rat hcn1  (Alomone Labs)

Journal: Experimental Animals

Article Title: Involvement of NMDA receptors in tremor expression in Aspa / Hcn1 double-knockout rats

doi: 10.1538/expanim.20-0025

Figure Lengend Snippet: NAA, NAAG, and glutamate contents in the brain regions of Aspa / Hcn1 double-knockout rats. A, NAA concentrations in various brain regions. B, NAAG concentrations in various brain regions. C, Glutamate concentrations in various brain regions. Grey and white bars represent wild-type (F344) and Aspa em34Kyo / Hcn1 em1Kyo double-knockout rats, respectively. Each bar represents the mean ± SEM. of 6 animals. *; P <0.05, **; P <0.01, ***; P <0.001. Hip; hippocampus, Cx; cerebral cortex, Md; medulla oblongata, Cb; cerebellum, Th; thalamus, Sp; spinal cord.

Article Snippet: F344- Aspa em34Kyo [ ] and F344- Hcn1 em1Kyo [ ] rats were supplied by the National BioResource Project-Rat [ ].

Techniques: Double Knockout

Journal: Experimental Animals

Article Title: Involvement of NMDA receptors in tremor expression in Aspa / Hcn1 double-knockout rats

doi: 10.1538/expanim.20-0025

Figure Lengend Snippet: An NMDA receptor antagonist suppressed tremor in Aspa / Hcn1 double-knockout rats. The total duration (left) and intensity (right) of body tremor in Aspa em34Kyo / Hcn1 em1Kyo double-knockout rats given LY341495 (A), NBQX (B), or d-CPPene (C). d-CPPene, an NMDA receptor antagonist, significantly suppressed both the duration and intensity of tremor throughout the observation period (60 min). *; P <0.05, **; P <0.01, ***; P <0.001.

Article Snippet: F344- Aspa em34Kyo [ ] and F344- Hcn1 em1Kyo [ ] rats were supplied by the National BioResource Project-Rat [ ].

Techniques: Double Knockout

Journal: Experimental Animals

Article Title: Involvement of NMDA receptors in tremor expression in Aspa / Hcn1 double-knockout rats

doi: 10.1538/expanim.20-0025

Figure Lengend Snippet: An NR2C/NR2D NMDA receptor antagonist suppressed tremor in Aspa / Hcn1 double-knockout rats. The total duration (left) and intensity (right) of body tremor in Aspa em34Kyo / Hcn1 em1Kyo double-knockout rats given Ro 25-6981 (A) or cis-PPDA (B). cis-PPDA, an antagonist of NMDA receptors containing the NR2C and NR2D subunits, significantly suppressed both the duration and intensity of tremor throughout the observation period (60 min). *; P <0.05, **; P <0.001.

Article Snippet: F344- Aspa em34Kyo [ ] and F344- Hcn1 em1Kyo [ ] rats were supplied by the National BioResource Project-Rat [ ].

Techniques: Double Knockout

Journal: The Journals of Gerontology Series A: Biological Sciences and Medical Sciences

Article Title: HCN as a Mediator of Urinary Homeostasis: Age-Associated Changes in Expression and Function in Adrenergic Detrusor Relaxation

doi: 10.1093/gerona/gly137

Figure Lengend Snippet: (A) HCN mRNA bladder expression and age. Hcn-1 mRNA levels measured by RT-qPCR were approximately 40% lower in WT old female bladders when compared to WT young controls, with no apparent change in Hcn-2 expression. Data were normalized to gene expression of young mice to give comparable fold changes by geometric averaging of multiple internal control genes. Comparisons made by t-test on log10 transformed data, *p ≤ .05 (n = 3 per group). Hcn-1 is decreased in old compared to young WT female bladders (p = .008), whereas no changes in Hcn-2 were observed. Bars show means (SD). (B) HCN bladder protein expression and age. (B1) Western blot analysis confirms the presence of higher levels of HCN1 as opposed to HCN2 protein in the mouse bladder. HCN1 protein expression declines with age. (B2) Ponceau S staining of the PVDF membrane prior to staining with HCN1 or HCN2 antibodies verifies equal protein loading of the young and old samples. (B3) HCN1 KO mice are missing a band slightly above the 100kDa molecular weight marker, corresponding to mouse HCN1 in bladder (marked with an asterisk in YWT). YWT = young wild-type female bladder; YKO = young knockout female bladder; OWT = Old wild-type female bladder; RT-qPCR = quantitative real-time polymerase chain reaction; HCN = Hyperpolarization activated, cyclic nucleotide gated channel.

