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Comparative Study
. 2007 Oct 24;27(43):11501-9.
doi: 10.1523/JNEUROSCI.3057-07.2007.

Cell-autonomous inhibition of alpha 7-containing nicotinic acetylcholine receptors prevents death of parasympathetic neurons during development

Martin Hruska  1 , Rae Nishi
Affiliations
Comparative Study

Cell-autonomous inhibition of alpha 7-containing nicotinic acetylcholine receptors prevents death of parasympathetic neurons during development

Martin Hruska et al. J Neurosci. .

Abstract

Neurotrophic molecules are key retrograde influences of cell survival in the developing nervous system, but other influences such as activity are also emerging as important factors. In the avian ciliary ganglion, half the neurons are eliminated between embryonic day 8 (E8) and E14, but it is not known how cell death is initiated. Because systemic application of alpha7-nicotinic acetylcholine receptor (nAChR) antagonists prevents this cell loss, we examined differences in receptor densities and responses of intracellular calcium to nicotine using the calcium-sensitive dye fura-2. In addition, we determined whether cell-autonomous inhibition of alpha7 activation in neurons prevented cell death. E8 neurons are heterogeneous with respect to alpha7-nAChR density, which leads to large increases in [Ca2+]i in some neurons; E8 neurons also exhibit a slower rate of Ca2+ decay after nicotinic stimulation than E13 neurons. Expressing alpha-bungarotoxin that is tethered to the membrane by a glycosylphosphatidylinositol linkage (GPIalpha btx) in ciliary ganglion neurons with the retroviral vector RCASBP(A) blocks increases in intracellular calcium induced by nicotine through alpha7-nAChRs and prevents neurons from dying. Expression of GPIalpha btx in surrounding non-neural tissues, but not in neurons, does not prevent cell loss. Furthermore, the GPIalpha btx is not efficiently expressed in the accessory oculomotor neurons, eliminating preganglionic inputs as another site for action of the antagonist. These results support the hypothesis that cholinergic inputs facilitate cell death in the developing autonomic nervous system by activating alpha7-nAChRs, possibly by leading to increases in intracellular calcium that exceed the threshold for cell survival.

