Journal: Journal of Pain Research

Article Title: α-lipoic acid suppresses neuronal excitability and attenuates colonic hypersensitivity to colorectal distention in diabetic rats

doi: 10.2147/JPR.S135017

Figure Lengend Snippet: ALA downregulated NaV1.7 and NaV1.8 expression. Notes: (A) Western blots for NaV1.7 of T13-L2 DRGs from NS- and ALA-treatment rats. Bar graph showed mean density relative to GAPHD for NaV1.7. ALA treatment greatly reduced expressions of NaV1.7 (n=4 for each group, ** p <0.01, compared with NS, two-sample t -test). (B) Western blots for NaV1.8 of T13-L2 DRGs from NS- and ALA-treatment rats. Bar graph showed mean density relative to β-actin for NaV1.8. ALA treatment greatly reduced expressions of NaV1.8 (n=5 for NS group, n=4 for ALA group, * p <0.05, compared with NS, two-sample t -test). Abbreviations: ALA, α-lipoic acid; DRG, dorsal root ganglion; NS, normal saline.

Article Snippet: The primary antibodies used to probe the target proteins included rabbit anti-NaV1.7 or anti-NaV1.8 (1:200, Alomone Labs, Jerusalem, Israel), rabbit anti-GAPDH (1:1000, Biotechnology Co., CHN), and mouse anti-actin (1:1000; Chemicon, Temecula, CA, USA).

Techniques: Expressing, Western Blot

Journal: Brain

Article Title: Integrative miRNA–mRNA profiling of human epidermis: unique signature of SCN9A painful neuropathy

doi: 10.1093/brain/awad025

Figure Lengend Snippet: Downregulated miRNA in epidermis of painful SCN9A-related neuropathy patients (NavNP). Microfluidic analysis of miRNA profiling in total RNA extracted from the epidermis of 11 NavNP patients and 7 healthy controls (HC) demonstrated a significant reduction of miR-30d-5p ( P -value 3.23 × 10 −4 , FC −5.83), miR-30a-5p [ P -value 4.40 × 10 −4 , fold change (FC) −4.95], miR-203a-3p ( P -value 4.40 × 10 −4 , FC −3.64), miR-181a-2-3p ( P -value 4.40 × 10 −4 , FC −2.21) expression in NavNP patients compared to HC. Bar graph indicates the 2 −(ΔΔCq) . The comparisons are made applying Wilcoxon rank sum test and corrected for Bonferroni multiple test. The fold change in expression was calculated as 2 −(ΔΔCq) . We provide the fold change reduction in expression in the NavNP group compared to HC applying the negative inverse of 2 −(ΔΔCq) .

Article Snippet: Primary antibody against Nav1.7 (Alomone labs, cat.no.

Techniques: Expressing

Journal: Brain

Article Title: Integrative miRNA–mRNA profiling of human epidermis: unique signature of SCN9A painful neuropathy

doi: 10.1093/brain/awad025

Figure Lengend Snippet: miR-30 family regulates Nav1.7 signalling in keratinocytes. ( A ) Representative confocal microscope image of epidermis. Nav1.7 (green) in keratinocytes of NavNP patients and HC. Scale bar = 20 µm. ( B ) Boxplot of mean Nav1.7 immunofluorescence intensity for two studied groups, NavNP patients and HC, respectively. NavNP patients show significantly higher Nav1.7 signal intensity than HC ( P = 8.798 × 10 −5 ). *** P < 0.001 according to Wilcoxon rank sum test. ( C ) Scatter correlation plot between mean Nav1.7 immunofluorescence intensity and NEDD4 expression values in NavNP patients and HC samples. ( D and E ) Scatter correlation plot between mean Nav1.7 immunofluorescence intensity and ( D ) miR-30a-5p and miR-30d-5p ( E ) expression values in NavNP patients and HC samples. Spearman coefficient and P -value are shown in the graph.

Article Snippet: Primary antibody against Nav1.7 (Alomone labs, cat.no.

Techniques: Microscopy, Immunofluorescence, Expressing

Journal: PLoS ONE

Article Title: Antisense-Mediated Knockdown of Na V 1.8, but Not Na V 1.9, Generates Inhibitory Effects on Complete Freund's Adjuvant-Induced Inflammatory Pain in Rat

doi: 10.1371/journal.pone.0019865

Figure Lengend Snippet: (A) Representative RT-PCR results of five isoforms of voltage-gated sodium channels. Amplicons of Na V 1.1, Na V 1.6, Na V 1.7, Na V 1.8, Na V 1.9 and ß-actin were 540 bp, 509 bp, 441 bp, 515 bp, 572 bp and 229 bp, respectively. (B) Averaged fold changes of mRNA expression as normalized with naive control (n = 3). (C) Double immunofluorescent labeling of DRG neurons by anti-Na V 1.8 (red) and anti-NF-200 (green) or anti-Na V 1.9 (red) and anti-NF-200 (green) antibodies. (D) The percentage of Nav1.8- and Nav1.9-positive profiles as a proportion of the total number of DRG neurons before and after CFA treatment (n = 8). (E) Western blotting examples of Na V 1.8 and Na V 1.9 in naive and CFA-treated DRG neurons. (F) Averaged protein expression of Na V 1.8 and Na V 1.9 between naive and CFA-treated DRGs (normalized with the internal control tubulin) (n = 3 for each group). CFA, complete Freund's adjuvant. * p<0.05, **p<0.01, ***p<0.001.

Article Snippet: The primary antibodies were mouse anti-Neurofilament 200 monoclonal antibody (NF-200, 1∶200, Sigma, USA), rabbit anti-rat Nav1.8 and Nav1.9 antibodies (1∶200, Alomone, Israel).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Labeling, Western Blot

Journal: Journal of Experimental Pharmacology

Article Title: Antihyperalgesic effects of ProTx-II, a Nav1.7 antagonist, and A803467, a Nav1.8 antagonist, in diabetic mice

doi: 10.2147/JEP.S79973

Figure Lengend Snippet: The protein levels of Nav1.7 ( A ) and Nav1.8 ( B ) in the dorsal root ganglion of nondiabetic and diabetic mice. Notes: Immunoblots are representative of the results for Nav1.7 and Nav1.8. The immunoblots of Nav1.7 and Nav1.8 were normalized by GAPDH. Each column represents the mean with standard error of eight mice.

Article Snippet: The membrane was immunoblotted overnight at 4°C with rabbit antibodies against Nav1.7 (1:500; Alomone Labs, Jerusalem, Israel) and Nav1.8 (1:500; Sigma-Aldrich Co, St Louis, MO, USA).

