Journal: bioRxiv

Article Title: Distinct activation thresholds of unmyelinated C-fiber afferents by dorsal root ganglion and peripheral nerve stimulation

doi: 10.64898/2026.02.02.703367

Figure Lengend Snippet: Analyses of two prior single-cell RNA-seq studies of mouse DRG neurons reveal widespread expression of NaV1.6 across both A- and C-fiber afferents , . (A) Violin plots showing NaV1.6 (Scn8a) mRNA expression levels across distinct DRG neuronal populations. NF, neurofilament; NP, non-peptidergic; PEP, peptidergic; TH, tyrosine hydroxylase. (B) Violin plot showing NaV1.6 mRNA expression in colonic DRG neurons. RPM, reads per million; TPM, transcripts per million.

Article Snippet: Tissue sections were incubated with rabbit antibodies against NaV1.6 (1:1000, Alomone) and co-stained with rat antibodies against GFP (1:1000, MBL International Corp) for 24 h at 4 °C, and then incubated with a mixture of secondary antibodies Alexa Fluor® 594-conjugated anti-rabbit IgG (1:200, Abcam) and Alexa Fluor® 488-conjugated anti-rat IgG (1:200, Abcam) for 2 h at room temperature.

Techniques: Single Cell, RNA Sequencing, Expressing

Journal: bioRxiv

Article Title: Distinct activation thresholds of unmyelinated C-fiber afferents by dorsal root ganglion and peripheral nerve stimulation

doi: 10.64898/2026.02.02.703367

Figure Lengend Snippet: Both large- and small-diameter DRG neurons show clustering of NaV1.6 in stem axons near the somata. (A) Representative DRG sections co-stained for NaV1.6 (red) and afferents (green) showing NaV1.6 clusters (indicated by white arrow heads) in the stem axon of both large- and small-diameter neurons. Scale bar: 20 μm. (B) Distance between the somata and the first NaV1.6 clustering in the stem axon. There was no significant difference between the large- and small-diameter groups (t-test, p = 0.58).

Article Snippet: Tissue sections were incubated with rabbit antibodies against NaV1.6 (1:1000, Alomone) and co-stained with rat antibodies against GFP (1:1000, MBL International Corp) for 24 h at 4 °C, and then incubated with a mixture of secondary antibodies Alexa Fluor® 594-conjugated anti-rabbit IgG (1:200, Abcam) and Alexa Fluor® 488-conjugated anti-rat IgG (1:200, Abcam) for 2 h at room temperature.

Techniques: Staining

Journal: bioRxiv

Article Title: Distinct activation thresholds of unmyelinated C-fiber afferents by dorsal root ganglion and peripheral nerve stimulation

doi: 10.64898/2026.02.02.703367

Figure Lengend Snippet: NaV1.6 clustering is present in A-fiber axons but absent in C-fiber axons of the sciatic nerve. (A) Representative immunostaining of NaV1.6 in sciatic nerve sections with sparsely labeled afferents following intrathecal AAV9 injection. Arrowheads indicate the presence of NaV1.6 clusters in Aβ- (Φ > 4.5 μm) and Aδ-fiber axons (1.5 μm < Φ < 4.5 μm), but not in C-fiber axons (Φ < 1.5 μm). (B) NaV1.6 cluster density (clusters per 100 μm) is significantly higher in Aβ-fiber axons than in Aδ-fiber axons (mean ± SEM; t test, p = 0.003). Scale bar: 10 μm.

Article Snippet: Tissue sections were incubated with rabbit antibodies against NaV1.6 (1:1000, Alomone) and co-stained with rat antibodies against GFP (1:1000, MBL International Corp) for 24 h at 4 °C, and then incubated with a mixture of secondary antibodies Alexa Fluor® 594-conjugated anti-rabbit IgG (1:200, Abcam) and Alexa Fluor® 488-conjugated anti-rat IgG (1:200, Abcam) for 2 h at room temperature.

Techniques: Immunostaining, Labeling, Injection

Journal: bioRxiv

Article Title: Distinct activation thresholds of unmyelinated C-fiber afferents by dorsal root ganglion and peripheral nerve stimulation

doi: 10.64898/2026.02.02.703367

Figure Lengend Snippet: NaV1.6 clustering in afferent nerve endings in the bladder. (A-B) Immunostaining of bladder tissue sections from VGLUT2/tdTomato mice showed the presence of NaV1.6 clustering, in some bladder afferent axons (putative nerve terminals) as indicated by white arow heads. (C–D) Non-terminal axons of bladder afferents lack detectable NaV1.6 staining. Scale bar: 20 μm.

Article Snippet: Tissue sections were incubated with rabbit antibodies against NaV1.6 (1:1000, Alomone) and co-stained with rat antibodies against GFP (1:1000, MBL International Corp) for 24 h at 4 °C, and then incubated with a mixture of secondary antibodies Alexa Fluor® 594-conjugated anti-rabbit IgG (1:200, Abcam) and Alexa Fluor® 488-conjugated anti-rat IgG (1:200, Abcam) for 2 h at room temperature.

