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Electronic Supplementary Material CELL & TISSUE RESEARCH Hyperpolarisation-activated and cyclic nucleotide-gated channels are differentially expressed in juxtaglomerular cells in the olfactory bulb of mice Hans-Ulrich Fried, U. Benjamin Kaupp and Frank Müller Supplementary Information Identification of immunohistochemical fingerprints (IFs). IFs were established in two morphologically distinct populations of cells. The first population included cells without thick dendritic tuft (PG- and SA-like cells), the second population included cells with a thick dendritic tuft (ET-like cells). In our analysis, we assumed that all cells in the GL are stained by at least one of our antibodies (see Fig. 6). Combinations of double and triple stainings were used to establish IFs. We could base our classification of IFs on combinations of double and triple stainings, because our initial experiments revealed that markers or HCN isoforms were observed in only few of all theoretically possible combinations of co-localization and that certain combinations of markers or HCN isoforms never occurred. In the following, we provide the experimental strategy to identify each IF. Note that only unequivocal staining differences (immunonegative versus immunopositive or weakly immunopositive versus very strong immunopositive) were used to establish the IFs. To keep the number of figures and panels reasonable, we show only positive staining results in Fig. 4, suppl. Fig. 4, and suppl. Fig. 5. 1 Experiments A – G define IFs in PG- and SA-like populations Experiment A: IF1 Initial experiments showed that strong HCN1 was never co-localized with TH, CCK, PV, CR, HCN2, or HCN3 in PG- or SA-like cells. HCN1/CB stainings showed that all HCN1-positive cells are either strongly HCN1positive (experiment A) or HCN1- and CB-double positive (experiment D). Thus, only the following triple stainings were needed to characterize the strong HCN1-positive cells: HCN1/HCN4/CB stainings (suppl. Fig. 4 a-panels) showed that all strong HCN1positive cells are HCN4- and CB-negative. HCN1/NOS/vilip1 stainings (suppl. Fig. 4 b-panels) showed that all strong HCN1positive cells are NOS- and vilip1-positive. Finally, HCN1/NOS/hippo stainings (suppl. Fig. 4 c-panels) showed that all strong HCN1-positive cells are NOS- and hippo-positive (hippo staining intensity of +/-M, see Table 2). Conclusion: all strongl HCN1-positive PG- or SA-like cells co-express NOS, vilip1, and hippo: IF1 Experiment B: IF2 Initial experiments showed that HCN4 was never co-localized with TH, CCK, vilip1, PV, CR, or HCN2 in PG- or SA-like cells. Thus, only the following triple stainings were needed to characterize IF2: HCN1/HCN4/CB stainings (suppl. Fig.4 a-panels) show: These cells include IF2 and IF10 HCN4: + to ++ cells are HCN1: - to +/and CB: - to +/- 2 HCN4: + cells are HCN1: - or + and CB: ++ to +++ These cells include IF5 and IF6 HCN4-positive cells can be subdivided into strongly CB-positive and CB-negative (+/-) populations. All HCN4-pos. / CB-neg. cells are HCN1-negative (- to +/-). HCN4/CB/hippo stainings (data not shown) show: All HCN4-pos./CB-negative cells are also hippo-negative HCN4/CB/NOS stainings (data not shown) show: HCN4: + to ++ CB: - cells are NOS: - HCN4: + CB: +/- cells are NOS: +++ These cells are IF10 HCN4-pos. / CB-neg. cells can be subdivided into NOS-negative and strongly NOSpositive populations. HCN4/NOS/HCN3 stainings (data not shown) show: All HCN4-pos./NOS-negative cells are HCN3-negative Conclusion: among the HCN4-positive PG-SA-like cells, there is one subpopulation that expresses only HCN4, but no other markers or HCN isoforms tested: IF2 Experiment C: IF3 and IF4 Initial experiments showed that CCK was never co-localized