Journal: Frontiers in Neuroscience

Article Title: Blockade of TRPC Channels Limits Cholinergic-Driven Hyperexcitability and Seizure Susceptibility After Traumatic Brain Injury

doi: 10.3389/fnins.2021.681144

Figure Lengend Snippet: Normalized fold change in TRPC1/4/5 mRNA (2 –ΔΔ CT ) in TBI mice compared to sham.

Article Snippet: Parietal cortex or dorsal hippocampus brain sections (−1.3 to −2.3 mm posterior to bregma) were probed with a TRPC4 mouse monoclonal antibody (1:1,000, Rockland #200-301-G54, RRID:AB_2611251 ) at 1:500 dilution and fluorescein isothiocyanate (FITC) donkey antimouse IgG (1:250, Jackson ImmunoResearch #715-095-150, RRID:AB_2340792 ).

Techniques:

Journal: Frontiers in Neuroscience

Article Title: Blockade of TRPC Channels Limits Cholinergic-Driven Hyperexcitability and Seizure Susceptibility After Traumatic Brain Injury

doi: 10.3389/fnins.2021.681144

Figure Lengend Snippet: Cell-type specific TRPC1, TRPC4, and TRPC5 channel upregulation in the hippocampus and cortex after CCI-TBI. (A,B) Representative Western immunoblots using (A) TRPC4 (∼120 kDa) and (B) TRPC5 (∼110 kDa) antibodies in sham and TBI cortex and hippocampus. Blots were normalized to β-actin protein (42 kDa) as loading control. (C,D) Summarized data for Western blot quantification of TRPC4 ( C , n = 7–13 animals per group) and TRPC5 ( D , n = 5–7 animals per group) from microdissected brain regions in mice 7 days after TBI. (E,F) Shown are summarized plots of percent difference in TRPC4 (E) and TRPC5 (F) protein between ipsilateral and contralateral hemispheres of microdissected regions from data in Panels (C,D) . All data bars represent the mean ± SEM. * p < 0.05 vs. sham of same subregion. † p < 0.05 vs. contralateral hemisphere of same subregion.

Article Snippet: Parietal cortex or dorsal hippocampus brain sections (−1.3 to −2.3 mm posterior to bregma) were probed with a TRPC4 mouse monoclonal antibody (1:1,000, Rockland #200-301-G54, RRID:AB_2611251 ) at 1:500 dilution and fluorescein isothiocyanate (FITC) donkey antimouse IgG (1:250, Jackson ImmunoResearch #715-095-150, RRID:AB_2340792 ).

Techniques: Western Blot, Control

Journal: Frontiers in Neuroscience

Article Title: Blockade of TRPC Channels Limits Cholinergic-Driven Hyperexcitability and Seizure Susceptibility After Traumatic Brain Injury

doi: 10.3389/fnins.2021.681144

Figure Lengend Snippet: Normalized TRPC4/TRPC5 protein in 7-day sham and TBI mice.

Article Snippet: Parietal cortex or dorsal hippocampus brain sections (−1.3 to −2.3 mm posterior to bregma) were probed with a TRPC4 mouse monoclonal antibody (1:1,000, Rockland #200-301-G54, RRID:AB_2611251 ) at 1:500 dilution and fluorescein isothiocyanate (FITC) donkey antimouse IgG (1:250, Jackson ImmunoResearch #715-095-150, RRID:AB_2340792 ).

Techniques:

Journal: Frontiers in Neuroscience

Article Title: Blockade of TRPC Channels Limits Cholinergic-Driven Hyperexcitability and Seizure Susceptibility After Traumatic Brain Injury

doi: 10.3389/fnins.2021.681144

Figure Lengend Snippet: Surges in neuronal activity following CCI-TBI are TRPC4/TRPC5-mediated. (A) Representative images of parietal cortex from sham and TBI TRAP mice that were administered 4-OHT at t = 12 h before procedure. Prefix “ c ” denotes contralateral, prefix “ i ” denotes ipsilateral. Red = cFos-tdTomato; blue = DAPI. (B) Representative images of hippocampal subregions from sham and TBI TRAP mice that were administered 4-OHT at t = 12 h before procedure. (C) Summarized quantification of cFos+ neuron density (neurons/0.1 mm 3 ) in sham and TBI TRAP mice activated at the time of TBI, as in Panels (A,B) . (D) Representative images taken from sham mice, TBI mice, and TBI mice also administered M084 (10 mg/kg) (TBI + M084) that were administered 4-OHT t = 7 days after procedure. (E) Summarized quantification of cFos+ neurons in sham, TBI, and TBI + M084 mice 7 days after procedure, as in Panel (D) . All data bars represent the mean ± SEM. * p < 0.05 vs. sham. # p < 0.05 vs. TBI cDG. † p < 0.05 vs. TBI of same region. Scale bars: 100 μm.

