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.

A serial multiplex immunogold labeling method for identifying peptidergic neurons in connectomes

“…We imaged the VNC region in each section at a resolution of 2.22 nm/pixel followed by stitching and alignment of the images. We found strong and localized labeling in only a small subset of neurites for each antibody ( Figure 2A , Shahidi, et al, 2015 ). In consecutive sections, the same neurite was often strongly labeled with the same antibody ( Figure 2A , FVa, layers 1–5).…”

“…In sections collected on different grids that were labeled with different antibodies, we found distinct patterns of neurite-specific labeling ( Figure 2B,C ). In many sections, we could observe dense core vesicles (DCVs) in the cytoplasm of the gold-labeled neurites, indicative of the peptidergic nature of these cells ( Figure 2A–F , Shahidi, et al, 2015 ). In high-resolution (0.22 nm/pixel) images, we could observe gold labeling associated with DCVs ( Figure 2D–F ), suggesting that our immunoEM procedure labeled mature neuropeptides residing inside these vesicles.…”

“…( D–F ) High-resolution micrographs of immunogold labeled, silver-enhanced gold particles (arrows), and unenhanced ultra small gold particles (arrowheads) Scale bar: ( A-C ) 1 μm; ( D– F) 200 nm. High-resolution images are available in ( Shahidi, et al, 2015 ). DOI: http://dx.doi.org/10.7554/eLife.11147.005 …”