Journal: Cell reports

Article Title: Optogenetic Activation of Striatopallidal Neurons Reveals Altered HCN Gating in DYT1 Dystonia.

doi: 10.1016/j.celrep.2020.107644

Figure Lengend Snippet: Figure 7. ConcanamycinA Treatment Modulates HCN1 and HCN2 Expression (A) Cartoon showing the mechanism of concanamycinA (concan) treatment upon vesicular release. (B) sIPSCs recorded in slices of Tor1a+/+ and Tor1a+/DGAG treated with saline (black traces) or with concan (red traces). (C) Boxplot summarizes the effect of concan on firing frequency in Tor1a+/+ and Tor1a+/DGAG. (D) Western blots showing immunoreactivity of HCN1 (untreated N = 5, treated N = 7) and HCN2 (untreated N = 6, treated N = 6) subunits from GPe extracts from slices of animals treated with saline or concan. Protein values were normalized to the corresponding expression of tubulin and actin, respectively. Bar plots illustrate the effect of treatment on HCN subunits level in Tor1a+/+ and Tor1a+/DGAG mice. Levels were normalized to the control (CTRL) level of Tor1a+/+. (E) Cell-attached recordings of autonomous pacemaking activity of GP neurons from Tor1a+/DGAG treated with control solution (black) or concan (red). Boxplots show effect of concan on firing frequency and CV. (F) Whole-cell recordings from GP neurons of Tor1a+/DGAG during optical striatal stimulation (blue lines, dual pulse, 20 Hz) after treatment with concan. Five superimposed traces were aligned on the striatal stimulation. Peri-stimulus time histogram (PSTH) shows firing pause and resetting phase triggered by striatal stimulation. Each dot represents single measurements.

Article Snippet: Protein extracts were stored at 80 C and 10 mg of each sample, in Page LDS sample buffer 4x (Invitrogen) was analyzed to detect: TA (1:1000, Abcam AB_2240792), PV (1:2000, Abcam AB_956206), GAD67 (1:2000, Sigma Aldrich AB_261978), GAD65 (1:1000, Sigma Aldrich AB_10762670), VGAT (1:1000 SySy AB_887871), Gephyrin (1:500, Proteintech AB_2247770), HCN2 (1:1000, Proteintech AB_10951108) HCN1 (1:1000, Proteintech AB_11182929), rabbit anti-D2R (1:1000, Millipore AB_2094980), antiactin (Sigma Aldrich AB_476744) as loading control.

Techniques: Expressing, Saline, Western Blot, Control, Activity Assay

Journal: bioRxiv

Article Title: HCN2 in cholinergic interneurons of the nucleus accumbens mediates reward response

doi: 10.1101/2021.09.21.460269

Figure Lengend Snippet: (A) Representative traces of HCN currents elicited by –150 mV in NAc ChIs from WT and cKO mice before (black, Ctrl) and after DA (1 μM, red). (B) Histograms showing the amplitude of HCN currents at -150 mV in control or DA-treated ChIs from NAc in WT (n = 8 neurons/ 4 mice) and cKO (n = 4 neurons/ 3 mice). Two-way RM ANOVA. Genotype X Treatment F [ , ] = 3.39; P= 0.09. Genotype F [ , ] = 5.90; P= 0.03. Treatment F [ , ] = 4.48; P= 0.06 * p < 0.05 by Bonferroni (C) Effect by aCSF without or with neostigmine (500 nM, arrow) on NAc ACh efflux in WT and HCN2 cKO (n= 7, 8 mice, respectively). (D) Means of ACh efflux in the first four bins with aCSF and last four bins with neostigmine. Two-way ANOVA RM. Genotype X Treatment F [ , ] = 0.8; P> 0.05. Genotype F [ , ] = 1.0; P> 0.05. Treatment F [ , ] = 11.33; P= 0.005. N.S. not significant vs. WT. (E) Line graph of ACh efflux during cocaine (1 µM) and neostigmine (500 nM) dialysis in WT and cKO (n= 7, 8), plotted as percentage of that during neostigmine. Two-way RM ANOVA. Genotype X Time F [3,39] = 0.65; P> 0.05. Genotype F [ , ] = 13.59; P= 0.002. Time F [ , ] = 1.29; P> 0.05. * p = 0.017 by Bonferroni. (F) Dot plot of means. ** p = 0.003 by unpaired t-test.

