Journal: bioRxiv

Article Title: Satellite Glial Cells Control Sensory Neuron Excitability via the Release of Fibulin-2

doi: 10.64898/2026.02.13.705760

Figure Lengend Snippet: A . Voltage protocols for measurement of different types of K + currents: total ( I Total ), K-type ( I K ) and A-type ( I A ) K + currents. B . Sample traces of voltage-dependent K + currents I total (left), I K (middle) and I A (right) evoked by the protocols in ( A ) from Control (upper panel) and rFibulin-2 treated DRG cells (lower panel). C . rFibulin-2 increases voltage-dependent K + currents I Total (left), I K (middle) and I A (right) in DRG cells. Insert bar graphs are K + currents at membrane potential of -10 mV (around voltage threshold level), indicating that rFibulin-2 decreases excitability mainly mediated by enhancement of I A conductance, which reduces input resistance. Number of cells tested from 3 independent experiments: control n = 10; rFibulin-2: n = 8. D . Phrixotoxin-1 (PaTx1) was used to isolate Kv4 current evoked by voltage ramp (-100 to +20 mV, 100 mV/s). Sample traces of ramp-evoked K + currents before (a) and during (b) application of PaTx1, and the PaTx1-sensitive current (c, c = a - b). Currents were normalized to membrane capacitance for better comparison. E . I-V curves were constructed from the ramp-evoked Kv4 current (mean current value over 0.1 mV intervals from averages of five trials for each cell to approximate quasi-steady-state current). Note PaTx1 significantly increases the Kv4 current when the membrane potentials are depolarized to positive values greater than -25 mV. Number of cells tested from 3 independent experiments: control n = 6; rFibulin-2: n = 6; T-test; * P < 0.05; ** P < 0.01. F . Representative western blot of control and Fibulin-2 KO DRG lysate analyzed for Fibulin-2 and Kv4.2. GAPDH is used as a loading control. G . Quantification of Kv4.2 expression in control and Fibulin-2 KO mice. n=3 WT and n=3 Fibulin-2 KO mice. T-test; ** P < 0.01. H . Representative western blot of control and Fibulin-2 KO DRG lysate analyzed for Fibulin-2 and Kv4.3. GAPDH is used as a loading control. I . Quantification of Kv4.3 expression in control and Fibulin-2 KO mice. n=3 WT and n=3 Fibulin-2 KO mice. T-test; *** P < 0.001 J . Fibulin-2 KO mice show hypersensitivity to mechanical stimuli compared to controls, measured by the Von Frey Test. 12 WT and 8 Fibulin-2 KO mice were used. Two-Way Anova. ∗p < 0.05, ∗∗p < 0.01, ***p<0.001. K . Fibulin-2 KO mice exhibit hypersensitivity to heat stimuli compared to controls, measured by the Hot-Plate test. 12 WT and 8 Fibulin-2 KO mice were used. Two-Way Anova. ∗p < 0.05, ∗∗p < 0.01, ***p<0.001. L . Fibulin-2 KO mice exhibit hypersensitivity to cold stimuli compared to controls, measured by the Cold-Plate test. 12 WT and 8 Fibulin-2 KO mice were used. Two-Way Anova. ∗p < 0.05, ∗∗p < 0.01, ***p<0.001. M . Representative immunofluorescence images of the hindpaw of control and Fibulin-2 KO mice immunostained for PGP9.5 (white) and DAPI (blue). Three sections from n=3 mouse per group were used. N . Quantification of intraepidermal nerve fiber density (IENFD). n=3 mice per genotype. T-test, ns- non-significant

Article Snippet: The Kv4 channel blocker Phrixotoxin-1 was from Alomone Labs.

