hypericin and hyperforin  (Cayman Chemical)

Journal: CNS Neuroscience & Therapeutics

Article Title: Congenital Hypothyroidism Dysregulates TRPC6 to Mediate Abnormal Dendritic Spine Growth of Hippocampal Neurons

doi: 10.1111/cns.70618

Figure Lengend Snippet: Analysis of TRPC6 expression in the hippocampal dentate gyrus of CH pups. (A) Schematic diagram of inducing CH rat pups. (B) Gene network of DEGs in hippocampal tissues of CH rat pups at P1, P7 and P21. (C) Heatmap of trpc6 , camkIV , creb and bdnf selected from DEGs in the hippocampus tissues of P1, P7, and P21 rat pups following CH. (D, E) Co‐localization of TRPC6 with NeuN + neurons in the hippocampus of CH rats at different stages by immunostaining. C1/7/21 indicates control, and D1/7/21 indicates CH. The rectangles in (D) indicate the magnification in (E). Scale bars: 200 μm in (D); 50 μm in (E). (F) The transcriptional levels of TRPC6 in the hippocampus of P1/7/21 rat pups by RT‐PCR. Quantities were normalized to endogenous gapdh . Experiments were performed in triplicates. Data are expressed as mean ± SEM, * p < 0.05, ** p < 0.01, and *** p < 0.001, two‐way analysis of variance followed by Tukey's post hoc test.

Article Snippet: For drug treatment, the TRPC6 activator hyperforin (0.3 μM—1 μM, MCE, Cat# HY‐116330A), TR antagonist 1 (5 μM, MCE, Cat# HY‐111443), or T3 (5 nM, MCE, Cat# HY‐A0070A) was added to the neuron culture medium for the desired experiments.

Techniques: Expressing, Immunostaining, Control, Reverse Transcription Polymerase Chain Reaction

Journal: CNS Neuroscience & Therapeutics

Article Title: Congenital Hypothyroidism Dysregulates TRPC6 to Mediate Abnormal Dendritic Spine Growth of Hippocampal Neurons

doi: 10.1111/cns.70618

Figure Lengend Snippet: Knockdown of TRPC6 affects dendritic spine growth of hippocampal neurons. (A) Immunostaining of TRPC6 in primary hippocampal neurons. (B) Detection of siRNA transfection efficiency by using 150 nM 5‐Cy3 oligonucleotide for 24 h. (C) Analysis of interference efficiency of three TRPC6 siRNA oligonucleotides using RT‐PCR, and the siRNA2 ( trpc6‐si2 ) was chosen for the subsequent experiments. Quantities were normalized to endogenous gapdh . Data are expressed as mean ± SEM, n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001, oneway analysis of variance followed by Dunnett's post hoc test. (D) Immunostaining of primary hippocampal granule neurons with MAP2 to detect dendritic spine density following trpc6‐si2 knockdown for 24 h. The rectangle indicates region magnified. (E) Statistical analysis of (D). Spine density was analyzed in triplicates each 15 fields. Scale bar: 10 μm in (A) and (D); 100 μm in (B). (F, G) Transcriptional analysis of camkIV and creb in primary hippocampal granule neurons after trpc6 knockdown for 24 h. Quantities were normalized to endogenous gapdh . Six pups of each group were used to isolate hippocampal neurons. Data are expressed as mean ± SEM, n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001, two‐tailed unpaired Student's t ‐test.

Article Snippet: For drug treatment, the TRPC6 activator hyperforin (0.3 μM—1 μM, MCE, Cat# HY‐116330A), TR antagonist 1 (5 μM, MCE, Cat# HY‐111443), or T3 (5 nM, MCE, Cat# HY‐A0070A) was added to the neuron culture medium for the desired experiments.

Techniques: Knockdown, Immunostaining, Transfection, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test

Journal: CNS Neuroscience & Therapeutics

Article Title: Congenital Hypothyroidism Dysregulates TRPC6 to Mediate Abnormal Dendritic Spine Growth of Hippocampal Neurons

doi: 10.1111/cns.70618

Figure Lengend Snippet: Effect of TRPC6 activator on the dendritic spine density of hippocampal DGCs. (A, B) Primary DGCs were cultured for 5 d, followed by incubation with 0.3 μM TRPC6 activator hyperforin (Hyp) for 24 h. The dendritic spine density was observed by immunofluorescence with MAP2 antibody. The rectangle indicates region magnified. Spine density was analyzed in triplicates each 15 fields. (C‐E) Transcriptional analysis of camkIV , creb and bdnf after treatment of the neurons with 1 μM hyperforin for 1 h. Quantities were normalized to endogenous gapdh . Scale bar: 10 μm in (A). Experiments were performed in triplicates. Data are expressed as mean ± SEM, * p < 0.05 and ** p < 0.01, two‐tailed unpaired Student's t ‐test.