Article Snippet: Proteins were detected using antibodies for HCN2 (14957) and β-Actin (4970, Cell Signaling Inc., Danvers, MA).

Techniques: Control

Journal: bioRxiv

Article Title: HCN2 in cholinergic interneurons of the nucleus accumbens mediates reward response

doi: 10.1101/2021.09.21.460269

Figure Lengend Snippet: (A) Basal levels of dopamine, DOPAC and HVA in WT and HCN2 cKO mice (n=6, 10 mice, n=6, 7 mice, and n=6, 7 mice, respectively), which were obtained as the average of three stable baseline samples before rewarding stimuli. DA t (10) = 0.1358, p > 0.05; DOPAC, t (7) = 0.7281, p > 0.05; HVA, t (10) = 0.1164, p > 0.05, by Welch’s t-test. (B-D). Responses to exposure to palatable food (B), an unfamiliar male mouse (C), and an unfamiliar female mouse (D) in WT and cKO mice (n = 5, 5 mice, n = 5, 6 mice and n = 5, 5 mice, respectively). The values obtained after rewarding stimuli were compared with the basal levels obtained as the average of three stable baseline samples (100%) using mixed linear models with time as a covariate, and post hoc Bonferroni. * p < 0.05, ** p < 0.01, *** p < 0.001 vs the basal levels. (B) Two Way ANOVA. Group X Time F [7, 64] = 3.04, P= 0.008. Group F [1, 64] = 26.57, P< 0.0001. Time F [7, 64] = 12.13, P< 0.0001. † p = 0.030 †† p = 0.0001 vs WT by Sidak. (C) Two Way ANOVA. Group X Time F [7, 72] = 2.27, P= 0.038. Group F [1, 72] = 1.50, P> 0.05. Time F [7, 72] = 3.59, P= 0.002. † p = 0.033 vs WT by Sidak. (D) Two Way ANOVA. Group X Time F [7, 64] = 3.30, P= 0.005. Group F [1, 64] = 29.48, P< 0.0001. Time F [7, 64] = 6.80, P< 0.0001. † p = 0.026, †† p = 0.001, ††† p < 0.001 vs. WT by Sidak. (E, F) Responses to NAc infusion of cocaine at the concentrations of 1 μM (E) and 10 μM (F) in WT and cKO mice (n= 5, 5 mice and n=5, 5 mice, respectively). (E) Two Way ANOVA. Group X Time F [7, 64] = 0.5866, P> 0.05. Group F [1, 64] = 4.54, P= 0.036. Time F [7, 64] = 10.82, P< 0.0001. (F) Two Way ANOVA. Group X Time interaction F [7, 64] = 0.20, P> 0.05. Group F [1, 64] = 0.61, P> 0.05. Time F [7, 64] = 29.09, P < 0.0001.

Article Snippet: Proteins were detected using antibodies for HCN2 (14957) and β-Actin (4970, Cell Signaling Inc., Danvers, MA).

Techniques:

Journal: bioRxiv

Article Title: HCN2 in cholinergic interneurons of the nucleus accumbens mediates reward response

