Journal: Translational Psychiatry

Article Title: Impact of subanesthetic ketamine delivered via AmyloLipid nanovesicle (ALN)-based intranasal system on biobehavioral responses in an animal model of PTSD

doi: 10.1038/s41398-026-03979-7

Figure Lengend Snippet: (1) Representative images of HCN1 immunoreactivity in the hippocampus which represents the difference analysis that used: ( A ) Quantitative morphometric analysis of HCN1 density in CA1 sublayers. B Quantitative morphometric analysis of HCN1 density in the stratum lacunosum moleculare. C Representative images of HCN1 immunoreactivity in the hippocampus across experimental groups. HCN1-positive cells in red; scale bar: 500 µm. D – E Quantitative morphometric analysis of HCN1 density in paraventricular nucleus (PVN). F Representative images of HCN1 immunoreactivity in PVN. HCN1-positive cells in red; scale bar: 500 µm. Data Presentation: ( A, B , and D ), show individual data points with mean ± SEM.

Article Snippet: Sections were fixed with ethanol, rinsed in phosphate-buffered saline (PBS), blocked with normal goat serum in PBS for 2 h, incubated overnight at 4 °C with primary antibodies against BDNF (1:300, ANT-010, Alomone Labs, Israel), NPY (1:100, AB-221145, Abcam, UK), or HCN1 (1:750, APC-056, Alomone Labs, Israel).

Techniques:

Journal: Nature neuroscience

Article Title: Pre-synaptic HCN1 channels regulate excitatory neurotransmission at select cortical synapses by altering Ca V 3.2 Ca 2+ channel activity

doi: 10.1038/nn.2757

Figure Lengend Snippet: a Morphology of typical mouse EC layer III pyramidal neuron. The scale represents 20 μm. b and c Example mEPSC recordings from wildtype (Wt) and HCN1−/− soma and dendrites before and after 15 min bath application of the HCN channel blocker, ZD7288 (ZD; 15 μM). The cumulative probability curves for each recording are displayed above the trace and on the right respectively. The average normalized mEPSCs obtained from the recordings are also presented. The scale shown in the upper panel of b and c applies to all traces within those panels. Graphs depicting the mean (filled squares) and standard error of Wt and HCN1−/− soma and dendritic mEPSC frequency in the absence and presence of ZD7288 are also shown. Open squares illustrate the mEPSC frequency from individual experiments. On the far left of each panel are the amplitude histograms for the total number of mEPSCs obtained from Wt soma (n=7), Wt dendrites (n=5), HCN1−/− soma (n=6) and HCN1−/− dendrites (n=5) with and without ZD7288. Superimposed on the histograms are Gaussian fits to demonstrate the peak amplitude of mEPSCs.

Article Snippet: Three antibodies were used: a monoclonal anti-HCN1 antibody was obtained from NeuroMab (USA); a polyclonal anti-HCN1 antibody was obtained from Alomone Labs (Israel); and a monoclonal Ca V 3.2 antibody was obtained from Neuromab.

Techniques:

Journal: Nature neuroscience

Article Title: Pre-synaptic HCN1 channels regulate excitatory neurotransmission at select cortical synapses by altering Ca V 3.2 Ca 2+ channel activity

doi: 10.1038/nn.2757

Figure Lengend Snippet: a , b Immunoparticles for HCN1 along the extrasynaptic plasma membrane (e.g. arrows) of dendritic spines (s) and shafts (Den) establishing synapses with excitatory terminals (b) in EC layer III. Pre-synaptic HCN1 subunits in excitatory terminals were mainly located in the active zone (b). c Although at lower proportion, immunoparticles for HCN1 were also observed in putative inhibitory axon terminals (it) (e.g. arrowheads), recognised by the shape of synaptic vesicles and the lack of a prominent postsynaptic density in the postsynaptic element, establishing synaptic contact with somata. d Immunoreactivity for HCN1 in EC layer III of HCN1 null mice as revealed at the electron microscopy level. No labelling could be detected in the null mice. ax, axon terminal; s, dendritic spine. e Quantitative analysis on the percentage of immunoparticles (n=2572) for HCN1 at post- and pre-synaptic sites in EC layer III, showed that 72% were localised at postsynaptic sites and 28% at presynaptic sites. Cyt, cytoplasm; N, nucleus. Scale bars: a , c and d , 0.5 μm; b , 0.2 μm.

