Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: The Dome Wall of Bladder Acts as a Pacemaker Site in Detrusor Instability in Rats

doi: 10.12659/MSM.904406

Figure Lengend Snippet: List of primers used in PCR.

Article Snippet: Tissues sections were incubated with primary antibody dilutions made up in 0.2% BSA/0.01 MPBS: goat anti-mouse c-KIT (1: 100, Santa Cruz, Germany), rabbit anti-mouse HCN2 (1: 100, Chemicon, Japan), and rabbit anti-mouse Cx43 (1: 100, Cell Signaling, USA) overnight at 4°C, respectively.

Techniques: Sequencing

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: The Dome Wall of Bladder Acts as a Pacemaker Site in Detrusor Instability in Rats

doi: 10.12659/MSM.904406

Figure Lengend Snippet: The expression of Connexin43. ( A ) HCN2 mRNAs and ( B ) protein expression in DI group. ( C ) Co-expression of HCN2 and c-kit-positive ICCs in the DI detrusor. * P <0.05 vs. other parts in DI group.

Article Snippet: Tissues sections were incubated with primary antibody dilutions made up in 0.2% BSA/0.01 MPBS: goat anti-mouse c-KIT (1: 100, Santa Cruz, Germany), rabbit anti-mouse HCN2 (1: 100, Chemicon, Japan), and rabbit anti-mouse Cx43 (1: 100, Cell Signaling, USA) overnight at 4°C, respectively.

Techniques: Expressing

Journal: British Journal of Pharmacology

Article Title: The hyperpolarization‐activated cyclic nucleotide‐gated 4 channel as a potential anti‐seizure drug target

doi: 10.1111/bph.15088

Figure Lengend Snippet: (A) Cartoon to illustrate: the cre construct in the SLICK‐H mouse line: the position of the lox p sites and the deletion of exon 4 in the HCN4 floxed mouse: and the experimental timeline. (b) Brain section showing typical Thy1 promoter eYFP brain expression in SLICK‐H mice (repeated in n = 3 mice, Scale bar = 600 μm). (c) Body weight of SLICK‐H and cHCN4KO mice 10 days after tamoxifen treatment. Data shown are individual values and mean ± SEM (SLICK‐H n = 23, cHCN4KO n = 25). (d) HCN mRNA expression in the cHCN4KO brain (n = 5) relative to the mean of the SLICK‐H control (n = 5). GAPDH was used as a reference gene for normalization and each run was analysed separately. HCN4 mRNA levels were reduced in the cHCN4KO mouse relative to SLICK‐H controls. The expression of HCN1 and HCN2 mRNA in thalamus and cortex tissue was unchanged. (e) Typical examples of protein expression on Western blots of HCN isoforms (100 mg of total protein per lane) in cHCN4KO (−) and SLICK‐H (+) mouse tissue. Depending on the brain region and the HCN isoform being analysed, the exposure time ranged between 5 and 400 s. HCN1 in thalamus was at background levels. (f) HCN protein expression in the cHCN4KO brain relative to the mean of the SLICK‐H control. Blots were analysed separately by ImageJ. (HCN1 protein expression levels of thalamus‐enriched tissue were too low to analyse). (Thalamus n = 6, Cortex n = 7). Exploratory Western blots of other isoforms showed no differences compared to SLICK‐H controls (Figure S1). F test analysis showed no significant differences in variance. No reduction in HCN4 expression was seen in one mouse. As this was outside confidence levels, it was excluded from analysis. Data expressed as individual values and mean ± SEM. * P < 0.05 versus SLICK‐H mean via Wilcoxon matched pairs signed‐rank test

Article Snippet: Total RNA was prepared by the TRIzol method, purified using the RNeasy mini kit (Qiagen, Hilden, Germany) and assayed for quality and quantity as previously described (Phillips et al., 2014 ). cDNA was prepared using random primers and the high‐fidelity cDNA Synthesis Kit (Roche Holding AG, Basel, Switzerland). qPCR was carried out on 10‐μg cDNA samples, in triplicate to ensure the reliability of single values, using hydrolysis probes HCN1 (Mm00468832_m1), HCN2 (Mm00468538_m1), HCN4 (Mm01176086_m1) and GAPDH (Mm99999915_g1).