Techniques: Western Blot

Journal: Molecular Pain

Article Title: Extracellular signal-regulated kinase phosphorylation enhancement and Na V 1.7 sodium channel upregulation in rat dorsal root ganglia neurons contribute to resiniferatoxin-induced neuropathic pain: The efficacy and mechanism of pulsed radiofrequency therapy

doi: 10.1177/17448069221089784

Figure Lengend Snippet: Upregulation of Na V 1.7 expression by RTX in cultured rat DRG neurons. Cells were treated without or with RTX (1 nM–1 μM) for 1 week in a culture medium. The cell lysates were subjected to western blot analysis using antibodies against Na V 1.7. (A) Representative western blot showing the RTX concentration-dependent upregulation of Na V 1.7. Blots shown are typically obtained from five independent experiments with similar results. (B) A quantitative densitometric analysis of Na V 1.7 expression ratio. Immunoreactivities were quantified using an immunoimage analyzer. Na V 1.7 levels were normalized to β-actin levels at each incubation time. Data of five experiments are expressed as mean ± standard deviation. DRG, dorsal root ganglia; RTX, resiniferatoxin.

Article Snippet: The membrane was subsequently incubated with a blocking solution (2% BSA in Tween-Tris-buffered saline [10 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.1% Tween-20]) and further incubated overnight at 4°C in 2% BSA with rabbit anti-Na V 1.7 polyclonal antibody (1:1000, ASC-008, Alomone Labs, Jerusalem, Israel) or Can Get Signal Solution-1 (TOYOBO, Osaka, Japan) with mouse anti-β-actin monoclonal antibody (1:5000, A5441, Sigma-Aldrich, St Louis, MO, USA).

Techniques: Expressing, Cell Culture, Western Blot, Concentration Assay, Incubation, Standard Deviation

Journal: Molecular Pain

Article Title: Extracellular signal-regulated kinase phosphorylation enhancement and Na V 1.7 sodium channel upregulation in rat dorsal root ganglia neurons contribute to resiniferatoxin-induced neuropathic pain: The efficacy and mechanism of pulsed radiofrequency therapy

doi: 10.1177/17448069221089784

Figure Lengend Snippet: Inhibition of RTX-induced Na V 1.7 expression upregulation using early PRF treatment in rat DRG, 5 weeks after RTX treatment. RT-PCR (A) and representative western blot (C) showing that the mRNA and protein levels of Na V 1.7 were upregulated by RTX; this Na V 1.7 upregulation was inhibited by PRF treatment. Samples were harvested 5 weeks after RTX or vehicle treatment (4 weeks after PRF in the RTX + early PRF group). This experimental design is shown in Fig. 2A. Quantitative densitometric analyses of Na V 1.7 expression ratio for RT-PCR (B) and western blot (D). Data are expressed as mean ± standard deviation, n = 6 samples (one animal per sample). RTX, resiniferatoxin; PRF, pulsed radiofrequency; RT-PCR, reverse transcription polymerase chain reaction; DRG, dorsal root ganglia.

Article Snippet: The membrane was subsequently incubated with a blocking solution (2% BSA in Tween-Tris-buffered saline [10 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.1% Tween-20]) and further incubated overnight at 4°C in 2% BSA with rabbit anti-Na V 1.7 polyclonal antibody (1:1000, ASC-008, Alomone Labs, Jerusalem, Israel) or Can Get Signal Solution-1 (TOYOBO, Osaka, Japan) with mouse anti-β-actin monoclonal antibody (1:5000, A5441, Sigma-Aldrich, St Louis, MO, USA).

Techniques: Inhibition, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Standard Deviation

Journal: Molecular Pain

Article Title: Extracellular signal-regulated kinase phosphorylation enhancement and Na V 1.7 sodium channel upregulation in rat dorsal root ganglia neurons contribute to resiniferatoxin-induced neuropathic pain: The efficacy and mechanism of pulsed radiofrequency therapy

doi: 10.1177/17448069221089784

Figure Lengend Snippet: Inhibition of RTX-induced Na V 1.7 expression upregulation by early tramadol treatment with late PRF therapy in rat DRG. RT-PCR (A) and representative western blot (C) showing that protein and mRNA Na V 1.7 levels were upregulated by RTX treatment; Na V 1.7 upregulation was inhibited by late PRF therapy with early tramadol administration. Samples were harvested at 9 weeks after RTX or vehicle treatment (4 weeks after PRF in the RTX + Tramadol + late PRF group). This experimental design is shown in . Quantitative densitometric analyses of the Na V 1.7 expression ratio for RT-PCR (B) and western blot (D). Data of six rats are expressed as mean ± SD. RTX, resiniferatoxin; PRF, pulsed radiofrequency; DRG, dorsal root ganglia; RT-PCR, reverse transcription polymerase chain reaction.

Article Snippet: The membrane was subsequently incubated with a blocking solution (2% BSA in Tween-Tris-buffered saline [10 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.1% Tween-20]) and further incubated overnight at 4°C in 2% BSA with rabbit anti-Na V 1.7 polyclonal antibody (1:1000, ASC-008, Alomone Labs, Jerusalem, Israel) or Can Get Signal Solution-1 (TOYOBO, Osaka, Japan) with mouse anti-β-actin monoclonal antibody (1:5000, A5441, Sigma-Aldrich, St Louis, MO, USA).

Techniques: Inhibition, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Molecular Pain

Article Title: Extracellular signal-regulated kinase phosphorylation enhancement and Na V 1.7 sodium channel upregulation in rat dorsal root ganglia neurons contribute to resiniferatoxin-induced neuropathic pain: The efficacy and mechanism of pulsed radiofrequency therapy

doi: 10.1177/17448069221089784

Figure Lengend Snippet: Development of RTX-induced neuropathic pain: a mechanism involving ERK and Na V 1.7 in DRG. (A) Schematic diagram of RTX-induced neuropathic pain pathophysiology. Binding of RTX to TRPV1 enhances ERK phosphorylation in DRG neurons, which may result in Na V 1.7 mRNA upregulation in the nucleus and Na V 1.7 expression upregulation. Enhanced ERK phosphorylation level returns to a steady state after RTX administration, whereas Na V 1.7 expression upregulation persists for several weeks, leading to a sustained painful condition. (B) In the early phase (days), PRF reduces pain by suppressing the RTX-induced enhancement of ERK phosphorylation and Na V 1.7 expression upregulation. (C) In the late phase (weeks), the enhanced ERK phosphorylation returns to a steady state level of phosphorylation. PRF therapy cannot suppress the RTX-induced pain, because it may not suppress Na V 1.7 expression upregulation. RTX, resiniferatoxin; PRF, pulsed radiofrequency; DRG, dorsal root ganglia; ERK, extracellular signal-regulated kinase; TRPV1, transient receptor potential vanilloid 1.

Article Snippet: The membrane was subsequently incubated with a blocking solution (2% BSA in Tween-Tris-buffered saline [10 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.1% Tween-20]) and further incubated overnight at 4°C in 2% BSA with rabbit anti-Na V 1.7 polyclonal antibody (1:1000, ASC-008, Alomone Labs, Jerusalem, Israel) or Can Get Signal Solution-1 (TOYOBO, Osaka, Japan) with mouse anti-β-actin monoclonal antibody (1:5000, A5441, Sigma-Aldrich, St Louis, MO, USA).

