Journal: bioRxiv

Article Title: Developmental HCN channelopathy results in decreased neural progenitor proliferation and microcephaly in mice

doi: 10.1101/2021.04.24.441237

Figure Lengend Snippet: (A) Quantitative RT-PCR analysis of Hcn1-Hcn4 mRNA expression levels in mouse forebrain normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA (2 -ΔCT , left y axis) and GAPDH and of the respective HCN subunit expression level at E13 (2 -ΔΔCT , right y axis). (B) Immunoblot analysis of total brain lysates with HCN subtype-specific antibodies at embryonic day E15 and at the adult age. (C) Schematic overview of the mouse lines expressing the dominant-negative HCN4 subunit (HCN-DN) in embryonic or adult mouse brains. The embryonic expression was controlled by the EMX1 or NEX promoters driving Cre-recombinase expression, which removed the floxed stop cassette preceding the tetracycline transactivator (tTA2). Postnatal expression of HCN-DN was driven by the CaMKII promoter. In the absence of doxycycline, HCN-DN, which has an N-terminal hemagglutinin (HA) tag, and eGFP were expressed from the bidirectional tetracycline-responsive promoter pBi Tet . (D) Summary statistics from voltage-clamp experiments shown in (E) revealed markedly reduced I h amplitudes in HCN-DN-expressing mutant cells (**, p < 0.01; one-way ANOVA with Tukey’s post-hoc test). (E) Doxycycline and genotype-dependent transgene expression in the brain was tested in in situ hybridization experiments using HCN-DN-specific probes (top row). Traces representative of current-clamp (middle row), or voltage-clamp recordings (bottom row), from CA1 pyramidal neurons in acute slice preparations from adult control (n = 14 cells; 4 mice), mutant (n = 11 cells; 4 mice), and doxycycline (dox)-treated mutant (mutant + dox, n = 9 cells; 2 mice) CaMKIIα-HCN-DN mice. The protocols are shown below the control traces. (F) Immunoblot detection of the HA-tag of the HCN-DN transgene in tissue lysates from mutant EMX1-HCN-DN brains at E10.5, 12.5, 14.5, at postnatal day (P) 0, and at E10.5 in a control brain. (G) eGFP expression in the whole embryo and head of E12.5 EMX1-HCN-DN mutants. Scale bars 1mm. Data are presented as mean ± s.e.m; Experiments in (A, B, F) represent individual examples. Images in (G) are representative of 5 animals.

Article Snippet: A TaqMan-PCR was performed with a commercially available primer and probe mix containing VICTM labeled GAPDH probe (GAPDH: Mm99999915_g1), and FAMTM labeled HCN subunit probe (HCN1: Mm00468832_m1; HCN2: Mm00468538_m; HCN3: Mm01212852_m; HCN4: Mm01176086_m1; Applied Biosystems).

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Dominant Negative Mutation, Mutagenesis, In Situ Hybridization, Control

Journal: Frontiers in Neuroanatomy

Article Title: Transmitter and ion channel profiles of saccadic omnipause neurons and cholinergic non-omnipause neurons in human nucleus raphe interpositus

doi: 10.3389/fnana.2025.1670220

Figure Lengend Snippet: Immunoperoxidase labeling of HCN subunits in human nucleus raphe interpositus (RIP). (A) Consecutive 5 μm thick coronal paraffin sections through RIP stained for HCN1 (left) and HCN2 (right). HCN1 (left) is labeled only in OPNs, which are ensheathed by perineuronal nets (red arrows) and lacks completely in cholinergic non-OPNs (green arrows) (middle). On the other hand, HCN2 (right) immunolabeling is found in both neuron groups. (B) HCN4 immunolabeling (right) is ubiquitously found both in OPNs (red arrows) in cholinergic non-OPNs (green arrows). Scale bar represents 50 μm.

Article Snippet: HCN4 subunit was detected with polyclonal guinea pig antibody (Cat #: APC-052-GP (formerly AGP-004); RRID: AB_2340957; Alomone Labs, Jerusalem, Israel), which recognizes an intracellular epitope at the N-terminus, corresponding to amino acid residues 119–155 of human HCN4.

Techniques: Labeling, Staining, Immunolabeling