title: A serial multiplex immunogold labeling method for identifying peptidergic neurons in connectomes.
abstract: Electron microscopy-based connectomics aims to comprehensively map synaptic connections in neural tissue. However, current approaches are limited in their capacity to directly assign molecular identities to neurons. Here, we use serial multiplex immunogold labeling (siGOLD) and serial-section transmission electron microscopy (ssTEM) to identify multiple peptidergic neurons in a connectome. The high immunogenicity of neuropeptides and their broad distribution along axons, allowed us to identify distinct neurons by immunolabeling small subsets of sections within larger series. We demonstrate the scalability of siGOLD by using 11 neuropeptide antibodies on a full-body larval ssTEM dataset of the annelid Platynereis. We also reconstruct a peptidergic circuitry comprising the sensory nuchal organs, found by siGOLD to express pigment-dispersing factor, a circadian neuropeptide. Our approach enables the direct overlaying of chemical neuromodulatory maps onto synaptic connectomic maps in the study of nervous systems.
authors.affiliation: Max-Planck-Institute for Developmental Biology, Tübingen, Germany.
authors.forename: Réza
authors.initials: R
authors.lastname: Shahidi
authors.affiliation: Max-Planck-Institute for Developmental Biology, Tübingen, Germany.
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Williams
authors.affiliation: Max-Planck-Institute for Developmental Biology, Tübingen, Germany.
authors.forename: Markus
authors.initials: M
authors.lastname: Conzelmann
authors.affiliation: Max-Planck-Institute for Developmental Biology, Tübingen, Germany.
authors.forename: Albina
authors.initials: A
authors.lastname: Asadulina
authors.affiliation: Max-Planck-Institute for Developmental Biology, Tübingen, Germany.
authors.forename: Csaba
authors.initials: C
authors.lastname: Verasztó
authors.affiliation: Max-Planck-Institute for Developmental Biology, Tübingen, Germany.
authors.forename: Sanja
authors.initials: S
authors.lastname: Jasek
authors.affiliation: Max-Planck-Institute for Developmental Biology, Tübingen, Germany.
authors.forename: Luis A
authors.initials: LA
authors.lastname: Bezares-Calderón
authors.affiliation: Max-Planck-Institute for Developmental Biology, Tübingen, Germany.
authors.forename: Gáspár
authors.identifier: http://orcid.org/0000-0001-8496-9836
authors.initials: G
authors.lastname: Jékely
mesh_terms: D000818:Animals; D063132:Connectome; D007150:Immunohistochemistry / Q000379:methods*; D046529:Microscopy, Electron, Transmission / Q000379:methods; D009474:Neurons / Q000737:chemistry* / Q000166:cytology*; D009479:Neuropeptides / Q000032:analysis*; D011077:Polychaeta / Q000166:cytology*; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D009479:Neuropeptides
keywords: Platynereis dumerilii; annelid; connectome; immunogold; neuropeptides; neuroscience