Techniques: Control, Membrane, Comparison, Construct, Western Blot, Expressing, Hot Plate Test, Immunofluorescence

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Disruption of cellular calcium homeostasis by duck Tembusu virus facilitates viral replication via AMPK pathway activation

doi: 10.3389/fcimb.2026.1743907

Figure Lengend Snippet: DTMUV infection increases cytoplasmic Ca 2+ levels in DEFs. (A) Flow cytometry profiles showing cytoplasmic Ca2+ levels in DEFs with and without DTMUV infection and probed with Flou-4AM. (B) Cytoplasmic Ca2+ levels of DEFs with (red) and without (blue) DTMUV infection (MOI = 0.1) for 6, 8, 10, or 12 hours, expressed as mean fluorescence intensity (MFI). (C) Cytoplasmic Ca2+ levels of DEFs with and without DTMUV infection and concurrent treatment with DMSO (control) verapamil or diltiazem hydrochloride. Data expressed as mean ± standard deviation (n = 3), analyzed using Student’s t-test; *p < 0.05, **p < 0.01, ****P<0.0001.

Article Snippet: The calcium channel blockers verapamil (Cat# HY-14275) and diltiazem hydrochloride (Cat# HY-14656), the AMPK inhibitor Compound C (Cat# HY-13418A), and calcium chelating agent BAPTA-AM (Cat# HY-100545) were purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Infection, Flow Cytometry, Fluorescence, Control, Standard Deviation

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Disruption of cellular calcium homeostasis by duck Tembusu virus facilitates viral replication via AMPK pathway activation

doi: 10.3389/fcimb.2026.1743907

Figure Lengend Snippet: VDCC blockers and a cytoplasmic Ca 2+ chelator reduce DTMUV particle production. (A, B) Analysis of plaque assays of DEFs infected with DTMUV and treated with verapamil (25 µM), diltiazem hydrochloride (50 µM), or DMSO (control; (A) ), and BAPTA-AM (25 µM) or DMSO (control; (B) ). Results expressed as the viral titer ratio (%) between each drug-treated group and the control group at 12, 24, and 36 hpi. Data expressed as mean ± standard deviation of triplicate samples, analyzed by two-way ANOVA with multiple comparisons. *p < 0.05, **p <0.01, ***p <0.001, ****p < 0.0001. Results shown are representative of three independent experiments.

Article Snippet: The calcium channel blockers verapamil (Cat# HY-14275) and diltiazem hydrochloride (Cat# HY-14656), the AMPK inhibitor Compound C (Cat# HY-13418A), and calcium chelating agent BAPTA-AM (Cat# HY-100545) were purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Infection, Control, Standard Deviation

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Disruption of cellular calcium homeostasis by duck Tembusu virus facilitates viral replication via AMPK pathway activation

doi: 10.3389/fcimb.2026.1743907

Figure Lengend Snippet: VDCC blockers and a cytoplasmic Ca 2+ chelator inhibit the replication step of DTMUV infection. (A) Viral entry assay of DEFs pretreated with DMSO, diltiazem (50 µM), or BAPTA-AM (25 µM) for 1 hour prior to DTMUV infection (MOI = 1) at 4°C for 1 hour and fusion at 37°C. Viral RNA levels in the cytoplasm were quantified by RT-qPCR at 2 hours post-infection (hpi), expressed as relative DTMUV mRNA levels between the drug-treated groups and the control group. (B) Viral replication assay of DEFs infected with DTMUV (MOI = 1) prior to treatment with DMSO (control), diltiazem hydrochloride (50 µM), or EAPTA-AM (25 µM) at 2 hpi, and RT-qPCR analysis of viral RNA replication in infected cells at 6 hpi, expressed as relative DTMUV mRNA levels between the drug-treated and control groups. (C) Plaque assay of viral release in DEFs cultured infected with DTMUV (MOI = 1) prior to treatment with DMSO (control), diltiazem hydrochloride (50 µM), or EAPTA-AM (25 µM) at 10 hpi and plating at 12 hpi. Results expressed as the viral titer ratio (%) between the drug-treated groups and the control group. (D) Viral replication assay of DEFs infected with DTMUV (MOI = 1) prior to treatment with DMSO (control) or alternative forms of verapamil (25 µM), diltiazem hydrochloride (50 µM), or BAPTA-AM (25 µM) at 1 hpi. Infected cells were harvested for RT-qPCR analysis of DTMUV mRNA levels at 8, 10, and 12 hpi, expressed as relative DTMUV mRNA levels between the drug-treated and control groups. Data expressed as mean ± standard deviation of triplicate samples, analyzed by one-way or two-way ANOVA with multiple comparisons; *p < 0.05, **p <0.01, ***p <0.001, ****p <0.0001. Data shown are representative of three independent experiments. ns: no significant difference.