Article Snippet: For drug treatment, the TRPC6 activator hyperforin (0.3 μM—1 μM, MCE, Cat# HY‐116330A), TR antagonist 1 (5 μM, MCE, Cat# HY‐111443), or T3 (5 nM, MCE, Cat# HY‐A0070A) was added to the neuron culture medium for the desired experiments.

Techniques: Cell Culture, Incubation, Immunofluorescence, Two Tailed Test

Journal: CNS Neuroscience & Therapeutics

Article Title: Congenital Hypothyroidism Dysregulates TRPC6 to Mediate Abnormal Dendritic Spine Growth of Hippocampal Neurons

doi: 10.1111/cns.70618

Figure Lengend Snippet: Effects of TRPC6 agonist on the number of neurons in DG and the growth of dendritic spines in CH pups. CH pups at 21 d were injected intraperitoneally with 2.5 mg/kg hyperforin for 7 d. (A) The morphology and number of neurons in the DG of the subjects were observed by Nissl's staining. The rectangle indicates the DG region magnified. Scale bars: 1000 μm; 50 μm or 20 μm in magnification. (B) Statistical analysis of (A), 15 fields in each section were subjected to the statistical analysis of neuron number. Data are expressed as mean ± SEM, n = 6, ** p < 0.01, two‐tailed unpaired Student's t ‐test. (C) Detection of the dendritic spine density of DGCs in the DG of the subjects following injection of hyperforin by Golgi staining. The rectangle indicates region magnified. Scale bar: 25 μm in (C). (D) Statistical analysis of (C). Spine density was analyzed in triplicates each 15 fields. Data are expressed as mean ± SEM, n = 3, * p < 0.05, ** p < 0.01, and *** p < 0.001, oneway analysis of variance followed by Dunnett's post hoc test.

Article Snippet: For drug treatment, the TRPC6 activator hyperforin (0.3 μM—1 μM, MCE, Cat# HY‐116330A), TR antagonist 1 (5 μM, MCE, Cat# HY‐111443), or T3 (5 nM, MCE, Cat# HY‐A0070A) was added to the neuron culture medium for the desired experiments.

Techniques: Injection, Staining, Two Tailed Test

Journal: CNS Neuroscience & Therapeutics

Article Title: Congenital Hypothyroidism Dysregulates TRPC6 to Mediate Abnormal Dendritic Spine Growth of Hippocampal Neurons

doi: 10.1111/cns.70618

Figure Lengend Snippet: Effect of TRPC6 activation on calcium transients in hippocampal neurons of rat pups. (A) Calcium transients in primary DGCs were stimulated with 10 μM hyperforin, and the cells were stained with Fluo‐8 AM (green) to indicate intracellular Ca 2+ level. Scale bar: 10 μm in (A). (B) The fluorescence intensity changes in (A). (C) Analysis of fluorescence intensity using neuronal calcium imaging within 232 s. (D) Detection of neuronal calcium transients following knockdown of trpc6 for 24 h and then treatment with hyperforin. Scale bar: 10 μm in (D). (E) The fluorescence intensity changes in (D). (F) Analysis of fluorescence intensity using neuronal calcium imaging following knockdown of trpc6 for 24 h and then treatment with hyperforin. The fluorescence intensity was quantified from 10 cells. Time‐lapse was collected every 8 s for a total of 232 s. Experiments were performed in triplicates. Data are expressed as mean ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001, two‐tailed unpaired Student's t ‐test.

Article Snippet: For drug treatment, the TRPC6 activator hyperforin (0.3 μM—1 μM, MCE, Cat# HY‐116330A), TR antagonist 1 (5 μM, MCE, Cat# HY‐111443), or T3 (5 nM, MCE, Cat# HY‐A0070A) was added to the neuron culture medium for the desired experiments.

Techniques: Activation Assay, Staining, Fluorescence, Imaging, Knockdown, Two Tailed Test

Journal: CNS Neuroscience & Therapeutics

Article Title: Congenital Hypothyroidism Dysregulates TRPC6 to Mediate Abnormal Dendritic Spine Growth of Hippocampal Neurons

doi: 10.1111/cns.70618

Figure Lengend Snippet: TRPC6 modulates the CaMKIV/CREB pathway in hippocampal tissue of CH pups and neurons. (A) Western blot analysis of CaMKIV, Egr‐3 and BDNF protein levels at hippocampal tissues from CH pups. CH pups at 21 d were injected intraperitoneally with 2.5 mg/kg hyperforin for 7 d. (B‐D) The quantitative analysis of (A). Quantities were normalized to endogenous GAPDH. Data are expressed as mean ± SEM, n = 3, * p < 0.05, ** p < 0.01, oneway analysis of variance followed by Tukey's post hoc test. (E) Western blot analysis of CREB in cytosolic and nuclear fraction of primary hippocampal neurons after transfection with trpc6‐si2 for 24 h, followed by stimulation of 5 nM T3 for 1 h. Quantities were normalized to endogenous GAPDH (cytoplasm) or Lamin B1 (nucleus). (F) The quantitative analysis of (E). Data are expressed as mean ± SEM, n = 3, * p < 0.05, two‐tailed unpaired Student's t ‐test. (G) Immunostaining of p‐CREB following the primary DGCs knockdown of trpc6 for 24 h and then treatment with 5 nM T3 for 1 h. The rectangle indicates region magnified. Scale bar: 100 μm, and 20 μm in magnification. (H) The analysis of fluorescence intensity in (G), n = 20, **** p < 0.0001, Mann‐Qhitney test.

