Discovery of leucokinin-like neuropeptides that modulate a specific parameter of feeding motor programs in the molluscan model, Aplysia

A better understanding of neuromodulation in a behavioral system requires identification of active modulatory transmitters. Here, we used identifiable neurons in a neurobiological model system, the mollusc Aplysia, to study neuropeptides, a diverse class of neuromodulators. We took advantage of two types of feeding neurons, B48 and B1/B2, in the Aplysia buccal ganglion that might contain different neuropeptides. We performed a representational difference analysis (RDA) by subtraction of mRNAs in B48 versus mRNAs in B1/B2. The RDA identified an unusually long (2025 amino acids) peptide precursor encoding Aplysia leucokinin-like peptides (ALKs; e.g. ALK-1 and ALK-2). Northern blot analysis revealed that, compared with other ganglia (e.g. the pedal-pleural ganglion), ALK mRNA is predominantly present in the buccal ganglion, which controls feeding behavior.Wethen used in situ hybridization and immunohistochemistry to localize ALKs to specific neurons, including B48. MALDI-TOF MS on single buccal neurons revealed expression of 40 ALK precursor-derived peptides. Among these, ALK-1 and ALK-2 are active in the feeding network; they shortened the radula protraction phase of feeding motor programs triggered by a command-like neuron. We also found that this effect may be mediated by the ALK-stimulated enhancement of activity of an interneuron, which has previously been shown to terminate protraction. We conclude that our multipronged approach is effective for determining the structure and defining the diverse functions of leucokinin-like peptides. Notably, theALKprecursor is the first verified nonarthropod precursor for leucokinin-like peptides with a novel, marked modulatory effect on a specific parameter (protraction duration) of feeding motor programs.