Cellular electrophysiology of DRG neurons
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) (). 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). shows the Na current of a large-diameter (35 µm) neuron measured under identical conditions. These Na currents began activating at relatively hyperpolarized voltages (≈ −60 mV) and rapidly inactivated with a time constant (τh) of 0.40 ± 0.13 ms (n=8). TTX inhibited the majority of this Na current (99.4 ± 2.0 %, n=14) consistent with the preferential expression of TTX-sensitive (TTX-S) Na channels in this population (). The whole-cell capacitance of these neurons ranged between 30 and 69 pF corresponding to a diameter of 38.9 ± 1.2 µm (n = 12).
Na currents of small and large DRG neurons
The normalized conductance was calculated from the peak Na currents of small and large neurons and plotted versus the test potential (). The smooth curves are fits of the data to a double Boltzmann function with midpoints (Vact) of −26 mV and −15 mV for the small neurons and −38 mV and −29 mV for the large neurons. Also plotted is the steady-state inactivation determined using 500 ms prepulses to voltages between −120 and −30 mV. The smooth curves are fits to a double Boltzmann with midpoints (Vin) of −66 mV and −40 mV for the small neurons and −103 mV and −68 mV for the large neurons. The activation and steady-state inactivation of both populations was a composite of two or more components suggesting that multiple Na channels contribute to the whole-cell currents in these neurons.
Voltage-dependent gating of DRG Na currents
The Na currents of these neurons were further characterized by individually measuring the properties of the TTX-S and TTX-R components. The TTX-S currents of the large neurons were isolated by subtracting the residual TTX-R current measured after the bath application of TTX from the total Na current. The normalized conductance and steady-state inactivation of these currents were determined and plotted versus the test potential (). The smooth curves are fits to Boltzmann functions with midpoints of activation (Vact
) and inactivation (Vin
) of −23 mV and −67 mV respectively (). The voltage-dependent gating of the isolated TTX-S current is similar to components of Na current measured in both the small (Vact
= −26 mV, Vin
= −66 mV) and large (Vact
= −29, Vin
= −8 mV) neurons (). This finding is consistent with work showing that TTX-S Na channels with similar gating properties are widely expressed in DRG neurons (Cummins et al., 2007
;Rush et al., 2007
The TTX-R current of small-diameter neurons was directly measured after bath application of TTX. The midpoint of activation (Vact) was −12 mV while the steady-state inactivation was best fitted with a double Boltzmann function with midpoints (Vin) of −76 mV and −30 mV (). The majority (90%) of the inactivation was associated with the more depolarized (Vin= −30 mV) component. The activation and inactivation of the isolated TTX-R current displayed properties similar to the predominant TTX-R current (Vact= −15 mV, Vin= −40 mV) preferentially expressed in small neurons ().
In addition to the slowly-inactivating TTX-R Na current, DRG neurons also express a persistent TTX-R component (Baker and Bostock, 1997
;Cummins et al., 1999
). This current was isolated by pre-treating with A-803467, a selective Nav1.8 channel inhibitor (Jarvis et al., 2007
), to reduce the more dominant slowly-inactivating TTX-R current (). Holding the neurons at relatively depolarized voltages (−60 mV) promotes the slow inactivation of the persistent Na current that only slowly recovers at hyperpolarized voltages (Cummins et al., 1999
). Applying short hyperpolarizing prepulses (−140 mV/25 ms) prior to the test pulses permitted the full recovery of the inactivating but not the persistent component (). shows the persistent TTX-R Na current obtained by subtracting the inactivating component (Panel B) from the total current (Panel A). The resulting Na currents slowly inactivated at −20 mV with a time constant (τh
) of 41 ± 6 ms, had a peak current density of 176 ± 29 pA/pF (n=8) and activated with a midpoint (V0.5
) of −39 mV (). These properties are similar to what has been previously reported for the persistent TTX-R Na currents of DRG neurons (Cummins et al., 1999
; Dib-Hajj et al., 2002
;Coste et al., 2004
;Priest et al., 2005
Voltage-dependent gating of the persistent TTX-R Na current
Single-cell analysis of the Na channel transcripts
To further characterize Na channel expression the mRNA present in individually harvested small and large DRG neurons was quantitatively measured using real-time PCR. The number of mRNA copies present in the cell lysates was obtained by comparing the cutoff cycle (Ct) values of the samples with those of known cDNA standards (Methods). compares the mRNA expression (copies/neuron) of small- (<25 µm) and large-diameter (>30 µm) neurons. Small neurons expressed mRNA encoding for both TTX-S (Nav1.7) and TTX-R (Nav1.8, Nav1.9) Na channels. Transcripts encoding for several other TTX-S isoforms (Nav1.1, Nav1.2, Nav1.6) were also detected in the small neurons but were present at comparatively low levels suggesting that Nav1.7 is the predominant TTX-S Na channel expressed in this population. The prevalent expression of Nav1.7, Nav1.8 and Nav1.9 channels is consistent with electrophysiology showing that small-diameter neurons express a combination of TTX-S and TTX-R Na currents (). This contrasted with the large-diameter neurons that displayed prominent TTX-S Na current () and expressed mRNA encoding for the TTX-S (Nav1.1, Nav1.6, Nav1.7) isoforms (). Paradoxically, these large neurons also displayed high levels of Nav1.8 mRNA () despite the absence of TTX-R Na current in the vast majority (80%) of these neurons. The expression of TTX-R Na channels in a subpopulation of the large neurons is examined in more detail in the next section.