Techniques: Binding Assay, Expressing

Journal: bioRxiv

Article Title: Schwann cells promote sensory neuron excitability during development

doi: 10.1101/2022.10.31.514415

Figure Lengend Snippet: ( Caption continued on next page .) ( A ) Images show Neurofilament and DAPI staining in immunopanned DRG neurons as described in or unpanned DRG neurons plated after dissociation without the panning steps. Even with the use of the mitotic inhibitor FUDR, unpanned DRG neuron cultures had significant contamination with non-neural cells, negatively stained for Neurofilament. ( B ) Untreated and SCCM treated DRG neurons exhibited statistically similar resting membrane potentials. n = 29 total cells from 5–7 distinct biological replicates per treatment group. ( C ) Bright field images indicate that DRG neuronal health is unaffected by Act-D treatment, consistent with prior studies. ( D ) The addition of Schwann cell growth media to DRG neurons was insufficient to increase expression of Na V 1.7 and Na V 1.8 transcripts, suggesting the effects of SCCM are specific to a Schwann cell-secreted molecule(s). Gray circles, mRNA number of an individual DRG neuron for the indicated genes; colored circles, the average # of mRNAs per cell in each biological replicate. Mean ± SEM is shown for the biological replicates (not significantly different in a paired t-test). n = 2 distinct biological replicates per group. ( E ) Immunopanned DRG neurons showed normal cell growth and neurofilament staining, all unaffected by SCCM treatment.

Article Snippet: Antibodies used in this study include the following: Neurofilament (Sigma-Aldrich, N4142, 1:1000), goat anti-rabbit IgG polyclonal antibody (CF™ 405M, 20181, 1:1000), Ptges3 (knockout validated, Origene technologies, TA803433, 1:1000), Mbp (knockout validated , Abcam, ab7349, 1:100), Na v 1.8 (knockout validated, Neuromab, SKU 75-166, 1:1000 for cultured cells, 1:200 for tissues [ ]), Na v 1.7 (knockout validated , Neuromab, SKU 75-103, 1:200 for tissues [ ]), Na v 1.1 (knockout validated , Alomone Labs, Asc-001, 1:1000), Na v 1.2 (knockout validated , Alomone Labs, Asc-002, 1:1000), Na v 1.6 (knockout validated , Alomone Labs, Asc-009, 1:1000), Na v 1.7 (knockout validated , Alomone Labs, Asc-008, 1:1000 for cultured cells, 1:200 for tissues [ ]), Na v 1.8 (knockout validated , Alomone Labs, ASC-016, 1:200 for tissues [ ]), Na v 1.5, knockout validated , Alomone Labs, ASC-005, 1:1000), Na v 1.9, Alomone Labs, AGP-030, 1:1000), and highly cross-absorbed Alexa Fluor 488-, 594-, or 647-labeled secondary antibodies (Thermo Fisher).

Techniques: Staining, Expressing

Journal: bioRxiv

Article Title: Schwann cells promote sensory neuron excitability during development

doi: 10.1101/2022.10.31.514415

Figure Lengend Snippet: ( Caption continued on next page .) ( A ) DESI-MS analysis of SCCM. The expected m/z of PGE 2 is 351.21770 and a m/z of 351.21824 ± 1.6 ppm was detected in the SCCM sample. ( B) SCCM and PGE 2 treatments enhanced Na V 1.6, Na v 1.7 and Na v 1.8 protein levels in DRG neurons. n = 80 to 328 cells from 1-4 distinct biological replicates per treatment group. Mean ± SEM is shown for cells. Untreated condition is replotted from , as these experiments were done concurrently. ( C ) SCCM and PGE 2 treatments did not cause a major change in protein expression of indicated genes, except a significant change in Na v 1.5 protein levels. Fluorescence levels were quantified using cellpose. Gray circles, fluorescent intensity of an individual DRG neuron for the indicated genes. n = 70 to 331 cells from 2-4 distinct biological replicates per treatment group. Mean ± SEM is shown for cells. p values compare cells in a one-way ANOVA and Tukey test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: Antibodies used in this study include the following: Neurofilament (Sigma-Aldrich, N4142, 1:1000), goat anti-rabbit IgG polyclonal antibody (CF™ 405M, 20181, 1:1000), Ptges3 (knockout validated, Origene technologies, TA803433, 1:1000), Mbp (knockout validated , Abcam, ab7349, 1:100), Na v 1.8 (knockout validated, Neuromab, SKU 75-166, 1:1000 for cultured cells, 1:200 for tissues [ ]), Na v 1.7 (knockout validated , Neuromab, SKU 75-103, 1:200 for tissues [ ]), Na v 1.1 (knockout validated , Alomone Labs, Asc-001, 1:1000), Na v 1.2 (knockout validated , Alomone Labs, Asc-002, 1:1000), Na v 1.6 (knockout validated , Alomone Labs, Asc-009, 1:1000), Na v 1.7 (knockout validated , Alomone Labs, Asc-008, 1:1000 for cultured cells, 1:200 for tissues [ ]), Na v 1.8 (knockout validated , Alomone Labs, ASC-016, 1:200 for tissues [ ]), Na v 1.5, knockout validated , Alomone Labs, ASC-005, 1:1000), Na v 1.9, Alomone Labs, AGP-030, 1:1000), and highly cross-absorbed Alexa Fluor 488-, 594-, or 647-labeled secondary antibodies (Thermo Fisher).

Techniques: Expressing, Fluorescence

Journal: bioRxiv

Article Title: Schwann cells promote sensory neuron excitability during development

