Light-induced calcium influx into retinal axons is regulated by presynaptic nicotinic acetylcholine receptor activity in vivo

Visual activity is thought to be a critical factor in controlling the devel opment of central retinal projections. Neuronal activity increases cytosoli c calcium, which was hypothesized to regulate process outgrowth in neurons. We performed an in vivo imaging study in the retinotectal system of albino Xenopus laevis tadpoles with the fluorescent calcium indicator calcium gre en 1 dextran (CaGD) to test the role of calcium in regulating axon arbor de velopment. We find that visual stimulus to the retina increased CaGD fluore scence intensity in retinal ganglion cell (RGC) axon arbors within the opti c tectum and that branch additions to retinotectal axon arbors correlated w ith a local rise in calcium in the parent branch. We find three types of re sponses to visual stimulus, which roughly correlate with the ON, OFF, and S USTAINED response types of RGC reported by physiological criteria. Imaging in bandscan mode indicated that patterns of calcium transients were nonunif orm throughout the axons. We tested whether the increase in calcium in the retinotectal axons required synaptic activity in the retina; intraocular ap plication of tetrodotoxin (10 mu M) or nifedipine (1 and 10 mu M) blocked t he stimulus-induced increase in RGC axonal fluorescence. A second series of pharmacological investigations was designed to determine the mechanism of the calcium elevation in the axon terminals within the optic tectum. Inject ion of bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid-AM (BAPTA-AM) (20 mM ) into the tectal ventricle reduced axonal calcium levels, supporting the i dea that visual stimulation increases axonal calcium. Injection of BAPTA (2 0 mM) into the tectal ventricle to chelate extracellular calcium also atten uated the calcium response to visual stimulation, indicating that calcium e nters the axon from the extracellular medium. Caffeine (10 mM) caused a lar ge increase in axonal calcium, indicating that intracellular stores contrib ute to the calcium signal. Presynaptic nicotinic acetylcholine receptors (n AChRs) may play a role in axon arbor development and the formation of the t opographic retinotectal projection. Injection of nicotine (10 mu M) into th e tectal ventricle significantly elevated RGC axonal calcium levels, wherea s application of the nAChR antagonist alpha BTX (100 nM) reduced the stimul us-evoked rise in RGC calcium fluorescence. These data suggest that light s timulus to the retina increases calcium in the axon terminal arbors through a mechanism that includes influx through nAChRs and amplification by calci um-induced calcium release from intracellular calcium stores. Such a mechan ism may contribute to developmental plasticity of the retinotectal system b y influencing both axon arbor elaboration and the strength of synaptic tran smission.