Article Snippet: Parietal cortex or dorsal hippocampus brain sections (−1.3 to −2.3 mm posterior to bregma) were probed with a TRPC4 mouse monoclonal antibody (1:1,000, Rockland #200-301-G54, RRID:AB_2611251 ) at 1:500 dilution and fluorescein isothiocyanate (FITC) donkey antimouse IgG (1:250, Jackson ImmunoResearch #715-095-150, RRID:AB_2340792 ).

Techniques: Activity Assay

Journal: Frontiers in Neuroscience

Article Title: Blockade of TRPC Channels Limits Cholinergic-Driven Hyperexcitability and Seizure Susceptibility After Traumatic Brain Injury

doi: 10.3389/fnins.2021.681144

Figure Lengend Snippet: TRPC4/TRPC5 channel activation artificially prolongs Ca 2+ influx in DGGCs after CCI-TBI. (A) Cumulative probability distribution of the peak amplitude of GCaMP6f fluorescence (ΔF/F) for each DGGC from sham (control) and iTBI slices during EA (1 μM, red) or EA + M084 (10 μM, gray) application. (B) Cumulative probability distribution of the Ca 2+ influx duration (in seconds) for each DGGC from control and iTBI slices during EA or EA + M084 application. (C,D) Histogram population distribution of control DGGC Ca 2+ influx events according to peak amplitude (C) and Ca 2+ event duration (D) . (E,F) Histogram population distribution of iTBI DGGC Ca 2+ influx events according to peak amplitude (E) and Ca 2+ event duration (F) . (G) Summarized means of peak fluorescence from data as in Panel (A) . (H) Summarized means of Ca 2+ influx duration from data as in Panel (B) . Red = EA alone, gray = EA + M084. All data bars represent the mean ± SEM. * p < 0.05 vs. EA alone from same procedure condition, † p < 0.05 vs. control of same drug condition.

Article Snippet: Parietal cortex or dorsal hippocampus brain sections (−1.3 to −2.3 mm posterior to bregma) were probed with a TRPC4 mouse monoclonal antibody (1:1,000, Rockland #200-301-G54, RRID:AB_2611251 ) at 1:500 dilution and fluorescein isothiocyanate (FITC) donkey antimouse IgG (1:250, Jackson ImmunoResearch #715-095-150, RRID:AB_2340792 ).

Techniques: Activation Assay, Fluorescence, Control

Journal: Scientific Reports

Article Title: Muscarinic receptor-induced contractions of the detrusor are impaired in TRPC4 deficient mice

doi: 10.1038/s41598-018-27617-5

Figure Lengend Snippet: ( A , B ) Representative tension recordings of spontaneous activity in detrusor strips taken from wild-type ( A ) and TRPC4 −/− mice ( B ). ( C,D ) Summary bar charts showing mean amplitude ( C ) and frequency ( D ) of spontaneous detrusor contractions in wild-type (WT, open bars) and TRPC4 −/− mice (filled bars). ( E , F ) Representative KCl (60 mM)-evoked contractions in wild-type ( E ) and TRPC4 −/− ( F ) detrusor strips. ( G ) Summary bar chart plotting mean KCl-evoked contraction amplitude, (measured as area under curve, mN.s) in wild-type (WT, open bars) and TRPC4 −/− detrusor strips. Error bars represent SEM. **** denotes p < 0.0001.

Article Snippet: Cells were incubated in anti-TRPC4 primary antibody (Alomone Labs, Israel) overnight at 4 °C.

Techniques: Activity Assay

Journal: Scientific Reports

Article Title: Muscarinic receptor-induced contractions of the detrusor are impaired in TRPC4 deficient mice

doi: 10.1038/s41598-018-27617-5

Figure Lengend Snippet: ( A ) Representative traces showing EFS-evoked contractions (1, 2, 4 & 8 Hz, respectively) in TRPC4 −/− detrusor strips before, during the presence of α,β-methylene ATP (10 µM) and during the presence of α,β-methylene ATP plus 4-DAMP (100 nM), respectively. ( B ) Summary bar charts showing mean amplitude of EFS-evoked contractions in TRPC4 −/− detrusor strips before (open bars), during the presence of α,β-methylene ATP (grey bars) and α,β-methylene ATP plus 4-DAMP (black bars). Error bars represent SEM. ** denotes p < 0.01 and *** denotes p < 0.001, respectively.

Article Snippet: Cells were incubated in anti-TRPC4 primary antibody (Alomone Labs, Israel) overnight at 4 °C.

Techniques:

Journal: Scientific Reports

Article Title: Muscarinic receptor-induced contractions of the detrusor are impaired in TRPC4 deficient mice

doi: 10.1038/s41598-018-27617-5

Figure Lengend Snippet: ( A,B ) Representative recordings showing EFS-evoked contractions (1, 2, 4 & 8 Hz, respectively) in wild-type ( A ) and TRPC4 −/− detrusor strips ( B ). ( C,D ) Summary bar charts showing mean amplitude of EFS-evoked contractions in wild-type (WT, open bars) and TRPC4 −/− preparations (filled bars), raw data ( C ) and normalised to the amplitude of a KCl-induced contraction in the same muscle strip ( D ). Error bars represent SEM. **** denotes p < 0.0001.

