4 9 anhydro tetrodotoxin  (Alomone Labs)

Journal: PLOS Biology

Article Title: Amyloid precursor protein modulates cerebellar Purkinje cell activity and motor function through regulation of Nav1.6 currents

doi: 10.1371/journal.pbio.3003513

Figure Lengend Snippet: (A) Representative traces for whole-cell recording of transient Na + currents ( I Na ) in PCs of App +/+ or App −/− mice. (B) Whole cell I – V curve, peak currents, and activation curves of transient I Na as shown in (A). App +/+ , n = 8; App −/− , n = 14 cells. (C, D) Representative traces and statistical bar-charts showing amplitude of transient I Na ( I NaT ) before or during bath application of (C) Nav1.6-specific blocker 4,9-anhydrotetrodotoxin (4,9-ahTTX, 200 nM) or (D) Nav1.1-specific inhibitor ICA-121431 (ICA, 350 nM). N = 6 cells for each group. The I Na1.6 or I Na1.1 were determined by subtracting the sodium currents recorded during bath application of specific blockers/inhibitors from that before drug application. (E) Representative persistent Na + currents recorded in PCs. The right panels show statistics of the peak persistent currents and voltage potentials eliciting peak persistent Na + currents. App +/+ , n = 6; App −/− , n = 12 cells. (F) Representative traces (the left panel) and statistics for whole-cell recording of resurgent I Na in PCs. The middle panel: Whole cell I – V curve of resurgent I Na ; the right panel: peak resurgent currents. App +/+ , n = 9; App −/− , n = 11 cells. Student t test; * P < 0.05; ** P < 0.01; ns, nonsignificant. Scale bars are indicated in the images. (G) APP co-immunoprecipitated with Nav1.6 but not Nav1.1. HEK293 cells co-expressing Flag-tagged APP and either Nav1.6 or Nav1.1-EGFP were lysed and immunoprecipitated (IP) with anti-Flag affinity beads (Smart-Lifesciences, #SA042001). Immunoblots (IB) were probed for: Nav1.6 (Alomone Labs #ASC-009), EGFP (Roche Applied Science, #11814460001) or Flag (AlpVHHs, #016-303-005). (H) Nav1.6 interacts with full-length APP (holo-APP), but not soluble APPα (sAPPα) and APP intracellular domain (AICD). HEK293 cells co-expressing Nav1.6 and Flag-tagged holo-APP, sAPPα, or AICD were lysed and IPed with anti-Flag affinity beads. IB were probed for Nav1.6 or Flag. Mock transfected controls are shown in the first lane. Input controls (7.5% lysate) shown below/beside corresponding lanes. Data representative of 3 biological replicates. The data underlying this Figure can be found in .

Article Snippet: Following a minimum recovery period of 4 days, mice received microinjections of either saline (0.5 μl/lateral), the Nav1.6 blocker 4,9-ahTTX (200 nM, 0.5 μl/lateral; MCE), or the Nav1.6 positive allosteric modulator PoTX (20 pM; 0.5 μl/lateral, MCE, HY-P10234A) 20 min before behavioral testing.

Techniques: Activation Assay, Immunoprecipitation, Expressing, Western Blot, Transfection

Journal: PLOS Biology

Article Title: Amyloid precursor protein modulates cerebellar Purkinje cell activity and motor function through regulation of Nav1.6 currents

doi: 10.1371/journal.pbio.3003513

Figure Lengend Snippet: (A–D) Electrophysiological characterization of PC spontaneous firing in acute cerebellar coronal slices (300-µm-thick). (A) Representative traces and quantification of spontaneous firing rate and spike amplitude before/during application of 200 nM 4,9-ahTTX in App +/+ mice. n = 7 cells. (B) Representative traces and quantification of spontaneous firing rate and spike amplitude before/during application of 30 nM PoTX (Nav1.6 positive allosteric modulator) in App −/− mice. n = 5 cells/group. (C, D) Representative traces and quantification of spontaneous firing rate and spike amplitude before/during application of 350 nM ICA (Nav1.1 inhibitor) in App +/+ (C) or App −/− (D) mice. Statistics: Paired t test; * P < 0.05; ** P < 0.01; *** P < 0.001 vs. baseline; ns, non-significant. n = 4 cells/group. Scale bars are indicated in the images. (E) Morphology verification of cerebellar cannula placement in a coronal section of mice cerebellum. (F) Open field locomotion: Total distance traveled (m) in App +/+ (4,9-ahTTX/vehicle) and App −/− (PoTX/vehicle) mice. Mice received bilateral microinjections of saline (vehicle), 200 nM 4,9-ahTTX, or 20 pM PoTX into the cerebellar cortex 20 min prior to testing. (G) Forelimb grip strength: maximal force/body weight in same cohorts as (F). Statistics (F, G): App +/+ + vehicle, n = 10; App +/+ + 4,9-ahTTX, n = 8; App −/− + vehicle, n = 8; App −/− + PoTX, n = 9; One-way ANOVA with Tukey post-hoc; * P < 0.05; ** P < 0.01, *** P < 0.001; ns, non-significant. ctrl, control; 4,9-ahTTX, 4,9-anhydrotetrodotoxin; ICA, ICA-121431; PoTX, poneratoxin. The data underlying this Figure can be found in .

