Spontaneous acetylcholine secretion from developing growth cones of Drosophila central neurons in culture: Effects of cAMP-pathway mutations

Abstract

We describe a novel bioassay system that uses Xenopus embryonic myocytes (myoballs) to detect the release of acetylcholine from Drosophila CNS neurons. When a voltage-clamped Xenopus myoball was manipulated into contact with cultured Drosophila 'giant' neurons, spontaneous synaptic current-like events were registered. These events were observed within seconds after contact and were blocked by curare and α-bungarotoxin, but not by TTX and Cd2+, suggesting that they are caused by the spontaneous quantal release of acetylcholine (ACh). The secretion occurred not only at the growth cone, but also along the neurite and at the soma, with significantly different release parameters among various regions. The amplitude of these currents displayed a skewed distribution. These features are distinct from synaptic transmission at more mature synapses or autapses formed in this culture system and are reminiscent of the transmitter release process during early development in other preparations. The usefulness of this co- culture system in studying presynaptic secretion mechanisms is illustrated by a series of studies on the cAMP pathway mutations, dunce (dnc) and PKA-RI, which disrupt a cAMP-specific phosphodiesterase and the regulatory subunit of cAMP-dependent protein kinase A, respectively. We found that these mutations affected the ACh current kinetics, but not the quantal ACh packet, and that the release frequency was greatly enhanced by repetitive neuronal activity in dnc, but not wildtype, growth cones. These results suggest that the cAMP pathway plays an important role in the activity-dependent regulation of transmitter release not only in mature synapses as previously shown, but also in developing nerve terminals before synaptogenesis.