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

Abstract

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.

Cultured dissociated primary dorsal root ganglion neurons from adult horses enable study of axonal transport

“…Dissociated primary dorsal root ganglia (DRG) cultures from laboratory rodents, usually derived from neonatal or embryonic donors, are widely used to study axon degeneration, regeneration, axonal transport impairment and basic mechanisms of sensory physiology and pain (Baccaglini & Hogan, 1983 ; Bilsland et al, 2010 ; Eva et al, 2012 ; Mellone et al, 2013 ; Osterloh et al, 2012 ). Furthermore, DRG cultures are used as models for neurite outgrowth and synapse formation during development and for growth factor‐dependent cell survival (Bayat et al, 2021 ; De Koninck et al, 1993 ; Malin et al, 2007 ; Nowicki et al, 2009 ; Wong et al, 2015 ; Wright & Snider, 1995 ). However, many disorders are unique to larger species, where extended axon length presents a far greater challenge for axonal transport than in rodents, and some have adult onset.…”