The codon-optimized human Na_V1.8 plasmid (pcDNA5-hNav1.8) was previously described (Faber et al., 2012). A cassette containing the HaloTag enzyme for covalent fluorophore linkage followed by a synthetic β4 transmembrane segment was fused to the N-terminus as previously described (Higerd-Rusli et al., 2022).

All animal preparations adhered to protocols approved by the Institutional Animal Care and Use Committee of the Veterans Administration Connecticut Healthcare System. DRG neurons were isolated from neonatal Sprague–Dawley rats and transfected with HaloTag-Na_V1.8 as previously described (Dib-Hajj et al., 2009; Higerd-Rusli et al., 2022). Studies of channel traffic used two microfluidic chambers separated by a physical barrier 450 μm in width with microgrooves to allow axons to extend from a soma chamber to the axon chamber (Xona Microfluidics, Research Triangle Park, NC) that were adhered to 50 mm glass-bottom dishes previously coated with poly-L-lysine and laminin as described (Akin et al., 2021). Transfected DRG neurons were applied to the somatic chambers and the growth medium (Neurobasal with 2% B27, 1% penicillin/streptomycin, 1% GlutaMAX, all from Thermo Fisher Scientific, Waltham, MA) was supplemented with 50 ng/ml NGF (Envigo, Indianapolis, IN) and GDNF (Preprotech, Windsor, NJ) the following day. The distal axonal chamber received medium with 100 ng/ml NGF and GDNF to attract growth of axons into this chamber. Cultures were treated as described below and analyzed on day 7 after preparation unless otherwise noted.

Either 24 h or 48 h before analysis, half the medium was removed from each culture dish or microfluidic chamber and replaced with fresh medium containing paclitaxel such that the final concentrations would be 25 nM or 125 nM. Control cultures received an identical volume of DMSO diluted in the same manner. Some experiments used a cocktail of inflammatory mediators applied in the same manner such that the final concentrations were: 1 μM bradykinin, 10 μM prostaglandin E2, 10 μM histamine, 10 μM 5-hydroxytryptamine, and 15 μM ATP (Hockley et al., 2014).

Each microfluidic chamber of a DRG culture was perfused with prewarmed normal imaging saline (recipe) for 3 min at a rate of 0.8 ml/min with a peristaltic pump. To visualize Na_V channels present at the surface of neuronal endings, the distal axon chamber was perfused with 1 ml of normal imaging saline containing 100 nM of cell-impermeant Janelia Fluor 635 (JF635i) and incubated for 15 min at 37°C. For analyzing the anterograde transport of channels, the somatic chamber was perfused and incubated at the same time with 1 ml of cell-permeable Janelia Fluor 549 (JF549). Both fluorophores were a gift of Luke Lavis and Jonathan B. Grimm, Janelia Farm Research Campus, Alexandria, VA (Grimm et al., 2015; Jonker et al., 2019) and are synthetic ligands that become covalently linked to the HaloTag enzyme fused to the extracellular terminus of the Na_V1.8 expression construct. Following incubation, each chamber was again washed with normal imaging saline for 3 min before confocal microscopy.

Axonal endings were identified by examining distal chambers of microfluidic dishes on an inverted Nikon Ti-Eclipse microscope (Nikon Instruments, Melville, NY) equipped with an incubation enclosure to maintain cultures at 37°C, and a Dragonfly spinning confocal disk system (Andor, Concord, MA) incorporating 561 nm and 635 nm diode lasers for high-speed imaging of living cells with limited phototoxicity. To quantify cell-surface channel expression, 0.2 μm optical sections of the axial extent of the endings were collected through a 60×/1.4 NA oil-immersion objective Nikon and a 700 nm ± 50 nm band-pass emission filter. The average JF635i fluorescence intensity of the distal 60 μm of an axon across the summed z-stack was used as the surface expression value, after subtracting the value obtained from an identically shaped region of interest in the adjacent background.

