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Hyperpolarization-activated cyclic nucleotide-gated channels in olfactory sensory neurons regulate axon extension and glomerular formation - PubMed

  • ️Fri Jan 01 2010

Hyperpolarization-activated cyclic nucleotide-gated channels in olfactory sensory neurons regulate axon extension and glomerular formation

Arie S Mobley et al. J Neurosci. 2010.

Abstract

Mechanisms influencing the development of olfactory bulb glomeruli are poorly understood. While odor receptors (ORs) play an important role in olfactory sensory neuron (OSN) axon targeting/coalescence (Mombaerts et al., 1996; Wang et al., 1998; Feinstein and Mombaerts, 2004), recent work showed that G protein activation alone is sufficient to induce OSN axon coalescence (Imai et al., 2006; Chesler et al., 2007), suggesting an activity-dependent mechanism in glomerular development. Consistent with these data, OSN axon projections and convergence are perturbed in mice deficient for adenylyl cyclase III, which is downstream from the OR and catalyzes the conversion of ATP to cAMP. However, in cyclic nucleotide-gated (CNG) channel knock-out mice OSN axons are only transiently perturbed (Lin et al., 2000), suggesting that the CNG channel may not be the sole target of cAMP. This prompted us to investigate an alternative channel, the hyperpolarization-activated, cyclic nucleotide-gated cation channel (HCN), as a potential developmental target of cAMP in OSNs. Here, we demonstrate that HCN channels are developmentally precocious in OSNs and therefore are plausible candidates for affecting OSN axon development. Inhibition of HCN channels in dissociated OSNs significantly reduced neurite outgrowth. Moreover, in HCN1 knock-out mice the formation of glomeruli was delayed in parallel with perturbations of axon organization in the olfactory nerve. These data support the hypothesis that the outgrowth and coalescence of OSN axons is, at least in part, subject to activity-dependent mechanisms mediated via HCN channels.

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Figures

Figure 1.
Figure 1.

Hyperpolarization-activated inward currents in dissociated mouse OSNs. A, Whole-cell recording in an acutely dissociated OSN; the arrow points to protruding cilia from the dendritic knob. B, Cells exhibit characteristic fast inward (asterisk) and late outward currents elicited by depolarizing steps (20 mV increments). C, Hyperpolarization-activated currents were elicited by voltage steps in 10 mV increments from −160 mV; only every other step is shown. D, The normalized conductance (G/Gmax) was fitted to a Boltzmann equation; the value of V1/2 = 130 ± 10 mV. E, The inward current was larger when the extracellular concentration of K+ was increased. The inset shows currents obtained with a hyperpolarizing step to −160 mV (filled circles, 5 m

m

; empty circles, 25 m

m

extracellular K+). F, The inward current was reduced by ZD 7288 (30 μ

m

) at all potentials tested in this cell. The inset shows currents obtained with a hyperpolarizing step to −200 mV (filled squares, control; empty squares, in ZD7288). The holding potential is −60 mV. The calibration bar is 20 ms and 100 pA for B, 200 ms and 100 pA for C, 100 ms and 100 pA for the inset in E, 100 ms and 20 pA for the inset in F.

Figure 2.
Figure 2.

HCNs are present in the OE during the early development of the olfactory system. A, RT-PCR from mouse OE for HCN1, 2, and 4. Numbers to the right are nucleotide length. Ad, Adult. B, Western blots from mouse OE. Numbers to the left are molecular weights. C, Quantification of protein levels across embryonic development for each HCN subunit. Asterisk indicates p < 0.05. Error bars, SEM.

Figure 3.
Figure 3.

HCN subunits are present in immature, developing OSNs at E13 and colocalize with GAP43. At P2, some colocalization (yellow) with GAP43 persists with HCN2 and HCN1. However, primarily HCN and OMP are coexpressed in the mature OSNs (OMP+) which are located apically in the epithelium. A–A″, E13; GAP-43. B–B″, P2; GAP-43. C–C″, P2; OMP. A–C, HCN1. A′–C′, HCN2. A″–C″, HCN4. Scale bars, 25 μm; scale bar in A applies to A–B″; scale bar in C applies to C–C″.