Article Snippet: Three antibodies were used: a monoclonal anti-HCN1 antibody was obtained from NeuroMab (USA); a polyclonal anti-HCN1 antibody was obtained from Alomone Labs (Israel); and a monoclonal Ca V 3.2 antibody was obtained from Neuromab.

Techniques: Clinical Proteomics, Membrane, Electron Microscopy

Journal: Nature neuroscience

Article Title: Pre-synaptic HCN1 channels regulate excitatory neurotransmission at select cortical synapses by altering Ca V 3.2 Ca 2+ channel activity

doi: 10.1038/nn.2757

Figure Lengend Snippet: a Traces from wildtype (Wt) and HCN1 null neurons respectively in the absence, presence and washout of Ca 2+ - free external solution. The outward holding current values are displayed above the traces. The mean and standard error of 9 individual experiments are shown in the bar graphs on the right. b Time course of the effects of applying Ca 2+ free solution. The average frequency per minute during 5 Wt and 4 HCN−/− recordings was calculated and normalised. c Example recordings from Wt neurons under control conditions, following application of Ca 2+ -free solution and Ca 2+ -free solution containing ZD7288. The outward holding current values at −70 mV are indicated above the traces. The graph on the right depicts the average frequency from 4 experiments. The scale bar shown in (a) applies to all traces within the figure. Significance (p < 0.05) is indicated by an asterisk.

Article Snippet: Three antibodies were used: a monoclonal anti-HCN1 antibody was obtained from NeuroMab (USA); a polyclonal anti-HCN1 antibody was obtained from Alomone Labs (Israel); and a monoclonal Ca V 3.2 antibody was obtained from Neuromab.

Techniques: Control

Journal: Nature neuroscience

Article Title: Pre-synaptic HCN1 channels regulate excitatory neurotransmission at select cortical synapses by altering Ca V 3.2 Ca 2+ channel activity

doi: 10.1038/nn.2757

Figure Lengend Snippet: a and b Recordings obtained in wildtype (Wt) and HCN−/− neurons in the absence and presence of the potent and selective T-type Ca 2+ channel blocker, TTA-A2 (500 nM). In Wt neurons, ZD7288 was additionally applied after TTA-A2 neurons too. The outward holding current at a holding potential of −70 mV are indicated above the traces. The cumulative probability curves for all the recordings are displayed on the right in each panel. The scale bar shown in a and b applies to all traces within a and b respectively. c Bar graph to demonstrate the effects of co-application of ZD7288 (15 μM) and Ca 2+ channel blockers for T- (TTA-A2 (500 nM), TTA-P2 (1 μM) and mibefradil (10 μM; Mib)), P/Q – (ω-agatoxin IVA (100 nM)), N- (ω -conotoxin GVIA (100 nM)), L- (nifedipine (2 mM)) and R- (SNX-482 (200 nM)) type Ca 2+ channels. The effects of treatment with ZD7288 alone are also shown. d Graph to illustrate the effects of the T-type (TTA-A2, TTA-P2 and mibefradil (Mib)) and R-type (SNX-482) Ca 2+ channel blockers on mEPSC frequency in HCN1 null EC layer III neurons. In c and d , the numbers of observations for each treatment are indicated above the individual bars. Asterisks indicate significance at p < 0.05.

Article Snippet: Three antibodies were used: a monoclonal anti-HCN1 antibody was obtained from NeuroMab (USA); a polyclonal anti-HCN1 antibody was obtained from Alomone Labs (Israel); and a monoclonal Ca V 3.2 antibody was obtained from Neuromab.

Techniques:

Journal: Nature neuroscience

Article Title: Pre-synaptic HCN1 channels regulate excitatory neurotransmission at select cortical synapses by altering Ca V 3.2 Ca 2+ channel activity

doi: 10.1038/nn.2757

Figure Lengend Snippet: a Two typical electron micrographs showing immunoreactivity for HCN1 (peroxidase reaction product) and Ca v 3.2 (immuoparticles) in wildtype (Wt) sections as detected using pre-embedding double-labelling methods at EM level. Immunogold particles for Ca V 3.2 were absent in Ca V 3.2 null ( Ca V 3.2−/− ) sections. Axon terminals and dendritic spines are indicated by b and s respectively. Scale bars: 0.2 μm. b Example recordings showing the effects of 15 min bath application of ZD7288 (ZD, 15 μM) on mEPSCs recorded from Ca V 3.2 null ( Ca V 3.2−/− ) EC layer III neurons and Wt neurons. The vertical and horizontal scale bars on the traces represent 10 pA and 2 s respectively. In values above the recordings indicate the outward holding currents at −70 mV. The cumulative probability curves are shown on the right. The average normalised mEPSC traces obtained from the traces before and after ZD7288 application are shown below the recordings. The graphs on the far right depict the individual (open squares) and mean (filled squares) frequency of mEPSCs obtained from Ca V 3.2 Wt (n=5) and null (n=6) in the absence and presence of ZD7288. Significance at p < 0.05 is indicated by an asterisk.

