The Journal of Neuroscience, October 24, 2007, 27(43):11501-11509; doi:10.1523/JNEUROSCI.3057-07.2007
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Development/Plasticity/Repair
Cell-Autonomous Inhibition of 
7-Containing Nicotinic Acetylcholine Receptors Prevents Death of Parasympathetic Neurons during Development
Martin Hruska and
Rae Nishi
Department of Anatomy and Neurobiology, University of Vermont College of Medicine, Burlington, Vermont 05405
Correspondence should be addressed to Dr. Rae Nishi, Department of Anatomy and Neurobiology, 149 Beaumont Avenue, Burlington, VT 05405. Email: rnishi{at}uvm.edu
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 
7-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 7 activation in neurons prevented cell death. E8 neurons are heterogeneous with respect to 7-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 -bungarotoxin that is tethered to the membrane by a glycosylphosphatidylinositol linkage (GPIbtx) in ciliary ganglion neurons with the retroviral vector RCASBP(A) blocks increases in intracellular calcium induced by nicotine through 7-nAChRs and prevents neurons from dying. Expression of GPIbtx in surrounding non-neural tissues, but not in neurons, does not prevent cell loss. Furthermore, the GPIbtx 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 7-nAChRs, possibly by leading to increases in intracellular calcium that exceed the threshold for cell survival.
Key words: glycosylphosphatidylinositol; -bungarotoxin; RCAS; calcium imaging; ciliary ganglion; retrovirus
Received July 5, 2007;
revised Aug. 17, 2007;
accepted Sept. 4, 2007.
Correspondence should be addressed to Dr. Rae Nishi, Department of Anatomy and Neurobiology, 149 Beaumont Avenue, Burlington, VT 05405. Email: rnishi{at}uvm.edu
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