with TH, NOS, hippo, CR, CB, HCN1, HCN2, or HCN4 in PG- or SA-like cells. Thus, only the following triple stainings were needed to characterize IF3 and IF4: Stainings with antibodies directed against HCN1/CCK/ and either vilip1 (suppl. Fig. 4 d-panels) or HCN3 (data not shown) showed that all CCK-positive cells are HCN1-negative 3 but vilip1- and HCN3-positive. The presence of HCN3 in IF3 and IF4 is shown in suppl. Fig. 4 e-panels and g-panels, respectively. Finally, HCN1/CCK/PV stainings (suppl. Fig. 4 f-panels) showed that CCK-positive cells are either PV-negative (IF3) or PV-positive (IF4). Conclusion: CCK-positive PG- or SA-like cells co-express vilip1 and HCN3, but segregate into PV-positive (IF4) and PV-negative (IF3) populations. Experiment D: IF5 and IF6 Initial stainings showed that CB was never co-localized with TH, CCK, NOS, vilip1, PV, CR, HCN2, or HCN3 in PG- or SA-like cells. Thus, only the following stainings were needed to characterize IF5 and IF6: CB/hippo stainings (suppl. Fig. 4 i-panels) showed that all CB-positive cells are also hippo-positive. CB/HCN1/HCN4 stainings (suppl. Fig. 4 a-panels) showed that all CB-positive cells are HCN4-positive but can be subdivided in HCN1-positive and HCN1-negative populations. Conclusion: CB-positive PG- or SA-like cells co-express hippo and HCN4, but segregate into HCN1-positive (IF5) and HCN1-negative (IF6) populations. Experiment E: IF7 and IF8 Initial experiments showed that CR was never co-localized with TH, CCK, hippo, PV, CB, HCN1, HCN2, HCN3, or HCN4 in PG- or SA-like cells. Thus, only the following triple staining was needed to characterize IF7 and IF8: CR/NOS/vilip1 stainings (suppl. Fig. 4 h-panels) showed that CR-positive PG- or SAlike cells can be subdivided in two population: IF7 is negative for NOS and vilip1, IF8 is positive for NOS and variable for vilip1. 4 Experiment F: IF9 Initial experiments showed that TH was never co-localized with CCK, NOS, vilip1 PV, CR, CB, HCN1, HCN2, HCN3, or HCN4 in PG- or SA-like cells. Thus, only a TH/hippo double staining was needed to characterize IF9: TH-positive PG- or SA-like cells (IF9) express hippo to a variable degree (- to ++). Note that, as varibility in hippo staining was very large, we did not subclassify cells into hippo-positive and hippo-negative cells. Experiment G: IF10 Inital experiments showed that NOS was never co-localized with TH, CCK, PV, CB, or HCN2 in cells with PG- or SA-like morphology. Thus, only the following triple stainings were needed to characterize IF10: HCN1/HCN4/NOS stainings (Fig.4b to e) show: NOS: +++ cells are HCN1: +++ and HCN4: - NOS: + to +++ cells are HCN1: and HCN4: - These cells are IF1 These cells are IF8 NOS: +++ cells are HCN1: +/and HCN4: + Three different populations of cells express NOS. From experiments “A“ and “E“, we know that two of the NOS-pos. populations (IF1 and IF8) do not express HCN3. 5 NOS/HCN3 stainings (Fig. 4c to h) show: These cells include IF1 and IF8 NOS: + to +++ HCN3: - NOS: +++ HCN3: ++ HCN3/CR and HCN3/hippo stainings (data not shown) show: All HCN3-pos. cells are CR- and hippo-negative CR/NOS/vilip1 stainings (suppl. Fig.4 h-panels) show: These cells are IF1 NOS: +++ CR: - cells are vilip1: + NOS: +++ CR: - cells are vilip1: - NOS-pos. / CR-neg. cells can be subdivided in vilip1-positive and vilip1-negative populations. From experiment “A“, we know that the vilip1-positive population must be IF1. Conclusion: NOS-positive PG- or SA-like cells fall into three populations (IF1, IF8, and IF10). NOS, HCN3, and HCN4 are exclusively co-localized in IF10. Both IF1 and IF8 do not express HCN3 or HCN4. Experiment G: IF11 HCN2 was not co-localized with any of our markers or other HCN isoforms in PG- or SA-like cells. Thus, HCN2 labeled a distinct cell