Developmental expression of the TTX-resistant voltage-gated sodium channels Nav1.8 (SNS) and Nav1.9 (SNS2) in primary sensory neurons

Abstract

The development of neuronal excitability involves the coordinated expression of different voltage-gated ion channels. We have characterized the expression of two sensory neuron-specific tetrodotoxin-resistant sodium channel alpha subunits, Na(v)1. (SNS/PN3) and Na(v)1.9 (SNS2/NaN), in developing rat lumbar dorsal root ganglia (DRGs). Expression of both Na(v)1.8 and Na(v)1.9 increases with age, beginning at embryonic day (E) 15 and E17, respectively, and reaching adult levels by postnatal day 7. Their distribution is restricted mainly to those subpopulations of primary sensory neurons in developing and adult DRGs that give rise to unmyelinated C-fibers (neurofilament 200 negative). Na(v)1.8 is expressed in a higher proportion of neuronal profiles than Na(v)1.9 at all stages during development, as in the adult. At E17, almost all Na(v)1.8-expressing neurons also express the high-affinity NGF receptor TrkA, and only a small proportion bind to IB4, a marker for c-ret-expressing (glial-derived neurotrophic factor-responsive) neurons. Because IB4 binding neurons differentiate from TrkA neurons in the postnatal period, the proportion of Na(v)1.8 cells that bind to IB4 increases, in parallel with a decrease in the proportion of Na(v)1.8-TrkA co-expressing cells. In contrast, an equal number of Na(v)1.9 cells bind IB4 and TrkA in embryonic life. The differential expression of Na(v)1.8 and Na(v)1.9 in late embryonic development, with their distinctive kinetic properties, may contribute to the development of spontaneous and stimulus-evoked excitability in small diameter primary sensory neurons in the perinatal period and the activity-dependent changes in differentiation they produce.

Fig. 1.

The developmental regulation of Na_v1.8 and Na_v1.9 expression in embryonic DRG. Northern blot analysis of Na_v1.8 (top) and Na_v1.9 (middle) α-subunit mRNA transcripts in lumbar DRG between developmental ages E15 and P0. Cyclophilin mRNA (CYC, bottom) is a loading control.

Fig. 2.

Quantitative analysis of Na_v1.8 and Na_v1.9 protein expression levels through embryonic and neonatal development. A, The percentage of Na_v1.8- or Na_v1.9-positive cell profiles as a proportion of the total number of DRG neuronal profiles in the developing DRG. Error bars represent SEM. (*p < 0.01, **p < 0.001, for values for Na_v1.8 vs Na_v1.9; ANOVA paired test). Levels of SNS and SNS2 protein expression are significantly different (p = 0.001; ANOVA) from E17 to P7, compared with adult Na_v1.8 and Na_v1.9 expression.B, The proportion of the Na_v1.8- and Na_v1.9-positive profiles expressed in neuronal subpopulations expressing NF 200, TrkA, and IB4 binding, with increasing development.

Fig. 3.

Distribution of Na_v1.8 changes with phenotypic changes in the developing DRG. Double immunocytochemistry of Na_v1.8 labeled (red), TrkA labeled (green), and composite image (far right panel) showing double labeled (yellow) DRG cells at specific developmental ages. Scale bars, 50 μm.

Fig. 4.

Distribution of Na_v1.9 in subpopulations of DRG neurons through development. Immunoreactivity of cells positive for Na_v1.9 (red), marker population (green), or both (yellow) (composite image) in Na_v1.9 colocalization studies with TrkA (left), IB4 (middle), and NF200 (right) in DRG neurons at developmental ages E17, P0, and P7. Scale bars, 50 μm.

Fig. 5.

A summary of significant events defining rat DRG sensory neuron development. A schematic representation showing a time scale of developmentally regulated events of a typical DRG is illustrated from embryonic age 11 (E11) to postnatal age 21 (P21) and in adult animals (left toright of the diagram; not to scale). This is an attempt to correlate the major changes that occur in the developing DRG, with particular emphasis on acquisition of neuronal electrical excitability and changes in the expression of voltage-gated sodium channels (VGSCs). The expression of the sodium channels may be regulated by the developmental expression of specific neurotrophic factors. The top (shaded)panel illustrates the distinct patterns of electrical activity in the developing DRG, marking the period of developmentally regulated ectopic spontaneous discharge from E16 to E20, and the earliest detection of a TTX-resistant (TTXr) sodium current. The second panel outlines key developmental events, including the birth of specific neuronal subpopulations; the large light A-cell population (future A fibers) are born from E11.5 to E14.5, in advance of the birth of small, dark C-cell neurons (giving rise to C-fibers) which occurs later at E13.5–E16.5. A third unidentified subpopulation of putative nonpeptide-containing neurons (RT97-negative and IB4-negative) are generated between E14 and E15. Almost immediately after neuronal birth, the onset of axon outgrowth begins from E14 onward, followed by peripheral innervation of both A- and C-fibers simultaneously from E14 and central target innervation from E15 or E18 for A- and C-fibers, (Figure legend continues.) (Figure legend continued.) respectively. Themiddle panel shows changes in mRNA expression of known TTX-sensitive (TTXs) and the TTX-resistant (TTXr) voltage-gated sodium channels (VGSC) in the DRG, which may be correlated with developmentally regulated changes in neurotrophic factors (second panel from bottom) or changes in neuronal phenotype, which is indicated by a shift in the expression of specific neurotrophic receptors (bottom panel).Dotted lines indicate that expression at earlier ages has not yet been investigated. Arrows in the text denote an increase or decrease in expression, respectively. References are indicated by numbers on right side of figure: 1.Fitzgerald, 1987; 2.Fitzgerald and Fulton, 1992; 3. Jackman and Fitzgerald, 2000; 4.Altman and Bayer, 1984; 5.Coggeshall et al., 1994; 6.Felts et al., 1997;7. Ernfors et al., 1992; 8.Ernfors et al., 1989; 9.ElShamy and Ernfors, 1996;10.Schecterson and Bothwell, 1992; 11.Sebert and Shooter, 1993; 12.Constantinou et al., 1994.13. Davis et al., 1987; 14.Rohrer et al., 1998; 15.Molliver et al., 1997b;16.Alvares and Fitzgerald, 1999; 17.Bennett et al., 1996; 18.Phillips and Armanini, 1996;19.Farinas et al., 1998; 20.Ehrhard and Otten, 1994; 21. Wright and Snider, 1996.