Article Snippet: Three antibodies were used: a monoclonal anti-HCN1 antibody was obtained from NeuroMab (USA); a polyclonal anti-HCN1 antibody was obtained from Alomone Labs (Israel); and a monoclonal Ca V 3.2 antibody was obtained from Neuromab.

Techniques:

Journal: Nature neuroscience

Article Title: Pre-synaptic HCN1 channels regulate excitatory neurotransmission at select cortical synapses by altering Ca V 3.2 Ca 2+ channel activity

doi: 10.1038/nn.2757

Figure Lengend Snippet: a and b Representative traces and their cumulative probability curves illustrating the effects of lowering the external K + concentration from 2.5 mM to 1.75 mM in wildtype (Wt) and HCN1 null neurons. The additional effects of mibefradil (Mib; 10 μM) on Wt mEPSCs in 1.75 mM K + are also presented. The outward holding currents at −70 mV are shown above the traces. An overlay of the average normalised EPSCs from Wt and HCN1−/− neurons under control conditions (black), with 1.75 mM K + (light grey) and in the presence of 1.75 mM K + and mibefradil (dark grey) are also displayed. The average mEPSC frequency (filled squares) as well as the individual frequency values obtained from each experiment (open squares) under these conditions in Wt and HCN1 null neurons are shown on the far right in each panel. The scale bar shown in a and b applies to all traces within a and b respectively. Asterisks represent significance at p < 0.05. c Graph showing the time course of the effects of reducing the K + concentration from 2.5 mM to 1.75 mM in Wt and HCN1−/− neurons. The average mEPSC frequency for each minute of the recording before and after 15 min bath application of 1.75 mM KCl is shown.

Article Snippet: Three antibodies were used: a monoclonal anti-HCN1 antibody was obtained from NeuroMab (USA); a polyclonal anti-HCN1 antibody was obtained from Alomone Labs (Israel); and a monoclonal Ca V 3.2 antibody was obtained from Neuromab.

Techniques: Concentration Assay, Control

Journal: Journal of neurochemistry

Article Title: Activation of rhodopsin gene transcription in cultured retinal precursors of chicken embryo: role of Ca(2+) signaling and hyperpolarization-activated cation channels.

doi: 10.1111/jnc.12624

Figure Lengend Snippet: Fig. 7 Developmental expression of hyper- polarization-activated channels (HCN)1 mRNA and protein in the chicken retina. (a) HCN1 mRNA levels in chicken retina at indicated developmental stages (real-time RT-PCR, mean SEM, n = 3). (b) HCN1 immunodetection on western blots of membrane proteins from chicken retina at indicated developmental stages (Cy3-coupled secondary antibody). Specific HCN1 signal indicated by arrow. Non-specific bands were also detected with the secondary antibody alone (not illustrated). (c, d) HCN1 immunodetection on sections of P1 chick retina (biotinylated secondary antibody, vectastain kit). (c) Low magnification image of the whole retina. (d), Higher magnification of photoreceptors with labeling in rod spherules. OLM, outer limiting membrane; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer; N, nuclei; IS, inner segments. Magnification bars: 50 lm in C; 10 lm in (d).

Article Snippet: The membranes were incubated (overnight, 4°C) with mousemonoclonal anti-HCN1 primary antibody (clone S70-28, Acris Antibodies, Herford, Germany) diluted 1 : 1000 in PBS-Tween, 1% BSA.

Techniques: Expressing, Quantitative RT-PCR, Immunodetection, Western Blot, Membrane, Labeling

Journal: bioRxiv

Article Title: Identification of novel pulmonary vein nodes as generators of ectopic arrhythmic foci for atrial fibrillation: An immuno-histochemical proof

doi: 10.1101/2021.06.10.447815

Figure Lengend Snippet: Immunohisto-chemistry using monoclonal HCN4 and HCN1 antibodies. a. and c. show immunostaining by HCN1 antibody at 40x, while b. (20x) and d. (40x) show immunostaining by HCN4 antibody on PV nodes. HCN (Hyperpolarisation-activated cyclic nucleotide–gated) channels.