Techniques: Construct, Expressing, Control, Western Blot

Journal: CNS Neuroscience & Therapeutics

Article Title: HCN channels in the lateral habenula regulate pain and comorbid depressive‐like behaviors in mice

doi: 10.1111/cns.14831

Figure Lengend Snippet: Four isoforms of HCN channels are enriched in the LHb. (A–D) Representative images of HCN channels (green), NeuN (red) and merged images of the HCN channel, and NeuN in LHb ( n = 9 slices from 3 mice; Up, scale bar, 200 μm, Bottom, scale bar, 20 μm). White arrows indicate the HCN channel and soma co‐labeled neurons and yellow arrows indicate the HCN channel distributing on the non‐neuronal sites. (A) HCN1; (B) HCN2; (C) HCN3; (D) HCN4. (E–H) Percentage of co‐localized NeuN + and HCN + neurons ( n = 9 slices from 3 mice). (E) HCN1 (48.82 ± 3.550%); (F) HCN2 (86.95 ± 2.160%); (G) HCN3 (87.71 ± 1.576%); (H) HCN4 (34.70 ± 4.315%). Data are shown as mean ± SEM.

Article Snippet: Thereafter, the membranes were incubated with rabbit polyclonal anti‐HCN1 (1:1000; Proteintech, USA), HCN2 (1:1000; proteintech, USA), HCN3 (1:500; Alomone Labs, Israel), HCN4 (1:500; Alomone Labs, Israel), and rabbit polyclonal anti‐β‐actin (1:1000; Proteintech, USA) antibodies overnight at 4°C.

Techniques: Labeling

Journal: CNS Neuroscience & Therapeutics

Article Title: HCN channels in the lateral habenula regulate pain and comorbid depressive‐like behaviors in mice

doi: 10.1111/cns.14831

Figure Lengend Snippet: Increased Ih and up‐regulation of HCN2 isoforms of the LHb in SNI 6w mice. (A) Pie charts illustrating the percentage of LHb neurons without Ih and with Ih in Sham and SNI 6w mice (Sham, n = 59 neurons without Ih and 24 neurons with Ih from 36 mice; SNI, n = 47 neurons without Ih and 32 neurons from 17 mice, p = 0.1210, Chi‐square test). (B) Proportion of three firing types of neurons without Ih and neurons with Ih in the LHb of Sham 6w and SNI 6w mice, respectively (without Ih neurons in Sham 6w mice: n = 26 silent neurons, n = 26 tonic neurons, n = 7 burst neurons; without Ih neurons in SNI 6w mice: n = 14 silent neurons, n = 19 tonic neurons, n = 14 burst neurons from 29 mice, p = 0.0051, Chi‐square test; with Ih neurons in Sham 6w mice: n = 14 silent neurons, n = 8 tonic neurons, n = 2 burst neurons; with Ih neurons in SNI 6w mice: n = 14 silent neurons, n = 13 tonic neurons, n = 5 burst neurons from 21 mice, p = 0.1027, Chi‐square test.). (C) Representative traces of Ih currents of LHb neurons in Sham and SNI 6w mice. (D) The I‐V curves showing the amplitude of Ih responding to different voltages of LHb neurons in Sham and SNI 6w mice (Sham, n = 24 neurons from 12 mice; SNI, n = 31 neurons from 15 mice, F (1.046,21.21) = 13.25, p (−70 mV) = 0.0188, p (−80 mV) = 0.0034, p (−90 mV) = 0.0009, p (−100 mV) = 0.0013, p (−110 mV) = 0.0011, p (−120 mV) = 0.0009, p (−130 mV) = 0.0009, two‐way ANOVA). (E) Amplitude of Ih at −130 mV of LHb neurons in Sham and SNI 6w mice (Sham, n = 24 neurons from 12 mice; SNI, n = 31 neurons from 15 mice, p = 0.0049, Mann–Whitney test). (F) Time constant of Ih of LHb neurons in Sham and SNI 6w mice (Sham, n = 24 neurons from 12 mice; SNI, n = 31 neurons from 15 mice, p < 0.0001, Mann–Whitney test). (G) Cm of LHb neurons with Ih in Sham and SNI 6w mice (Sham, n = 24 neurons from 12 mice; SNI, n = 31 neurons from 15 mice, p = 0.0576, Mann–Whitney test). (H) Ih current density versus voltage in Sham and SNI 6w mice (Sham, n = 24 neurons from 12 mice; SNI, n = 31 neurons from 15 mice, F (0.9957,20.20) = 7.095, p (−60 mV) = 0.0282, p (−70 mV) = 0.0252, p (−80 mV) = 0.0140, p (−90 mV) = 0.0106, p (−100 mV) = 0.0142, p (−110 mV) = 0.0147, p (−120 mV) = 0.0133, p (−130 mV) = 0.0141, two‐way ANOVA). (I) Ih current density at −130 mV in Sham and SNI 6w mice (Sham, n = 24 neurons from 12 mice; SNI, n = 31 neurons from 15 mice, p = 0.0019, Mann–Whitney test). (J) Activation curves in Sham and SNI 6w mice. (K) Half‐activation voltage of Ih in Sham and SNI 6w mice (Sham, n = 11 neurons from 8 mice; SNI, n = 16 neurons from 8 mice, p = 0.0079, Mann–Whitney test). (L–O), Representative images (UP) and quantitative analysis (Bottom) of western blot showing the protein level of HCN1‐4 from Sham and SNI mice. (L) HCN1 ( n = 5 for each group, p = 0.3807, unpaired t ‐test). (M) HCN2 ( n = 7 for each group, p = 0.0305, unpaired t ‐test). (N) HCN3 ( n = 6 for each group, p = 0.3416, unpaired t ‐test). (O) HCN4 ( n = 6 for each group, p = 0.9736, unpaired t ‐test). Data are shown as mean ± SEM or Min to Max. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Thereafter, the membranes were incubated with rabbit polyclonal anti‐HCN1 (1:1000; Proteintech, USA), HCN2 (1:1000; proteintech, USA), HCN3 (1:500; Alomone Labs, Israel), HCN4 (1:500; Alomone Labs, Israel), and rabbit polyclonal anti‐β‐actin (1:1000; Proteintech, USA) antibodies overnight at 4°C.

