Functional Characterization Of A Neuropeptide Receptor Exogenously Expressed In Aplysia Neurons

Neuropeptides are one of the most diverse classes of neurotransmitters/neuromodulators in animals,and their receptors are mostly a class of G-protein coupled receptors that play a fundamental role in the functions of neural circuits underlying behaviors.However,functions of neuropeptide receptors are poorly understood.Previous work by Jing et al 2010 has identified the precursor for a neuropeptide Aplysia ATRP(ap ATRP),and shown that ap ATRP is widely distributed in the Aplysia feeding circuit,and one of the major actions of ap ATRP is to enhance excitability of feeding motoneurons B61/B62 in the buccal ganglion.Recently,initially based on bioinformatics,our collaborator,Dr.Checco,cloned one receptor for ap ATRP,that he named ap ATRPR.In addition to all L-amino acid form of ap ATPR(L-ap ATPR),Dr.Checco also identified a novel D-amino-acid containing peptide(DAACP)form of ap ATPR,D2-ap ATPR.He expressed the receptor in a cell line,and showed that both L-ap ATRP and D2-ap ATRP are active at the receptor.Here,we describe work characterizing the functions of ap ATRPR in Aplysia neurons.In a first series of experiments,we showed that both L-ap ATRP and D2-ap ATRP enhanced excitability of B61/B62,matching the effects of these ligands on ap ATRPR in the cell line.This is consistent with the notion that ap ATRPR may mediate the effects of these ligands on B61/B62 neurons.To provide further evidence supporting this idea,we need first demonstrate that ap ATRPR may indeed mediate the increase in excitability of a neuron that does not express ap ATRP.Among a number of buccal neurons that we tested,we found that B1/B2 neurons did not show an increase in excitability in response to perfusion of L-ap ATPR.To express exogenous genes in Aplysia neurons,we used p NEX3,a plasmid vector provided to us by our collaborator Dr.Kaang.We constructed and amplified p NEX3-EGFP and p NEX3-ap ATRPR.We separated B1/B2 neurons into two groups: control group and experimental group.B1/B2 neurons in control group were injected with p NEX3-EGFP,whereas B1/B2 neurons in experimental group were injected with both p NEX3-EGFP and p NEX3-ap ATRPR.After incubation for 1-3 days,the green fluorescence indicates the expression of EGFP and suggests the coexpression of EGFP and ap ATRPR.We applied L-ap ATRP to B1/B2 neurons that showed the green fluorescence.We found that B1/B2 neurons in the experimental group could be excited by L-ap ATRP,whereas B1/B2 neurons in the control group couldn’t.In conclusion,we provided evidence indicating that ap ATRPR is sufficient to mediate excitability increase to L-ap ATRP in Aplysia neurons.Future work to knockdown ap ATPRR likely endogenously expressed in B61/B62 neurons is needed to provide the necessary evidence.