Probing functional properties of nociceptive axons using a microfluidic culture system

PLoS One. 2013 Nov 20;8(11):e80722. doi: 10.1371/journal.pone.0080722. eCollection 2013.

Abstract

Pathological changes in axonal function are integral features of many neurological disorders, yet our knowledge of the molecular basis of axonal dysfunction remains limited. Microfluidic chambers (MFCs) can provide unique insight into the axonal compartment independent of the soma. Here we demonstrate how an MFC based cell culture system can be readily adapted for the study of axonal function in vitro. We illustrate the ease and versatility to assay electrogenesis and conduction of action potentials (APs) in naïve, damaged or sensitized DRG axons using calcium imaging at the soma for pharmacological screening or patch-clamp electrophysiology for detailed biophysical characterisation. To demonstrate the adaptability of the system, we report by way of example functional changes in nociceptor axons following sensitization by neurotrophins and axotomy in vitro. We show that NGF can locally sensitize axonal responses to capsaicin, independent of the soma. Axotomizing neurons in MFC results in a significant increase in the proportion of neurons that respond to axonal stimulation, and interestingly leads to accumulation of Nav1.8 channels in regenerating axons. Axotomy also augmented AP amplitude following axotomy and altered activation thresholds in a subpopulation of regenerating axons. We further show how the system can readily be used to study modulation of axonal function by non-neuronal cells such as keratinocytes. Hence we describe a novel in vitro platform for the study of axonal function and a surrogate model for nerve injury and sensitization.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Action Potentials / drug effects
  • Animals
  • Axons / drug effects
  • Axons / physiology*
  • Axotomy
  • Biological Assay
  • Calcium / metabolism
  • Capsaicin / pharmacology
  • Cell Communication / drug effects
  • Cells, Cultured
  • Coculture Techniques
  • Electric Stimulation
  • Female
  • Ganglia, Spinal / drug effects
  • Ganglia, Spinal / injuries
  • Ganglia, Spinal / pathology
  • Keratinocytes / cytology
  • Keratinocytes / drug effects
  • Male
  • Mice, Inbred C57BL
  • Microfluidics / instrumentation
  • Microfluidics / methods*
  • Models, Biological
  • Nerve Growth Factor / pharmacology
  • Nociception* / drug effects
  • Patch-Clamp Techniques
  • Rats, Wistar
  • Sensory Receptor Cells / drug effects
  • Sensory Receptor Cells / pathology
  • Sodium Channel Blockers / pharmacology
  • Synaptic Transmission / drug effects
  • Synaptic Transmission / physiology
  • TRPV Cation Channels / metabolism

Substances

  • Sodium Channel Blockers
  • TRPV Cation Channels
  • Trpv1 protein, rat
  • Nerve Growth Factor
  • Capsaicin
  • Calcium

Grant support

The authors would like to declare that this work has been partly funded by a grant from Pfizer Neusentis, UK to King’s College London as part of the Pfizer KCL Pain Lab collaboration based in the Wolfson Centre for Age-Related Diseases, King’s College London. Ramin Raouf, Clare Farmer, Christoforos Tsantoulas, Katsuhiro Baba, and Patricia Machado were employed by Pfizer. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.