doi: 10.1101/2022.10.31.514415

Figure Lengend Snippet: ( A ) Cartoon depicts size fractionation and mass spectrometry steps that identified PGE 2 as the excitability-inducing molecule in SCCM. ( B ) PGE 2 treatment (1 μM, 16-28 hr) increased Na V expression in DRG neurons, similar to SCCM. n = 6 distinct biological replicates per treatment group. Bottom images: Incubation with neutralizing PGE 2 antibody or PGE 2 receptor (EP1-EP4) antagonists blocked the SCCM-induced transcriptional increase in Na V s (see also representative images for DMSO-treated DRG neurons in ). n = 4-6 distinct biological replicates per treatment group. ( C) PGE 2 treatment enhanced Na V protein levels in DRG neurons. Images show immunohistochemistry for indicated genes in single representative DRG neurons that were either untreated (top) or treated with PGE 2 overnight (bottom) (see also quantification in ). ( D and E ) Quantification of the RNAscope results shown in (B), using FishQuant. Controls were either untreated cells or cells treated with DMSO vehicle (solvent for PGE 2 ). Mean ± SEM is shown for biological replicates. p values compare biological replicates in a paired t-test (D) or mixed-effect analysis (E). ( F and G ) DRG neurons treated with PGE 2 (1 μM, 16-28 hr) fired significantly more action potentials at suprathreshold current injections and exhibited a decrease in the firing threshold, similar to SCCM. Incubation with neutralizing PGE 2 antibody or PGE 2 receptor (EP1–EP4) antagonists blocked the excitability-inducing effect of SCCM. DRG neurons treated with 1 μM PGD 2 , a constitutional isomer of PGE 2 , remained hypoexcitable (see also ). Mean ± SEM is shown for cells, p values compare cells in a one-way ANOVA and Tukey test. n = 20-29 total cells from 3–7 biological replicates. Untreated and SCCM conditions are replotted from and , as these experiments were done concurrently. ( H-I ) Injection of dmPGE 2 into the P0 sciatic nerve increased Na V 1.7 and Na V 1.8 transcript levels in lumbar DRG neurons compared to controls (the third fluorescence channel was reserved for Neun ; see ). Results were quantified using FishQuant (I). Gray circles, Na V mRNA count in a single DRG neuron; colored circles, the average of cells in each biological replicate. Mean ± SEM is shown for biological replicates. p values compare biological replicates in cells in a one-way ANOVA and Tukey test. n = 3 mice per treatment group (at least 45 DRG neurons per mouse). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: Antibodies used in this study include the following: Neurofilament (Sigma-Aldrich, N4142, 1:1000), goat anti-rabbit IgG polyclonal antibody (CF™ 405M, 20181, 1:1000), Ptges3 (knockout validated, Origene technologies, TA803433, 1:1000), Mbp (knockout validated , Abcam, ab7349, 1:100), Na v 1.8 (knockout validated, Neuromab, SKU 75-166, 1:1000 for cultured cells, 1:200 for tissues [ ]), Na v 1.7 (knockout validated , Neuromab, SKU 75-103, 1:200 for tissues [ ]), Na v 1.1 (knockout validated , Alomone Labs, Asc-001, 1:1000), Na v 1.2 (knockout validated , Alomone Labs, Asc-002, 1:1000), Na v 1.6 (knockout validated , Alomone Labs, Asc-009, 1:1000), Na v 1.7 (knockout validated , Alomone Labs, Asc-008, 1:1000 for cultured cells, 1:200 for tissues [ ]), Na v 1.8 (knockout validated , Alomone Labs, ASC-016, 1:200 for tissues [ ]), Na v 1.5, knockout validated , Alomone Labs, ASC-005, 1:1000), Na v 1.9, Alomone Labs, AGP-030, 1:1000), and highly cross-absorbed Alexa Fluor 488-, 594-, or 647-labeled secondary antibodies (Thermo Fisher).

Techniques: Fractionation, Mass Spectrometry, Expressing, Incubation, Immunohistochemistry, Injection, Fluorescence

Journal: bioRxiv

Article Title: Schwann cells promote sensory neuron excitability during development

doi: 10.1101/2022.10.31.514415

Figure Lengend Snippet: ( Caption continued on next page .) ( A to D ) RNAscope shows expression of Na V 1.6 , Na V 1.7 and Na V 1.8 transcripts in DRG neurons treated with SCCM from Flox ( Ptges3 fl/fl ) or cKO ( Ptges3 fl/fl ;Dhh CRE/+ ) mice. Micrographs of SCCM treatment from cHET ( Ptges3 fl/+ ;Dhh CRE/+ ), Dhh CRE/+ and WT mice are not shown but are also quantified in ( E to G ). ( E to G ) Conditioned media from Ptges3 conditional knockout Schwann cells (cKO) failed to enhance neuronal Na V 1.8 expression, contrary to Schwann cells collected from controls (Flox, Dhh CRE/+ , or WT). We detected a slight but significant change in Na V 1.6 and Na V 1.7 expression in DRG neurons following the addition of cKO media; however, this effect was lower than the fold increase observed with WT, Flox, or Dhh CRE/+ SCCM addition. The addition of PGE 2 to cKO media rescued the loss of Na v expression-inducing effect in cKO SCCM. Gray circles, mRNA number of an individual DRG neuron for the indicated genes. Mean ± SEM of all cells; p values compare cells in a one-way ANOVA and Tukey test. Significant increases in comparison to untreated DRG neurons or between groups indicated by brackets are shown. n = 61–195 DRG neurons from 2–4 biological replicates per group. p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: Antibodies used in this study include the following: Neurofilament (Sigma-Aldrich, N4142, 1:1000), goat anti-rabbit IgG polyclonal antibody (CF™ 405M, 20181, 1:1000), Ptges3 (knockout validated, Origene technologies, TA803433, 1:1000), Mbp (knockout validated , Abcam, ab7349, 1:100), Na v 1.8 (knockout validated, Neuromab, SKU 75-166, 1:1000 for cultured cells, 1:200 for tissues [ ]), Na v 1.7 (knockout validated , Neuromab, SKU 75-103, 1:200 for tissues [ ]), Na v 1.1 (knockout validated , Alomone Labs, Asc-001, 1:1000), Na v 1.2 (knockout validated , Alomone Labs, Asc-002, 1:1000), Na v 1.6 (knockout validated , Alomone Labs, Asc-009, 1:1000), Na v 1.7 (knockout validated , Alomone Labs, Asc-008, 1:1000 for cultured cells, 1:200 for tissues [ ]), Na v 1.8 (knockout validated , Alomone Labs, ASC-016, 1:200 for tissues [ ]), Na v 1.5, knockout validated , Alomone Labs, ASC-005, 1:1000), Na v 1.9, Alomone Labs, AGP-030, 1:1000), and highly cross-absorbed Alexa Fluor 488-, 594-, or 647-labeled secondary antibodies (Thermo Fisher).

Techniques: Expressing, Knock-Out

Journal: bioRxiv

Article Title: Schwann cells promote sensory neuron excitability during development

