Localization of HCN1 channels to presynaptic compartments: novel plasticity that may contribute to hippocampal maturation

Abstract

Increasing evidence supports roles for the current mediated by hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, I(h), in hippocampal maturation and specifically in the evolving changes of intrinsic properties as well as network responses of hippocampal neurons. Here, we describe a novel developmental plasticity of HCN channel expression in axonal and presynaptic compartments: HCN1 channels were localized to axon terminals of the perforant path (the major hippocampal afferent pathway) of immature rats, where they modulated synaptic efficacy. However, presynaptic expression and functions of the channels disappeared with maturation. This was a result of altered channel transport to the axons, because HCN1 mRNA and protein levels in entorhinal cortex neurons, where the perforant path axons originate, were stable through adulthood. Blocking action potential firing in vitro increased presynaptic expression of HCN1 channels in the perforant path, suggesting that network activity contributed to regulating this expression. These findings support a novel developmentally regulated axonal transport of functional ion channels and suggest a role for HCN1 channel-mediated presynaptic I(h) in hippocampal maturation.

Figure 1.

Age-specific and pathway-selective localization of HCN1 channels in the ML of the hippocampal DG. a, Horizontal section through the hippocampal formation of an 11-d-old (P11) rat: HCN1 immunoreactivity is found in the ML at this age (arrows); note also the characteristic channel distribution in the dendritic fields of CA1 and subiculum (Sub) (Lörincz et al., 2002; Brewster et al., 2007). b, A higher magnification view of the DG in a demonstrates that HCN1 expression in the ML is confined to the mML (arrows), where perforant path fibers deriving from medial (med) EC terminate. Little HCN1 signal was detected in the outer (oML) and inner molecular layer (iML), the termination zones of entorhinal afferents from lateral (lat) EC and DG commissural fibers, respectively. c–f, HCN1 expression in the ML decreases with maturation and is less in suprapyramidal mML of a P15 rat (c, arrows) compared with the signal on P11 (b, arrows). Signal is further reduced on P25 (d, arrow) and is virtually absent on P90 (e, f, arrows). Note that on P15 and P25, HCN1 signal is stronger in the less mature infrapyramidal blade of DG (c, d, arrowheads) compared with the maturationally advanced suprapyramidal blade (c, d, arrows). f, Higher magnification of the DG in e. GCL, Granule cell layer; Hil, hilus; PaSub, parasubiculum; PrSub, presubiculum. Asterisks denote hippocampal fissure. Scale bar: (in f) a, e, 400 μm; c, d, 150 μm; b, f, 50 μm.

Figure 2.

HCN1, but not HCN2 or HCN4, channels localize to the ML in immature rats. a, b, Antigen specificity of the HCN1 antisera used in this study was tested with tissue from HCN1-deficient (HCN1^−/−) mice: no HCN1 immunosignal was detectable in hippocampal sections from these mice (b), whereas the HCN1 pattern in the hippocampus from wild-type (WT; P11) mice resembled that in rats of the corresponding age (a; compare with Fig. 1a). c, d, Antisera specific for the HCN2 (c) or HCN4 (d) isoform revealed characteristic expression patterns of these isoforms in the P11 rat hippocampus, but no immunoreactivity was detected in the ML (arrows), suggesting that HCN channel expression in the ML is specific for the HCN1 isoform. Hil, Hilus; GCL, granule cell layer; Sub, subiculum; sp, stratum pyramidale. Asterisks denote hippocampal fissure. Arrows in a and b indicate ML. Dashed lines in c and d delineate borders of the GCL. Scale bar (in d): a, b, 350 μm; c, d, 100 μm.

Figure 3.

HCN1 channel expression in the ML requires an intact perforant path. a, As in P11 rat hippocampus in vivo (Fig. 1a), an HCN1-immunoreactive band resided in the ML (arrows) of combined entorhinohippocampal slice cultures that were explanted on P4 and cultured for 7 d. b, Transection of the perforant path in these cultures (after 4 d in vitro; dashed lines) completely abolished HCN1 immunoreactivity in the ML (arrows), suggesting that perforant path integrity is required for the expression of HCN1 channels in the ML. c, d, Higher magnification views of the DG in a and b, respectively. GCL, Granule cell layer; Hil, hilus. Asterisks denote hippocampal fissure. Scale bar (in d): a, c, 350 μm; b, d, 50 μm.

