Functional and Molecular Analysis of Proprioceptive Sensory Neuron Excitability in Mice

Jessica F Madden; Olivia C Davis; Kieran A Boyle; Jacqueline A Iredale; Tyler J Browne; Robert J Callister; Douglas W Smith; Phillip Jobling; David I Hughes; Brett A Graham

doi:10.3389/fnmol.2020.00036

Functional and Molecular Analysis of Proprioceptive Sensory Neuron Excitability in Mice

Front Mol Neurosci. 2020 May 5:13:36. doi: 10.3389/fnmol.2020.00036. eCollection 2020.

Authors

Jessica F Madden^{1

2}, Olivia C Davis³, Kieran A Boyle³, Jacqueline A Iredale^{1

2}, Tyler J Browne^{1

2}, Robert J Callister^{1

2}, Douglas W Smith^{1

2}, Phillip Jobling^{1

2}, David I Hughes³, Brett A Graham^{1

2}

Affiliations

¹ School of Biomedical Sciences and Pharmacy, Faculty of Health and Medicine, University of Newcastle, Callaghan, NSW, Australia.
² Hunter Medical Research Institute (HMRI), New Lambton Heights, NSW, Australia.
³ Institute of Neuroscience Psychology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom.

Abstract

Neurons located in dorsal root ganglia (DRG) are crucial for transmitting peripheral sensations such as proprioception, touch, temperature, and nociception to the spinal cord before propagating these signals to higher brain structures. To date, difficulty in identifying modality-specific DRG neurons has limited our ability to study specific populations in detail. As the calcium-binding protein parvalbumin (PV) is a neurochemical marker for proprioceptive DRG cells we used a transgenic mouse line expressing green fluorescent protein (GFP) in PV positive DRGs, to study the functional and molecular properties of putative proprioceptive neurons. Immunolabeled DRGs showed a 100% overlap between GFP positive (GFP+) and PV positive cells, confirming the PVeGFP mouse accurately labeled PV neurons. Targeted patch-clamp recording from isolated GFP+ and GFP negative (GFP-) neurons showed the passive membrane properties of the two groups were similar, however, their active properties differed markedly. All GFP+ neurons fired a single spike in response to sustained current injection and their action potentials (APs) had faster rise times, lower thresholds and shorter half widths. A hyperpolarization-activated current (I_h) was observed in all GFP+ neurons but was infrequently noted in the GFP- population (100% vs. 11%). For GFP+ neurons, I_h activation rates varied markedly, suggesting differences in the underlying hyperpolarization-activated cyclic nucleotide-gated channel (HCN) subunit expression responsible for the current kinetics. Furthermore, quantitative polymerase chain reaction (qPCR) showed the HCN subunits 2, 1, and 4 mRNA (in that order) was more abundant in GFP+ neurons, while HCN 3 was more highly expressed in GFP- neurons. Likewise, immunolabeling confirmed HCN 1, 2, and 4 protein expression in GFP+ neurons. In summary, certain functional properties of GFP+ and GFP- cells differ markedly, providing evidence for modality-specific signaling between the two groups. However, the GFP+ DRG population demonstrates considerable internal heterogeneity when hyperpolarization-activated cyclic nucleotide-gated channel (HCN channel) properties and subunit expression are considered. We propose this heterogeneity reflects the existence of different peripheral receptors such as tendon organs, muscle spindles or mechanoreceptors in the putative proprioceptive neuron population.

Keywords: HCN channel; Ih; action potential; dorsal root ganglia; parvalbumin; proprioception; sensory neuron.