Article Snippet: Membranes were incubated in 1:1,000 rat anti-HCN1 or 1:250 rat anti-HCN2 antibody in TBST+5% nonfat milk at 4°C (EMD Millipore, MAB5594, and MAB5596, respectively).

Techniques: Expressing, Quantitative RT-PCR, Gene Expression, Control, Transformation Assay, Western Blot, Staining, Membrane, Molecular Weight, Marker, Knock-Out, Real-time Polymerase Chain Reaction

Journal: The Journals of Gerontology Series A: Biological Sciences and Medical Sciences

Article Title: HCN as a Mediator of Urinary Homeostasis: Age-Associated Changes in Expression and Function in Adrenergic Detrusor Relaxation

doi: 10.1093/gerona/gly137

Figure Lengend Snippet: RNAScope to localize Hcn in bladder. Representative light microscopy images of RNAScope from two young wild-type (WT) female mice and two old WT female mice. Each dot is reflective of one RNA molecule. Panels A and B are probed for Hcn1 and Hcn2 mRNA. Panel A shows 20× images, showing differential distribution of Hcn1 mRNA to detrusor, and more diffuse but more sparse distribution of Hcn2 mRNA. Panel B shows 40× images providing further detail of detrusor and mucosal distributions of Hcn1 and Hcn2 mRNA. Panel C shows positive and negative control images. Online version in color.

Article Snippet: Membranes were incubated in 1:1,000 rat anti-HCN1 or 1:250 rat anti-HCN2 antibody in TBST+5% nonfat milk at 4°C (EMD Millipore, MAB5594, and MAB5596, respectively).

Techniques: RNAscope, Light Microscopy, Negative Control

Journal: The Journal of Clinical Investigation

Article Title: Regularizing firing patterns of rat subthalamic neurons ameliorates parkinsonian motor deficits

doi: 10.1172/JCI99986

Figure Lengend Snippet: (A–D) LV-Hcn1-shRNA, LV-Hcn2-shRNA, LV-Hcn3-shRNA, and LV-Hcn4-shRNA effectively downregulated the expression of Hcn1, Hcn2, Hcn3, and Hcn4 mRNAs and proteins (n = 6 from 6 independent experiments) in the STN. (E) LV-Hcn2-oe upregulated the expression of Hcn2 mRNAs and proteins (n = 6 from 6 independent experiments). (F–H) Effects of downregulation and overexpression of the HCN2 channel in the STN on motor deficits of turning behavior (F, n = 12), adhesive-removal test (G, n = 10), and locomotor footprints (H, n = 10) in PD rats with sham operation, saline injection, and histamine injection. Downregulation of the HCN2 channel significantly increased the apomorphine-induced turnings, prolonged contralesional adhesive-removal time, and shortened bilateral stride length, whereas downregulation of the HCN1, HCN3, or HCN4 channel had no effect on these motor deficits. Only the downregulation of HCN2 rather than the HCN1, HCN3, or HCN4 channel blocked the amelioration of turnings, removal time, and stride length of PD rats induced by microinjection of histamine into the STN. Overexpression of the HCN2 channel in STN not only decreased the turnings, reduced removal time, and enlarged bilateral stride length of PD rats, but also improved the histamine-induced amelioration in these motor behaviors. Data are represented as mean ± SEM. ***P < 0.001, 1-way (A–E) or 2-way ANOVA (F–H) with Newman-Keuls post hoc test.

Article Snippet: To downregulate the expression of individual HCN channel subtypes in the STN, 4 shRNA lentiviruses for Hcn ( Hcn1 to Hcn4 ) targeting rat HCN mRNA were constructed (Genechem).

Techniques: shRNA, Expressing, Over Expression, Adhesive, Saline, Injection, Microinjection

Journal: The Journal of comparative neurology

Article Title: Dopamine D1 receptor expression is bipolar cell type-specific in the mouse retina

doi: 10.1002/cne.23932

Figure Lengend Snippet: Primary antibodies

Article Snippet: HCN1 clone N70/28 , Fusion protein amino acids 778- 910 at C-terminus of rat HCN1 , NeuroMab, 75–110, Mouse monoclonal , AB_2115181 , 1:200.

