Journal: eLife

Article Title: Striatal cholinergic interneuron pause response requires Kv1 channels, is absent in dyskinetic mice, and is restored by dopamine D5 receptor inverse agonism

doi: 10.7554/eLife.102184

Figure Lengend Snippet: ( A ) Schematic representation of the tested hypotheses. DP: depolarization; HP: hyperpolarization; Glu: glutamate; DA: dopamine. ( B ) Representative recordings of striatal cholinergic interneurons (SCINs) in response to optogenetic activation of thalamic terminals, with or without margatoxin (MgTx; Kv1.3 channel blocker) or dendrotoxin (DTx; Kv1.1 and Kv1.6 channels blocker) in the bath. ( C ) Pause duration/baseline ISI of SCINs that responded with 1, 2, 3, or 4 spikes to optogenetic activation of thalamic terminals, with or without MgTx 3 nM, 30 nM, or DTx 100 nM, in the bath (two-way RM ANOVA interaction ns, treatment *p=0.0051; control-MgTx 30 nM: *p=0.0005; control-MgTx 3 nM: ns; control-DTX: *p=0.0462; MgTx 30 nM-MgTx 3 nM: *p<0.0001; MgTx 30 nM-DTX: ns; MgTx 3 nM-DTX: *p=0.0027). ( D ) Baseline ISI of SCINs recorded with or without MgTx 3 nM, 30 nM, or DTx (one-way ANOVA, ns). ( E ) Burst duration of SCINs that responded with 2, 3, or 4 spikes to optogenetic activation of thalamic terminals, with or without MgTx 3 nM, 30 nM, or DTx 100 nM, in the bath (two-way RM ANOVA, ns). ( F ) Representative recordings of SCINs in response to optogenetic activation of thalamic terminals, with or without Ba 2+ (10 µM; Kir2.2 channel blocker at this concentration) or XE 991 (10 µM; Kv7 channel blocker) in the bath. ( G ) Pause duration/baseline ISI of SCINs that responded with 1, 2, 3, or 4 spikes to optogenetic activation of thalamic terminals, with or without 10 µM XE 991 or 10 µM Ba 2+ (two-way RM ANOVA, ns). ( H ) Baseline ISI of SCINs recorded with or without Ba 2+ or XE 991 (one-way ANOVA, ns). ( I ) Burst duration of SCINs that responded with 2, 3, or 4 spikes to optogenetic activation of thalamic terminals, for the above conditions (two-way RM ANOVA, ns). Mean ± SEM; n=7–16 cells per group, from >5 mice. Figure 2—source data 1. Contribution of the Kv1 current to the pause response.

Article Snippet: The following stock solvents and final concentrations were used: distilled H 2 O for BaCl (10 μM), ZD7288 (30 μM), mecamylamine (10 μM, RBI), methiothepin (10 μM), quinpirole (1 μM, 10 μM), and sumanirole (10 μM); DMSO for CNQX (40 μM, Tocris), PIC (100 μM), SKF81297 (2 μM), SCH23390 (10 μM), sulpiride (10 μM, Santa Cruz Biotechnology), XE991 (10 μM), and clozapine (10 μM, Rospaw Laboratory); and the manufacturer’s recommended storage buffer (0.1% BSA, 100 mM NaCl, 10 mM Tris pH 7.5, 1 mM EDTA) for MgTx (3 nM and 30 nM, Alomone Labs) and g-DTx (100 nM, Alomone Labs).

Techniques: Activation Assay, Control, Concentration Assay

Journal: Cell Death Discovery

Article Title: Therapeutic potential of voltage-dependent potassium channel subtype 1.3 blockade in alleviating macrophage-related renal inflammation and fibrogenesis

doi: 10.1038/s41420-025-02508-7

Figure Lengend Snippet: A Respective western blotting image and densitometry analysis of ERK/NF-κB signaling after transfection with LV-Kv1.3 in THP-1 cells. Data were expressed as mean ± SD (n = 4). B The phosphorylation signal data are depicted in the form of stacked histogram overlaid plots. The mean fluorescence intensity (MFI) of the ERK1/2 and NF-κB p65 phosphorylation signal was expressed as the mean ± SD (n = 3). C Effects of MgTx, PD98059, and Licochalcone B on Kv1.3 and ERK/NF-κB signaling in LV-Kv1.3-transduced THP-1 cells were analyzed by western blotting. Data were expressed as mean ± SD (n = 3). D Respective western blotting image and densitometry analysis of ERK/NF-κB signaling in HK-2 cells co-cultured with the supernatant from LV-Kv1.3-transduced THP-1 cells for 48 h. Data were expressed as mean ± SD (n = 4). E Respective western blotting image and densitometry analysis of ERK/NF-κB signaling in mice subjected to UUO (7 days post-surgery) and IRI. Data were expressed as mean ± SD (n = 4).

