Distribution of HCN channel isoforms in the olfactory bulb
The distribution of HCN channels in the olfactory bulb was examined with 12 different antibodies against the four HCN channel isoforms (Table 1). Each HCN isoform was recognized by three antibodies generated in different species. In addition, for each HCN isoform, two antibodies were generated against different epitopes. The specificity and suitability of antibodies was ensured by three criteria. (1) Western blots of proteins from olfactory bulb and from HEK 293 cells that expressed either one of the four HCN isoforms. After treatment with PNGaseF to remove protein glycosylation, the antibodies recognized proteins of the expected molecular weight of 102, 94, 86, and 130 kDa for HCN1, HCN2, HCN3, and HCN4, respectively (Fig. 1a; Müller et al. 2003). (2) Independent antibodies directed against the same HCN isoform produced identical staining patterns as demonstrated by the superposition of images (Fig. 1b, c, Electronic Supplementary Material, Fig. 1). (3) In HCN1 and HCN3 knockout animals, no staining was observed with antibodies against the deleted isoform (Electronic Supplementary Material, Fig. 2; HCN1 knockout mice: Nolan et al. 2003; HCN3 knockout mice: gift of Martin Biel, Ludwig-Maximilians Universität, Munich, Germany).
Each HCN isoform showed a characteristic expression pattern in the various layers of the olfactory bulb (Fig. 1c, e–g). The HCN staining originated from somata and neuronal processes alike. Each HCN isoform was found only in subsets of cells. Therefore, staining intensities differed between different layers. HCN1 was strongly expressed in the glomerular layer (GL), followed by some weaker expression in the internal plexiform layer (IPL) and the granule cell layer (GrL). Only weak staining was observed in the external plexiform layer (EPL). For HCN2, the most intense staining was observed in individual cell bodies distributed across all but the olfactory nerve layer (AL). HCN3 was strongly expressed in the IPL and the outer part of the EPL. Strong HCN4 staining was observed in all layers; in the AL, it appeared to be the only HCN isoform. Thus, in the GL, abundant expression of all four HCN isoforms was observed. In the following, we restrict our analysis to the GL.
HCN isoforms are expressed in many different combinations
In the GL, antibodies against all HCN isoforms stained the somatic and dendritic plasma membrane (Fig. 2). Moreover, the specific glia cell marker GFAP (glial fibrillary acidic protein) did not co-localize with HCN staining, suggesting that the majority of HCN channels were present in neurons (Electronic Supplementary Material, Fig. 6). Cells stained by HCN antibodies fell into two groups. The first group was characterized by relatively small somata and thin dendritic processes (Fig. 2e–o). This group comprised both PG and SA cells. The second group displayed large somata and usually possessed only one thick primary tuft, which ramified within a single glomerulus, features characteristic of ET cells (Fig. 2p–s, see also Materials and methods).
Triple- and double-labeling revealed subpopulations of PG- and SA-like cells that expressed either a single HCN isoform or combinations of HCN isoforms (Fig. 2e–s). Because compelling evidence (staining intensity of + or stronger in Table 2) for the co-expression of HCN2 with the other isoforms was lacking (Fig. 2e–k), triple-staining with HCN1, HCN3, and HCN4 antibodies was sufficient to reconstruct a complete co-expression pattern of HCN channels (see Fig. 2l–s, Table 2). PG- and SA-like cells were identified that expressed only HCN1 (Fig. 2l–o, green arrowheads) or HCN4 (Fig. 2p–s, dark blue arrowheads). Moreover, HCN1 and HCN3 co-localized (Fig. 2l–o, orange arrowhead) in one population of cells, and in another, HCN1 and HCN4 were co-localized (Fig. 2l–o, red arrowhead). Thus, some cell populations expressed a single HCN channel isoform only (HCN1, HCN2, or HCN4), whereas others co-expressed HCN1/3 or HCN1/4. Co-expression of HCN isoforms occurred more often in ET-like cells than in PG- and SA-like cells. The following combinations were observed: HCN1/3, HCN3/4, and HCN1/3/4 (Fig. 2a–d, p–s). This demonstrates, for the first time at the protein level, the expression of three HCN channel isoforms within the same neuron. Remarkably, in the GL, we could find no convincing evidence for the expression of HCN3 channels alone. Even in the entire main olfactory bulb, exclusive HCN3 staining was rarely observed (see Fig. 2a–d, outer EPL).
Staining patterns in somata and dendritic processes, e.g., the glomerular neuropil (region inside the dashed white outlines of glomeruli in Fig. 2), were similar. Dendrites harboring only HCN1, HCN2, or HCN4 were common, whereas dendrites expressing only HCN3 were rare. Furthermore, HCN2 by and large was expressed on its own. Co-localization of HCN1, HCN3, and HCN4 existed in all possible combinations (HCN1/3, HCN1/4, HCN3/4, and HCN1/3/4). Relatively thick dendritic processes in the outer EPL (Fig. 2a–d) showed the co-localization of either HCN3/4 (purple dendritic processes in Fig. 2d) or of HCN1/3/4 (white dendritic processes in Fig. 2d). By visual inspection of staining, we observed that the thick dendritic processes originated from the somata of ET-like cells and extended laterally into the EPL. Thus, they probably represented lateral dendrites of at least two different ET cell populations. One ET cell population expressed HCN3/4, whereas another population expressed HCN1/3/4.