doi: 10.1101/2022.10.31.514415

Figure Lengend Snippet: ( A to H ) RNAscope and immunostaining of NaV expression in vivo in Ptges3-Flox or Ptges3-cKO mice. Left panels: RNAscope images show expression of Na V 1.7 and Na V 1.8 transcripts in lumbar DRG neurons at P0 (A) and P28 (E) (see also , , and for E16 time point and supporting data). Number of Na V 1.7 and Na V 1.8 mRNAs per cell quantified using FishQuant from P0 (B) and P28 (D). n = 4-9 mice per group. Right panels: immunohistochemistry for Na V 1.7 and Na V 1.8 in lumbar DRG neurons at P0 (C) and P28 (G). Cellular fluorescence was quantified using Cellpose. Gray circles, mRNA count or fluorescence intensity in a DRG neuron; pink circles, the average of cells in each mouse. n = 2-5 mice per group. Mean ± SEM is shown for biological replicates (mice). p values compare biological replicates in Mann-Whitney test (B and D) and an unpaired t-test (F and H). ( I to K ) Calcium imaging of acutely purified DRG neurons from Ptges3-Flox or Ptges3-cKO mice. Images show fluorescent intensity of the calcium indicator (Fluo-4,AM) in DRG neurons acutely isolated (within 2 hours of purification) from Ptges3-Flox (top) or Ptges3-cKO (bottom) mice at baseline, during the addition of VTD and 20 seconds (s) after VTD addition (I). Representative neuron traces (K) and the maximum difference between the florescence after stimulation and during baseline (max delta F/F) (panel L) are shown. n = 3 distinct biological replicates (mice) per group, 8-26 cells per mouse. Mean ± SEM is shown for biological replicates. p values compare biological replicates in a Mann-Whitney test (B, D, and F) or an unpaired t-test (H). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: Antibodies used in this study include the following: Neurofilament (Sigma-Aldrich, N4142, 1:1000), goat anti-rabbit IgG polyclonal antibody (CF™ 405M, 20181, 1:1000), Ptges3 (knockout validated, Origene technologies, TA803433, 1:1000), Mbp (knockout validated , Abcam, ab7349, 1:100), Na v 1.8 (knockout validated, Neuromab, SKU 75-166, 1:1000 for cultured cells, 1:200 for tissues [ ]), Na v 1.7 (knockout validated , Neuromab, SKU 75-103, 1:200 for tissues [ ]), Na v 1.1 (knockout validated , Alomone Labs, Asc-001, 1:1000), Na v 1.2 (knockout validated , Alomone Labs, Asc-002, 1:1000), Na v 1.6 (knockout validated , Alomone Labs, Asc-009, 1:1000), Na v 1.7 (knockout validated , Alomone Labs, Asc-008, 1:1000 for cultured cells, 1:200 for tissues [ ]), Na v 1.8 (knockout validated , Alomone Labs, ASC-016, 1:200 for tissues [ ]), Na v 1.5, knockout validated , Alomone Labs, ASC-005, 1:1000), Na v 1.9, Alomone Labs, AGP-030, 1:1000), and highly cross-absorbed Alexa Fluor 488-, 594-, or 647-labeled secondary antibodies (Thermo Fisher).

Techniques: Immunostaining, Expressing, In Vivo, Immunohistochemistry, Fluorescence, MANN-WHITNEY, Imaging, Purification, Isolation

Journal: bioRxiv

Article Title: Schwann cells promote sensory neuron excitability during development

doi: 10.1101/2022.10.31.514415

Figure Lengend Snippet: ( A ) UMAP visualization of DRG scRNA-seq data in Ptges3-Flox mice at P4. ( B ) Cell identity composition heatmaps of CGRP and proprioceptor DRG neuron subtype populations. Dark red indicates high relative cell density. ( C ) Number of CGRP and proprioceptor DRG neurons are reduced by half in Ptges3-cKO mice. ( D to G ) RNAscope shows expression of Neun, Pvalb, or Calca , and Na V 1.8 transcripts in lumbar DRG neurons at P0 to label proprioceptor or CGRP neurons, respectively. Pvalb + or Calca + neuron numbers are normalized to Neun + cells to calculate the percentage of proprioceptor or CGRP DRG neurons respectively. n = 3-5 mice, p values compare biological replicates in an unpaired t-test. ( H and I ) Bar plots indicate expression of Na V 1.7 and Na V 1.8 in CGRP and proprioceptor DRG subpopulations, from scRNA-seq data. p values compare cells in an unpaired t-test. ( J and K ) Hot plate and Hargreaves tests indicate that paw withdrawal latencies in response to noxious heat was significantly lengthened in Ptges3-cKO mice. n = 14, 20 mice (hot plate); 32, 17 mice (Hargreaves). Control littermates in (K) were either Ptges3 fl/fl or Ptges3 fl/+ mice. ( L ) Pain response following 1% PFA injection into the hind paw indicated the typical biphasic response. The first phase (acute pain) was unaffected, but the second phase (inflammatory pain) was reduced by ~40% in Ptges3-cKO mice. n = 9-11 mice (also see ). ( M ) Rotarod assay (32 RPM, constant speed) indicates decreased fall latency in Ptges3-cKO mice. n = 18, 17 mice. ( N ) Precise foot placement is severely impacted in Ptges3-cKO mice recorded in the horizontal ladder test with unevenly placed rungs. n = 16 mice for each group. p values compare biological replicates (triangles-females, circles-males) in an unpaired t-test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: Antibodies used in this study include the following: Neurofilament (Sigma-Aldrich, N4142, 1:1000), goat anti-rabbit IgG polyclonal antibody (CF™ 405M, 20181, 1:1000), Ptges3 (knockout validated, Origene technologies, TA803433, 1:1000), Mbp (knockout validated , Abcam, ab7349, 1:100), Na v 1.8 (knockout validated, Neuromab, SKU 75-166, 1:1000 for cultured cells, 1:200 for tissues [ ]), Na v 1.7 (knockout validated , Neuromab, SKU 75-103, 1:200 for tissues [ ]), Na v 1.1 (knockout validated , Alomone Labs, Asc-001, 1:1000), Na v 1.2 (knockout validated , Alomone Labs, Asc-002, 1:1000), Na v 1.6 (knockout validated , Alomone Labs, Asc-009, 1:1000), Na v 1.7 (knockout validated , Alomone Labs, Asc-008, 1:1000 for cultured cells, 1:200 for tissues [ ]), Na v 1.8 (knockout validated , Alomone Labs, ASC-016, 1:200 for tissues [ ]), Na v 1.5, knockout validated , Alomone Labs, ASC-005, 1:1000), Na v 1.9, Alomone Labs, AGP-030, 1:1000), and highly cross-absorbed Alexa Fluor 488-, 594-, or 647-labeled secondary antibodies (Thermo Fisher).

Techniques: Expressing, Injection

Journal: PLoS ONE

Article Title: Selective optogenetic activation of Na V 1.7–expressing afferents in Na V 1.7-ChR2 mice induces nocifensive behavior without affecting responses to mechanical and thermal stimuli

doi: 10.1371/journal.pone.0275751

Figure Lengend Snippet: Establishment of different lines of transgenic mice (a) and distribution of ChR2-EYFP channels in DRG (b). (a) Na V 1.7 iCre/+ ;Ai32/+ mice were created by crossing homozygous Na V 1.7–iCre mice with heterozygous Ai32 mice. Subsequently, Na V 1.7 iCre/+ ;Ai32/+, Na V 1.7 iCre/iCre ;Ai32/+, Na V 1.7 iCre/+ ;Ai32/Ai32, and Na V 1.7 iCre/iCre ;Ai32/Ai32 mice were created by crossing Na V 1.7 iCre/+ ;Ai32/+ mice with each other. The four genotypes of mice used in the study are highlighted in yellow. (b) A typical immunohistochemical image showing of ChR2-EYFP in the DRG, the dorsal horn of spinal cord, and glabrous skin of Na V 1.7 iCre/+ ;Ai32/+ mouse was shown. Green and red fluorescence indicates ChR2-EYFP and Na V 1.7, respectively. ChR2–EYFP were expressed on Na V 1.7-expressing DRG neurons. Green fluorescence (ChR2–EYFP expression) can be observed in the dorsal horn. ChR2–EYFP is localized in free nerve endings in the lower and upper dermis of glabrous skin.