The same axonal endings measured for cell-surface Na_V1.8 levels were also analyzed for anterograde transport of Na_V1.8-containing vesicles that had been labeled with JF549 applied to the somatic chamber ~20 min prior. Images were acquired on the Dragonfly spinning disk system mentioned above using the same 60× objective and a 600 nm ± 50 nm band-pass filter. For each axon, 200 images at 100 ms exposure time and 2-frame averaging were acquired with an iXon888 EM-CCD camera (Andor). Images were processed by subtracting the average fluorescence intensity of a background region of interest in each image from the entire image in ImageJ (NIH), and individual axons with moving vesicles were identified. The ImageJ plugin KymographClear (Mangeol et al., 2016) was used to generate kymographs of the fluorescence intensity of each pixel along the axon versus time in milliseconds. These kymographs were then passed to KymoButler (Jakobs et al., 2019), an algorithm developed for automated extraction of the trajectories for particles at least 3 pixels in diameter from kymographs. For these presumptive labeled transport vesicles, Kymobutler calculates the average velocity, measures the fluorescence, and identifies pauses. All analyses included only those vesicles with net anterograde motion of at least 0.1 μm/s. Summary statistics, significance testing, and visualizations of these datasets were conducted by custom routines written in Python 3.8, which are available online at https://github.com/ycnrr.

For electrophysiological studies, neonatal rat DRG neurons were plated into coverslips coated with poly-L-lysine and laminin in medium supplemented with 50 ng/ml NGF and GDNF. Cells were incubated with 25 nM Paclitaxel or an equivalent volume of DMSO for control. Following 24 h of treatment, coverslips were taken for voltage-clamp recordings.

Patch pipettes were fabricated from borosilicate glass (World Precision Instruments, Sarasota, FL) using a P-97 puller (Sutter Instruments, Novato, CA) and fire-polished for a resistance of 0.8–1.2 megaohms when filled with internal solution. The pipette internal solution contained (in mM): 140 CsF, 10 NaCl, 1.1 EGTA, 10 HEPES, 20 Dextrose (pH 7.3 with CsOH, adjusted to 310 mOsm/l with dextrose). External bath solution contained (in mM): 140 NaCl, 20 TEA-Cl, 3 KCl, 1 CaCl₂, 1 MgCl₂, 10 HEPES, 5 Sucrose, 0.1 CdCl₂, 0.001 TTX (pH 7.3 with NaOH, adjusted to 320 mOsm/l with sucrose).

DRG neurons between 25 and 30 μM were selected due to the relatively high expression of Na_V1.8 in neurons of this size (Shields et al., 2012). Macroscopic currents were recorded in voltage-clamp mode using an EPC-10 amplifier and the PatchMaster Next program (HEKA Electronik, Lambrecht, Germany). Sodium currents were recorded in the whole-cell configuration. Cells with a leak current >200 pA were excluded. Series resistance compensation of 80–90% was applied to reduce voltage error. Cells were excluded if the voltage error exceeded 5 mV. Recordings were sampled at 50 kHz through a low-pass Bessel filter of 10 kHz. After achieving the whole-cell configuration, a 5-min delay was applied to allow adequate time for the pipette solution and cytoplasmic milieu to equilibrate.

As PTX-treated neurons were maintained in culture for over 48 h, extensive neurite outgrowth caused inadequate space clamp. To isolate somatic sodium currents from inadequately clamped axonal currents, a voltage pre-pulse protocol, as described by Milescu et al. (2010) was implemented. Briefly, neurons were held at a potential of −80 mV (to inactivate non-Na_V1.8 TTX-R channels). Then, patched cells were pre-pulsed to an individualized potential for 8 ms to inactivated axonal sodium currents. This prepulse potential was found to be the most negative voltage that could trigger an axonal spike without activating somatic channels – usually −35 to −25 mV. This was then followed by returning the cells to a hyperpolarized interpulse potential for 1 ms to allow for somatic but not axonal channel recovery. The interpulse potential was determined as the most negative potential that would not recover inadequately clamped sodium channels – usually between −100 and −120 mV. Finally, somatic sodium currents were triggered by subsequent 100 ms test pulses from −80 to +40 mV in 5 mV increments.

