Different Neuropeptides Are Expressed in Different Functional Subsets of Cholinergic Excitatory Motorneurons in the Nematode Ascaris suum

Abstract

Neuropeptides are known to have dramatic effects on neurons and synapses; however, despite extensive studies of the motorneurons in the parasitic nematode Ascaris suum, their peptide content had not yet been described. We determined the peptide content of single excitatory motorneurons by mass spectrometry and tandem mass spectrometry. There are 2 subsets of ventral cord excitatory motorneurons, each with neuromuscular output either anterior or posterior to their cell body, mediating forward or backward locomotion, respectively. Strikingly, the two sets of neurons contain different neuropeptides, with AF9 and 6 novel peptides (As-NLP-21.1-6) in anterior projectors, and the 6 afp-1 peptides in addition to AF2 in posterior projectors. In situ hybridization confirmed the expression of these peptides, validating the integrity of the dissection technique. This work identifies new components of the functional behavioral circuit, as well as potential targets for anti-parasitic drug development.

An Endoplasmic Reticulum ATPase Safeguards Endoplasmic Reticulum Identity by Removing Ectopically Localized Mitochondrial Proteins

“…To test whether catp-8 is required for general secretory/sorting proteins, we examined two other PVD membrane/secreted proteins in the catp-8 mutant background. HPO-30 is a four-pass transmembrane protein that functions in PVD as a co-receptor of DMA-1 to establish dendritic arbors (Zou et al, 2018), and NLP-21 is a neuropeptide (Konop et al, 2015). We found no difference between wild-type and the catp-8 mutant ( Figures S6A and S6B), indicating that catp-8 is not required for biogenesis of general transmembrane or secreted protein.…”

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.…”

“…Our results show that the simplification of the mixture of peptides present in a single neuron compared with that in a ganglion makes a significant reduction in problems due to ion suppression. 3,5,6…”