Techniques: MANN-WHITNEY, Activation Assay, Western Blot

Journal: CNS Neuroscience & Therapeutics

Article Title: HCN channels in the lateral habenula regulate pain and comorbid depressive‐like behaviors in mice

doi: 10.1111/cns.14831

Figure Lengend Snippet: Specific knockdown of HCN2 channels decreases the LHb neuronal excitability. (A) Schematic illustration for virus injection (left), representative figure for virus injection in the LHb (right) (scale bar, 100 μm). (B) Representative traces of Ih in the LHb neurons. (C) The I‐–V curves showing the amplitude of Ih responding to different voltages of LHb neurons in virus injection mice (scramble, n = 24 neurons from 8 mice; SNI, n = 13–15 neurons from 8 mice, F (7.256) = 8.625, p (−60 mV) = 0.0309, p (−70 mV) = 0.0004, p (−80 mV) = 0.0002, p (−90 mV) = 0.0003, p (−100 mV) = 0.0002, p (−110 mV) = 0.0004, p (−120 mV) = 0.0003, p (−130 mV) = 0.0011, two‐way ANOVA). (D ) Amplitude of Ih at −130 mV of LHb neurons in scramble and shRNA‐ Hcn2 mice (scramble, n = 24 neurons from 8 mice; SNI, n = 15 neurons from 8 mice, p = 0.0036, Mann–Whitney test). (E) Pie charts showing the percentage of three firing types of LHb neurons in scramble and shRNA‐ Hcn2 mice (scramble, n = 2 burst neurons, 18 tonic neurons and 5 silent neurons from 8 mice; shRNA‐ Hcn2 , n = 1 burst neurons, 20 tonic neurons, and 16 silent neurons from 8 mice, p = 0.1334, Chi‐square test). (F, G) Representative traces (F) and statistical data (G) of spontaneous firing frequency of the LHb tonic‐firing neurons in scramble and shRNA‐ Hcn2 mice (scramble, n = 18 neurons from 8 mice; SNI, n = 20 neurons from 8 mice, p = 0.0002, Mann–Whitney test). H–J, Summary of the passive membrane properties of LHb neurons in scramble and shRNA‐ Hcn2 mice (scramble, n = 25 neurons from 8 mice, shRNA‐ Hcn2 , n = 37 neurons from 8 mice). (H) RMP ( p = 0.3442, Mann–Whitney test). (I) Rheobase current ( p = 0.0001, Mann–Whitney test). (J) Rin ( p = 0.0002, Mann–Whitney test). (K, L) Summary of the active membrane properties of LHb neurons in scramble and shRNA‐ Hcn2 mice (scramble, n = 25 neurons from 8 mice, shRNA‐ Hcn2 , n = 37 neurons from 8 mice). (K) AP threshold ( p = 0.0183, unpaired t ‐test). (L) AP amplitude ( p = 0.0556, unpaired t ‐test). (M, N) Representative traces of the spike of LHb neurons responded to 20 pA, 60 pA and 100 pA in scramble and shRNA‐ Hcn2 mice. (M) Frequency‐current (F–I) curves showing the number of spikes of LHb neurons responded to a series of 1 s current pulses from 0 pA to 100 pA with 20 pA steps in scramble and shRNA‐ Hcn2 mice (scramble, n = 25 neurons from 8 mice, shRNA‐ Hcn2 , n = 37 neurons from 8 mice, F (5,300) = 0.9407, p (0 pA) < 0.0001, p (20 pA) = 0.0008, p (40 pA) = 0.0012, p (60 pA) = 0.0081, p (80 pA) = 0.0201, p (100 pA) = 0.0405, two‐way ANOVA). Data are shown as mean ± SEM or Min to Max. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Thereafter, the membranes were incubated with rabbit polyclonal anti‐HCN1 (1:1000; Proteintech, USA), HCN2 (1:1000; proteintech, USA), HCN3 (1:500; Alomone Labs, Israel), HCN4 (1:500; Alomone Labs, Israel), and rabbit polyclonal anti‐β‐actin (1:1000; Proteintech, USA) antibodies overnight at 4°C.