Article Snippet: MgTx, PD98059 and Licochalcone B were obtained from MedChemExpress (HY-P1280, HY-12028 and HY-N0373, respectively).

Techniques: Western Blot, Transfection, Phospho-proteomics, Fluorescence, Cell Culture

Journal: Journal of neurochemistry

Article Title: Inhibition of cGMP-Signalling Rescues Retinal Ganglion Cells From Axotomy-Induced Degeneration.

doi: 10.1111/jnc.70072

Figure Lengend Snippet: FIGURE 1 | The NO/cGMP/PKG pathway in the retina, targets and inhibitors. (a) Retinal expression of enzymes and proteins involved in nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) signalling. Staining performed on post-natal (P) day 12 retina, except for sGC staining per- formed on P24 retina. Neuronal nitric oxide synthase (nNOS, magenta), NOS activity (purple), soluble guanylyl cyclase (sGC; cyan), cGMP accu- mulation (yellow), protein kinase G 1β (PKG1β; green), and Kv1.3- and Kv1.6-channels (red) were all detected within the ganglion cell layer (GCL). Several of these enzymes/proteins were also expressed in the outer- and inner nuclear layer (ONL/INL) and the retinal pigment epithelium (RPE). The TUNEL assay (orange) was used to detect dying cells in the GCL, DAPI (grey) was used as nuclear counterstain. (b) In the prototypic NO- signalling pathway, NOS generates NO to activate sGC and trigger cGMP production. Exceedingly high cGMP-levels may lead to an overactivation of PKG, which phosphorylates and likely activates voltage-gated potassium channels belonging to the Kv1 family (i.e., Kv1.3 and Kv1.6). Excessive potassium influx may trigger retinal ganglion cell (RGC) death. Different components of this pathway can be interrogated using specific inhibitors, such as 7-nitroindazole (7-NI) for NOS, H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one (ODQ) for sGC, CN238 for PKG and Margatoxin (MrgX) for Kv1.3/Kv1.6. Scale bar: 50 μm.

Article Snippet: To assess the involvement of NO/cGMP/PKG signalling in RGC degeneration, the following drug treatments were used: 15 μM 7- nitroindazole (7- NI) (cat. no. N7778; Sigma–Aldrich), 0.5 μM 1H- [1,2,4] oxadiazolo [4,3- a] quinoxalin- 1- one (ODQ) (cat. no. O3636; Sigma–Aldrich), 50 μM RP- 8- Br- pMe- PET- cGMPS (CN238; cat. no. P 007; Biolog Life Science Institute GmbH & Co, Bremen, Germany), and 50 nM Margatoxin (MrgX) (cat. no. STM- 325; Alomone Labs, Jerusalem, Israel).

Techniques: Expressing, Staining, Activity Assay, TUNEL Assay

Journal: Journal of neurochemistry

Article Title: Inhibition of cGMP-Signalling Rescues Retinal Ganglion Cells From Axotomy-Induced Degeneration.