doi: 10.1101/2021.09.21.460269

Figure Lengend Snippet: (A) Study design and representative microscope images showing the localization of the fiber trace (arrows) and the dLight1.3 GFP in the NAc. AC, anterior commissure. Scale bars, 500 µm. (B) Representative photometric traces between 5s before and 10s after intraperitoneal injection of saline or cocaine (1.5 mg/kg). Arrows indicate injection times, dashed line represents %ΔF/F= 0. (C) Perievent time heatmap of individual samples(top) and histogram of group means (bottom) of saline or cocaine in n= 3 mice / group. (D) Effect of cocaine (1.5 mg/kg) injections in WT (n= 3 mice) on fluorescent peak amplitude (top) and area (AUC, bottom). * p = 0.018, *** p = 0.0003, vs. saline by unpaired t-test. (E) Study design and representative traces of two consecutive injections of cocaine (1.5 mg/kg) in WT and HCN2 cKO mice. (F) Changes in fluorescent peak amplitude and area by consecutive cocaine injections in WT (n= 8 mice) and cKO (n= 6 mice). Amplitude Two Way ANOVA RM, F genotype X treatment [ , ] = 7.39, P= 0.019. ** p = 0.007 between genotypes by Sidak. Area Two Way ANOVA RM, F genotype X treatment [ , ] = 1.053, P> 0.05. F genotype [ , ] = 6.65, P= 0.024. F treatment [ , ] = 0.53, P> 0.05. * p = 0.026 between genotypes by Sidak. (G) Study design and representative traces following injections of cocaine (1.5 mg/kg) followed by saline or donepezil (DON, 0.2 or 2 mg/kg) in WT and HCN2 cKO. (H) Perievent time heatmap and histogram of averages by saline (n= 3, WT), donepezil 0.2 mg/kg (n= 3, WT) or 2 mg/ kg in WT or cKO (n= 4 mice each). (I) Changes in fluorescent peak amplitude and area in WT. One Way ANOVA (amplitude), P= 0.0002, *** p < 0.001 by Tukey. One Way ANOVA (area), P= 0.003, ** p < 0.01 by Tukey. (J) Effect of donepezil (2 mg/kg) injections in WT and cKO (n= 4, 4 mice) on fluorescent peak amplitude and area. ** p = 0.004, *** p < 0.001 by unpaired t-test.

Article Snippet: Proteins were detected using antibodies for HCN2 (14957) and β-Actin (4970, Cell Signaling Inc., Danvers, MA).

Techniques: Microscopy, Injection, Saline

Journal: bioRxiv

Article Title: HCN2 in cholinergic interneurons of the nucleus accumbens mediates reward response

doi: 10.1101/2021.09.21.460269

Figure Lengend Snippet: (A) Distance traveled in WT and cKO during 60 min after cocaine (15 mg/kg, n= 6, 5) or saline (n= 8, 7). Cocaine Two Way ANOVA RM, F genotype X time [5, 45] = 7.68, P< 0.0001, F genotype [ , ] = 14.57, P= 0.004. F time [ , ] = 9.98, P= 0.001. Saline Two Way ANOVA RM, F genotype X time [5, 65] = 4.94, P< 0.0007, F genotype [ , ] = 1.87, P> 0.05. F time [ , ] = 20.92, P< 0.0001. (B) CPP design. Black indicates paradigm phases and their day numbers, and cyan indicates tests names and their day numbers. Acq, acquisition. E, extinction. R, reinstatement. (C) Time spent in each chamber in WT and cKO (n= 8, 5) during the different tests. Two Way ANOVA RM, F genotype X phase [18, 128] = 4.96, P< 0.0001, F genotype [ , ] = 4.75, P= 0.010. F phase [3, 61] = 0.84, P= 0.476. **** p < 0.0001,*** p < 0.001, ** p < 0.01, * p < 0.05 by Tukey. (D) Representative low (left) and high (right) magnification immunohistochemical images following stereotaxic injection of AAV.FLEX.HCN2 in NAc of cKO mouse. Arrows indicate HCN2 labeling in ChI. Scale bars, 500 μm (left) and 50 μm (right). AC, anterior commissure. (E) Time spent in each chamber during CPP acquisition test in cKO mice injected with AAV.FLEX.GFP (n= 9) or AAV.FLEX.HCN2 (n= 11). Two Way ANOVA, F AAV X chamber [ , ] = 5.10, P= 0.037, F AAV [ , ] = 3.73, P> 0.05. F chamber [ , ] = 12.08, P= 0.004. * p = 0.011 by Sidak. (F) Correlation between time in the saline-associated chamber and number of HCN2 immunopositive ChIs in cKO mice injected with AAV.FLEX.HCN2 (n= 6, indicated as full red circles in E). * p = 0.021 by Pearson.

