Using high-throughput barcode sequencing to efficiently map connectomes

Nucleic Acids Res. 2017 Jul 7;45(12):e115. doi: 10.1093/nar/gkx292.

Abstract

The function of a neural circuit is determined by the details of its synaptic connections. At present, the only available method for determining a neural wiring diagram with single synapse precision-a 'connectome'-is based on imaging methods that are slow, labor-intensive and expensive. Here, we present SYNseq, a method for converting the connectome into a form that can exploit the speed and low cost of modern high-throughput DNA sequencing. In SYNseq, each neuron is labeled with a unique random nucleotide sequence-an RNA 'barcode'-which is targeted to the synapse using engineered proteins. Barcodes in pre- and postsynaptic neurons are then associated through protein-protein crosslinking across the synapse, extracted from the tissue, and joined into a form suitable for sequencing. Although our failure to develop an efficient barcode joining scheme precludes the widespread application of this approach, we expect that with further development SYNseq will enable tracing of complex circuits at high speed and low cost.

MeSH terms

  • Animals
  • Calcium-Binding Proteins
  • Cell Adhesion Molecules, Neuronal / genetics*
  • Cell Adhesion Molecules, Neuronal / metabolism
  • Connectome / methods*
  • Embryo, Mammalian
  • Gene Expression Regulation
  • Genes, Reporter
  • Genetic Vectors / chemistry
  • Genetic Vectors / metabolism
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • HEK293 Cells
  • High-Throughput Nucleotide Sequencing
  • Hippocampus / cytology
  • Hippocampus / metabolism*
  • Humans
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism
  • Mice
  • Neural Cell Adhesion Molecules / genetics*
  • Neural Cell Adhesion Molecules / metabolism
  • Neurons / cytology
  • Neurons / metabolism*
  • Plasmids / chemistry
  • Plasmids / metabolism
  • Polymerase Chain Reaction / methods
  • Primary Cell Culture
  • RNA / genetics*
  • RNA / metabolism
  • Sindbis Virus / genetics
  • Sindbis Virus / metabolism
  • Synapses / metabolism
  • Synaptic Transmission
  • Transfection

Substances

  • Calcium-Binding Proteins
  • Cell Adhesion Molecules, Neuronal
  • Luminescent Proteins
  • Neural Cell Adhesion Molecules
  • Nrxn1 protein, mouse
  • neuroligin 1
  • red fluorescent protein
  • Green Fluorescent Proteins
  • RNA