Adult mouse dorsal root ganglia neurons form aberrant glutamatergic connections in dissociated cultures

PLoS One. 2021 Mar 3;16(3):e0246924. doi: 10.1371/journal.pone.0246924. eCollection 2021.


Cultured sensory neurons can exhibit complex activity patterns following stimulation in terms of increased excitability and interconnected responses of multiple neurons. Although these complex activity patterns suggest a network-like configuration, research so far had little interest in synaptic network formation ability of the sensory neurons. To identify interaction profiles of Dorsal Root Ganglia (DRG) neurons and explore their putative connectivity, we developed an in vitro experimental approach. A double transgenic mouse model, expressing genetically encoded calcium indicator (GECI) in their glutamatergic neurons, was produced. Dissociated DRG cultures from adult mice were prepared with a serum-free protocol and no additional growth factors or cytokines were utilized for neuronal sensitization. DRG neurons were grown on microelectrode arrays (MEA) to induce stimulus-evoked activity with a modality-free stimulation strategy. With an almost single-cell level electrical stimulation, spontaneous and evoked activity of GCaMP6s expressing neurons were detected under confocal microscope. Typical responses were analyzed, and correlated calcium events were detected across individual DRG neurons. Next, correlated responses were successfully blocked by glutamatergic receptor antagonists, which indicated functional synaptic coupling. Immunostaining confirmed the presence of synapses mainly in the axonal terminals, axon-soma junctions and axon-axon intersection sites. Concisely, the results presented here illustrate a new type of neuron-to-neuron interaction in cultured DRG neurons conducted through synapses. The developed assay can be a valuable tool to analyze individual and collective responses of the cultured sensory neurons.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Communication
  • Cell Culture Techniques / methods*
  • Cells, Cultured
  • Electric Stimulation
  • Ganglia, Spinal / cytology*
  • Ganglia, Spinal / metabolism
  • Mice
  • Mice, Transgenic
  • Receptors, Glutamate / metabolism
  • Synaptophysin / metabolism*
  • Vesicular Glutamate Transport Protein 2 / genetics*


  • Receptors, Glutamate
  • Slc17a6 protein, mouse
  • Synaptophysin
  • Syp protein, mouse
  • Vesicular Glutamate Transport Protein 2

Grants and funding

This work is funded by Boğaziçi University Research Fund to author AG under the Project Code 8080D. During the experiments some facilities of REMER (Istanbul Medipol University, Regenerative and Restorative Medicine Center) were used. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.