Article Snippet: For drug treatment, the TRPC6 activator hyperforin (0.3 μM—1 μM, MCE, Cat# HY‐116330A), TR antagonist 1 (5 μM, MCE, Cat# HY‐111443), or T3 (5 nM, MCE, Cat# HY‐A0070A) was added to the neuron culture medium for the desired experiments.

Techniques: Western Blot, Injection, Transfection, Two Tailed Test, Immunostaining, Knockdown, Fluorescence

Journal: CNS Neuroscience & Therapeutics

Article Title: Congenital Hypothyroidism Dysregulates TRPC6 to Mediate Abnormal Dendritic Spine Growth of Hippocampal Neurons

doi: 10.1111/cns.70618

Figure Lengend Snippet: Effects of TRs inhibitors on the expression of TRPC6 and dendritic spine growth in hippocampal neurons. (A‐C) Primary hippocampal neurons were treated with T3 (5 nM) for 1 h, followed by addition of TR antagonist 1 (5 μM) for 12 h, RT‐PCR analysis of trpc6 , creb and bdnf . Quantities were normalized to endogenous gapdh . (D, E) Morphological and density changes of dendritic spines after treatment of hippocampal neurons with TR antagonist 1 (5 μM) for 12 h. The rectangle indicates region magnified. Scale bar: 10 μm in (D). Data are expressed as mean ± SEM, n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001, two‐tailed unpaired Student's t ‐test.

Article Snippet: For drug treatment, the TRPC6 activator hyperforin (0.3 μM—1 μM, MCE, Cat# HY‐116330A), TR antagonist 1 (5 μM, MCE, Cat# HY‐111443), or T3 (5 nM, MCE, Cat# HY‐A0070A) was added to the neuron culture medium for the desired experiments.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test

Journal: CNS Neuroscience & Therapeutics

Article Title: Congenital Hypothyroidism Dysregulates TRPC6 to Mediate Abnormal Dendritic Spine Growth of Hippocampal Neurons

doi: 10.1111/cns.70618

Figure Lengend Snippet: Effects of TRPC6 agonist hyperforin on body weight and NOR of CH pups. (A) Schematic diagram of behavioral test for the CH rat pups. CH pups at 21 d were intraperitoneally injected with hyperforin (2.5 mg/kg), and behavioral tests were performed at 7 d later. (B, C) Body weight of CH pups with or without treatment of TRPC6 agonist. (D‐F) Locomotor trajectories of pups in the NOR test. (G) Recognition index of pups in the NOR test. Data are expressed as mean ± SEM, n = 9, * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001, oneway analysis of variance followed by Tukey's post hoc test.

Article Snippet: For drug treatment, the TRPC6 activator hyperforin (0.3 μM—1 μM, MCE, Cat# HY‐116330A), TR antagonist 1 (5 μM, MCE, Cat# HY‐111443), or T3 (5 nM, MCE, Cat# HY‐A0070A) was added to the neuron culture medium for the desired experiments.

Techniques: Injection

Journal: CNS Neuroscience & Therapeutics

Article Title: Congenital Hypothyroidism Dysregulates TRPC6 to Mediate Abnormal Dendritic Spine Growth of Hippocampal Neurons

doi: 10.1111/cns.70618

Figure Lengend Snippet: Effect of TRPC6 agonist hyperforin on cognitive function of CH pups. (A‐C) MWM experiment to test the locomotor trajectories of the rat pups. (D) Escape latency of the pups in the probing trial of MWM test. (E) Number of target island crossings in the probing trial. (F) Times staying in island in the probing trial. (G) Number of target quadrant crossings in the probing trial. (H) Average swimming speed in the probing trial. (I) Total swimming distance in the probing trial. Data are expressed as mean ± SEM, n = 9, * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001, oneway analysis of variance followed by Tukey's post hoc test or Kruskal‐Wallis test.

Article Snippet: For drug treatment, the TRPC6 activator hyperforin (0.3 μM—1 μM, MCE, Cat# HY‐116330A), TR antagonist 1 (5 μM, MCE, Cat# HY‐111443), or T3 (5 nM, MCE, Cat# HY‐A0070A) was added to the neuron culture medium for the desired experiments.

Techniques: