Using Multidisciplinary Approaches To Characterize Glutamatergic Neurons And Their Functions In Aplysia Neural Circuits

Understanding circuit function requires the characterization of component neurons and their neurotransmitters. Previous work on radula protraction in the Aplysia feeding circuit demonstrated that critical neurons initiate feeding via cholinergic excitation. In contrast, it is less clear how retraction is mediated at the interneuronal level. In particular, glutamate involvement was suggested, but wasn’t directly confirmed.A conventional approach to study glutamatergic neurons and their functions is to use pharmacological reagents. However, this approach has its own issues because although these pharmacological reagents have been wildly studied in vertebrates, when used in invertebrates, these reagents may not be as specific. Another important approach is to identify molecules involved in neurotransmission, e.g., vesicular glutamate transporters. While vesicular glutamate transporters are specifically present in glutamatergic neurons, the approach is limited as functional study of glutamatergic neurons still requires additional methods, such as pharmacological methods. So it is necessary to combine multidisciplinary approaches to identify glutamatergic neurons and study their functions.Here, we studied a suspected glutamatergic retraction interneuron, B64. Firstly, we performed the bioinformatics analysis of a cDNA sequence cloned from B64 by our collaborators previously. Bioinformatics suggests that not only the sequence but also the hydrophobic property of the cDNA are highly similar with those reported vesicular glutamate transporter (VGLUTs) in other species. So we named it Aplysia vesicular glutamate transporter (ApVGLUT). Intriguingly, phylogenic tree shows that ApVGLUT is more closely related to mammalian VGLUTs than to Drosophila and C. elegans VGLUTs. Then we utilized a recombinant vector (also from our cooperators) to express a new protein which indeed transports glutamate in an ATP and proton gradient-dependent manner and the result suggests that ApVGLUT is a newly discovered glutamate transporter. Further, using immunocytochemistry, we demonstrated that B64 is ApVGLUT positive, supporting the idea that it is glutamatergic. Pharmacological data also indicated that both EPSPs and IPSPs elicited by B64 are mediated by glutamate. Thus, B64 utilizes glutamate as a primary neurotransmitter to control radula retraction. More generally, our systematic approaches based on multidisciplinary approach may facilitate analyses of transmitter actions in small circuits with identifiable neurons.