Article Snippet: Following a minimum recovery period of 4 days, mice received microinjections of either saline (0.5 μl/lateral), the Nav1.6 blocker 4,9-ahTTX (200 nM, 0.5 μl/lateral; MCE), or the Nav1.6 positive allosteric modulator PoTX (20 pM; 0.5 μl/lateral, MCE, HY-P10234A) 20 min before behavioral testing.

Techniques: Saline, Control

Journal: PLOS Biology

Article Title: Amyloid precursor protein modulates cerebellar Purkinje cell activity and motor function through regulation of Nav1.6 currents

doi: 10.1371/journal.pbio.3003513

Figure Lengend Snippet: (A) Schematic of the viral injection strategy to achieve APP overexpression in PCs and trans- synaptic tracing of downstream neurons in the DCN. (B) Representative example of fluorescence-assisted selection of cells for patch-clamp recordings. Images from an acute cerebellar slice observed in differential interference contrast (DIC, left), or fluorescence mode (EGFP, right). (C) Left panel: Representative traces of sIPSCs in the PC-projected DCN neurons held at 0 mV. Brain slices of App +/+ + vec, App −/− + vec and App −/− + APP mice were recorded; GABA B -receptor antagonist picrotoxin (PTX, 100 μM) was perfused at the end of the recordings to validate IPSCs. Right panel: statistics of IPSCs frequency and amplitude, n = 10 cells for each group. (D) IPSCs recorded from the PC-projected DCN neurons with 4,9-ahTTX (200 nM), n = 8 cells for each group. (E) Miniature IPSCs (mIPSCs) recorded from PC-projected DCN neurons in the presence of TTX (1 μM), n = 8 for each group. Scale bars as presented in the images. A 5-min recording segment from a stable period, defined as commencing at least 10 min after obtaining whole-cell access, was used for statistical analysis. Data are represented as the mean with SEM (C, D IPSCs amplitude) or the median with interquartile range (D IPSCs frequency, E). One-way ANOVA and Kruskal–Wallis test. * P < 0.05; ** P < 0.01; *** P < 0.001; ns, non-significant. The data underlying this Figure can be found in .

Article Snippet: Following a minimum recovery period of 4 days, mice received microinjections of either saline (0.5 μl/lateral), the Nav1.6 blocker 4,9-ahTTX (200 nM, 0.5 μl/lateral; MCE), or the Nav1.6 positive allosteric modulator PoTX (20 pM; 0.5 μl/lateral, MCE, HY-P10234A) 20 min before behavioral testing.

Techniques: Injection, Over Expression, Fluorescence, Selection, Patch Clamp

Journal: bioRxiv

Article Title: Developmental mechanisms contributing to non-linear firing dynamics in spinal motoneurons of the postnatal mouse

doi: 10.64898/2026.03.12.711366

Figure Lengend Snippet: (A) Representative traces of a leak-subtracted and filtered PIC measured in voltage clamp before (black) and after (red) application of the NaV1.6 blocker 4,9 Anhydro-tetrodotoxin (4,9 AH-TTX; 200 nM). (B) 4,9 AH-TTX decreased PIC amplitude and depolarized PIC onset voltage (C) at both pre- (n=10; P7-9) and post- (n=12; P10-13) weight bearing stages. (D) Representative traces of the membrane potential and repetitive firing during a triangular depolarizing current ramp before (black) and after (red) application of 4,9 AH-TTX. (E) 4,9 AH-TTX increased recruitment-derecruitment hysteresis (Delta I) but did not alter the proportion of firing types (F) at pre- and post-weight bearing stages. 4,9 AH-TTX increased the current at firing onset on the ascending limb of the ramp (G) and increased current at firing offset on descending limb of the ramp (H) at both pre-and post-weight bearing stages. Data are presented as individual data points and were analyzed using a 2 factor ANOVA with drug and stage as factors. P values are reported when significant differences were detected. Hedge’s g values indicated small (0.2-0.49), medium (0.5-0.79) and large (>0.8) effect sizes.

Article Snippet: Nifedipine (20 μM; Tocris) was used to assess the contribution of L-type calcium channels, and 4,9 - Anhydro Tetrodotoxin (200 nM; Tocris) to assess the contribution of NaV1.6 channels to PIC and hysteresis.

Techniques: Membrane