Mass Spectrometry of Single GABAergic Somatic Motorneurons Identifies a Novel Inhibitory Peptide, As-NLP-22, in the Nematode Ascaris suum

Abstract

Neuromodulators have become an increasingly important component of functional circuits, dramatically changing the properties of both neurons and synapses to affect behavior. To explore the role of neuropeptides in Ascaris suum behavior, we devised an improved method for cleanly dissecting single motorneuronal cell bodies from the many other cell processes and hypodermal tissue in the ventral nerve cord. We determined their peptide content using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). The reduced complexity of the peptide mixture greatly aided the detection of peptides; peptide levels were sufficient to permit sequencing by tandem MS from single cells. Inhibitory motorneurons, known to be GABAergic, contain a novel neuropeptide, As-NLP-22 (SLASGRWGLRPamide). From this sequence and information from the A. suum expressed sequence tag (EST) database, we cloned the transcript (As-nlp-22) and synthesized a riboprobe for in situ hybridization, which labeled the inhibitory motorneurons; this validates the integrity of the dissection method, showing that the peptides detected originate from the cells themselves and not from adhering processes from other cells (e.g., synaptic terminals). Synthetic As-NLP-22 has potent inhibitory activity on acetylcholine-induced muscle contraction as well as on basal muscle tone. Both of these effects are dose-dependent: the inhibitory effect on ACh contraction has an IC50 of 8.3 × 10–9 M. When injected into whole worms, As-NLP-22 produces a dose-dependent inhibition of locomotory movements and, at higher levels, complete paralysis. These experiments demonstrate the utility of MALDI TOF/TOF MS in identifying novel neuromodulators at the single-cell level.Graphical AbstractᅟElectronic supplementary material The online version of this article (doi:10.1007/s13361-015-1177-z) contains supplementary material, which is available to authorized users.

Different Bioactive Neuropeptides are Expressed in Two Sub-Classes of GABAergic RME Nerve Ring Motorneurons in Ascaris suum

“…With these modifications, staining of cells in the lateral lines were overall more intense, and preparations now revealed light staining in RMEV, and to a lesser extent RMED (Figure 10b), and consistent staining in ventral cord motorneurons DE2 and VE2. 5,6 The new protocol produced similar expression levels and intensity of afp-4 /AF2 in RMEV in ISH (13/29 preparations) and MS (11/20 cells), but RMED was easily lost during the whole mount dissection and washing and was only observed in 2/19 preparations. Given the weak and variable expression of afp-4 by both techniques and the improved detection after increased exposure to proteinase K, it appears that afp-4 is expressed at low levels in these cells near the detection limit for both techniques.…”

“…We synthesized digoxigenin (DIG)-labeled antisense probes for afp-2 (AF5), afp-4 (AF2), afp-15 (AF7, 39, 40), afp-16 (AF42–45 and PepQV), and Asu-nlp-58 (Asu-NLP-58.1, 58.2) and looked for expression in whole-mount preparations as previously described. 5,6…”

“…To further investigate afp-4 expression in RMEV and RMED, we synthesized a riboprobe to the same 223-nucleotide portion of the afp-4 transcript used previously 8 but implemented the optimized ISH protocol used to examine neuropeptide expression in the ventral cord motorneurons. 5,6 First, we increased the concentration and exposure to proteinase K to digest more of the thick hypodermis, thereby increasing the permeability of the tissue, and second, we allowed staining to continue overnight. With these modifications, staining of cells in the lateral lines were overall more intense, and preparations now revealed light staining in RMEV, and to a lesser extent RMED (Figure 10b), and consistent staining in ventral cord motorneurons DE2 and VE2.…”

“…A. suum has the additional experimental advantage that its neurons are large enough that single identified neurons can be isolated by manual dissection, and their neuropeptide content can be analyzed by mass spectrometry (MS). 3–6 The peptides can then be sequenced by tandem MS, all from a single neuron. 3–6 The peptide-encoding transcripts can also be detected in identified neurons by in situ hybridization in whole-mount preparations, 4–8 and the sequences of the transcripts give an independent prediction of peptide sequences based on the well-substantiated proteolytic cleavages of precursor proteins at dibasic (or sometimes monobasic) amino acids 9 followed by the trimming of the C-terminal basic amino acids and subsequent oxidation of C-terminal glycine residues to generate C-terminal amides.…”

“…1,7,14,15 This is perhaps not surprising because we previously found several major apparent differences in the cellular expression of peptides encoded by homologous genes in A. suum and C. elegans . 3–6 However, the cellular expression of peptides in C. elegans depended on the use of reporter genes rather than on the direct detection of gene products (the peptides themselves or their encoding mRNAs) as in A. suum . 19 Confirmation by independent techniques is badly needed in C. elegans .…”