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.
Highlights
A comprehensive understanding of nervous system function requires knowledge of precise neuronal connectivity and the unique combinations of molecules expressed by each neuron
We identified several other neurons that received a few synapses from the SNnuch cells, including two interneurons (INRGWa-dcr1, INRGWa-dcl1) that were identified by serial multiplex immunogold labeling (siGOLD) labeling to express the RGWa neuropeptide (Figure 9A,B)
We introduced siGOLD, a method to molecularly identify specific neurons in large serial electron microscopy (EM) datasets based on the immunogold labeling of subsets of sections followed by serial reconstruction of the tagged neurons
Summary
A comprehensive understanding of nervous system function requires knowledge of precise neuronal connectivity and the unique combinations of molecules expressed by each neuron. Connectomics using serial-sectioning electron microscopy (EM) aims to map the synapse-level connectivity of entire neural circuits (Morgan and Lichtman, 2013). One important class of molecules are neuromodulators, including monoamines and neuropeptides, that actively shape the output of circuits by modifying synaptic function and neuron excitability (Bargmann, 2012; Bargmann and Marder, 2013; Bucher and Marder, 2013; Marder, 2012). The direct mapping of specific neuromodulators to their source cells in synapse-level anatomical maps would help to integrate connectomic and neuromodulatory perspectives, thereby enriching our understanding of circuit function. Fixation-resistant protein tags compatible with EM procedures and observable by correlative light and electron microscopy (CLEM) or directly by immunogold labeling (immunoEM), such as the smGFPs (Viswanathan et al, 2015), have been developed.
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