title: Three independent techniques localize expression of transcript afp-11 and its bioactive peptide products to the paired AVK neurons in Ascaris suum: in situ hybridization, immunocytochemistry, and single cell mass spectrometry.
abstract: We utilized three independent techniques, immunocytochemistry (ICC), single cell mass spectrometry (MS), and in situ hybridization (ISH), to localize neuropeptides and their transcripts in the nervous system of the nematode Ascaris suum . AF11 (SDIGISEPNFLRFa) is an endogenous peptide with potent paralytic effects on A. suum locomotory behavior. A highly specific antibody to AF11 showed robust immunostaining for AF11 in the paired AVK neurons in the ventral ganglion. We traced the processes from the AVK neurons into the ventral nerve cord and identified them as ventral cord interneurons. MS and MS/MS of single dissected AVKs detected AF11, two previously characterized peptides (AF25 and AF26), seven novel sequence-related peptides, including several sharing a PNFLRFamide C-terminus, and peptide NY, a peptide with an unrelated sequence. Also present in a subset of AVKs was AF2, a peptide encoded by the afp-4 transcript. By sequencing the afp-11 transcript, we discovered that it encodes AF11, all the AF11-related peptides detected by MS in AVK, and peptide NY. ISH detected the afp-11 transcript in AVK neurons, consistent with other techniques. ISH did not detect afp-11 in the ALA neuron, although both ICC and MS found AF11 in ca. 30% of ALAs. All 10 AF11-related peptides reduced acetylcholine-induced muscle contraction, but they differed in their rate of reversal of inhibition after removal of the peptide.
authors.affiliation: Neuroscience Training Program, University of Wisconsin-Madison, Madison, WI 53706, USA.
authors.forename: Jessica L
authors.initials: JL
authors.lastname: Jarecki
authors.forename: India R
authors.initials: IR
authors.lastname: Viola
authors.forename: Kari M
authors.initials: KM
authors.lastname: Andersen
authors.forename: Andrew H
authors.initials: AH
authors.lastname: Miller
authors.forename: Megan A
authors.initials: MA
authors.lastname: Ramaker
authors.forename: Martha M
authors.initials: MM
authors.lastname: Vestling
authors.forename: Antony O
authors.initials: AO
authors.lastname: Stretton
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017165:Ascaris suum / Q000166:cytology / Q000235:genetics; D045888:Ganglion Cysts / Q000235:genetics; D005786:Gene Expression Regulation; D007150:Immunohistochemistry; D017403:In Situ Hybridization / Q000379:methods*; D008051:Lipid Bilayers / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D009474:Neurons / Q000737:chemistry / Q000378:metabolism*; D009479:Neuropeptides / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D009924:Organ Culture Techniques; D013570:Synaptic Membranes / Q000235:genetics; D014158:Transcription, Genetic / Q000502:physiology*
chemical_list: D008051:Lipid Bilayers; D009479:Neuropeptides