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 development of central retinal projections. Neuronal activity increases cytosolic 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 green 1 dextran (CaGD) to test the role of calcium in regulating axon arbor development. We find that visual stimulus to the retina increased CaGD fluorescence intensity in retinal ganglion cell (RGC) axon arbors within the optic tectum and that branch additions to retinotectal axon arbors correlated with a local rise in calcium in the parent branch. We find three types of responses to visual stimulus, which roughly correlate with the ON, OFF, and SUSTAINED response types of RGC reported by physiological criteria. Imaging in bandscan mode indicated that patterns of calcium transients were nonuniform throughout the axons. We tested whether the increase in calcium in the retinotectal axons required synaptic activity in the retina; intraocular application of tetrodotoxin (10 microM) or nifedipine (1 and 10 microM) blocked the 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. Injection 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 idea that visual stimulation increases axonal calcium. Injection of BAPTA (20 mM) into the tectal ventricle to chelate extracellular calcium also attenuated the calcium response to visual stimulation, indicating that calcium enters the axon from the extracellular medium. Caffeine (10 mM) caused a large increase in axonal calcium, indicating that intracellular stores contribute to the calcium signal. Presynaptic nicotinic acetylcholine receptors (nAChRs) may play a role in axon arbor development and the formation of the topographic retinotectal projection. Injection of nicotine (10 microM) into the tectal ventricle significantly elevated RGC axonal calcium levels, whereas application of the nAChR antagonist alphaBTX (100 nM) reduced the stimulus-evoked rise in RGC calcium fluorescence. These data suggest that light stimulus to the retina increases calcium in the axon terminal arbors through a mechanism that includes influx through nAChRs and amplification by calcium-induced calcium release from intracellular calcium stores. Such a mechanism may contribute to developmental plasticity of the retinotectal system by influencing both axon arbor elaboration and the strength of synaptic transmission.
Edwards
J A
JA
Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA.
Cline
H T
HT
D000818:Animals; D001370:Axonal Transport / Q000187:drug effects; D001369:Axons / Q000378:metabolism* / Q000648:ultrastructure; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D000697:Central Nervous System Stimulants / Q000494:pharmacology; D002614:Chelating Agents / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D020439:Growth Cones / Q000502:physiology; D007202:Indicators and Reagents / Q000378:metabolism; D007814:Larva; D008027:Light; D018722:Nicotinic Agonists / Q000494:pharmacology; D018733:Nicotinic Antagonists / Q000494:pharmacology; D010775:Photic Stimulation; D011978:Receptors, Nicotinic / Q000187:drug effects / Q000378:metabolism*; D017661:Receptors, Presynaptic / Q000378:metabolism*; D012160:Retina / Q000166:cytology / Q000378:metabolism*; D013779:Tetrodotoxin / Q000494:pharmacology; D013997:Time Factors; D014982:Xenopus laevis
D002121:Calcium Channel Blockers; D000697:Central Nervous System Stimulants; D002614:Chelating Agents; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D018722:Nicotinic Agonists; D018733:Nicotinic Antagonists; D011978:Receptors, Nicotinic; D017661:Receptors, Presynaptic; D013779:Tetrodotoxin; D002118:Calcium