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Figures

Figure 1.
Figure 1.
Cell-surface α7-nAChRs on developing ciliary ganglion neurons. A–G, Acutely dissociated neurons were incubated while alive with αbtx–Alexa 488 and then imaged (A–C) or subjected to flow cytometry (D–G). E13 neurons (B, C) were fixed and stained for somatostatin immunoreactivity to identify choroid neurons. A, At E8, some neurons express a large number of α7-nAChRs (arrow), whereas others have very low numbers (arrowhead). B, At E13, α7-nAChRs are clustered on ciliary neurons (arrows). C, E13 choroid neurons have evenly distributed receptors on their cell surface. Similar results were obtained from three additional experiments. Images were captured as optical stacks on an Olympus epifluorescence microscope and deconvolved on a DeltaVision workstation. Scale bar, 15 μm. D–G, Acutely dissociated E6–E10 ciliary ganglion neurons were colabeled with the neuronal-specific Q211 antibody and αbtx–Alexa 488, and at each age, 40,000 cells were analyzed by flow cytometry. Axes for all four graphs are identical to those labeled in F. Cells in the upper right quadrant are neurons (Q211 positive) that express α7-nAChRs (αbtx binding). D, E, The majority of neurons label at a tightly clustered level of αbtx intensity; however, at E6 (D) and E7 (E), there is a subpopulation of cells with high αbtx–Alexa 488 binding (arrowhead) and a subpopulation with very low αbtx–Alexa 488 binding (arrow). F, At E9, all neurons bind αbtx, but few neurons still exhibit high αbtx binding (arrowhead). G, By E10, virtually all neurons have relatively homogeneous αbtx–Alexa 488 binding.
Figure 2.
Figure 2.
Calcium influx through α7-nAChRs during ciliary ganglion development. Acutely dissociated E8 neurons were loaded with the ratiometric calcium indicator dye fura-2 and stimulated by perfusing with 10 μm nicotine. A, Cell responses to two applications of nicotine (Nic1 and Nic2) for 20 s with 5 min washout between them are shown. B, The mean Nic1 and Nic2 Ca2+ responses are not significantly different, indicating that nAChRs recover from desensitization during the 5 min washout (p > 0.05, Student's t test; n = 14 neurons). C, Nicotine-induced Ca2+ response is significantly reduced by treatment with MLA (n = 34 neurons; ***p < 0.0001, one-way ANOVA) and completely eliminated by treatment with MLA and DHβE (n = 30 neurons; p < 0.0001, one-way ANOVA). D, At E8, α7-nAChR-specific Ca2+ responses are distributed over a wide range (mean, 0.09 ± 0.007; n = 41), whereas E9 Ca2+ responses are skewed to the left with majority of Ca2+ responses clustered around mean (0.06 ± 0.004; n = 80). Compared with E9 neurons, 20 s application of 10 μm nicotine induces larger increases in [Ca2+]i in E8 neurons (p < 0.0002, Student's t test). E, F, The rate of Ca2+ decay was measured in E8 and E13 acutely dissociated neurons. Traces were normalized to the percentage of peak Ca2+ amplitude to detect changes in the time course of Ca2+ decay. E, At E8, Ca2+ decays back to baseline at a much slower rate than at E13 (E8: τ = 48.9 ± 0.98 s, n = 33 from 3 different ganglia; E13: τ1 = 17.5 ± 0.42 s, τ2 = 175.6 ± 91.4 s, n = 6 from 5 different ganglia). F, Treatment with MLA eliminates the differences in the rate of Ca2+ decay between E8 and E13 neurons. Values represent mean ± SEM. AU, Arbitrary units.
Figure 3.
Figure 3.
GPIαbtx expression in ciliary ganglion neurons after injection with RCASBP(A) retrovirus. A, Diagram of GPIαbtx cassette, containing the mature αbtx sequence linked to the lynx1 consensus sequence (seq) for addition of a GPI anchor. B, C, Dissociated E8 ciliary ganglion neurons infected with control (B, open) RCASBP(A) or RCASBP(A)–GPIαbtx (C) were incubated with anti-αbtx while alive at 4°C to label cell-surface αbtx (green), fixed, and colabeled with neuronal-specific Hu C/D antibody (red) to demonstrate efficacy of infection. Only neurons infected with the RCASBP(A)–GPIαbtx are labeled with the antibody. Scale bar, 10 μm. D, GPIαbtx-infected neurons exhibit a significant reduction in calcium influx via α7-nAChRs compared with the open-infected neurons (***p < 0.0001, Student's t test). GPIαbtx reduces the peak Ca2+ amplitude to the same extent as exogenously applied αbtx and exogenous application of αbtx does not further reduce peak calcium amplitude in GPIαbtx-infected neurons. Values represent mean ± SEM of results from ≥20 neurons for each treatment from three separate experiments.
Figure 4.
Figure 4.
The majority of GPIαbtx is not released from cell membranes. A–D, Uninfected or GPIαbtx-infected E8 neurons were incubated alone or coincubated for 24 h and labeled while alive with αbtx–Alexa 488 to identify cell-surface α7-nAChRs. B, D, Uninfected neurons label brightly with αbtx–Alexa 488 whether they are incubated alone (B) or coincubated with GPIαbtx-infected neurons (D), demonstrating that few of the cell-surface α7-nAChRs are occupied by GPIαbtx that has been released from the membrane. C, GPIαbtx-infected neurons do not label with αbtx–Alexa 488, showing that cell-surface α7-nAChRs are either occupied by membrane-tethered αbtx or they fail to traffic to the cell surface when GPIαbtx is expressed. Scale bar, 15 μm.
Figure 5.
Figure 5.
Cell-autonomous inhibition with GPIαbtx rescues ciliary ganglion neurons from cell death. A–F, Embryos were infected with open RCASBP(A) or RCASBP(A)–GPIαbtx at 36 h of development (stage 8–9; A–C) or 48 h of development (stage 10–13; D–F). Immunoreactivity for p27gag (green) is observed in neurons (red) of ciliary ganglia from embryos infected at stage 8–9 (A, C; arrows indicate few uninfected neurons), whereas only surrounding non-neural tissue is immunoreactive for p27gag in embryos infected at stage 10–13 (D, F). Scale bars: (in F) 200 μm; inset, 50 μm. G, Serially sectioned E14 ganglia were labeled with neuronal-specific Islet-1 antibody (nuclear; arrows) and somatostatin antibody (cytoplasmic; arrowheads) to identify choroid neurons. Scale bars, 50 μm. H, The total number of neurons (Islet-1 positive) and the number of choroid neurons (somatostatin positive) in sections stained as in G were counted by design-based stereology, and the number of ciliary neurons was inferred by subtracting the number of choroid neurons from the total. Expression of GPIαbtx prevents cell death of ciliary (**p < 0.01, one-way ANOVA) and choroid (***p < 0.001, one-way ANOVA) neurons. Expression of GPIαbtx only in surrounding non-neural tissues does not prevent cell death. Values represent mean ± SEM.
Figure 6.
Figure 6.
Presynaptic α7-nAChRs from AON are unlikely to mediate death of ciliary ganglion neurons. A, Neurons in the preganglionic AON from embryos infected at stage 8–9 were identified with Hu C/D antibody. B, Only surrounding glia, but not Hu C/D-positive cells, label with p27gag antibody, indicating that AON neurons are not infected. C, Overlay of the Hu C/D immunoreactivity (red) with the p27gag immunoreactivity (green). Only a few neurons show p27gag immunoreactivity (arrows). Scale bars: (in C) 200 μm; inset, 50 μm.
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