Techniques: Immunostaining, Staining

Journal: bioRxiv

Article Title: Distinct activation thresholds of unmyelinated C-fiber afferents by dorsal root ganglion and peripheral nerve stimulation

doi: 10.64898/2026.02.02.703367

Figure Lengend Snippet: NEURON simulation of ePNS and DRG stimulation of a C-fiber afferent. (A) Action potentials evoked by DRG stimulation initiates close to the soma and propagates bi-directionally to both peripheral and central axons. (B) Action potentials evoked by ePNS of the peripheral axon propagates to soma and central axon. (C) The clustering of NaV1.6 in the stem axon strongly decreased the threshold of DRG stimulation to evoke APs. In contrast, comparable increases in NaV1.8 conductance within the clustering zone had no effect on the threshold, whereas increases in NaV1.7 conductance produced only a modest reduction. (D) Spatial plot along the soma and stem axon to reveal the site of spike initiation at the NaV1.6 clustering zone. Five spatial membrane voltage traces have 0.05 msec intervals (from 1 to 5).

Article Snippet: Tissue sections were incubated with rabbit antibodies against NaV1.6 (1:1000, Alomone) and co-stained with rat antibodies against GFP (1:1000, MBL International Corp) for 24 h at 4 °C, and then incubated with a mixture of secondary antibodies Alexa Fluor® 594-conjugated anti-rabbit IgG (1:200, Abcam) and Alexa Fluor® 488-conjugated anti-rat IgG (1:200, Abcam) for 2 h at room temperature.

Techniques: Produced, Membrane

Journal: The FASEB Journal

Article Title: Human Dorsal Root Ganglia Neuronal Cell Line to Study Nociceptive Signaling: A New Pipeline for Pain Therapy

doi: 10.1096/fj.202503698R

Figure Lengend Snippet: Key nociceptive ion channel abundance, localization, and activity in HD10.6 cells. (A, B) D0 and D10 HD10.6 cells were fixed and stained for DAPI (blue), NF200 (green), and nociceptive ion channel TRPV1 (A) or NaV1.7 (B) in red. Representative images shown from three independent experiments. Scale bar 50 μm; filled arrows indicate examples of signal at soma whereas empty arrows indicate signal within axons. (C–F) Calcium influx in response to buffer only (BUF., blue) as well as during and following 10 μM TRPV1 agonist capsaicin (CAP., C, D, orange) or 1 μM NaV1.7 agonist OD1 (E, F, orange) was recorded from differentiated HD10.6 cells with Ca 2+ indicator Fluo‐4 AM (5 μM). Buffer was applied for 2 min and followed by the application of capsaicin or OD1 for 30 s. After up to 4 additional minutes of buffer, 50 mM KCl was applied for 5 s at the termination of the experiment to verify cell viability. Representative traces in response to respective stimuli shown (C,E), as well as the maximal normalized intensity (D,F). Replicates from three independent experiments. Statistical comparisons were made using Wilcoxon signed‐rank test due to non‐normality. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: NaV1.7 , Alomone Labs ASC‐008 , 1:100.

Techniques: Activity Assay, Staining

Journal: The FASEB Journal

Article Title: Human Dorsal Root Ganglia Neuronal Cell Line to Study Nociceptive Signaling: A New Pipeline for Pain Therapy

doi: 10.1096/fj.202503698R

Figure Lengend Snippet: Culture of HD10.6 cells into microfluidic devices to study peripheral administration. (A, B) D0 HD10.6 cells were seeded into the soma compartment of a microfluidic chamber, and media was changed to differentiation media 24 h post‐seeding. A 2X nerve growth factor (NGF) gradient was used to guide axons towards the axon‐only compartment. At D14, the cultures within the chamber were fixed and stained for neuronal marker β3 tubulin (magenta), marker for NaV1.7 PTX‐488 (green), and DAPI (blue). Empty arrows point to axons traveling through microgrooves. (A) captures a representative image of the soma compartment and axons within the proximal end of the microgrooves, whereas (B) shows the axon‐only compartment and distal microgrooves. Scale bar 50 μm. Respective compartments are highlighted in green within the right‐hand animated microfluidic chambers; images of microfluidic chambers created with BioRender.com . (C, D) D10 HD10.6 cells in 24‐well cell culture wells were infected with AAV9‐mCherry. 3 days following infection, cells were fixed and stained for β3 tubulin (green), mCherry (red), and DAPI (blue) The percentage of mCherry+ HD10.6 cells based on the total number of cells identified by DAPI and β3 tubulin‐positive staining is demonstrated in (C). A representative image of AAV9‐mCherry‐treated (1E9, top) and untreated (bottom) HD10.6 cells at this time‐point is shown in (D). (E) 3 days following AAV9‐mCherry infection in the peripheral compartment of HD10.6 cells cultured in microfluidic chambers, the cultures were fixed and stained for β3 tubulin (green), mCherry (red), and DAPI (blue). Representative image of soma (top) and peripheral terminal compartments (bottom) demonstrate mCherry signal within the soma and axons of HD10.6 cells. Replicates from three independent experiments; statistical comparisons were made using an unpaired non‐normal t ‐test. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: NaV1.7 , Alomone Labs ASC‐008 , 1:100.

Techniques: Staining, Marker, Cell Culture, Infection