Differential and age-dependent expression of hyperpolarization-activated, cyclic nucleotide-gated cation channel isoforms 1-4 suggests evolving roles in the developing rat hippocampus

Abstract

Hyperpolarization-activated cation currents (I(h)) are found in several brain regions including thalamus and hippocampus. Important functions of these currents in promoting synchronized network activity and in determining neuronal membrane properties have been progressively recognized, but the molecular underpinnings of these currents are only emerging. I(h) currents are generated by hyperpolarization-activated, cyclic nucleotide-gated cation channels (HCNs). These channel proteins are encoded by at least four HCN genes, that govern the kinetic and functional properties of the resulting channels. Because of the potential impact of I(h)-mediated coordinated neuronal activity on the maturation of the functional hippocampal network, this study focused on determining the expression of the four members of the HCN gene family throughout postnatal hippocampal development at both the regional and single cell level.The results of these experiments demonstrated that HCNs 1, 2 and 4 are differentially expressed in interneuronal and principal cell populations of the rat hippocampal formation. Expression profiles of each HCN isoform evolve during postnatal development, and patterns observed during early postnatal ages differ significantly from those in mature hippocampus. The onset of HCN expression in interneurons of the hippocampus proper precedes that in the dentate gyrus, suggesting that HCN-mediated pacing activity may be generated in hippocampal interneurons prior to those in the hilus. Taken together, these findings indicate an age-dependent spatiotemporal evolution of specific HCN expression in distinct hippocampal cell populations, and suggest that these channels serve differing and evolving functions in the maturation of coordinated hippocampal activity.

Fig. 1

Expression patterns of the four HCN mRNA isoforms in mature hippocampus. By the end of the third postnatal week, mRNA expression patterns of each HCN isoform resemble the adult patterns. Coronal sections of dorsal hippocampus from 24 day old rats, shown here, were subjected to in situ hybridization histochemistry for each of the HCN mRNAs: (A) HCN1 mRNA: robust expression is visible in the CA1 pyramidal cell layer (arrows), involving both pyramidal cells and in interneurons (see also Fig. 2B). In contrast, only interneurons express HCN1 in the CA3 pyramidal cell layer (see also Fig. 2A) and no HCN1 mRNA expression is observed in dentate gyrus (DG) granule cells. (B) HCN2 mRNA is homogenously expressed throughout the principal layers. (C) HCN3 mRNA hybridization signal is negligible. (D) HCN4 mRNA expression pattern in hippocampus generally resembles that of HCN2 mRNA. Differential thalamic (Th) expression patterns confirm probe specificity: virtually no HCN1 and HCN3 hybridization signals are detected in adult thalamus. In addition, HCN2 (B) and HCN4 (D), both expressed in thalamic nuclei, are distinguished by the selective expression of HCN4 mRNA in the medial habenula (arrow in D). Note: the findings for P24 were confirmed in P60 rats (not shown). Scale bar = 250 μm.

Fig. 2

HCN isoform expression in hippocampal interneurons is specific to neurochemically and anatomically defined populations. (A, B) HCN1 mRNA: (A) combined HCN1 mRNA (blue) and parvalbumin immunocytochemistry (brown) demonstrates co-expression of this HCN isoform in the vast majority of CA3 basket cells (arrows) in the 24 day old rat, while no expression is evident in CA3 pyramidal cells. In contrast to co-localization with PV-containing interneurons in principal cell layers, (B) shows little overlap of HCN1 mRNA (blue) and somatostatin expression (brown) in interneurons of CA1 stratum oriens (arrowheads). Thus, these two panels indicate that HCN1 mRNA is expressed in CA1 (B) but not CA3 (A) pyramidal cells, as well as in typically large-sized neurons in or closely adjacent to the pyramidal layer (arrows in A and B), which are likely basket and chandelier cells. (C, D) HCN2 mRNA: (C) combined HCN2 mRNA (blue) and parvalbumin immunocyto-chemistry (brown, arrows) reveals that HCN2 is rarely expressed in basket and chandelier cells, while (D) it is frequently expressed in somatostatin-labeled neurons at the O/A border in CA1 (O/A neurons; arrows). Abbreviations: s.o., stratum oriens; s.p., stratum pyramidale; s.r., stratum radiatum. Scale bar = 25 μm.