Article Snippet: The calcium channel blockers verapamil (Cat# HY-14275) and diltiazem hydrochloride (Cat# HY-14656), the AMPK inhibitor Compound C (Cat# HY-13418A), and calcium chelating agent BAPTA-AM (Cat# HY-100545) were purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Infection, Quantitative RT-PCR, Control, Viral Replication Assay, Plaque Assay, Cell Culture, Standard Deviation

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Disruption of cellular calcium homeostasis by duck Tembusu virus facilitates viral replication via AMPK pathway activation

doi: 10.3389/fcimb.2026.1743907

Figure Lengend Snippet: DTMUV-mediated AMPK activation is markedly diminished by treatment with VDCC blockers or a cytoplasmic Ca 2+ chelator. (A) Western blotting of pAMPKα (Thr172) in DEFs infected with DTMUV (MOI = 1) and harvested at the indicated time points. (B) Immunoblotting analysis of pAMPKα (Thr172) levels in DEFs treated with DMSO (control), verapamil (25 µM; Vera), diltiazem hydrochloride (50 µM; Dilt), or BAPTA-AM (25 µM; BAP), with and without DTMUV infection (MOI = 1) for 12 hours.

Article Snippet: The calcium channel blockers verapamil (Cat# HY-14275) and diltiazem hydrochloride (Cat# HY-14656), the AMPK inhibitor Compound C (Cat# HY-13418A), and calcium chelating agent BAPTA-AM (Cat# HY-100545) were purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Activation Assay, Western Blot, Infection, Control

Journal: Frontiers in Cellular Neuroscience

Article Title: Glutamate load fosters spreading depolarization under osmotic stress in brain slices

doi: 10.3389/fncel.2025.1722194

Figure Lengend Snippet: Mechanistic summary of swelling-induced glutamate release and neuronal receptor activation during spreading depolarization. Under physiological conditions, astrocytes maintain extracellular glutamate and osmotic homeostasis via aquaporin-4 channels (AQP4), Na + -K + -2Cl − cotransporter 1 (NKCC1) and volume-regulated anion channel (VRAC), thereby preserving normal neuronal excitability. Osmotic stress promotes astrocyte swelling which activates VRAC to release glutamate, elevating extracellular glutamate and overstimulating neuronal NMDA N-methyl-D-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate receptors—this cascade fosters the occurrence of spreading depolarization. Pharmacological blockade—using DCPIB (VRAC blocker), Bumetanide (NKCC1 inhibitor), TGN-020 (AQP4 inhibitor), along with NMDA antagonist MK-801 and AMPA/kainate antagonist CNQX—attenuates astrocyte swelling, reduces glutamate accumulation and alleviates spreading depolarization. The figure was created in https://BioRender.com .

Article Snippet: Another set of slices was exposed to the volume-regulated anion channel (VRAC) blocker 4-(2-Butyl-6,7-dichloro-2-cyclopentyl-indan-1-on-5-yl) oxobutyric acid (DCPIB; ; Tocris; 20 μM; n = 15).