Article Snippet: Incubation with primary monoclonal antibodies against HCN1 (Novus Biologicals, nbp1-22450, dilution 1μg/ml) and HCN4 (Abcam, ab32675 dilution 1:100) was performed for 2 hours at room temperature followed by an overnight incubation at 4°C.

Techniques: Immunohistochemistry, Immunostaining

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Downregulation of the HCN1 Channel Alleviates Anxiety‐ and Depression‐Like Behaviors in Mice With Cerebral Ischemia–Reperfusion Injury by Suppressing the NLRP3 Inflammasome

doi: 10.1161/JAHA.124.038263

Figure Lengend Snippet: A , TTC staining shows that hippocampus tissue was damaged and atrophic cerebral ischemia–reperfusion (n=3). White areas in the mouse brain slices represented ischemic infarction area. B , Representative bands of HCN1 and HCN2 in Western blotting analysis (n=4). C , Statistical results showed that the protein expression of HCN1 in the ipsilateral hippocampus of PSD mice was significantly increased (n=4). D , The protein expression of HCN2 in each group did not show significant differences. E , Double immunofluorescence staining showed that the HCN1 channel was predominantly colocalized with neurons, which was rarely colocalized with astrocytes or microglia. HCN1 (red), neuron marker NeuN (green), astrocyte marker GFAP (green), and microglia marker Iba‐1 (green). Scale bar=20 μm. n=3 per group. ** P <0.01 vs sham group, ## P <0.01 vs PSD group. HCN indicates hyperpolarization‐activated cyclic nucleotide‐gated cation channel; MCAO, middle cerebral artery occlusion; PSD, poststroke depression; NeuN (neuron‐specific nuclear protein); Iba‐1 (Ionized calcium binding adaptor molecule 1); GFAP (glial fibrillary acidic protein); and TTC, 2,3,5‐triphenyltetrazolium chloride.

Article Snippet: Antibodies against HCN1 (NBP1‐22450) and HCN2 (NBP2‐12895) were purchased from Novus (Cambridge, MA).

Techniques: Staining, Western Blot, Expressing, Double Immunofluorescence Staining, Marker, Binding Assay

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Downregulation of the HCN1 Channel Alleviates Anxiety‐ and Depression‐Like Behaviors in Mice With Cerebral Ischemia–Reperfusion Injury by Suppressing the NLRP3 Inflammasome

doi: 10.1161/JAHA.124.038263

Figure Lengend Snippet: A , Representative bands of HCN1 protein expression in 3 cell types. B , Selective HCN channel inhibitor ZD7288 treatment (ranging from 2.5 to 50 μmol/L) could significantly enhance the SY5Y cell viability after OGD/R injury by CCK‐8 detection (n=6). ZD7288 was dissolved in DMSO to a concentration of 100 mmol/L and then diluted to the required concentrations with DMEM. In the ZD7288 treatment group, cells were incubated with ZD7288 at the beginning of OGD treatment. C , The intracellular K + concentration was detected by a K + fluorescence probe PBFI‐AM. ZD7288 treatment could ameliorate the reduction of intracellular K + concentration induced by OGD/R (n=6). D , Representative fluorescence images of K + probe PBFI‐AM (blue) staining and bright‐field photograph. Scale bar=50 μm. n=6 per group. ** P <0.01 vs control group, ## P <0.01 vs OGD/R group. CCK‐8 indicates cell counting kit‐8; DMSO, dimethyl sulfoxide; HCN, hyperpolarization‐activated cyclic nucleotide‐gated cation channel; OGD/R, oxygen–glucose deprivation/reoxygenation; and PBFI‐AM (potassium‐binding benzofuran isophthalate acetoxymethylester).

Article Snippet: Antibodies against HCN1 (NBP1‐22450) and HCN2 (NBP2‐12895) were purchased from Novus (Cambridge, MA).