Article Snippet: For Na V 1.7 staining, sections of DRG were incubated in 0.1% Triton X-100 and 5% goat serum in PBS at room temperature for 4 h, followed by incubation with anti-Na V 1.7 polyclonal antibody (rabbit anti-rat, 1:250; catalog #ASC-008, Alomone Labs, Jerusalem, Israel) at 4°C with overnight agitation.

Techniques: Transgenic Assay, Immunohistochemical staining, Fluorescence, Expressing

Journal: PLoS ONE

Article Title: Selective optogenetic activation of Na V 1.7–expressing afferents in Na V 1.7-ChR2 mice induces nocifensive behavior without affecting responses to mechanical and thermal stimuli

doi: 10.1371/journal.pone.0275751

Figure Lengend Snippet: Paw withdrawal test (von Frey test) (a) and plantar test (b). (a) The von Frey test was performed with the wild type (WT) and mice of the four genotypes. The hind paw withdrawal data were analyzed using one-way ANOVA. All results are calculated as mean ± SD of 10 or more animals. Individual results for each strain are as follows: WT (B6J), 4.4 ± 0.7 g; Na V 1.7 iCre/+ ;Ai32/+, 4.8 ± 0.4 g; Na V 1.7 iCre/iCre ;Ai32/+, 4.5 ± 0.8 g; Na V 1.7 iCre/+ ;Ai32/Ai32, 4.0 ± 1.2 g; and Na V 1.7 iCre/iCre ;Ai32/Ai32, 4.3 ± 0.7 g. (b) The plantar test was performed with the WT and mice of the four genotypes. The data were analyzed using one-way ANOVA. All results are calculated as mean ± SD of 10 or more animals. Individual results for each strain were as follows: WT (B6J), 5.7 ± 0.8 s; Na V 1.7 iCre/+ ;Ai32/+, 5.5 ± 1.2 s; Na V 1.7 iCre/iCre ;Ai32/+, 6.6 ± 1.7 s; Na V 1.7 iCre/+ ;Ai32/Ai32, 6.6 ± 2.2 s; and Na V 1.7 iCre/iCre ;Ai32/Ai32, 6.3 ± 1.2 s.

Article Snippet: For Na V 1.7 staining, sections of DRG were incubated in 0.1% Triton X-100 and 5% goat serum in PBS at room temperature for 4 h, followed by incubation with anti-Na V 1.7 polyclonal antibody (rabbit anti-rat, 1:250; catalog #ASC-008, Alomone Labs, Jerusalem, Israel) at 4°C with overnight agitation.

Techniques:

Journal: PLoS ONE

Article Title: Selective optogenetic activation of Na V 1.7–expressing afferents in Na V 1.7-ChR2 mice induces nocifensive behavior without affecting responses to mechanical and thermal stimuli

doi: 10.1371/journal.pone.0275751

Figure Lengend Snippet: ChR2 expression in DRG neurons as measured by RT-PCR. β–Actin was used as a positive control to confirm successful protein extraction and equal loading of samples. All data are calculated as mean ± SD of 5 animals. * P < 0.001, compared with Na V 1.7 iCre/+ ;Ai32/+ mice. † P = 0.007 and # P = 0.006, compared with Na V 1.7 iCre/iCre ;Ai32/+ mice.

Article Snippet: For Na V 1.7 staining, sections of DRG were incubated in 0.1% Triton X-100 and 5% goat serum in PBS at room temperature for 4 h, followed by incubation with anti-Na V 1.7 polyclonal antibody (rabbit anti-rat, 1:250; catalog #ASC-008, Alomone Labs, Jerusalem, Israel) at 4°C with overnight agitation.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Positive Control, Protein Extraction, Mouse Assay

Journal: bioRxiv

Article Title: Equivalent excitability through different sodium channel subtypes and implications for analgesia by subtype-selective drugs

doi: 10.1101/2022.10.04.510784

Figure Lengend Snippet: (A) Sample voltage clamp recordings show that sodium current was almost completely abolished by the Na V 1.8 inhibitor PF-24 (1 μM). Peak current was significantly reduced by PF-24 (F 1.72 =12.651, p<0.012, two-way RM ANOVA; n=7). Another Na V 1.8 inhibitor, A-803467, had a similar effect (see ). (B) PF-24 significantly altered spiking pattern (χ 2 =5.14, p=0.0233, McNemar test) and reduced firing rate (F 1,42 =11.946, p=0.011, two-way RM ANOVA; n=8). (C) PF-24 significantly increased rheobase (Z 15 =2.783, p=0.003, Wilcoxon rank test) and reduced spike height (T 15 =3.151, p=0.007, paired t-test) but did not affect resting membrane potential (T 15 =0.304, p=0.765, paired t-test). The Na V 1.7 inhibitor PF-71 had negligible effects at DIV0 (see ). (D) A computational model reproduced the effect of Na V 1.8 on spiking pattern (also see ). The PF-24 effect was simulated as a ~85% reduction in Na V 1.8 . *, p<0.05; **; p<0.01; Student-Newman-Keuls post-hoc tests in A and B.

Article Snippet: After another 3x rinse with PBS, neurons were treated with 10% normal goat serum for 30 min followed with rabbit primary Na V 1.7 antibody (1:200, ASC-008, Alomone) or Na V 1.8 antibody (1:200, ASC-028, Alomone) in PBS with 0.1% Tritween-20 and 1% BSA for 1 h. For some of the coverslips, primary antibodies were replaced with control peptides (ASC008AG1040 for Na V 1.7 and ASC016AG0640 for Na V 1.8) provided by Alomone as negative controls.

Techniques:

Journal: bioRxiv

Article Title: Equivalent excitability through different sodium channel subtypes and implications for analgesia by subtype-selective drugs

doi: 10.1101/2022.10.04.510784

Figure Lengend Snippet: (A) Sample voltage clamp recordings show that sodium current was reduced by the Na V 1.7 inhibitor PF-71 (30 nM) and by the Na V 1.1/1.3 inhibitor ICA (1 μM). Peak current was significantly reduced by PF-71 and ICA (F 2,192 =26.361, p<0.001, two-way RM ANOVA; n=9). (B) PF-71 and ICA both significantly altered spiking pattern (χ 2 =4.17, p=0.041 and χ 2 =7.11, p=0.0077, respectively, McNemar tests) and significantly reduced firing rate (F 1.54 =40.659, p<0.001, n=10 and F 1.78 =35.156, p<0.001, n=14, respectively, two-way RM ANOVAs). (C) PF-71 significantly increased rheobase (Z 18 =3.464, p<0.001, Wilcoxon rank test) and decreased spike height (T 18 =7.946, p<0.001, paired t-test). ICA did not significantly alter rheobase (Z 18 =1.248, p=0.225) but did reduce spike height (T 18 =3.243, p=0.005). Neither drug affected resting membrane potential (T 15 =1.681, p=0.113 for PF-71; T 18 =-1.132, p=0.272 for ICA, paired t-test). The Na V 1.8 antagonist PF-24 had negligible effects at DIV4-7 (see ). (D) A computational model reproduced the combined effects of Na V 1.3 and Na V 1.7 on spiking pattern (also see ). PF-71 effect was simulated as a 70% reduction in Na V 1.7 . ICA effect was simulated as a 90% reduction in Na V 1.3 . *, p<0.05; **, p<0.01; Student-Newman-Keuls post-hoc tests in A and B.

