Single-cell analysis of sodium channel expression in dorsal root ganglion neurons
Introduction
The dorsal root ganglion (DRG) is composed of a heterogeneous population of neurons that convey a variety of sensory information from peripheral and visceral tissues to the central nervous system. DRG neurons have been classified according to the size of their cell bodies, duration and amplitude of their action potentials, the extent of myelination, expression of neurofilaments and their axonal conduction velocities (Fornaro et al., 2008, Goldstein et al., 1991, Harper and Lawson, 1985, Lawson et al., 1993, Lee et al., 1986, Yoshida and Matsuda, 1979, Lawson, 2002). These studies indicate that the small-diameter DRG neurons (< 30 μm) give rise to slowly-conducting unmyelinated (C) or thinly myelinated (Aδ) nerve fibers associated with thermoception and pain (Harper and Lawson, 1985). Large-diameter DRG neurons (> 30 μm) have been linked to rapidly-conducting myelinated A fibers (Aα and Aβ), such as those typically associated with proprioception and low-threshold mechanoreception.
The small- and large-diameter neurons of the DRG express distinct populations of voltage-gated Na channels that govern the initiation and conduction of action potentials (Cummins et al., 2007, Dib-Hajj et al., 2009, Rush et al., 2007). DRG Na currents have been broadly classified based on voltage-dependence, kinetics and pharmacology into rapidly-gating tetrodotoxin-sensitive (TTX-S), slowly-gating TTX-resistant (TTX-R) and persistent TTX-R components (Caffrey et al., 1992, Elliott and Elliott, 1993, Kostyuk et al., 1981, Roy and Narahashi, 1992). At least five distinct Na channel isoforms are known to be expressed in the DRG (Black et al., 1996, Amaya et al., 2000, Dib-Hajj et al., 1998). Nav1.7 (PN1) encodes for a rapidly-gating TTX-S Na channel that is preferentially expressed in the dorsal root and sympathetic ganglia (Toledo-Aral et al., 1997, Black et al., 1996, Sangameswaran et al., 1997). Nav1.7, along with several other isoforms (Nav1.1, Nav1.2, and Nav1.6) accounts for the TTX-S Na current observed in most DRG neurons. Nav1.8 (PN3) encodes for a slowly-gating TTX-R Na current that is primarily expressed in small DRG neurons and produces the majority of the depolarizing inward current during action potentials (Akopian et al., 1996, Blair and Bean, 2002, Sangameswaran et al., 1996). The Nav1.9 channel (NaN) encodes for a slowly-gating Na channel that underlies a persistent TTX-R current in small DRG neurons (Dib-Hajj et al., 1998, Tate et al., 1998, Dib-Hajj et al., 2002). The differential expressions of these Na channels in small and large DRG neurons coupled with isoform-specific differences in voltage-dependence and gating kinetics are important determinants of neuronal excitability and the transmission of sensory information.
The goal of this study was to investigate Na channel expression in small- (< 25 μm) and large-diameter (> 30 μm) neurons acutely dissociated from the rat DRG. A combination of cellular electrophysiology and single-cell RT-PCR were used to compare the pharmacology and gating properties of the endogenous Na currents with the Na channel transcripts expressed in the same neurons. The neurons were further classified based on the expression of cytoplasmic neurofilaments (peripherin, NF200) and a cell adhesion molecule involved in myelination (Necl-1). The data indicate that small unmyelinated neurons express a combination of TTX-S (Nav1.7) and TTX-R (Nav1.8, Nav1.9) isoforms while large myelinated neurons preferentially express TTX-S (Nav1.1, Nav1.6, Nav1.7) channels.
Section snippets
Cellular electrophysiology of DRG neurons
Fig. 1A shows the whole-cell Na current of a small-diameter (25 μm) neuron acutely dissociated from the rat DRG. The currents began activating around − 40 mV and inactivated with a time constant (τh) of 0.97 ± 0.03 ms at + 20 mV (n = 13). Tetrodotoxin (TTX) was a relatively weak inhibitor of this Na current reducing the peak amplitude by 19.7 ± 1.0% (n = 13) (Fig. 1B). The whole-cell capacitances of these small neurons ranged between 15 and 24 pF corresponding to a cell body diameter of 24.9 ± 0.5 μm (n = 13).
Discussion
DRG sensory neurons express multiple Na channel isoforms that substantially differ in voltage-dependence, kinetics and pharmacology (Cummins et al., 2007, Lai et al., 2004, Rush et al., 2007). These Na channels are differentially expressed in subpopulations of DRG neurons where they contribute to excitability and regulate action potential firing. Previous studies have employed a variety of techniques including electrophysiology, immunohistochemistry and in situ hybridization to investigate Na
Conclusions
This study employed electrophysiology and single-cell RT-PCR to investigate Na channel expression in identified populations of DRG sensory neurons. The ability to quantitatively analyze multiple transcripts from single neurons enabled a comparison of the pharmacology and gating properties of the endogenous Na currents with the Na channel isoforms expressed in the same neurons. Small-diameter (< 25 μm) DRG neurons displaying prominent TTX-R Na currents co-expressed both TTX-S and TTX-R isoforms
DRG cell culture
Seven day old rat pups (male and female) were anaesthetized with isoflurane before decapitation and the dorsal root ganglia (DRG) harvested from all accessible levels. Ganglia were incubated for 30 min at 37 °C in 2 ml of HBSS/HEPES containing 1.5 mg/ml collagenase (Sigma-Aldrich, St. Louis, MO) followed by 1 mg/ml trypsin (Sigma-Aldrich) for an additional 30 min. Trypsin was removed and the ganglia transferred to L-15 Leibovitz media supplemented with 1% fetal bovine serum (Gibco), 2 mM glutamine, 24
Acknowledgments
The authors would like to thank Steve Malinowski for technical assistance. This work was supported by a grant from the National Institute of General Medical Sciences (GM078244).