Techniques: Knockdown, Virus, Injection, shRNA, MANN-WHITNEY, Membrane

Journal: CNS Neuroscience & Therapeutics

Article Title: HCN channels in the lateral habenula regulate pain and comorbid depressive‐like behaviors in mice

doi: 10.1111/cns.14831

Figure Lengend Snippet: Bilateral knockdown of HCN2 channels in the LHb ameliorate pain and comorbid depressive‐like behaviors in SNI 6w mice. (A) Schematic illustration for virus injection and timeline of experimental procedures. (B) Representative and magnified images of AAV‐shRNA‐ Hcn2 ‐GFP or AAV‐shRNA‐scramble‐GFP co‐labeled with NeuN in the LHb. (C) Representative images (UP) and quantitative analysis (Bottom) of Western blot showing the protein level of HCN2 from the LHb of scramble and shRNA‐ Hcn2 mice ( n = 6 for each group, p = 0.0061, unpaired t test). (D) Effects of the HCN2 knockdown on mechanical pain threshold in scramble and shRNA‐ Hcn2 mice (scramble, n = 16 mice, shRNA‐ Hcn2 , n = 11 mice, F (2,59) = 0.1924, p (6w) = 0.0006, Two‐way ANOVA). (E, F) Effects of the HCN2 knockdown on immobility time in FST and TST. (E) FST (scramble, n = 8 mice, shRNA‐ Hcn2 , n = 5 mice, p = 0.0236, unpaired t test). (F) TST (scramble, n = 7 mice, shRNA‐ Hcn2 , n = 5 mice, p = 0.0002, unpaired t test). (G) Representative images of activity tracking of mice in OFT. (H) Left, time in center region with 5 min in OFT (scramble, n = 8 mice, shRNA‐ Hcn2 , n = 6 mice, p = 0.1419, Mann–Whitney test). Right, total distance traveled within 30 min in OFT (scramble, n = 8 mice, shRNA‐ Hcn2 , n = 6 mice, p = 0.3831, unpaired t test). (I) Representative activity tracking of mice in EPM within 5 min. (J) Left: time in open arms (scramble, n = 8 mice, shRNA‐ Hcn2 , n = 8 mice, p = 0.5555, unpaired t test). Right: open arms entries (scramble, n = 8 mice, shRNA‐ Hcn2 , n = 8 mice, p = 0.7914, unpaired t test). Data are shown as mean ± SEM or Min to Max. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Thereafter, the membranes were incubated with rabbit polyclonal anti‐HCN1 (1:1000; Proteintech, USA), HCN2 (1:1000; proteintech, USA), HCN3 (1:500; Alomone Labs, Israel), HCN4 (1:500; Alomone Labs, Israel), and rabbit polyclonal anti‐β‐actin (1:1000; Proteintech, USA) antibodies overnight at 4°C.

Techniques: Knockdown, Virus, Injection, shRNA, Labeling, Western Blot, Activity Assay, MANN-WHITNEY