Article Snippet: After another 3x rinse with PBS, neurons were treated with 10% normal goat serum for 30 min followed with rabbit primary Na V 1.7 antibody (1:200, ASC-008, Alomone) or Na V 1.8 antibody (1:200, ASC-028, Alomone) in PBS with 0.1% Tritween-20 and 1% BSA for 1 h. For some of the coverslips, primary antibodies were replaced with control peptides (ASC008AG1040 for Na V 1.7 and ASC016AG0640 for Na V 1.8) provided by Alomone as negative controls.

Techniques:

Journal: bioRxiv

Article Title: Equivalent excitability through different sodium channel subtypes and implications for analgesia by subtype-selective drugs

doi: 10.1101/2022.10.04.510784

Figure Lengend Snippet: (A) Inhibiting Na V 1.7 with PF-71 (30 nM) did not alter spiking pattern (χ 2 =0.00, p=1.00, McNemar test) or reduce firing rate (F 1.30 =5.805, p=0.061, two-way RM ANOVA, n=6) in DIV0 neurons; in fact, firing rate was slightly increased. (D-E) PF-71 did not affect rheobase (Z 9 =0.677, p=0.578, Wilcoxon rank test) but did reduce spike height (T 9 =3.759, p=0.004, paired-t-test).

Article Snippet: After another 3x rinse with PBS, neurons were treated with 10% normal goat serum for 30 min followed with rabbit primary Na V 1.7 antibody (1:200, ASC-008, Alomone) or Na V 1.8 antibody (1:200, ASC-028, Alomone) in PBS with 0.1% Tritween-20 and 1% BSA for 1 h. For some of the coverslips, primary antibodies were replaced with control peptides (ASC008AG1040 for Na V 1.7 and ASC016AG0640 for Na V 1.8) provided by Alomone as negative controls.

Techniques:

Journal: bioRxiv

Article Title: Equivalent excitability through different sodium channel subtypes and implications for analgesia by subtype-selective drugs

doi: 10.1101/2022.10.04.510784

Figure Lengend Snippet: (A) The computational model predicts that the Na V 1.8 conductance, which is “necessary” for repetitive spiking at DIV0 can, in principle, be replaced by Na V 1.7 (left), and vice versa at DIV4-7 (right). (B) Replacement experiments involved inhibiting native channels pharmacologically and then introducing virtual conductances using dynamic clamp. At DIV0 (left), inhibiting native Na V 1.8 (with PF-24) converted neurons to transient spiking, but introducing virtual Na V 1.7 reverted neurons to repetitive spiking (in 3 of 3 neurons tested). At DIV4-7, inhibiting native Na V 1.7 (with PF-71) converted the neuron to transient spiking, but introducing virtual Na V 1.8 reverted neurons to repetitive spiking (in 4 of 4 neurons tested). Parameters for virtual channels were identical to simulations except for the maximal conductance density, which was titrated in each cell. (C) Voltage (top) for first (left) and second (right) spikes in the DIV0 model aligned with voltage activation curves for each Na V isoform (bottom). Dashed line shows voltage threshold (defined as V where dV/dt reaches 5 mV/ms). (D) Conductance plotted against voltage to create a phase portrait (top) showing Na V conductance at different phases of the spike. Inset shows full voltage range; main graph zooms in on voltages near threshold. Bottom plots show current plotted over the same voltage range. For the first spike, Na V 1.7 (orange) mediates nearly all perithreshold inward current. For the second spike, voltage threshold is increased and Na V 1.8 (green) mediates nearly all perithreshold inward current because Na V 1.7 has inactivated (see ). ( E-F ) In the DIV4-7 model, Na V 1.7 (orange) and Na V 1.3 (maroon) contribute to initiation of all spikes whereas the contribution of Na V 1.8 is negligible.

Article Snippet: After another 3x rinse with PBS, neurons were treated with 10% normal goat serum for 30 min followed with rabbit primary Na V 1.7 antibody (1:200, ASC-008, Alomone) or Na V 1.8 antibody (1:200, ASC-028, Alomone) in PBS with 0.1% Tritween-20 and 1% BSA for 1 h. For some of the coverslips, primary antibodies were replaced with control peptides (ASC008AG1040 for Na V 1.7 and ASC016AG0640 for Na V 1.8) provided by Alomone as negative controls.

Techniques: Activation Assay

Journal: bioRxiv

Article Title: Equivalent excitability through different sodium channel subtypes and implications for analgesia by subtype-selective drugs

doi: 10.1101/2022.10.04.510784

Figure Lengend Snippet: (A) Sample response at DIV0 showing that a virtual Na V 1.8 conductance applied with dynamic clamp restored repetitive spiking after inhibiting native Na V 1.8 channels with PF-24. This restoration was repeated in 3 of 3 neurons tested. (B) Sample recording at DIV4-7 showing that a virtual Na V 1.7 conductance restored repetitive spiking after inhibiting native Na V 1.7 channels with PF-71. This restoration was repeated in 4 of 4 neurons tested.

Article Snippet: After another 3x rinse with PBS, neurons were treated with 10% normal goat serum for 30 min followed with rabbit primary Na V 1.7 antibody (1:200, ASC-008, Alomone) or Na V 1.8 antibody (1:200, ASC-028, Alomone) in PBS with 0.1% Tritween-20 and 1% BSA for 1 h. For some of the coverslips, primary antibodies were replaced with control peptides (ASC008AG1040 for Na V 1.7 and ASC016AG0640 for Na V 1.8) provided by Alomone as negative controls.

Techniques:

Journal: bioRxiv

Article Title: Equivalent excitability through different sodium channel subtypes and implications for analgesia by subtype-selective drugs

doi: 10.1101/2022.10.04.510784

Figure Lengend Snippet: (A) In the DIV0 model, Na V 1.7 contributed to the first spike but its inactivation meant that all subsequent spikes relied exclusively on Na V 1.8. (B) In the DIV4-7 model, despite some inactivation of Na V 1.3 (red) and Na V 1.7 (green), the remaining current was still large enough to produce a net inward current sufficient to support repetitive spiking.

Article Snippet: After another 3x rinse with PBS, neurons were treated with 10% normal goat serum for 30 min followed with rabbit primary Na V 1.7 antibody (1:200, ASC-008, Alomone) or Na V 1.8 antibody (1:200, ASC-028, Alomone) in PBS with 0.1% Tritween-20 and 1% BSA for 1 h. For some of the coverslips, primary antibodies were replaced with control peptides (ASC008AG1040 for Na V 1.7 and ASC016AG0640 for Na V 1.8) provided by Alomone as negative controls.