Article Snippet: Cells were incubated in anti-TRPC4 primary antibody (Alomone Labs, Israel) overnight at 4 °C.

Techniques: Stripping Membranes

Journal: Scientific Reports

Article Title: Muscarinic receptor-induced contractions of the detrusor are impaired in TRPC4 deficient mice

doi: 10.1038/s41598-018-27617-5

Figure Lengend Snippet: ( A,B ) Representative carbachol (CCh, 300 nM)-induced contraction of wild-type ( A ) and TRPC4 −/− detrusor strips ( B ). ( C,D ) Summary bar charts showing mean amplitude of CCh-induced contractions in wild-type (WT, open bars) and TRPC4 −/− preparations (filled bars), raw data ( C ) and normalised to the amplitude of a KCl-induced contraction in the same muscle strip ( D ). Error bars represent SEM. *** denotes p < 0.001 and ****p < 0.0001.

Article Snippet: Cells were incubated in anti-TRPC4 primary antibody (Alomone Labs, Israel) overnight at 4 °C.

Techniques: Stripping Membranes

Journal: Scientific Reports

Article Title: Muscarinic receptor-induced contractions of the detrusor are impaired in TRPC4 deficient mice

doi: 10.1038/s41598-018-27617-5

Figure Lengend Snippet: ( A,B ) Representative neostigmine (1 μM)-induced contraction of wild-type ( A ) and TRPC4 −/− detrusor strips ( B ). Solid red lines in A&B represent single exponential fits (τ). ( C,D ) Summary bar charts showing mean amplitude of neostigmine-evoked contractions ( C ) and rate of contraction (1/τ) ( D ) in wild-type (WT, open bars) and TRPC4 −/− preparations (filled bars). Error bars represent SEM. ** denotes p < 0.01 and ****p < 0.0001, respectively.

Article Snippet: Cells were incubated in anti-TRPC4 primary antibody (Alomone Labs, Israel) overnight at 4 °C.

Techniques:

Journal: Scientific Reports

Article Title: Muscarinic receptor-induced contractions of the detrusor are impaired in TRPC4 deficient mice

doi: 10.1038/s41598-018-27617-5

Figure Lengend Snippet: ( A,B ) Representative recording ( A ) and summary bar chart ( B ) showing the effect of ML204 (10 μM) on EFS-evoked contractions (2, 4 & 8 Hz, respectively) in TRPC4 −/− detrusor strips. ( C,D ) Representative recording ( C ) and summary bar chart ( D ) showing the effect of ML204 (10 μM) on CCh-induced contractions in TRPC4 −/− detrusor strips. Error bars represent SEM. **** denotes p < 0.0001. Note that the duration of the EFS stimulus in these experiments was five minutes, in contrast to two minutes for the experiments shown in Fig. .

Article Snippet: Cells were incubated in anti-TRPC4 primary antibody (Alomone Labs, Israel) overnight at 4 °C.

Techniques:

Journal: Scientific Reports

Article Title: Muscarinic receptor-induced contractions of the detrusor are impaired in TRPC4 deficient mice

doi: 10.1038/s41598-018-27617-5

Figure Lengend Snippet: ( A,B ) Representative recordings showing EFS-evoked contractions (1, 2, 4 & 8 Hz, respectively) in wild-type ( A ) and TRPC4 −/− detrusor strips ( B ), before and during the presence of the TRPC5 inhibitor, clemizole hydrochloride. ( C ) Summary bar charts showing mean amplitude of EFS-evoked contractions in the presence of clemizole hydrochloride (normalised to the control response at each frequency) in wild-type (open bars) and TRPC4 −/− preparations (filled bars). Error bars represent SEM. *** denotes p < 0.001 and **** denotes p < 0.0001.

Article Snippet: Cells were incubated in anti-TRPC4 primary antibody (Alomone Labs, Israel) overnight at 4 °C.

Techniques:

Journal: Scientific Reports

Article Title: Muscarinic receptor-induced contractions of the detrusor are impaired in TRPC4 deficient mice

doi: 10.1038/s41598-018-27617-5

Figure Lengend Snippet: ( A – D ) Summary quantitative PCR data showing mean expression levels of TRPC1 ( A ), TRPC3 ( B ), TRPC5 ( C ) and TRPC6 ( D ) (relative to β-actin) in wild-type (WT, open bars) and TRPC4 −/− preparations (filled bars). ( E – G ) Representative confocal photomicrographs confirming that TRPC4 immunoreactivity was present in freshly isolated smooth muscle cells from wild-type detrusor, but not TRPC4 −/− preparations.

Article Snippet: Cells were incubated in anti-TRPC4 primary antibody (Alomone Labs, Israel) overnight at 4 °C.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Isolation