependent memory formation in Lymnaea stagnalis?

To investigate Lymna-DF-amide 2's role in CREB-dependent memory formation:

• Microinjection of recombinant Lymna-DF-amide 2 into identified neurons known to express CREB, such as the cerebral giant cell

• Measurement of phosphorylated CREB levels following peptide application using phospho-specific antibodies

• Electrophysiological assessment of synaptic plasticity in the presence of the peptide

• Combination with CRE oligonucleotide to determine if peptide effects are mediated through CREB-dependent transcription

• Behavioral conditioning paradigms in the presence of peptide or antagonists

• RNA-seq analysis to identify transcriptional changes induced by the peptide

CREB has been identified as a key component in consolidating learned behavior into long-term memory in Lymnaea , making the potential interaction with Lymna-DF-amide 2 particularly relevant for learning and memory research.

• How can recombinant Lymna-DF-amide 2 be used to investigate evolutionary relationships between invertebrate and vertebrate neuropeptide systems?

Evolutionary relationships between neuropeptide systems can be investigated through:

• Receptor cross-reactivity studies using recombinant Lymna-DF-amide 2 on vertebrate CCK/gastrin receptors

• Comparative structural analysis of peptide-receptor binding domains across species

• Functional substitution experiments in heterologous expression systems

• Phylogenetic analysis of peptide precursor and receptor sequences

• Comparative behavioral or physiological assays across diverse animal phyla

• Developmental expression pattern comparisons during embryogenesis

Research has already established that Lymna-DF-amide 1 does not affect trout gallbladder, which responds to both CCK and gastrin , suggesting functional divergence despite structural similarities.

• What methodological approaches can determine if environmental contaminants alter Lymna-DF-amide 2 expression or function?

Environmental influence on Lymna-DF-amide 2 can be assessed through:

• Exposure studies using controlled concentrations of environmental contaminants such as antidepressants

• Quantitative PCR to measure changes in precursor mRNA expression

• Mass spectrometry-based peptidomics to quantify peptide levels

• Behavioral assays measuring locomotion or other behaviors following exposure

• Immunohistochemical analysis to detect changes in peptide distribution patterns

• Electrophysiological recordings to assess altered neuronal responses

• Comparison with effects on other neuropeptide systems

Studies have demonstrated that Lymnaea stagnalis can absorb and store compounds from water, making it an excellent model for investigating how environmental factors influence neuropeptide systems . Recent research has shown differential impacts of antidepressants like fluoxetine and venlafaxine on snail locomotion , which might involve neuropeptide signaling pathways.

• What are the methodological considerations for studying peptide degradation rates of Lymna-DF-amide 2 in different neural compartments?

Studying compartment-specific peptide degradation requires:

• Pulse-chase experiments with isotopically labeled peptides

• Subcellular fractionation techniques to isolate different neuronal compartments

• Microdialysis sampling from specific regions of the central nervous system

• Development of degradation-resistant analogs for comparative stability studies

• Identification of specific peptidases involved in Lymna-DF-amide processing

• In vivo imaging of fluorescently labeled peptides to track degradation kinetics

This is particularly relevant given the evidence that some neuropeptides in Lymnaea neurons undergo differential sorting and degradation, as demonstrated with large electrondense granules (LEG) in type I ELH-producing neurons .

• How can structure-activity relationship studies of recombinant Lymna-DF-amide 2 inform peptide-based drug design?

Structure-activity relationship studies can be conducted by:

• Systematic alanine scanning to identify critical residues for activity

• N- and C-terminal truncation series to determine minimal active fragment

• Strategic substitutions at positions Xaa and Yaa to assess their contribution to bioactivity

• Incorporation of non-natural amino acids to enhance stability or receptor selectivity

• Conformational constraint introduction through cyclization or bridge formation

• Computational molecular modeling to predict structural determinants of activity

This approach can identify key pharmacophore elements that might be transferred to novel therapeutics targeting related receptors in higher organisms.