population (IF11) that is not detected by any other marker. Conclusions of experiments A to G Using antibodies against HCN1, HCN2, HCN4, CCK, CB, CR, TH, and NOS as starting points of our analysis, we have identified 11 different IFs in experiments A to G. Further stainings with antibodies not used as a starting point (i.e. vilip1, hippo, PV, and 6 HCN3) revealed no further IFs in PG-and SA-like cells (data not shown). Because, we showed that our antibodies most likely stain all juxtaglomerular cells, IF1 to 11 are the only exciting immunohistochemical fingerprints in PG-and SA-like cell. However, we can not exclude that the IFs defined here can be further subdivided using additonal antibodies. Experiments I - V define IFs in ET-like populations Inital experiments showed that cells with ET-like morphology 1. do not express HCN2; 2. are either positive for CCK or for TH (there are no ET-like cells that are either negative or positive for both CCK and TH). Experiment I: IF12 HCN1/CCK/NOS stainings (suppl. Fig.5 a-panels) show: IF12 CCK: + to ++ cells are HCN1:+ to ++ and NOS: + to ++ IF13 CCK: + to ++ cells HCN1: +/NOS: + to ++ IF14 IF15 IF16 CCK: + to ++ cells CCK: + to ++ cells CCK: HCN1: + to ++ HCN1: HCN1: NOS: +/NOS: NOS: + This triple staining showed five different staining patterns (IF12 to IF16). Only IF12 is HCN1- and NOS-positive. Stainings with antibodies against HCN1/NOS/ and either HCN4, hippo, vilip1, or HCN3 showed that all HCN1- and NOS-positive cells are HCN3- and HCN4-positive but hippo- and vilip1-negative. Localization of HCN3 and HCN4 in IF12 cells is shown in suppl. Fig. 5 c-panels and e-panels, respectively. Conclusion: CCK-positive ET-like cells fall in four populations (IF12, IF13, IF14, and IF15). IF12 is characterized by co-expression of CCK, NOS, HCN1, HCN3, and HCN4. 7 Experiment II: IF13 HCN1/CCK/NOS stainings (suppl. Fig.5 a-panels) show: IF12 CCK: + to ++ cells are HCN1:+ to ++ and NOS: + to ++ IF13 CCK: + to ++ cells HCN1: +/NOS: + to ++ IF14 IF15 IF16 CCK: + to ++ cells CCK: + to ++ cells CCK: HCN1: + to ++ HCN1: HCN1: NOS: +/NOS: NOS: + This triple staining showed five different staining patterns (IF12 to IF16). Two populations (IF13 and IF16) are NOS-positive but HCN1-negative. HCN1/NOS/vilip1 stainings (data not shown) show: All HCN1-neg. / NOS-pos. cells are vilip-negative HCN1/NOS/HCN3 stainings (suppl. Fig. 5 b-panels) show: HCN1: +/NOS: + to ++ cells are HCN3: + HCN1: NOS: + cells are HCN3: +/- These cells are IF16 HCN1-neg. / NOS-pos. cells can be subdivided into HCN3-positive and HCN3negative populations. For the following reason, the HCN3-positive cells are IF13, while the HCN3-negative cells are IF16. TH/HCN3 double stainings show that all TH-negative cells express HCN3. Note that all ET-like cells do either express CCK or TH, TH-negative cells must be CCK-positive. Therefore, IF13 must be HCN3-positive. HCN1/NOS/HCN4 stainings (suppl. Fig. 5 e-panels) show: HCN1: +/NOS: + to ++ cells are HCN4: + to ++ HCN1: NOS: + cells are HCN4: +/- These cells are IF16 HCN1-neg. / NOS-pos. cells can be subdivided into HCN4-positive and HCN4negative populations. TH/HCN4 double stainings show that only TH-negative cells (thereby CCK-positive) express HCN4. Thus, IF13 must be HCN4-positive. 