Fig 2: Quantitative polymerase chain reaction (qPCR) ?Ct analysis for hyperpolarization-activated cyclic nucleotide-gated channel (HCN channel) subunits in proprioceptive DRG cells. (A) Box and whisker plot show group data comparing ?Ct for GFP expression in GFP+ (green) and GFP- (blue) samples (normalized to ß actin expression). The low GFP ?Ct in GFP+, but not GFP-, confirm high expression in this sample and the reliability of this sampling approach. (B) Box whisker plots plot shows group data comparing ?Ct values for HCN1–4 in GFP+ (green) and GFP- (blue) samples. The expression of all HCN subunits was significantly different between GFP+ and GFP- groups (where *p < 0.05). HCN 2 had the greatest overall expression in both samples whilst HCN 3 and 4 were both expressed at much lower levels. Expression levels were highest for HCN 2, then HCN 1 and HCN 4 in the GFP+ sample and these values all exceeded corresponding values in the GFP- samples. In contrast, HCN 3 expression was lower in the GFP+ sample vs. GFP- cells.

Fig 3: Expression of HCN1 in PV-positive cells of the hippocampus at P56. (A, E, I): The strongest labeling for HCN1 was observed in the sl-m and alveus of the CA. Note also that its expression was gradually stronger in the sp, and sr of the CA2 (E) and CA3 (I) compared to the CA1 (A). (A, B, D): PV-positive cells located in the sp of the CA1 did not show HCN1 immunolabeling. On the other hand, in the CA2, CA3, and hilus of the DG most PV-immunopositive cells were labeled with HCN1. A-B, D: yellow arrows indicate PV-positive/HCN1-negative cells. E-F, H-J, L-N, P: white arrows indicate PV-positive/HCN1-positive cells. C, G, K, O: DAPI staining. Scale bars = 20 µm.

Fig 4: HCN channels are present on GABAergic terminals in the mPFC. (A) Low-magnification confocal images showing double stained with HCN channels (red) and GAD65 (green), a GABAergic terminal marker. The squares illustrate the cells in layers 5–6 of the mPFC. Scale bar: 40 µm. (B,C) Single-plane confocal images showing the HCN1-ir (B1), HCN2-ir (B2), HCN4-ir (B3), and GAD65-ir (C1-C3) at high magnification. GAD65-ir appears in punctuate structures distributed in the neuropil, as well as around unlabeled pyramidal cell soma (C1-C3). (D) Merging of the paired images (B1 and C1), (B2 and C2), and (B3 and C3) shows that the puncta of GAD65-ir surround the cell bodies of HCN1-ir (B1), HCN2-ir (B2), and HCN4-ir (B3) cells. Partially overlapping areas of red (HCN) and green (GAD65) profiles showing yellow. The arrowheads indicate double-labeled cells. Scale bar: 20 µm.

Fig 5: Localization pattern of HCN channel isoforms in the glomerular layer. a–d Low magnification of triple-labeling for HCN1 (green, RTQ-7C3), HCN3 (red, HCN3 Shigemoto), and HCN4 (blue, PG2-1A4) in the external plexiform layer (EPL) and glomerular layer (GL). e–o Example of various populations of PG- and SA-like cells. Three cell populations express only a single HCN isoform (overlays in h, k, o: green arrowhead HCN1, blue barred arrow HCN2, dark blue arrowhead HCN4). This is demonstrated with triple-labeling (e–h) for HCN1 (green HCN1a), HCN2 (blue HCN2a), and HCN4 (red PG2-1A4), double-labeling (i–k) for HCN2 (green QQA-1A6) and HCN3 (red HCN3 Shigemoto), and triple-labeling (l–o) for HCN1 (green HCN1a), HCN3 (red HCN3a4), and HCN4 (blue PG2-1A4). The cell population expressing only HCN4 is also visible in s (dark blue arrowheads). Co-localization of either HCN1 (green HCN1a) and HCN3 (red HCN3 Shigemoto) or HCN1 (green HCN1a) and HCN4 (blue PG2-1A4) in two additional cell populations (orange arrowhead, red arrowhead, respectively) is shown in o. p–s Three ET-like cell populations with different repertoires of HCN isoforms (white arrow HCN1, HCN3, and HCN4, orange arrow HCN1 and HCN3, purple arrow HCN3 and HCN4) can be detected in the triple-staining for HCN1 (green HCN1a), HCN3 (red HCN3 Shigemoto), and HCN4 (blue PG2-1A4). PG-like cells expressing only HCN4 are indicated (dark blue arrowheads). From these stainings, it is not apparent which of the cell populations identified in later experiments (Figs. 3, 4, 5) are stained. However, by using the results of all staining performed, one can unequivocally identify the cell types here. For convenience, cell populations in this and the following figures are labeled with the uniform color and arrow code shown in Table 2. Glomeruli are indicated by an asterisk and delineated by a dashed white line. Bars 50 µm (d), 10 µm (h, k, o, s)