Threshold-free population analysis identifies larger DRG neurons to respond stronger to NGF stimulation

PLoS One. 2012;7(3):e34257. doi: 10.1371/journal.pone.0034257. Epub 2012 Mar 27.

Abstract

Sensory neurons in dorsal root ganglia (DRG) are highly heterogeneous in terms of cell size, protein expression, and signaling activity. To analyze their heterogeneity, threshold-based methods are commonly used, which often yield highly variable results due to the subjectivity of the individual investigator. In this work, we introduce a threshold-free analysis approach for sparse and highly heterogeneous datasets obtained from cultures of sensory neurons. This approach is based on population estimates and completely free of investigator-set parameters. With a quantitative automated microscope we measured the signaling state of single DRG neurons by immunofluorescently labeling phosphorylated, i.e., activated Erk1/2. The population density of sensory neurons with and without pain-sensitizing nerve growth factor (NGF) treatment was estimated using a kernel density estimator (KDE). By subtraction of both densities and integration of the positive part, a robust estimate for the size of the responsive subpopulations was obtained. To assure sufficiently large datasets, we determined the number of cells required for reliable estimates using a bootstrapping approach. The proposed methods were employed to analyze response kinetics and response amplitude of DRG neurons after NGF stimulation. We thereby determined the portion of NGF responsive cells on a true population basis. The analysis of the dose dependent NGF response unraveled a biphasic behavior, while the study of its time dependence showed a rapid response, which approached a steady state after less than five minutes. Analyzing two parameter correlations, we found that not only the number of responsive small-sized neurons exceeds the number of responsive large-sized neurons--which is commonly reported and could be explained by the excess of small-sized cells--but also the probability that small-sized cells respond to NGF is higher. In contrast, medium-sized and large-sized neurons showed a larger response amplitude in their mean Erk1/2 activity.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Algorithms
  • Animals
  • Cells, Cultured
  • Extracellular Signal-Regulated MAP Kinases / metabolism
  • Ganglia, Spinal / physiology*
  • Male
  • Models, Biological
  • Models, Neurological
  • Models, Statistical
  • Nerve Growth Factor / metabolism*
  • Neurons / physiology*
  • Phosphorylation
  • Rats
  • Rats, Sprague-Dawley
  • Reproducibility of Results
  • Sensory Receptor Cells / metabolism
  • Signal Transduction
  • Time Factors

Substances

  • Nerve Growth Factor
  • Extracellular Signal-Regulated MAP Kinases