Fig. 3

Expression of HCN1–4 mRNAs in the hippocampus of the 5 day old rat. In the early postnatal hippocampus, HCN expression patterns differ significantly from those seen later: (A) HCN1 mRNA is strongly expressed in the pyramidal cell layer, involving pyramidal cells in CA1 and – unlike in the adult – also in CA3. (B) The homogenous expression of HCN2 mRNA throughout the pyramidal cell layer, observed in mature hippocampus (Fig. 1B), is already established by P5. (C) As found in the adult, HCN3 mRNA is poorly detectable in the early postnatal hippocampus. (D) HCN4 mRNA expression in the P5 rat is confined to the CA1 region (arrows), whereas expression of this isoform is robust also in the CA3 pyramidal cell layer of the mature hippocampus (see Fig. 1D). No HCN isoform is expressed in the dentate gyrus (DG, neither in granule cells nor in interneurons) during this age. Differential expression in thalamic nuclei (Th) is evident. Interestingly, HCN1 is prominently expressed in the anterodorsal thalamic nucleus (arrow in A) during the first and second postnatal weeks, while this isoform is not found in adult thalamus. HCN2 and HCN4 expression in thalamus generally approximates the mature pattern (Fig. 1B, D). Scale bar = 250 μm.

Fig. 4

Evolution of the HCN1 mRNA expression pattern in CA3 of the developing hippocampus. (A) Prominent expression of HCN1 mRNA is observed as early as P2 in pyramidal neurons (s.p.). However, only faint staining in stratum radiatum (arrows) suggests the onset of this isoform’s expression in a few interneurons at that age. (B) By P5, distinct interneuronal expression was evident in stratum radiatum (arrows). Note that during this age, pyramidal cells still express high levels of the HCN1 isoform. (C) By P18, a large number of interneurons, mostly associated with the pyramidal layer, express HCN1 mRNA. However, pyramidal cells no longer express this isoform. Abbreviations: s.p., stratum pyramidale; s.r., stratum radiatum. Scale bar = 50 μm.

Fig. 5

Differential expression of HCN mRNAs in developing CA1 interneurons and pyramidal cells. Cell specificity of HCN isoform expression was evident already by P11. (A) in situ hybridization for HCN1 demonstrates that this isoform is expressed in pyramidal cells, as well as in interneurons of stratum radiatum (arrowhead), but rarely in those of stratum oriens (s.o.). In addition, HCN1 mRNA is visible in typical large neurons at the base of the pyramidal cell layer (arrows), suggesting early onset of HCN1 mRNA expression in basket and chandelier cells. (B) Unlike HCN1, HCN2 mRNA is frequently expressed in interneurons of stratum oriens. This expression is evident already on P5 (arrows in top panel), and is robust by P11 (arrows in bottom panel). (C) HCN4 mRNA expression (arrows) resembles that of HCN2, but limited quantitative analysis suggests that fewer interneurons express this isoform compared with HCN2 mRNA (see text). Abbreviations: s.o., stratum oriens; s.p., stratum pyramidale; s.r., stratum radiatum. Scale bar (A, C) = 40 μm; (B) 50 μm.

Fig. 6

Evolution of HCN expression patterns in the dentate gyrus. In general, the onset of HCN mRNA expression in the dentate gyrus is delayed compared with the hippocampus proper. (A) HCN2 mRNA (as well as other isoforms, not shown) is not detectable in granule cells or in interneurons on P5. (B) By P11, HCN1 mRNA is expressed in interneurons, but not in granule cells. As demonstrated in other hippocampal regions, HCN1 mRNA is particularly prominent in interneurons associated with the principal (granule) cell layer (gcl, arrows). (C) In contrast to HCN1, HCN2 mRNA is more prevalent in interneurons residing in the deep hilus (h, arrows), and is also weakly detectable in granule cells. Scale bar = 60 μm.