Discovery Of Two Novel Aplysia Neuropeptide Families And Their Mechanisms Of Actions In The Feeding Circuit

In both invertebrates and vertebrates,neuropeptides are the most diverse class of neuromodulators with a variety of functions in the CNS and periphery.Neuropeptides play roles in behaviors by acting on the intrinsic properties(e.g.,excitability)and synaptic connections of specific neuronal elements in neural circuits.In the present work,we used an experimentally advantageous model system,the Aplysia feeding circuit,to study modulatory effect of neuropeptides on behaviors.We focus on two types of neuropeptides,leucokinin-like peptides and SPTR-related peptides.We elucidated their modulatory roles for the feeding programs and the underlying mechanisms in the feeding network.This study includes three parts:First,we studied the leucokinin-like peptides.Previous studies of the leucokinins family of peptides have been primarily conducted in arthropods,where they had myoactivity in the hindgut,increased diuretic activity and regulated meal size in insects.However,no prior studies have examined the effects of leucokinins on any central pattern generators(CPGs)for behavior.Here,we identified an Aplysia leucokinin-like peptides(ALKs)precursor for the first time.The precursor is one of the longest neuropeptide precursors characterized to date(consisting of 2025 amino acids),which encodes up to 40 ALKs.We then demonstrated that they could modulate a specific parameter of the feeding program,that is,shortening the protraction duration.We also demonstrated that ALKs act on one specific CPG neuron(B64)to produce the modulatory effects.Second,we identified a novel family of peptides,the Aplysia SPTR-Gene Family-Derived Peptides(apSPTR-GF-DPs).The SPTR precursor was first discovered in Lymnaea,which,like Aplysia,is a gastropod mollusc.More recent work has identified SPTR Gene Family(SPTR-GF)precursors in other molluscs and in several annelids,but no specific function was found.Aplysia SPTR precursor was previously identified by the EST method,but no further study is available.We,for the first time,demonstrated the expression of apSPTR-GF-DPs in Aplysia central nervous system(CNS)and focused on apSPTR-GF-DP2.We demonstrated that apSPTR-GF-DP2 shortened the protraction duration of the feeding motor program,similar to ALKs.In addition,we also showed that CBI-12(Cerebrobuccal interneuron-12)is the neuronal source of apSPTR-GF-DP2,and both apSPTR-GF-DP2 and CBI-12 targeted the interneuron B20.Importantly,comparison between the underlying mechanisms of CBI-12 peptides:apSPTR-GF-DP2 and FCAP/CP2,reveals a form of molecular degeneracy for peptidergic function.Third,since the above two peptide families both act on the protraction duration of the feeding program,we have conducted in-depth studies of the circuit mechanisms.Previous studies identified two protraction terminators,the retraction-phase intemeurons B64 and CBI-5/6,in the Aplysia feeding circuit.Earlier,we found that B64 is the direct target of ALKs,and identified the protraction neuron B20 as the indirect target of apSPTR-GF-DP2.Here,we found that apSPTR-GF-DP2 could increase the excitability of CBI-5/6.However,if the protraction terminator were to work,there should be a corresponding change in its spike timing.We found that apSPTR-GF-DP2 advanced CBI-5/6 spike timing.Similarly,ALKs also advanced B64 spike timing.In short,we,for the first time,discovered that neuropeptides not only could enhance the excitability of inteneurons,but also advance spike timing of the same intemeurons,thereby altering the motor output.In summary,we identified the functions of two novel neuropeptide families in the Aplysia feeding circuit(the CPG)and elucidated their actions on spike timing.Because neuropeptides are widely present in other animals,and most behaviors are mediated by CPGs,the novel modulatory mechanisms we identified may provide insights into the peptidergic neuromodulatory studies in other animals,including vertebrates.