Techniques: Expressing, CCK-8 Assay, Concentration Assay, Incubation, Fluorescence, Staining, Control, Cell Counting, Binding Assay

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Downregulation of the HCN1 Channel Alleviates Anxiety‐ and Depression‐Like Behaviors in Mice With Cerebral Ischemia–Reperfusion Injury by Suppressing the NLRP3 Inflammasome

doi: 10.1161/JAHA.124.038263

Figure Lengend Snippet: A , Schematic diagram of the AAV microinjection site in the hippocampus. The microinjection site in the left hippocampus of mice was AP: −2.3 mm, ML: 2.0 mm, DV: −2.0 mm. B , Representative fluorescence images of AAV (green) microinjection in the hippocampal slices (n=3). Four weeks after virus injection, the mice brains containing the hippocampus were sectioned coronally at 10 μm by a freezing microtome. Green fluorescence was observed on multiple hippocampal slices attached to the microinjection site. C , Representative blots of HCN1 and HCN2 proteins in the left hippocampus. D , Quantitative analysis showed that the protein expression level of HCN1 in the left hippocampus was markedly reduced 4 weeks after AAV‐Hcn1 microinjection (n=3). E , The protein expression level of HCN2 in the hippocampus was not affected by AAV‐Hcn1 microinjection. n=3 per group. ** P <0.01 vs AAV‐Ctrl group. AAV indicates adeno‐associated virus; AP, anteroposterior; DAPI, dihydrochloride; DV, dorsoventral; HCN, hyperpolarization‐activated cyclic nucleotide‐gated cation channel; and ML, mediolateral.

Article Snippet: Antibodies against HCN1 (NBP1‐22450) and HCN2 (NBP2‐12895) were purchased from Novus (Cambridge, MA).

Techniques: Microinjection, Fluorescence, Virus, Injection, Expressing

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Downregulation of the HCN1 Channel Alleviates Anxiety‐ and Depression‐Like Behaviors in Mice With Cerebral Ischemia–Reperfusion Injury by Suppressing the NLRP3 Inflammasome

doi: 10.1161/JAHA.124.038263

Figure Lengend Snippet: A , Schematic diagram for the experimental timeline. B , The mean speed of mice in each group did not show significant differences in the open field test (n=8). C , Compared with the PSD+AAV‐Ctrl group, time spent in the central area was significantly enhanced by microinjection with AAV‐Hcn1 in the ipsilateral hippocampus of PSD mice (n=8). D , Microinjection of AAV‐Hcn1 into the ipsilateral hippocampus of PSD mice tended to improve the preference index in the sucrose preference test compared with the PSD group (n=8). E , In the social contact test, the time spent in the stranger area was markedly increased after AAV‐Hcn1 microinjection in PSD mice. F , Representative graphs of mice trajectories in the open field test (n=8). The red dot was the starting point of the mouse, and the blue dot was the end point of the mouse. G , Representative graphs of mice trajectories in the social contact test. The red dot was the starting point of the mouse, and the blue dot was the end point of the mouse. H , Representative bands of HCN1 and NLRP3 inflammasome‐related proteins in the ipsilateral hippocampus. Compared with the PSD + AAV‐Ctrl group, the protein expressions of HCN1 ( I ), NLRP3 ( J ), and ASC ( K ) in the ipsilateral hippocampus were significantly decreased by AAV‐Hcn1 microinjection in PSD mice (n=4). Meanwhile, AAV‐Hcn1 microinjection tended to decrease the protein expressions of cleaved‐caspase‐1 ( L ) and GSDMD‐N ( M ) in the ipsilateral hippocampus of PSD mice (n=4). N , The protein expression of IL‐1β in the ipsilateral hippocampus was significantly decreased by AAV‐Hcn1 microinjection in PSD mice (n=4). * P <0.05 and ** P <0.01 vs sham + AAV‐Ctrl group, # P <0.05 and ## P <0.01 vs PSD+AAV‐Ctrl group. ASC indicates apoptosis‐associated speck‐like protein containing a caspase activation and recruitment domain; AAV, adeno‐associated virus; GSDMD, gasdermin D; HCN, hyperpolarization‐activated cyclic nucleotide‐gated cation channel; IL‐1β, interleukin‐1β; MCAO, middle cerebral artery occlusion; NLRP3, nucleotide‐binding domain‐like receptor protein 3; and PSD, poststroke depression.

Article Snippet: Antibodies against HCN1 (NBP1‐22450) and HCN2 (NBP2‐12895) were purchased from Novus (Cambridge, MA).

Techniques: Microinjection, Expressing, Activation Assay, Virus, Binding Assay