Nonvesicular release of acetylcholine is required for axon targeting in theDrosophilavisual system

Abstract

We report evidence for a developmental role of acetylcholine in axon pathfinding in the Drosophila visual system. Acetylcholine was detected on photoreceptor axons during their navigation to target sites in the brain, a time well before the formation of functional synapses. The pattern of photoreceptor axon projections was severely disrupted when acetylcholine synthesis or metabolism was altered or eliminated, or when transgenic ␣-bungarotoxin, a nicotinic acetylcholine receptor antagonist, was expressed in the developing eye or brain. The requirement for acetylcholine signaling exists before photoreceptor neurons form synaptic connections and does not require the function of vesicular acetylcholine transporter protein. That this early effect of acetylcholine is mediated through nonvesicular release is further supported by the observation that transgenic expression of tetanus toxin, a blocker of neurotransmitter release via synaptic vesicles, did not cause similar photoreceptor axon projection defects. These observations support the notion that a form of acetylcholine secretion mediates the behavior of growth cones during axon pathfinding.I t has been generally thought that the release of neurotransmitter at axon terminals reflects synaptic activity and contributes to the development of synaptic connectivity via activitydependent processes. However, a growing body of evidence suggests that neurotransmitters function in early development in processes that can be independent of synapses or synaptic activity, such as cell proliferation, differentiation, migration, axon outgrowth, and axon branching. In these roles, neurotransmitters are apparently released by mechanisms that are distinct from the conventional synaptic vesicular pathway (1, 2). For example, it has been shown that the neurotransmitters ␥-aminobutyric acid and glutamate can be released in a Ca 2ϩ -and soluble N-ethylmaleimide-sensitive factor attachment protein receptor-independent manner, before synapse formation (3).The effects of loss of acetylcholine synthesis on branching patterns of motor axons is particularly interesting in light of the earlier observation that neurotransmitters may function as chemical signals in axon pathfinding. Experiments on isolated embryonic chick and Xenopus neurons (4, 5) and Drosophila CNS neurons (6) have indicated that the transmitter acetylcholine is synthesized very early in neural development and is present on growing axons well before they reach their target fields or establish functional synapses. Experiments in culture have supported the notion that acetylcholine may have a role in axon navigation. An acetylcholine gradient can cause a growth cone to change direction (7). These studies raise the possibility that nonsynaptic release of neurotransmitter might play a role in regulating growth cone behavior before synaptogenesis.We have undertaken an investigation of the role of small molecule neurotransmitters in the development of the Drosophila visual system. In this system, light transduction depends on synthe...

The Vesicular Acetylcholine Transporter Is Required for Neuromuscular Development and Function

“…Previous experiments with munc18-1 null mice, which have no regulated secretion of a neurotransmitter, also suggested that NMJ development is reg- ulated by synaptic vesicle exocytosis, although for these mutants, it has not been established whether ACh synthesis and storage are affected (28). A number of previously reported studies suggested that distinct pathways of ACh secretion might exist at cholinergic synapses (56,60,61,64). Moreover, vesamicol-independent ACh release, presumably from synaptic vesicles, can be detected in response to pharmacological treatments (2,7,10,11,46).…”

“…Quantal ACh release, comparable to that seen in developing nerve terminals, has been detected in myocytes and fibroblasts in culture, which presumably do not express VAChT (14,24). More recently, it was found that the correct targeting of Drosophila photoreceptor axons is disrupted in flies with null mutations in ChAT (64). Remarkably, the inactivation of VAChT did not produce the same result (64).…”

“…More recently, it was found that the correct targeting of Drosophila photoreceptor axons is disrupted in flies with null mutations in ChAT (64). Remarkably, the inactivation of VAChT did not produce the same result (64). The result suggests that the release of ACh during development is not dependent on VAChT, perhaps because it is nonvesicular or because vesicular storage can occur without VAChT.…”

Selective antagonism of muscarinic receptors is neuroprotective in peripheral neuropathy

“…Selective antagonism of muscarinic receptors is neuroprotective in peripheral neuropathy opment (22,23). In mammals, cell bodies of sensory neurons synthesize and secrete ACh (24), express a peripheral form of choline acetyltransferase (pChAT), exhibit ChAT activity, have low acetylcholinesterase (AChE) activity, and express multiple muscarinic receptors including muscarinic Ach type 1 receptor (M 1 R) (25)(26)(27).…”

Acetylcholine negatively regulates development of the neuromuscular junction through distinct cellular mechanisms

“…For example, the neurotransmitter acetylcholine (ACh) is released from embryonic motor neurons (MNs) at the neuromuscular junction (NMJ), and it negatively regulates survival, axon branching, and synapse formation (4)(5)(6)(7). ACh is also released from developing neurons even before they arrive at their target, and it acts in an autocrine and/or paracrine fashion to regulate growth (8), pathfinding (9), spontaneous activity (10), and target selection (11). Whereas the NMJ has served as an excellent model to elucidate the process of synapse formation, the cellular and molecular mechanisms by which ACh regulates this process are still unclear; this is in part because the expression of ACh receptor (AChR) subtypes varies over space and time during the development of this synapse.…”