doi: 10.1111/jnc.70072

Figure Lengend Snippet: FIGURE 4 | Short-term NO/cGMP/PKG pathway inhibition reduces RGC degeneration. Retinal explant cultures derived from wild-type (WT) mice at post-natal day 12 were treated for 24 h with 15 μM 7-NI, 0.5 μM ODQ, 50 μM CN238 or 50 nM MrgX; that is, inhibitors targeting NOS, sGC, PKG, Kv1.3 and Kv1.6, respectively. Non-treated (NT) retina served as control; DAPI (grey) was used as nuclear counterstain. (a, b) TUNEL assay (red) and quantification of TUNEL-positive cells within the ganglion cell layer (GCL). A significant reduction of TUNEL-positive cells was noticed with all inhibiting drugs, except with 7-NI. (c, d) RBPMS-immunostaining (green) and counts of RBPMS-positive retinal ganglion cells (RGCs). MrgX significantly preserved RGCs. (e, f) Calretinin-immunostaining (cyan) and counts of calretinin-positive amacrine cells in the GCL. ODQ and CN238 treatments appeared to reduce GCL amacrine cell numbers. Quantifications indicate positive cells/mm2; data points in bar graphs represent retinal explants from different animals; testing was performed on n = 5–16 animals. Error bars: mean with SD; treatments were compared to NT using the non-parametric Kruskal–Wallis test followed by the two-stage step-up method of Benjamini, Krieger and Yekutieli as post hoc test; signif- icance levels: *p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001. INL, inner nuclear layer; ONL, outer nuclear layer; scale bar: 50 μm.

Article Snippet: To assess the involvement of NO/cGMP/PKG signalling in RGC degeneration, the following drug treatments were used: 15 μM 7- nitroindazole (7- NI) (cat. no. N7778; Sigma–Aldrich), 0.5 μM 1H- [1,2,4] oxadiazolo [4,3- a] quinoxalin- 1- one (ODQ) (cat. no. O3636; Sigma–Aldrich), 50 μM RP- 8- Br- pMe- PET- cGMPS (CN238; cat. no. P 007; Biolog Life Science Institute GmbH & Co, Bremen, Germany), and 50 nM Margatoxin (MrgX) (cat. no. STM- 325; Alomone Labs, Jerusalem, Israel).

Techniques: Inhibition, Derivative Assay, Control, TUNEL Assay, Immunostaining

Journal: Journal of neurochemistry

Article Title: Inhibition of cGMP-Signalling Rescues Retinal Ganglion Cells From Axotomy-Induced Degeneration.

doi: 10.1111/jnc.70072

Figure Lengend Snippet: FIGURE 5 | Forty-eight-hour inhibition of NO/cGMP/PKG-signalling preserves RGC viability. Retinal explant cultures derived from wild-type (WT) mice at post-natal day 12 were treated for 48 h with 15 μM 7-NI, 0.5 μM ODQ, 50 μM CN238, or 50 nM MrgX, targeting NOS, sGC, PKG, Kv1.3 and Kv1.6, respectively. Non-treated (NT) retina served as control; DAPI (grey) was used as nuclear counterstain. (a, b) TUNEL assay (red) and quan- tification of TUNEL-positive cells within the ganglion cell layer (GCL). A significant reduction of TUNEL-positive cells was noticed with CN238 and MrgX. (c, d) RBPMS-immunostaining (green) and counts of RBPMS-positive retinal ganglion cells (RGCs). ODQ and MrgX significantly preserved RGCs. (e, f) Calretinin-immunostaining (cyan) and counts of calretinin-positive amacrine cells in the GCL. 7-NI and ODQ treatments markedly reduced GCL amacrine cell count. Quantifications indicate positive cells/mm2; data points in bar graphs represent retinal explants from different animals; testing was performed on n = 5–17 animals. Error bars: mean with SD; treatments were compared to NT using the non-parametric Kruskal– Wallis test followed by the two-stage step-up method of Benjamini, Krieger and Yekutieli as post hoc test; significance levels: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. INL, inner nuclear layer; ONL, outer nuclear layer.

Article Snippet: To assess the involvement of NO/cGMP/PKG signalling in RGC degeneration, the following drug treatments were used: 15 μM 7- nitroindazole (7- NI) (cat. no. N7778; Sigma–Aldrich), 0.5 μM 1H- [1,2,4] oxadiazolo [4,3- a] quinoxalin- 1- one (ODQ) (cat. no. O3636; Sigma–Aldrich), 50 μM RP- 8- Br- pMe- PET- cGMPS (CN238; cat. no. P 007; Biolog Life Science Institute GmbH & Co, Bremen, Germany), and 50 nM Margatoxin (MrgX) (cat. no. STM- 325; Alomone Labs, Jerusalem, Israel).

Techniques: Inhibition, Derivative Assay, Control, TUNEL Assay, Immunostaining, Cell Counting