Techniques: Purification, Recombinant

Journal: The Journal of comparative neurology

Article Title: Dopamine D1 receptor expression is bipolar cell type-specific in the mouse retina

doi: 10.1002/cne.23932

Figure Lengend Snippet: Evidence that HCN1 antibody labels type 5 cells and colocalized with tdTomato. (A) HCN1 signaling was observed in the IPL near the outer edge of the ON ChAT band. (B) Verification of CtBP antibody. CtBP2/Ribeye antibodies labeled ribbon synapses in mouse retinal sections. Puncta of ribbon synapses in photoreceptors are labeled in the OPL, while ribbon synapses in bipolar cell terminals are labeled throughout the IPL. Both anti-CtBP2 from Santa Cruz (B1) and anti-Ribeye from Synaptic Systems (B2; Table 1) showed similar pattern that are also consistent with the immunostaining reported in mouse retina with different Ribeye antibody (Figure 1; (tom Dieck et al., 2005). (C) HCN1 signaling overlaps with a ribbon synapse marker, CtBP, showing labeled puncta. (D) High power view showing HCN1 staining and CtBP puncta from the dashed box illustrated in B. All panels are single digital section images (0.3 µm thick). From a through d, images were taken from one side to another side of the HCN1 strong staining every 1 µm. Many CtBP puncta colocalized with HCN1, indicating that HCN1 colocalized with bipolar cell terminals. (E) Strong HCN1 staining also colocalized with tdTomato staining immediately next to the ON ChAT band. (F) 2D cross-correlation coefficient analysis of CtBP puncta and HCN1 signaling. The majority of CtBP2 puncta colocalized with HCN1 staining (n=25 puncta with HCN1 colocalization; upper). A 90° rotation of the HCN1 image reduced the correlation coefficient (lower panel) indicating that HCN1/CtBP2 colocalization was not random. (G) 2D cross-correlation coefficient analysis of tdTomato and HCN1 signaling. Correlation was detected (n=20 images; upper panel), whereas no correlation was observed when one channel was rotated 90° (lower panel). (H) CtBP puncta were expressed as the proportion of puncta colocalized with HCN1 or not colocalized with HCN1 (1217 puncta were analyzed from 12 images, n=3 animals). (I) CtBP puncta were expressed as the proportion of puncta colocalized with tdTomato or not colocalized with tdTomato. (955 puncta were analyzed from 9 images, n=3 animals).

Article Snippet: HCN1 clone N70/28 , Fusion protein amino acids 778- 910 at C-terminus of rat HCN1 , NeuroMab, 75–110, Mouse monoclonal , AB_2115181 , 1:200.

Techniques: Labeling, Immunostaining, Marker, Staining

Journal: Neuroscience

Article Title: Morphological and physiological analysis of type-5 and other bipolar cells in the Mouse Retina

doi: 10.1016/j.neuroscience.2015.12.016

Figure Lengend Snippet: HCN1 immunoreactivity and type-5 cell terminals. (A) Western blot of mouse anti-HCN1 antibody against mouse cortex. The arrow shows the reported molecular weight for HCN1 at ~100 kDa. (B) HCN1 labeled the entire IPL. Bright immunoreactivity was observed right above the ON ChAT band where type-5 cells terminate. (B′) The bright HCN1 and ChAT staining was magnified. The bright HCN1 signaling was colocalized with CtBP2 antibody, a ribbon synapse marker, indicating that the bright HCN1 colocalized with bipolar cell terminals. (C) Neurobiotin was injected to a Type-5-1 cell, which was not colocalized with HCN1 bright signaling (n=5). (D) Neurobiotin was injected to a Type-5-2 cell, which was colocalized with HCN1 bright signaling (n=5). (E) Neurobiotin was injected to a XBC. HCN1 bright signaling also colocalized with its terminals (n=8). Immunohistochemical studies shown in (B) and (B′) were conducted using thin sections (14 μm thick). Panels in (C) through (E) were performed in thick slice sections (250 μm thick). Lower panels of (C) through (E) were captured images of a few digital sections (~1 μm thick). (F) 2D cross-correlation coefficient analysis was performed between type 5-2 axon terminals and HCN1 staining using single digital sections (0.3 μm thick). Terminals of type 5-2 cells were colocalized with HCN1 strong staining (n=20 images, left). The colocalization was not random because when the HCN1 image was rotated 180º, the correlation coefficient was reduced (right panel). (G) 2D cross-correlation coefficient analysis also revealed that XBC axon terminals and HCN1 staining colocalized (n=10 images, left). The colocalization was not random because when the HCN1 image was rotated 180º, the correlation coefficient was reduced (right panel).

Article Snippet: HCN1 clone N70/28 , Rat HCN1 amino acids 778–910 , NeuroMab, 75–110 Mouse monoclonal , AB_2115181 , 1:200.

Techniques: Western Blot, Molecular Weight, Labeling, Staining, Marker, Injection, Immunohistochemical staining