An electron microscopic method to identify peptidergic neurons in connectomes

Abstract

Animal nervous systems are complex networks of connections between diverse types of neurons and effector tissues. Mapping the connections of molecularly identified neuron types at the synaptic level would greatly enhance our understanding of the structure and function of the nervous system.

In this thesis, I created a large serial EM dataset for neuronal network analysis, circuit reconstruction and stereotypy studies in the nervous system of the larvae of the marine annelid Platynereis dumerilii. I also developed an innovative method to assign molecular identities to peptidergic neurons directly in the EM dataset. I used serial-section transmission electron microscopy (ssTEM) combined with serial multiplex immunogold labeling (siGOLD) to molecularly identify multiple different neuron types. siGOLD is the method of labeling subsets of sections with various antibodies to reveal the molecular identities of specific neurons. I used neuropeptide antibodies to establish this method, taking advantage of the high immunogenicity of neuropeptides and their broad distribution throughout the axons. I demonstrate the effectiveness of siGOLD by using 11 neuropeptide antibodies to specifically label several distinct types of peptidergic neurons on a full-body larval ssTEM dataset of a Platynereis larva. siGOLD was also applied in the reconstruction of a peptidergic circuit comprising the sensory nuchal organs that express the circadian neuropeptide pigment-dispersing factor (PDF). Overall, this approach enables the direct overlaying of chemical neuromodulatory maps onto synaptic connectomic maps in the study of nervous systems. The full-body Platynereis EM dataset provided evidence for stereotypy between neuronal circuit anatomy and function in individuals of the same species. It also provided a database that can be reconstructed into a full-body connectome in the near future.