Figure 4.

HCN1 channels localize to perforant path axon terminals. a, b, EM analyses using pre-embedding silver intensified immunogold (a) and postembbeding immunogold techniques (b) identified HCN1 channels predominately in perforant path terminals (arrows) in the middle molecular layer of immature rats (P10). To a lesser extent, HCN1 channels were also detected in postsynaptic structures of dentate granule cells (arrowheads). Scale bar (in b): a, 0.4 μm; b, 0.2 μm. c, Whole-cell patch-clamp recordings illustrate that a 2 s duration hyperpolarizing pulse from −60 to −130 mV elicited little I_h in the DG granule cells of immature rats. Note that mean amplitude and current density of I_h in granule cells (n = 12; 4 rats) reached <5% of the values detected in CA1 pyramidal cells (n = 4; 3 rats) at the same age. These data suggest that there is minimal expression of functional HCN channels in postsynaptic elements of the perforant path-granule cell synapse.

Figure 5.

I_h channels in immature perforant path terminals are functional and contribute to synaptic properties. a, b, Repetitive stimulation (10 stimuli) of MPP in slices from early postnatal rats (P10–P14) resulted in STD (filled circles) of fEPSP recorded in the mML. Application of I_h channel blockers (open circles) to the bath solution modulated STD in a frequency-dependent manner: STD was increased in the presence of ZD7288 (10 μm; a), and zatebradine (20 μm; b), respectively, when stimuli were given at 20 Hz (*p < 0.05 for both 2D7288 and zatebradine), but not after stimulation with 1, 5, or 10 Hz. c, HCN channel blockade did not affect STD after MPP stimulation in slices from adult rats (P50–P80). d, Similarly, I_h-blockade did not alter STD in slices from early postnatal rats, when the LPP was stimulated (note the characteristic initial facilitation of LPP-evoked EPSPs). e, A comparison of fEPSP_8–10/fEPSP₁ ratios after 20 Hz stimulation of MPP illustrates the age dependency of ZD7288- and zatebradine-induced effects on STD: no difference was observed after I_h blockade in the adult slices, suggesting that little I_h was activated in adult MPP, consistent with our neuroanatomical data. f, In general, STD after 20 Hz was significantly more pronounced in slices from adult compared with slices from immature rats (*p < 0.05). Note that this difference was abolished in the presence of the I_h blockers (see e). Insets in a–d show the first and the tenth control EPSP superimposed with the corresponding EPSP obtained in the presence of I_h blocker.

Figure 6.

HCN1 expression is not downregulated with age in the EC layer II stellate cells, the cells of origin of perforant path. a–c, In situ hybridization (ISH) using S³⁵-labeled probes indicated expression of HCN1 mRNA in layer II of the medial (med) EC (arrows) in both immature (P11; a) and adult rat (P90; b), and quantitative analysis of radioactive ISH-signal suggested that expression levels were not different at these ages (c). d, e, Single-cell resolution ISH using digoxigenin-labeled probes demonstrated that layer II stellate cells expressed HCN1 mRNA robustly in both immature (d) and adult medial EC (e). f, g, Representative Western blots (f) and quantitative analysis of HCN1 protein levels (relative to actin, g) further confirmed that HCN1 expression was not decreased in adult compared with immature medial EC. Sub, Subiculum; lat EC, lateral EC. Scale bar (in e): a, b, 500 μm; d, 50 μm; e, 60 μm.

Figure 7.

HCN1 expression in the ML is regulated by neuronal activity. a–c, To examine whether neuronal activity influences expression of HCN1 in the ML, combined entorhinohippocampal slices (P4 explanted; 7 d in vitro) were incubated from day 2 to day 7 in vitro with 0.1 μm TTX in the culture medium. TTX treatment resulted in a significant (∼26%) increase of HCN1 immunoreactivity in the ML (measured as optical density) compared with untreated sister cultures (c; *p = 0.02). d–f, HCN1 mRNA expression in layer II of the medial EC (med EC, arrows) was not altered in TTX-treated (e) compared with untreated (d, f) cultures, indicating that the stronger HCN1 signal in the ML after TTX treatment was not the result of increased HCN1 expression in the cells of origin. GCL, Granule cell layer; Hil; hilus; Sub, subiculum. Asterisks denote hippocampal fissure. Scale bar (in e): a, b, 100 μm; d, e, 400 μm.