Article Snippet: Proteins were detected using antibodies for HCN2 (14957) and β-Actin (4970, Cell Signaling Inc., Danvers, MA).

Techniques: Saline, Immunohistochemical staining, Injection, Labeling

Journal: bioRxiv

Article Title: HCN2 in cholinergic interneurons of the nucleus accumbens mediates reward response

doi: 10.1101/2021.09.21.460269

Figure Lengend Snippet: (A) Representative immunohistochemical images depicting GFP labeling following stereotaxic injection of AAV.FLEX.GFP in the NAc of ChAT Cre mouse. Scale bars, 500 μm. AC, anterior commissure. (B) Time spent in the saline- and cocaine-associated chambers during CPP acquisition (Acq) extinction (E1) and cocaine-induced reinstatement (R1) tests in ChAT Cre mice injected with AAV.FLEX.GFP (n= 8) or AAV.FLEX.HCN2 (n= 11). Two Way ANOVA, F phase X chamber [6, 64] = 5.25, P= 0.0002. F phase [2, 71] = 4.58, P= 0.014. F chamber [3, 36] = 25.41, P< 0.0001. ** p < 0.01 by Tukey. (C-E) Chemogenetic manipulation of NAc ChIs. (C) Representative immunohistochemical images depicting mCherry labeling following stereotaxic injection of AAV2-hSyn-DIO-mCherry to the NAc of ChAT Cre mouse. Scale bars, 500 μm. AC, anterior commissure; LV, lateral ventricle. (D) Study design. CPP conditioning started three weeks after the stereotaxic AAV injection. CNO (4 mg/kg) was injected to all animals on days 7 and 9 of the conditioning phase, 15 min before cocaine. (E) Time spent in the saline- and cocaine-associated chambers during the CPP acquisition test (Acq) in ChAT Cre mice injected with Gi-DREADD, Gs-DREADD or mCherry control (n= 5 mice in each group). Two Way ANOVA, F genotype X chamber [2, 24] = 7.13, P= 0.0037. F genotype [2, 24] = 5.03, P= 0.015. F chamber [1, 24] = 118.20, P< 0.0001. * p = 0.038, ** p = 0.0012 by Tukey.

Article Snippet: Proteins were detected using antibodies for HCN2 (14957) and β-Actin (4970, Cell Signaling Inc., Danvers, MA).

Techniques: Immunohistochemical staining, Labeling, Injection, Saline, Control

Journal: Molecular Medicine Reports

Article Title: Implantation of engineered conduction tissue in the rat heart

doi: 10.3892/mmr.2019.9933

Figure Lengend Snippet: Immunostaining for connexin-40, connexin-43, HCN2, connexin-45, HCN4 and cTnT in the transplanted ECTs (n=26) and BCSs (n=24). Representative staining for (A) connexin-40, (B) connexin-43, (C) HCN2, (D) connexin-45, (E) HCN4 and (F) cTnT in the rats transplanted with ECTs and BCSs at days 20, 60, and 90 after implantation. The transplanted tissue cells were identified by labeling with CM-Dil (red). Immunostaining for connexin-40, connexin-43, HCN2, connexin-45, HCN4 and cTnT in the implantation site was shown by the secondary antibodies (green). The nuclei were counterstained with DAPI (blue). ECTs, engineered conduction tissues; BCSs, blank collagen sponges.

Article Snippet: Excess solution was discarded and the tissues were incubated with rabbit anti-connexin-40 (cat. no. 36-4900; 1:200; Invitrogen; Thermo Fisher Scientific, Inc.), connexin-43 (cat. no. C6219; 1:150; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), connexin-45 (cat. no. PA5-79311; 1:200; Invitrogen; Thermo Fisher Scientific, Inc.), HCN2 (cat. no. ab19346; 1:200; Abcam), Hcn4 (cat. no. AB5808; 1:200; Chemcon International, Inc.; EMD Millipore) and cTnT (cat. no. ab45932; 1:200; Abcam) at 4°C overnight.