References (52)
- et al.
Diversity of expression of the sensory neuron-specific TTX-resistant voltage-gated sodium ion channels SNS and SNS2
Mol. Cell. Neurosci.
(2000) - et al.
Spinal sensory neurons express multiple sodium channel alpha-subunit mRNAs
Brain Res. Mol. Brain Res.
(1996) - et al.
Changes in the expression of tetrodotoxin-sensitive sodium channels within dorsal root ganglia neurons in inflammatory pain
Pain
(2004) - et al.
Three types of sodium channels in adult rat dorsal root ganglion neurons
Brain Res.
(1992) - et al.
Functional role of the C-terminus of voltage-gated sodium channel Na(v)1.8
FEBS Lett.
(2004) - et al.
Gating and modulation of presumptive NaV1.9 channels in enteric and spinal sensory neurons
Mol. Cell. Neurosci.
(2004) - et al.
The roles of sodium channels in nociception: implications for mechanisms of pain
Pain
(2007) - et al.
NaN/Nav1.9: a sodium channel with unique properties
Trends Neurosci.
(2002) - et al.
Voltage-gated sodium channels in pain states: role in pathophysiology and targets for treatment
Brain Res. Rev.
(2009) - et al.
Neuronal intermediate filament expression in rat dorsal root ganglia sensory neurons: an in vivo and in vitro study
Neuroscience
(2008)
Heterologous expression and functional analysis of rat Nav1.8 (SNS) voltage-gated sodium channels in the dorsal root ganglion neuroblastoma cell line ND7-23
Neuropharmacology
Ionic currents in the somatic membrane of rat dorsal root ganglion neurons-I. Sodium currents
Neuroscience
Primary sensory neurones: neurofilament, neuropeptides, and conduction velocity
Brain Res. Bull.
Structure and function of a novel voltage-gated, tetrodotoxin-resistant sodium channel specific to sensory neurons
J. Biol. Chem.
A novel tetrodotoxin-sensitive, voltage-gated sodium channel expressed in rat and human dorsal root ganglia
J. Biol. Chem.
A tetrodotoxin-resistant voltage-gated sodium channel expressed by sensory neurons
Nature
Low-threshold, persistent sodium current in rat large dorsal root ganglion neurons in culture
J. Neurophysiol.
Roles of tetrodotoxin (TTX)-sensitive Na+ current, TTX-resistant Na+ current, and Ca2+ current in the action potentials of nociceptive sensory neurons
J. Neurosci.
A novel persistent tetrodotoxin-resistant sodium current in SNS-null and wild-type small primary sensory neurons
J. Neurosci.
NaN, a novel voltage-gated Na channel, is expressed preferentially in peripheral sensory neurons and down-regulated after axotomy
Proc. Natl Acad. Sci.
The TTX-resistant sodium channel Nav1.8 (SNS/PN3): expression and correlation with membrane properties in rat nociceptive primary afferent neurons
J. Physiol.
Characterization of TTX-sensitive and TTX-resistant sodium currents in small cells from adult rat dorsal root ganglia
J. Physiol.
The presence and role of the tetrodotoxin-resistant sodium channel Na(v)1.9 (NaN) in nociceptive primary afferent neurons
J. Neurosci.
Localization of the tetrodotoxin-resistant sodium channel NaN in nociceptors
NeuroReport
A structural scaffolding of intermediate filaments in health and disease
Science
Comparative study of the distribution of the alpha-subunits of voltage-gated sodium channels in normal and axotomized rat dorsal root ganglion neurons
J. Comp. Neurol.
Cited by (122)
Schisandrin B from Schisandra chinensis alleviated pain via glycine receptors, Nav1.7 channels and Cav2.2 channels
2024, Journal of EthnopharmacologyModeling chemotherapy induced peripheral neuropathy (CIPN) in vitro: Prospects and limitations
2020, Experimental NeurologyCitation Excerpt :Therefore, mixed co-cultures must be considered less artificial compared to pure neuronal cell cultures. Mixed cultures, on the other hand, may require advanced single cell analysis techniques in addition to immunostaining, such as single cell RT-PCR (Ho and O'Leary, 2011) or a quantitative automated microscopy (Andres et al., 2010), when signaling cascades and response to stimuli are investigated. Markers that are often used to label specific cell populations are β III tubulin for neurons and S100 for Schwann cells.
5.08 - Molecular Biology of the Nociceptor/Transduction
2020, The Senses: A Comprehensive Reference: Volume 1-7, Second Edition