The rich expression patterns of HCN channels indicate that many different cell populations exist. In the following, we relate HCN channel expression patterns to previously described cell populations by combining immunostaining for markers and HCN.
Immunohistochemical characterization of juxtaglomerular cells expressing HCN channels
We used antibodies against eight cellular marker proteins to characterize populations of juxtaglomerular cells expressing HCN channels. Seven of these marker proteins, including tyrosine hydroxylase (TH), nitric oxide synthase (NOS), the neuropeptide cholecystokinin (CCK), the calcium-binding proteins visinin-like protein 1 (vilip1; also known as neurocalcin α), parvalbumin (PV), calretinin (CR), and calbindin-D28K (CB), have been previously used to distinguish between subpopulations of juxtaglomerular cells (Baker et al. 1983; Brinon et al. 1992, 1997, 1998; Kosaka et al. 1994; Liu and Shipley 1994; Bastianelli and Pochet 1995; Crespo et al. 1997; Bernstein et al. 2003; Gutierrez-Mecinas et al. 2005; Parrish-Aungst et al. 2007; Kosaka and Kosaka 2007b). In general, the staining patterns that we obtained for these marker proteins were similar to those published. In addition, we employed antibodies against hippocalcin, another calcium-binding protein, which proved useful as another marker to distinguish between different juxtaglomerular cells. To the best of our knowledge, hippocalcin antibodies have not been used before for staining of the olfactory bulb. A lower magnification of a representative hippocalcin staining is shown in Electronic Supplementary Material, Fig. 3. We used marker and HCN channel antibodies in more than 140 combinations. We found highly reproducible co-localization patterns in the different juxtaglomerular cell populations (for details see Electronic Supplementary Material); these patterns are referred to as IFs in the following.
In many cases, marker antibodies stained cells intensely, thereby revealing even small anatomical details that allowed us to distinguish between populations of PG and SA cells. PG cells are neurons with small somata and usually have one to two dendrites that ramify extensively in a single (sometimes two) neighboring glomeruli. In contrast, SA cells are larger than most PG cells. Often, SA cells have more than two dendrites, which do not ramify in glomeruli (Table 3) but rather reach across several adjacent glomeruli to contact other juxtaglomerular cells (Pinching and Powell 1971).
In PG-like cells, at least nine different IFs were identified (IF1–IF9) that showed multiple expression patterns of HCN channels (Fig. 3, Table 2, Electronic Supplementary Material, Fig. 4). In two IFs, strong staining for one HCN isoform was present (IF1 and IF2). In four other IFs, moderate staining for either one HCN isoform (IF6) or several HCN isoforms was observed (IF3–IF5), and the remaining IFs seemed to be devoid of HCN staining (IF7–IF9). A detailed description of IFs found in PG-like cells is given in the Electronic Supplementary Material.
Two IFs were identified in SA-like cell populations (IF10 and IF11). We found strong HCN staining in both IFs. Strong staining for NOS and HCN3, but weak staining for HCN4 was detected in IF10 (Fig. 4b–h, Table 2). Dendrites of cells with the IF10 usually reached across several adjacent glomeruli (Fig. 4a).
IF11 (labeling with HCN2 antibodies only) was found in a second SA-like cell population. The cell type could not be classified unequivocally, because distal dendrites were not stained (independent observers classified these cells as PG-like or SA-like). Based on the soma size alone, these cells might represent SA cells or large PG cells (for a comparison, see Table 3). However, whereas PG cells usually had one or two dendrites that ramified within glomeruli, cells with IF11 had more than three dendrites that could not be traced into glomeruli but rather seemed to surround them (Fig. 4i–k). Therefore, we suggest classifying these cells as SA-like cells. A detailed description of IFs found in SA-like cells is given in the Electronic Supplementary Material.
We observed TH-positive cells at the border to the EPL; these probably corresponded to SA cells recognized previously (Kosaka et al. 1998). Within the GL, these cells could not be distinguished unequivocally from the large number of TH-expressing juxtaglomerular cells and, therefore, were not further analyzed.
Six different IFs were identified in ET-like cells (Fig. 5, Electronic Supplementary Material, Fig. 5). IF12–IF15 represented different combinations of staining for HCN isoforms, whereas IF16 and IF17 were virtually devoid of HCN channel staining. IF12–IF15 contained staining for CCK but differed in their staining for HCN1, HCN4, NOS, vilip1, and hippo (Table 2, Fig. 5a–d, Electronic Supplementary Material, Fig. 5). IF12 featured staining with HCN1, HCN3, and HCN4 antibodies. In IF13 and IF15, HCN3 was found together with HCN4, whereas in IF14, HCN3 was present with HCN1 (Electronic Supplementary Material, Fig. 5). Cells with IF12, IF13, and IF15 possessed lateral dendrites (Table 3).