8 NOS/HCN4/hippo stainings (data not shown) show: All NOS- and HCN4-positive cells are hippo-negative Conclusion: of the four CCK positive ET-like cell populations, IF13 is characterized by co-expression of NOS, HCN3, and HCN4. Experiment III: IF14 HCN1/CCK/NOS stainings (suppl. Fig.5 a-panels) show: IF12 CCK: + to ++ cells are HCN1:+ to ++ and NOS: + to ++ IF13 IF14 CCK: + to ++ cells CCK: + to ++ cells HCN1: +/HCN1: + to ++ NOS: + to ++ NOS: +/- IF15 IF16 CCK: + to ++ cells CCK: HCN1: HCN1: NOS: NOS: + This triple staining showed five different staining patterns (IF12 to IF16). Only IF14 is HCN1-positive and NOS-negative (around detection threshold). Stainings with antibodies directed against HCN1/NOS/ and either HCN3, HCN4, vilip1, or hippo showed that all HCN1-positive and NOS-negative cells are HCN3-, vilip1-, and hippo-positive but HCN4-negative (suppl. Fig. 5 e-panels). That IF14 cells are HCN3-, vilip1-, and hippo-positive is shown in suppl. Fig. 5 b-, d-, and g-panels, respectively. Conclusion: in the four CCK positive ET-like cell populations co-expression of vilip1, hippo, HCN1, and HCN3 is only seen in IF14. Experiment IV: IF15 HCN1/CCK/NOS stainings (suppl. Fig.5 a-panels) show: IF12 CCK: + to ++ cells are HCN1:+ to ++ and NOS: + to ++ IF13 IF14 IF15 CCK: + to ++ cells CCK: + to ++ cells CCK: + to ++ cells HCN1: +/HCN1: + to ++ HCN1: NOS: + to ++ NOS: +/NOS: - IF16 CCK: HCN1: NOS: + This triple staining showed five different staining patterns (IF12 to IF16). Only IF13 and IF15 are HCN1-negative and CCK-positive. 9 Stainings with antibodies directed against HCN1/CCK/ and either vilip1, HCN3 or HCN4 showed that all HCN1-negative and CCK-positive cells are vilip1-negative but HCN3and HCN4-positive. That HCN3 and HCN4 are expressed in IF15 cells is shown in suppl. Fig. 5 f-panels. Finally, the HCN1/CCK/hippo staining showed that HCN1-negative and CCKpositive cells segregate into hippo-negative and hippo-positive populations. We know from experiment II that IF13 is hippo-negative, thus, IF15 must be hippo-positive. Conclusion: Of the four CCK positive ET-like cell populations co-expression of hippo, HCN3, and HCN4 is only observed in IF15. Experiment V: IF16 and IF17 TH/hippo/NOS stainings (suppl. Fig. 5 i-panels and j-panels) showed that TH-positive ET-like cells segregate in NOS-positive (IF16) and NOS-negative (IF17) populations. Both populations had a variable expression of hippo (staining intensity of – to ++). Further experiments showed that TH was never co-localized with CCK, vilip1, HCN1. Suppl. Fig. 1 Specificity of HCN stainings in the GL revealed by double labelings with antibodies recognizing different epitopes of HCN1 (a to c), HCN2 (d to f), HCN3 (g to h), and HCN4 (j to l), respectively. Antibodies (see table 1) are specified in each panel. In each of the double labelings antibodies directed against the same HCN isoform yielded qualitatively identical staining patterns. This indicates specificity of antibodies with cross-reactivity below background levels. Scale bars = 10 m Suppl. Fig. 2 Specificity of HCN1 and HCN3 antibodies revealed in knockout animals. a and b: Representative stainings with antibodies directed against HCN1 (HCN1) in wild type (a) 10 and HCN1 knockout (b) animals. c and d: Representative stainings with antibodies directed against HCN3 (HCN3 Shigemoto) in wild type (c) and HCN3 knockout (d) animals. No staining is observed in the knockout animals indicating specificity of antibodies with crossreactivity below background levels. Scale bars = 100 m Suppl. Fig. 3 Staining with antibodies directed against hippocalcin (hippo) at lower magnification. Hippo staining is found in several layers of the olfactory bulb (GL, EPL, IPL, and GrL). No staining is visible in the AL and ML. EPL, external plexiform layer; GL, glomerular layer; GrL, granule cell layer; IPL, internal plexiform layer; ML, mitral cell layer; AL, olfactory nerve layer. Scale bars = 100 m Suppl. Fig. 4 Immunohistochemical identification of PG-like cell populations. a-panels (a, a’, a’’, and a’’’): Example of stained PG-like cells in a lower magnification. Triple labeling for HCN1 (green, HCN1), HCN4 (red, PG2-1A4), and CB (blue, CabPneu) reveals IF1 (green arrowhead, only HCN1 positive), IF2 (blue arrowhead, only HCN4 positive), IF5 (red arrowhead, HCN1, HCN4, and CB positive), and IF6 (light turquoise arrowhead, HCN4 and CB positive). b- and c-panels: Immunohistochemical identification of cells with IF1. IF1 cells (green arrowhead) are characterized by the co-localization of HCN1, NOS, vilip1, and hippo as seen in two triple labelings (b-panels: HCN1 in green, NOS in red, and vilip1 in blue; antibodies used were: HCN1, NOSmono, and vilip1 c-panels: HCN1 in green, NOS in red, and hippo in blue; antibodies used were: HCN1 NOSmono, and hippo). d-, e-, f-, and g-panels: Immunohistochemical identification of cells with IF3 and IF4. Both, IF3 (light orange arrowhead) and IF4 (dark orange arrow head) feature co-expression of HCN1, HCN3, CCK, and vilip1. The HCN1 staining is often just above background and therefore not clearly visible in all stainings. PV is only detectable in IF4. The co-localization patterns in IF3 and IF4 are shown in four triple stainings (d-panels: HCN1 in green, CCK in red, and vilip1 in 11 blue; antibodies used were: HCN1, CCK, and vilip1; e-panels: CCK in green, HCN3 in red, and PV in blue; antibodies used were: CCK, HCN3 Shigemoto, and PV f-panels: HCN1 in green, CCK in red, and PV in blue; antibodies used were: HCN1, CCK, and PV g-panels: HCN1 in green, HCN3 in red, and PV in blue; antibodies used were: HCN1, HCN3 Shigemoto, and PV). h-panels: Immunohistochemical identification of cells with IF7 and IF8. Triple labeling for CR (green, CAL), NOS (red, NOSmono), and vilip1 (blue, vilip1) reveals IF7 (black arrowhead, only CR positive) and IF8 (grey arrowhead, CR, NOS, and vilip1 positive). i-panels: Immunohistochemical identification of cells with IF5 and IF6. Triple labeling for HCN1 (green, HCN1), hippo (red, hippo), and CB (blue, CabPneu) reveals IF5 (red arrowhead, HCN1, hippo, and CB positive) and PG6 (light turquoise arrowhead, hippo and CB positive). Note: cells with IF5 and IF6 have also been characterized in the a-panels. jpanels: Immunohistochemical identification of cells with IF9. Co-localization of TH (green, TH) and hippo (red, hippo) reveals IF9 (white arrowhead, TH and hippo positive). Colour codes of arrowheads same as in table 2. The a-panels and h-panels are also shown in Fig. 3. Scale bars = 10 m Suppl. Fig. 5. Immunohistochemical identification of ET-like cell populations. a-, b-, c-, d-, e-, f-, g-, and h-panels: Immunohistochemical identification of cells with IF12, IF13, IF14, and IF15 cells. a-panels: The triple staining for HCN1 (green, HCN1), CCK (red, CCK), and NOS (blue, NOSmono) reveals four different CCK-positive IFs in ET-like cells. IF12 (white arrows): strong co-expression of HCN1, CCK, and NOS. IF13 (dark purple arrow): coexpression of CCK and NOS, little or no HCN1. IF14 (orange arrow): co-expression of HCN1, CCK, and only weakly NOS. IF15 (light purple arrow): only CCK expression. bpanels: The triple staining for HCN1 (green, HCN1), HCN3 (red, HCN3 Shigemoto), and NOS (blue, NOSpoly) shows the expression of HCN3 in cells with IF14 (orange arrow, 12 identified by moderate HCN1 and weak NOS staining). A weak HCN3 expression is also seen in cells with IF13 (dark purple arrow, identified by strong NOS and no HCN1). c-panels: The triple staining for HCN1 (green, HCN1), HCN3 (red, HCN3 Shigemoto), and vilip1 (blue, vilip1) shows the strong expression of vilip1 in cells with IF14 (HCN1 (green) and HCN3 (red) positive) and weak expression of vilip1 in cells with IF12 (also HCN1 (green) and HCN3 (red) positive). In a vilip1 and NOS double staining the strong vilip1 expression in weakly NOS-positive cells (IF14) was confirmed (data not shown). d-panels: In the staining for HCN1 (green, HCN1), CCK (red, CCK), and vilip1 (blue, vilip1), the sensitivity of the detector for vilip1 was adjusted to match the