Techniques: Activation Assay, Preserving

Journal: Journal of Neurochemistry

Article Title: Sevoflurane Inhibits Layer 5 Pyramidal Neurons via Kv1.2‐Dependent Modulation of Subthreshold Currents

doi: 10.1111/jnc.70360

Figure Lengend Snippet: Application of the Kv1.2 antagonist Tityustoxin‐Κα partially decrease effects of sevoflurane. (A) Voltage responses to hyperpolarizing and depolarizing current steps in control conditions, after sevoflurane and after additional Tityustoxin Κα (100 nM, sevo + TsTX‐Κα) application for type A (red; TsTX‐Κα, n = 8) and type B (blue; TsTX‐Κα, n = 5) L5 PNs. Arrow depicts hyperpolarization magnitude, arrowhead points to the sag or rebound AP. (B) Tityustoxin‐Κα application increased the firing frequency initially decreased by sevoflurane marginally for type A PNs but more so in type B PNs, but could not recover firing frequency to baseline frequency. Asterisks (*) show significant difference between aCSF and sevoflurane + TsTX‐Κα; significance levels * p < 0.05, ** p < 0.01, ** p < 0.001. (C) Boxplots showing that the average membrane potential depolarization of type A PNs could not be recovered by TsTX‐Κα. (D) Boxplots showing that the average input resistance of type B PNs was recovered by TsTX‐Κα. (E) Boxplots of average Δsag also show TsTX‐Κα to reverse sevoflurane effects. Boxplots show median (horizontal bar), interquartile range (IQR, box), and 1.5× IQR values. Full statistical report in Table .

Article Snippet: In some experiments, the Kv1.2 channel blocker rTityustoxin‐Kα (TsTX‐Kα, Alomone Labs, Cat#: STT‐360, 100 nM) was added to aCSF pre‐bubbled with sevoflurane.

Techniques: Control, Membrane

Journal: Journal of Neurochemistry

Article Title: Sevoflurane Inhibits Layer 5 Pyramidal Neurons via Kv1.2‐Dependent Modulation of Subthreshold Currents

doi: 10.1111/jnc.70360

Figure Lengend Snippet: Sevoflurane shifts Kv1.2 activation to more hyperpolarized voltages, suppressing subthreshold currents in L5 pyramidal neurons. (A) Pharmacology schematics. (B) Current responses to 7 subthreshold voltage steps (−77 to −47 mV, 5 mV increments, 500 ms duration) of type A (all groups, n = 7 neurons) and type B (all groups, n = 5 neurons) PNs in the presence of aCSF, sevoflurane and sevoflurane + Tityustoxin‐Kα. Bottom: The digitally subtracted trace of sevoflurane trace from the Sevo + TsTX‐Κα trace, showing the TsTX‐Κα current enhancement was similar for the two subtypes. (C) Current voltage‐dependency (I–V) plots for average steady state currents ( I ss ) in response to subthreshold voltage steps in aCSF, after application of sevoflurane and after additional application of Tityustoxin‐Kα (TsTX‐Kα). Arrows denote reversal potential of subthreshold currents in the presence of aCSF (type A—red, type B—blue) and sevo (black). Arrowheads show no difference in average current in response to a −50 mV step in aCSF vs. Sevo + TsTX‐Κα. (D) I–V plots as in ‘C’ for type B PNs. (E) Z ‐scores and −log 10 (P FDR ) plots from mixed effects linear model for each 5 mV voltage bin (1: −77 to −72 mV; 6: −52 to −47 mV) for current amplitudes, and 15 mV bin comparisons (−77 to −62 mV and −62 to −47 mV) for I–V slope analysis. Dotted lines indicate standard significance thresholds and the line at z = 0, which represents no effect of the treatment. All error bars represent SEM. Full statistical report in Table .

Article Snippet: In some experiments, the Kv1.2 channel blocker rTityustoxin‐Kα (TsTX‐Kα, Alomone Labs, Cat#: STT‐360, 100 nM) was added to aCSF pre‐bubbled with sevoflurane.

Techniques: Activation Assay