I-V relationships generated by the voltage protocol were fit according to the following equation:

Where I is the peak current for the test potential V, V_rev is the reversal potential, G_max is the maximum channel conductance, V_1/2 is the half-maximal activation potential, and k_G is the slope factor.

DRG neurons were cultured and treated with either 25 nM PTX or an equivalent volume of DMSO for 24 h as done for voltage-clamp recordings (above). Total RNA was then isolated according to the directions in the RNeasy Plus Mini Kit (Qiagen, Germantown, MD). Complementary DNA was produced using iScript™ Reverse Transcription Supermix (Bio-Rad, Hercules, CA), and one tenth of each reaction was used in 20 μl Bio-Rad PrimePCR™ probe assays for beta actin (qRnoCIP0050804) or Scn10a (qRnoCIP0027343) with SsoAdvanced supermix on the Bio-Rad CFX96 Touch System. Thermal cycling parameters included a 30s initial denaturation step at 95°C, followed by 40 cycles of 15 s at 95°C and 30s at 60°C. RNA quality was verified with the PrimePCR™ RNA Quality Probe Assay. Three independent dishes were processed in parallel for each treatment condition. Relative expression levels were derived from the ΔΔC_t method and normalizing to beta actin using Bio-Rad CFX Manager software.

We have previously shown that surface level of Na_V1.7 channels at the endings of DRG axons in culture is elevated by paclitaxel (PTX) treatment, which was paralleled by increased transport of anterogradely moving vesicles containing higher numbers of channels (Akin et al., 2021). We first used this methodology to determine if this was also true for Na_V1.8 channels, as suggested by its cotransport with Na_V1.7 channels (Higerd-Rusli et al., 2022). Indeed, increased levels of surface level of Na_V1.8 in distal axonal ends of DRG neurons were apparent after 24 h of treatment with either 25 nM or 125 nM PTX (Figure 1).

Increased levels of Na_V1.8 channels at the surface of axonal endings could arise from increased stability of channels at the plasma membrane or enhanced delivery of channels. The microfluidic chamber culture system provides an ideal method to distinguish between these possibilities by monitoring the traffic of channel-containing vesicles as they migrate in real time. The average velocity of Na_V1.8-containing vesicles, defined as the distance traveled over the duration of the imaging epoch, was significantly enhanced by PTX treatment (median (95% confidence interval): 0.79 (0.68–0.89) μm/s for control, 1.25 (1.12–1.40) for 25 nM, and 1.13 (1.04–1.22) for 125 nM; Figure 2A). The median vesicle fluorescence intensity at the 25 nM dose was greater than that of control cultures, suggesting increased loading of Na_V1.8 channels into vesicles (median (95% confidence interval): 164.7 (147.1–183.4) for control, 200.6 (190.5–210.1) for 25 nM PTX, and 179.0 (172.5–189.1) for 125 nM PTX; Figure 2B).

Because the average vesicular velocity measured over the entire imaging period does not reflect any irregular changes in vesicular movement, we also recorded the frequency and duration of vesicle pauses, both of which were decreased by PTX (Figure 3). Moreover, there were more vesicles detected under PTX treatment conditions in general. The number of vesicles traversing half of the imaged axon per minute, termed the vesicular flux, was significantly increased at the 125 nM dose (Figure 4). Therefore, PTX treatment not only increased the surface levels of Na_V1.8 channels through increased channels per vesicle, but through increased vesicles per axon and accelerated delivery with fewer and shorter pauses along the transport route.

To examine potential functional consequences of PTX treatment on Na_V1.8 surface levels in DRG neurons, we examined the Na_V1.8-dependent current density in DRG neuron cultures after treating cells for 24 h with 25 nM PTX. Endogenous Na_V1.8-dependent current density was unaffected by PTX treatment (Figure 5). This is consistent with the observation that expression of endogenous Na_V1.8-encoding mRNA by quantitative RT-PCR was 1.04 ± 0.26 (average ± s.d.) fold in PTX-treated cultures relative to 1.01 ± 0.13 in controls.