strong staining in IF14 (orange arrow; note the pronounced dendritic tuft in the ET like cells shown here). Expression of vilip1 in cells with IF12 is much weaker and, therefore, not visible at this detector setting. e-panels: The triple staining for HCN1 (green, HCN1), HCN4 (red, PG2-1A4), and NOS (blue, NOSpoly) shows that HCN4 is found in IF12 (white arrow, HCN1 and strong NOS expression) and IF13 (purple arrow, strong NOS and no HCN1 expression), but not in IF14 (orange arrow, weak NOS and strong HCN1 expression). f-panels: A staining with four primary antibodies HCN4 (green, PG2-1A4), HCN3 and NOS (both red, HCN3 Shigemoto and NOSpoly, respectively), and TH (blue, TH) is shown. Although using the same secondary antibody, due to the difference in subcellular localization, NOS (cytoplasmic staining) and HCN3 (plasma membrane staining) could be clearly differentiated. HCN3 and HCN4 are co-expressed in cells with IF15 (light purple arrow). g-panels: Triple staining for HCN1 (green, HCN1), HCN4 (red, PG2-1A4), and hippo (blue, hippo). Hippo is present in IF14 (orange arrow, strong HCN1 and no HCN4 expression). h-panels: Triple staining for HCN1 (green, HCN1), CCK (red, CCK), and hippo (blue, hippo). Expression of hippo is found in a cell positive for CCK but negative for HCN1, a feature characteristic for either IF13 or IF15 (light purple arrow). Because hippo is not found in strongly NOS-positive ET cells (i.e. IF13), the expression pattern of the cell (CCK and hippo, but no HCN1) is consistent with IF15 (data not 13 shown). The second CCK-positive but HCN1-negative cell in this panel (purple arrow, IF13) is negative for hippo. i- and j-panels: Immunohistochemical identification of cells with IF16 and IF17. IF16 and IF17 are positive for TH and hippo (IF16, blue arrow; IF17 grey arrow). The triple staining for TH (green, TH), hippo (red, hippo), and NOS (blue, NOSgt) shows that all three markers are found in IF16 (blue arrow, i-panels). In a triple staining with the same set of antibodies, IF17 is positive for TH and hippo but negative for NOS (j-panels). Colour codes of arrows same as in table 2. The d-, i-, and j-panels are also shown in Fig. 5. Scale bars = 10 m Suppl. Fig. 6. Double stainings with antibodies directed against GFAP (green) and HCN channels (red) show that no HCN staining is colocalized with GFAP. Thus, the majority of HCN1 (a), HCN2 (b), HCN3 (c), and HCN4 (d) channels are found in neurons rather then astrocytes. Antibodies used were: GFAP and HCN1(a), GFAP and QQA-1A6 (b), GFAP and HCN3 (c), and GFAPab and PG2-1A4 (d). Arrows in c point to HCN3 positive cells. Scale bar = 100 m Suppl. Fig. 7. 3D reconstructions of confocal z-stacks. a: Reconstructions of two CB positive (CabPneu) PG-like cells (arrowhead) with one or two dendrites, which ramify within the same glomerulus. b: Reconstruction of a NOS positive (NOSpoly) SA-like cell (arrow) with a long reaching dendrite. Three other cells (asterisk) are in direct vicinity of the SA-like cell body or dendrite and were not perfectly reconstructed as separate objects. c: Reconstruction of three vilip1 positive PG-like cells (arrowheads) with several dendrites, and an ET-like cell (arrow) with a thick primary tuft. d: Reconstruction of a NOS positive (NOSpoly) ET-like cell (arrow) with a tick primary tuft (single asterisk) and a thick lateral dendrite (double asterisk). Scale bars = 10 m 14 Suppl. Table 1. Number of cells and glomeruli counted to average number of cells per glomerulus and section in table 3. IF IF1 IF2 IF3 IF4 IF5 IF6 IF7 IF8 IF9 IF10 IF11 IF12 IF13 IF14 IF15 IF16 IF17 cells counted across number of glomeruli 717 371 562 410 191 258 522 521 318 180 145 142 95 272 88 36 125 350 190 301 355 190 190 121 121 140 476 343 194 194 301 150 140 140 15