Techniques: Immunostaining, Staining, Labeling

Journal: bioRxiv

Article Title: HCN domain is required for HCN channel expression and couples voltage- and cAMP-dependent gating mechanisms

doi: 10.1101/2020.03.02.973560

Figure Lengend Snippet: HCND is essential for the surface expression of HCN channels. a , Representative Immunoblot of the biotinylated protein fraction (upper half) for untransfected (control), WT and ΔHCND mHCN2 transfected HEK 293 cells probed with HCN2 specific antibodies. Representative immunoblot of the whole-cell lysates (lower half) from untransfected (control), WT and ΔHCND mHCN2 transfected HEK 293 cells probed with α-tubulin antibodies. b , Quantification of unglycosylated channel expression in untransfected (control), WT and ΔHCND mHCN2 transfected HEK 293 cells. n = 3.

Article Snippet: The HCN channels were detected using a rabbit anti-HCN2 primary antibody (Boster, A02804) that recognizes the C-terminal sequence of HCN2 channels and a goat anti-rabbit secondary antibody (Abcam, ab6721).

Techniques: Expressing, Western Blot, Control, Transfection

Journal: bioRxiv

Article Title: HCN domain is required for HCN channel expression and couples voltage- and cAMP-dependent gating mechanisms

doi: 10.1101/2020.03.02.973560

Figure Lengend Snippet: Interactions between the HCND and C-linker/CNBD affect voltage-dependent gating of HCN2 channels. a , Enlarged view of the interaction interface between the HCND and the C-linker/CNBD in HCN1 channels. The interacting residues E436 and Q440 on the C-linker, H517 on the CNBD and R112 on the HCND are shown as sticks. b , Representative currents from E478A_Q482A_H559A (3M) mutant mHCN2 channels. E478, Q482 and H559 correspond to E436, Q440 and H517 in HCN1 channels. c , Averaged conductance-voltage relationship for the currents from WT (black circles, n of 9) and 3M mutant (red circles, n of 10) mHCN2 channels. Lines correspond to the fits with the Boltzmann function with the V 1/2 of −91.3 ± 0.3 mV for WT and −97.1 ± 0.3 mV for the 3M mutant, and s of 13.8 ± 0.2 and 11.9 ± 0.3 for 3M mutant mHCN2 channels.

Article Snippet: The HCN channels were detected using a rabbit anti-HCN2 primary antibody (Boster, A02804) that recognizes the C-terminal sequence of HCN2 channels and a goat anti-rabbit secondary antibody (Abcam, ab6721).

Techniques: Mutagenesis

Journal: bioRxiv

Article Title: HCN domain is required for HCN channel expression and couples voltage- and cAMP-dependent gating mechanisms

doi: 10.1101/2020.03.02.973560

Figure Lengend Snippet: Interactions between the HCND and C-linker/CNBD affect cAMP-dependent gating of HCN2 channels. Representative currents from WT ( a ) and 3M mutant ( b ) mHCN2 channels recorded at −90 mV in the absence (black) and presence (red) of 10 μM cAMP. c , Plots of the percent increase in tail currents versus the cAMP concentration for WT (black circles) and 3M (red circles) mutant mHCN2 channels. The tail currents were recorded at −40 mV after the test pulse to −90 mV. Lines correspond to the fits with the Hill equation with the Kd of 0.1 ± 0.04 μM for WT and 0.06 ± 0.2 μM for 3M mutant mHCN2 channels. n ≥ 5 for each condition. d , Plots of deactivation time constants for tail currents recorded at −40 mV after the test pulse to −90 mV for WT and 3M mutant channels in the presence and absence of 10 μM cAMP, as indicated. n = 6 for WT and n = 5 for 3M mutant channels. The data with and without cAMP for WT and 3M channels were compared using paired Student t-test. The data between WT and 3M mutant channels were compared using unpaired Student t-test. * P < 0.02.

Article Snippet: The HCN channels were detected using a rabbit anti-HCN2 primary antibody (Boster, A02804) that recognizes the C-terminal sequence of HCN2 channels and a goat anti-rabbit secondary antibody (Abcam, ab6721).

Techniques: Mutagenesis, Concentration Assay