Techniques:

Journal: bioRxiv

Article Title: Equivalent excitability through different sodium channel subtypes and implications for analgesia by subtype-selective drugs

doi: 10.1101/2022.10.04.510784

Figure Lengend Snippet: Despite TTX having negligible effects in DIV0 neurons according to our initial analysis (see ), simulation results in predicted that the first spike was nonetheless initiated by Na V 1.7. By extension, this predicted that TTX should cause a depolarizing shift in voltage threshold for the first spike. Analysis of the experimental data confirmed this to be true, with threshold (mean±SEM) increasing from −33.7±1.4 mV at baseline to −28.3±1.4 mV after TTX (T 24 =-3.19, p =0.004, paired t-test). Confirmation of this unexpected prediction helps further validate our model neuron.

Article Snippet: After another 3x rinse with PBS, neurons were treated with 10% normal goat serum for 30 min followed with rabbit primary Na V 1.7 antibody (1:200, ASC-008, Alomone) or Na V 1.8 antibody (1:200, ASC-028, Alomone) in PBS with 0.1% Tritween-20 and 1% BSA for 1 h. For some of the coverslips, primary antibodies were replaced with control peptides (ASC008AG1040 for Na V 1.7 and ASC016AG0640 for Na V 1.8) provided by Alomone as negative controls.

Techniques:

Journal: bioRxiv

Article Title: Equivalent excitability through different sodium channel subtypes and implications for analgesia by subtype-selective drugs

doi: 10.1101/2022.10.04.510784

Figure Lengend Snippet: (A) Both Na V 1.8 and Na V 1.7 mRNA levels (relative to a housekeeping gene (HKG), see Methods) decreased significantly between DIV0 and DIV4-7 (factor 1: time, F 1,12 =56.677, p<0.001, factor 2: Na V isoform, F 1,12 =17.952, p=0.001, two-way ANOVA and Student-Newman-Keuls post-hoc tests on log transformed data, n=4 mice per time point) but more so for Na V 1.8 than for Na V 1.7 (interaction: time x isoform, F 1,12 = 11.455, p=0.005). The differential reduction translated into a significantly higher Na V 1.8: Na V 1.7 ratio on DIV0 than at DIV4-7 (T 6 =21.375, p<0.001, unpaired t-test). These changes may account for Na V 1.8 becoming unnecessary for repetitive spiking at DIV4-7 but cannot account for Na V 1.7 becoming necessary. (B) Immunoreactivity (IR) for Na V 1.8 protein exceeded Na V 1.7-IR at DIV0, but the opposite was true on DIV4-7. Na V -IR was measured relative to YFP-IR in the same cell, and then each cell’s Na V 1.8:YFP ratio was considered relative to the average Na V 1.7:YFP ratio in the co-processed coverslip (left) or average Na V 1.8:YFP ratio was considered relative to the average Na V 1.7:YFP ratio in the same animal (right). Both ratios were >1 at DIV0 but decreased significantly at DIV4-7 (U=78, p<0.001, n=37 for DIV0, n=40 for DIV4-7, Mann-Whitney test (left) and T 6 =4.046, p=0.007, unpaired t-test (right)). (C) Chronically applied cercosporamide (10 μM) mitigated the changes in Na V 1.8- and Na V 1.7-IR at DIV5 (Na V 1.8: H 3 =157.95, p<0.001; Na V 1.7: H 3 =80.662, p<0.001; One-way ANOVA on ranks, Dunn’s post-hoc tests, p<0.05 for all pairs). Panel on the right shows data normalized to baseline (DIV0) to emphasize relative changes.

Article Snippet: After another 3x rinse with PBS, neurons were treated with 10% normal goat serum for 30 min followed with rabbit primary Na V 1.7 antibody (1:200, ASC-008, Alomone) or Na V 1.8 antibody (1:200, ASC-028, Alomone) in PBS with 0.1% Tritween-20 and 1% BSA for 1 h. For some of the coverslips, primary antibodies were replaced with control peptides (ASC008AG1040 for Na V 1.7 and ASC016AG0640 for Na V 1.8) provided by Alomone as negative controls.

Techniques: Transformation Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: Equivalent excitability through different sodium channel subtypes and implications for analgesia by subtype-selective drugs

doi: 10.1101/2022.10.04.510784

Figure Lengend Snippet: ( A ) Sample responses in DIV0 neurons from mice injected with CFA three days earlier. In 12 cells tested, PF-71 converted 5 neurons to transient spiking ( i ), encouraged repetitive spiking in 4 neurons ( ii ), and had no effect in 3 neurons ( iii ), thus highlighting increased heterogeneity after CFA. ( B ) At DIV0, the effect of PF-71 differed significantly between CFA and control neurons, converting 42% (5 of 12) CFA neurons from repetitive to transient spiking vs 0% (0 of 9) control neurons (p=0.0451, Fisher Exact test). Applying PF-24 to neurons that continued to spike repetitively after PF-71 had little effect on CFA neuron, converting only 13% (1 of 7) of CFA neurons vs 88% (7 of 8) of control neurons (p=0.001, Fisher Exact test). Together these results argue that Na V 1.7 contributes more and Na V 1.8 contributes less to nociceptor excitability after inflammation. (C) At DIV0, PF-71 significantly increased resting membrane potential (T 11 =-3.530, p=0.005, paired t-test) and rheobase (Z 11 =2.186, p=0.024, Wilcoxon rank test), and significantly decreased spike height (T 11 =4.413, p=0.001, paired t-test) in CFA neurons. Further addition of PF-24 significantly changed rheobase (Z 9 =2.176, p=0.023, Wilcoxon rank test) and action potential amplitude (T 9 =3.237, p=0.01, paired t-test) but did not affect resting membrane potential (T 9 =1.049, p=0.321, paired t-test). (D) Paw inflammation caused by CFA significantly altered thermal sensitivity (Hargreaves: F 5,65 =19.556, p<0.001, two-way RM ANOVA) and mechanical sensitivity (von Frey: F 4,52 =16.786, p<0.001). When given three days after CFA, PF-71 significantly reversed the altered sensitivities (Hargreaves: T 8 =-7.296, p<0.001; von Frey: T 8 =-4.341, p=0.002; paired t-tests) but had no effect in naive mice (Hargreaves: T 5 =-0.141, p=0.894; von Frey: T 5 =1.000, p=0.363). Insets show values for each animal before and 2 hours after PF-71 injection. *, p<0.05; **, p<0.01; Student-Newman-Keuls post-hoc tests.

Article Snippet: After another 3x rinse with PBS, neurons were treated with 10% normal goat serum for 30 min followed with rabbit primary Na V 1.7 antibody (1:200, ASC-008, Alomone) or Na V 1.8 antibody (1:200, ASC-028, Alomone) in PBS with 0.1% Tritween-20 and 1% BSA for 1 h. For some of the coverslips, primary antibodies were replaced with control peptides (ASC008AG1040 for Na V 1.7 and ASC016AG0640 for Na V 1.8) provided by Alomone as negative controls.

Techniques: Injection