Expression of Na channel transcripts in DRG neurons
mRNA Expression(Copies per Neuron)
A particular strength of the single-cell measurements is that it permits analysis of multiple Na channel transcripts from individual neurons thereby providing a quantitative assessment of isoform expression at the cellular level. Small and large neurons were individually harvested and assayed for TTX-S (Nav1.1, Nav1.6, Nav1.7) and TTX-R (Nav1.5, Nav1.8, Nav1.9) Na channels. Small neurons highly expressed Nav1.7, but comparatively few copies of Nav1.1 or Nav1.6 mRNA (). This contrasted with the large neurons that broadly expressed mRNA for all three of the TTX-S isoforms. shows a similar analysis of the TTX-R Na channels. The small neurons highly expressed Nav1.8 and Nav1.9 while the large neurons expressed variable quantities of Nav1.8 and low levels of Nav1.9. Nav1.5 was detected in very few of the neurons (14%) and was present at comparatively low copy numbers (11 ± 5 copies/neuron, n=71).
Nav1.8 channels are highly expressed in a subpopulation of the large neurons
Whole-cell recordings indicated that the Na currents of small neurons were relatively insensitive to externally applied TTX while large neurons preferentially expressed TTX-S Na current (). This conflicted with transcript analysis indicating that TTX-R Nav1.8 channel mRNA was present at comparably high levels in both populations (). However, single-cell analysis shows that Nav1.8 mRNA displays an unusually broad distribution in the large neurons (). The majority (78%) expressed Nav1.8 mRNA at moderate levels (1016 ± 120 copies/neuron) while the remaining fraction (22%) displayed considerably higher copy numbers (7439 ± 525 copies/neuron, n=20). A similar broad distribution of Nav1.8 mRNA in larger-diameter neurons has been observed using in situ
hybridization (Fukuoka et al., 2008
). Together these data suggest that Nav1.8 channels may be highly expressed in a subpopulation of the large neurons.
shows an example of a large-diameter (40 µm) neuron expressing TTX-R Na current. The cell bodies of neurons expressing this current had diameters typical of large neurons (38.8 ± 1.4 µm) but had Na currents that were weakly inhibited by TTX (24.3 ± 4.5%, n=14). To further investigate this unique population the TTX-R current of large neurons were normalized to the whole-cell capacitance (pA/pF) and plotted as a frequency histogram (). Consistent with two populations the TTX-R current histograms were bimodal with peaks at 17 and 232 pA/pF. This contrasted with the current density histograms of the small neurons that displayed a single peak around 379 pA/pF, consistent with a more uniform distribution of TTX-R channels in this population ().
Expression of TTX-R Na current in large neurons
The molecular basis of this 10-fold difference in TTX-R current density was further investigated using Na current measurements to determine the TTX sensitivity of large neurons before harvesting and mRNA analysis. The voltage-dependent activation (V0.5 = −11.7 ± 1.2 mV, n=6) and inactivation time constants (τh = 1.2 ± 0.2 ms at +20 mV, n=6) of the TTX-R Na currents in these large neurons were similar to what was previously observed for the predominant TTX-R current of small neurons (V0.5 = −11.7 ± 0.8 mV, τh = 1.0 ms) suggesting that Nav1.8 expression may underlie these currents. shows that the real-time PCR amplification of Nav1.8 mRNA from large neurons segregates into two populations consistent with low and high expression of Nav1.8 channels. Quantitative analysis showed that large neurons displaying TTX-R current (157 ± 44 pA/pF, n=5) expressed Nav1.8 mRNA at 5-fold higher levels than those with small TTX-R currents (22 ± 11 pA/pF, n=6). Although Nav1.9 mRNA was also elevated in this population, no persistent TTX-R current was observed and Nav1.9 expression levels (671 ± 160 copies/neuron, n=10) were nearly 5-fold lower than what is typically observed in small neurons (2854 ± 238 copies/neuron, n=71) where these currents are routinely observed.
Molecular markers of small unmyelinated and large myelinated neurons
Cytoplasmic neurofilaments contribute to the neuronal cytoskeleton, provide structural integrity and are important determinants of the axonal diameter and the velocity of impulse conduction along peripheral nerve fibers (Hoffman et al., 1985
;Fuchs and Cleveland, 1998
). The heavy neurofilament NF200 (200 kDa) and intermediate neurofilament peripherin (57 kDa) are preferentially expressed in large and small DRG neurons respectively and have proved to be useful markers for distinguishing between these populations (Goldstein et al., 1991
;Fornaro et al., 2008
). Consistent with these expectations peripherin mRNA was found to be present at 1.7-fold higher levels in small neurons while NF200 mRNA was 20-fold higher in the large neurons ().
Expression of peripherin, NF200 and Necl-1
Necls are a family of cell adhesion molecules that mediate axonal-glial interactions (Spiegel et al., 2007
;Maurel et al., 2007
). Heterophilic interaction between Necl-1 of peripheral neurons and Necl-4 of Schwann cells promotes the formation of myelin sheaths. shows that Necl-1 mRNA is preferentially expressed in large neurons. The combination of NF200 and Necl-1 mRNA suggests that these large neurons may give rise to thick myelinated axons. Conversely, the small neurons express peripherin but comparatively little NF200 or Necl-1 suggesting that these neurons may be associated with thin unmyelinated axons.