Figure 3.

Subcellular localization of HCN1 channels in developing hippocampus. (A, B) Low-magnification photographs demonstrate that HCN1 channels are localized in the dendritic field of CA1 pyramidal cells as early as P2 (A) found in both stratum radiatum as well as in stratum lacunosum-moleculare (slm,B). On P11, but not later, HCN1 signal is also observed in the medial molecular layer (mml) of DG, where the signal is likely within the axons of the perforant path (Bender, Kretz, and others 2005). (C, D) Dual labeling of HCN1 (red) and the dendritic marker MAP-2 (green) on P2 (C) and P11 (D) reveals that most HCN1 coresides with the dendritic marker. In contrast, dual labeling of HCN1 with presynaptic markers of afferents to the CA1 dendrites failed to demonstrate a presynaptic location of HCN1 in VGLUT1-containing terminals, whereas a modest colocalization of the HCN1 immunoreactivity with GABAergic terminals (GAD65) was found on P11 but not on P2 (D). (E) By P18, the HCN1 distribution pattern strongly resembles the mature pattern (F) characterized by a robust expression of HCN1 channels in the distal dendritic field of CA1 (slm) and by pronounced HCN1 signal in CA3 (arrows in B,E,F). (G, H) HCN1 signal in the pyramidal cell layer (particularly in CA3) is mainly attributable to expression of the channels in axons of basket cells. Dual labeling of HCN1 (red) and PV (green in G) or CCK (green in H) reveals that HCN1 frequently localizes to axonal terminals of PV-expressing basket cells (G) but never to terminals that contain CCK (H). sp, stratum pyramidale; so, stratum oriens; asterisks demarcate the hippocampal fissure. Confocal microscopy virtual slice thicknesses:(A,B,E,F) = 15 μm (surface scanning), (C, D) (MAP-2-HCN1) and (G, H) = 1 μm. (D) GAD65 and VGLUT dual labeling with HCN1 = 0.5 μm. Scale bar:(A,B,E,F) = 900 μm; (C) = 27 μm (left panels), 13.5 μm (right panel); (D) = 27 μm (left and right panels), 13.5 μm (middle panel); (G, H) = 40 μm (left panels), 20 μm (right panel).