Figure 4.
Figure 4.

HCN subunits are coexpressed in OSNs at P2. A, HCN1 (green). B, HCN2 (blue). C, HCN4 (red). D, RGB image of A–C. Scale bar, 25 μm (in A).

Figure 5.
Figure 5.

HCNs are present in cultured primary OSNs. Incubation with the HCN channel blocker loperamide or ZD7288 reduces neurite outgrowth and branching. A–C, Cultured primary OSNs labeled with HCN1 (A), HCN2 (B), and HCN4 (C). D–G, Primary cultured OSNs labeled with NCAM. D, OSNs cultured in control conditions. E, OSNs cultured with 20 μ

m

loperamide. F–G, High magnification of boxed areas in D and E, respectively. H, Total neurite length (all neurite segments). I, Number of mean branch layers (1°, 2°, or 3° neurites). J, Number of branch points. K, Number of neurite roots. L, Number of extremities. M, Neurite field area. Scale bars: A–E, 10 μm; F, G, 2 μm. Error bars, SEM. *p < 0.05, statistically significant difference from controls.

Figure 6.
Figure 6.

Cultured primary OSNs from control and HCN1−/− mice challenged with 20 μ

m

loperamide or 30 μ

m

ZD 7288. Cells from HCN1−/− mice have significantly reduced neurite length compared to cells from B6129SF/2 mice (Student's t test, p < 0.05) in control conditions. In control mice loperamide and ZD 7288 reduce neurite outgrowth 46% (ANOVA, p < 0.0001). In HCN1−/− mice the effect is only 36% (ANOVA, p < 0.01), suggesting that HCN channels are responsible for the inhibitor effect. Error bars, SEM.

Figure 7.
Figure 7.

HCN1−/− mice have delayed glomerular formation. A, B, At E17 protoglomeruli normally seen in WT mice (A) have yet to form in HCN1−/− mice (B). C–F, At P4 glomeruli can be seen in WT and HCN1−/− mice. However, in the HCN1−/− mice glomeruli are poorly formed and lack discrete boundaries (D, F). Scale bar (in F), 100 μm in A–D; 200 μm in E and F.

Figure 8.
Figure 8.

Glomerular layer abnormalities persist in adult HCN1−/− mice. A, WT mice have discrete, round glomeruli. B, HCN1−/− mice have irregularly shaped glomeruli that are not well circumscribed. Scale bar for A and B (in A), 25 μm.

Figure 9.
Figure 9.

Glomerular size and distribution are perturbed in P4 HCN1−/− mice. A, Binned distribution shows the frequency of glomerular area (μm2) among dorsal glomeruli in WT and HCN1−/− mice. B, VGlut2-labeled sections from similar regions in WT and HCN1−/− mice; note the radial stacking of glomeruli in the HCN1−/− mice. Scale bar, 100 μm. Error bars, SEM.

Figure 10.
Figure 10.

HCN1−/− peripherin-positive OSN axons are not confined to the outer nerve layer of the developing OB. A–C, OSN nerve layer from WT mice. NCAM (red) labels OSN axons of the inner and outer nerve layer, while peripherin (green) labeling is restricted to the outer nerve layer; nuclear marker DRAQ5 is in blue. D–F, In HCN1−/− mice peripherin-labeled axons crossed into the inner nerve layer. Scale bar for A–F (in F), 50 μm.

Figure 11.
Figure 11.

In HCN1−/− mice MOR28 glomeruli change their location but have normal coalescence. A, The histogram shows the change in position relative to the first section containing AOB in the WT compared to HCN1−/− mice. B–E, OB sections from P0 mice labeled with MOR28 and DRAQ5. B, C, Left and right medial WT glomeruli, respectively. D, E, Left and right medial HCN1−/− glomeruli, respectively. RM, Right medial; LM, left medial; RL, right lateral; LL, left lateral. Scale bar for B–E (in E), 50 μm. Error bars, SEM. *p < 0.05, statistically significant difference from controls.

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