Neuropeptides, as pervasive intercellular signaling molecules in the CNS, modulate a variety of behavioral systems in both protostomes and deuterostomes. Allatostatins are neuropeptides in arthropods that inhibit the biosynthesis of juvenile hormones. Based on amino acid sequences, they are divided into three different types in arthropods: allatostatin A, allatostatin B, allatostatin C. Allatostatin C (AstC) was first isolated from Manduca sexta, and it has an important conserved feature of a disulfide bridge formed by two cysteine residues. Moreover, AstC appears to be the ortholog of mammalian somatostatin, and it has functions in common with somatostatin, such as modulating feeding behaviors. The AstC signaling system has been widely studied in arthropods, but minimally studied in molluscs. In this study, we seek to identify the AstC signaling system in the marine mollusc Aplysia californica. We cloned the AstC precursor from the cDNA of Aplysia. We predicted a 15-amino acid peptide with a disulfide bridge, i.e., AstC, using NeuroPred. We then cloned two putative allatostatin C-like receptors and through NCBI Conserved Domain Search we found that they belonged to the G protein-coupled receptor (GPCR) family. In addition, using an inositol monophosphate 1 (IP1) accumulation assay, we showed that Aplysia AstC could activate one of the putative receptors, i.e., the AstC-R, at the lowest EC50, and AstC without the disulfide bridge (AstC') activated AstC-R with the highest EC50. Moreover, four molluscan AstCs with variations of sequences from Aplysia AstC but with the disulfide bridge activated AstC-R at intermediate EC50. In summary, our successful identification of the Aplysia AstC precursor and its receptor (AstC-R) represents the first example in molluscs, and provides an important basis for further studies of the AstC signaling system in Aplysia and other molluscs.

Behavioral variability often arises from variable activity in the behavior-generating neural network. The synaptic mechanisms underlying this variability are poorly understood. We show that synaptic noise, in conjunction with weak feedforward excitation, generates variable motor output in the Aplysia feeding system. A command-like neuron (CBI-10) triggers rhythmic motor programs more variable than programs triggered by CBI-2. CBI-10 weakly excites a pivotal pattern-generating interneuron (B34) strongly activated by CBI-2. The activation properties of B34 substantially account for the degree of program variability. CBI-10– and CBI-2–induced EPSPs in B34 vary in amplitude across trials, suggesting that there is synaptic noise. Computational studies show that synaptic noise is required for program variability. Further, at network state transition points when synaptic conductance is low, maximum program variability is promoted by moderate noise levels. Thus, synaptic strength and noise act together in a nonlinear manner to determine the degree of variability within a feedforward network.

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Neuropeptides act mostly on a class of G-protein coupled receptors, and play a fundamental role in the functions of neural circuits underlying behaviors. However, physiological functions of some neuropeptide receptors are poorly understood. Here, we used the molluscan model system Aplysia and microinjected the exogenous neuropeptide receptor apATRPR (Aplysia allatotropin-related peptide receptor) with an expression vector (pNEX3) into Aplysia neurons that did not express the receptor endogenously. Physiological experiments demonstrated that apATRPR could mediate the excitability increase induced by its ligand, apATRP (Aplysia allatotropin-related peptide), in the Aplysia neurons that now express the receptor. This study provides a definitive evidence for a physiological function of a neuropeptide receptor in molluscan animals.