IF16 and IF17 featured staining with antibodies directed against TH (Fig. 5e–l). Co-localization of CCK and TH was never observed. IF16 contained NOS staining, whereas IF17 was devoid of it. No lateral dendrites were identified in cells with TH staining. A detailed description of IFs found in ET-like cells is given in the Electronic Supplementary Material.
The combination of classical markers and HCN channel antibodies allowed us to identify cell populations that show one of at least 17 IFs. Four cell populations expressed only a single HCN isoform, two cell populations were stained by HCN antibodies only, six cell populations co-expressed several HCN channel isoforms, and five cell populations lacked HCN channels.
Frequency and soma size of populations of juxtaglomerular cells
For a full characterization of the immunohistochemically different cell populations in the GL, we estimated their frequency per glomerulus (see Materials and methods) and related it to the total number of cells per glomerulus as visualized by TOPRO-3, a dye that intercalates into the DNA of all cells (Table 3).
We counted cells across 81 adjacent glomeruli in three animals and estimated a total of 705±136 cells per glomerulus, in good agreement with an analysis by Parrish-Aungst et al. (2007) who recorded ∼680 cells per glomerulus. On average, 40% of cells in the GL were PG cells (267±25 cells per glomerulus), as determined by counting cells across at least 121 adjacent glomeruli per PG-like cell population in at least three animals (see Materials and methods). SA-like cells and ET-like cells represented only 1% and 5% of cells, respectively: 9±1 SA-like cells per glomerulus (≥343 adjacent glomeruli in ≥3 animals per cell population), and 32±6 ET-like cells per glomerulus (≥138 adjacent glomeruli in ≥3 animals per cell population).
The estimate critically depended on whether all juxtaglomerular cell populations were identified. To estimate the fraction of juxtaglomerular cells that escaped staining, we compared, in the same section, the number of immunohistochemically labeled cells with the number of TOPRO-3-stained cells. The combination of antibodies directed against TH, NOS, CB, vilip1, CR, HCN1, HCN2, and HCN4 was sufficient to stain all juxtaglomerular cell populations identified in this study (see Table 2). GFAP staining was used to distinguish glia cells from neurons. The majority of glia cells in the GL are GFAP-positive astrocytes (Bailey and Shipley 1993; Kimelberg 2004). None of the staining for markers or HCN channels co-localized with the GFAP staining.
If many antibodies are combined and visualized by using secondary antibodies conjugated to the same dye, the equivalent detection of strong and weak staining is difficult. Thus, weakly stained cells may escape detection. To circumvent this problem, we visualized strongly and weakly staining antibodies with different dyes and adjusted detection settings to match the staining intensity. In combined TOPRO-3 and antibody staining (Fig. 6), we counted cells across 51 adjacent glomeruli (two animals) and identified 2.9% cells not labeled by the antibody mixture. The majority of these cells (2%) were positioned at the border to the AL or EPL, probably representing cells of the AL and EPL rather than those of the GL. The few cells in the GL not labeled by antibodies (0.9%) may have represented GFAP-negative glia cells (Kimelberg 2004). When one of the nine antibodies was omitted, the number of unstained cells was significantly enhanced (data not shown). Together, these results demonstrate that the vast majority, possibly even all, of the juxtaglomerular cells have been stained by our markers.
Populations of juxtaglomerular cells not only differed in IFs, but also in their morphology. Significant differences in the soma size of populations of PG-like cells were apparent, in agreement with previous reports (e.g., Kosaka and Kosaka 2007b; Parrish-Aungst et al. 2007; Pinching and Powell 1971; Qin et al. 2005).
To quantify the soma size, we determined the apparent cell diameter from the measured circumference (Table 3). Mean diameters of PG-like cells (IF1-IF9) were between 6.5 μm and 9.1 μm; most diameters were ca. 8 μm. By pair-wise Kruskal-Wallis tests with all possible combinations of mean diameters, we observed that most diameters of these PG-like cells were statistically different from those of other PG-, SA-, and ET-like cells: 94 of 108 possible pair-wise combinations between different PG-like cells, PG-like and SA-like cells, and PG-like and ET-like cells had a P-value well below 0.001. However, cells with the IF7 and IF8 (P=0.98) and cells with the IF2, IF6, and IF9 (P>0.76) had similar diameters. The soma diameters of the two SA-like populations were similar (cells with IF10: 9.4±1.1 μm; cells with IF11: 8.9±0.7 μm), although this difference was statistically significant (P=0.035). ET-like cells had soma diameters ranging from 11.6 μm to 12.6 μm. Pair-wise comparisons of cells with IF12/IF17 and IF13/IF16 showed similar diameters (P>0.76). Cells with IF14 and IF16 differed in their somatic diameters (P<0.02); all other possible comparisons of ET-like cell diameters were neither statistically different nor statistically similar (0.1<P<0.65). Soma diameters resembled the diameters of PG, SA, and ET cells as described in classical Golgi-impregnated studies (Pinching and Powell 1971).