While the role of cholinergic neurotransmission from motoneurons is well established during neuromuscular development, whether it regulates central nervous system development in the spinal cord is unclear. Zebrafish presents a powerful model to investigate how the cholinergic system is set up and evolves during neural circuit formation. In this study, we carried out a detailed spatiotemporal analysis of the cholinergic system in embryonic and larval zebrafish. In 1-day-old embryos, we show that spinal motoneurons express presynaptic cholinergic genes including choline acetyltransferase (chata), vesicular acetylcholine transporters (vachta, vachtb), high-affinity choline transporter (hacta) and acetylcholinesterase (ache), while nicotinic acetylcholine receptor (nAChR) subunits are mainly expressed in interneurons. However, in 3-day-old embryos, we found an unexpected decrease in presynaptic cholinergic transcript expression in a rostral to caudal gradient in the spinal cord, which continued during development. On the contrary, nAChR subunits remained highly expressed throughout the spinal cord. We found that protein and enzymatic activities of presynaptic cholinergic genes were also reduced in the rostral spinal cord. Our work demonstrating that cholinergic genes are initially expressed in the embryonic spinal cord, which is dynamically downregulated during development suggests that cholinergic signaling may play a pivotal role during the formation of intra-spinal locomotor circuit.

Survival of animals is dependent on the correct selection of an appropriate behavioral response to competing external stimuli. Theoretical models have been proposed and underlying mechanisms are emerging to explain how one circuit is selected among competing neural circuits. The evolutionarily conserved forebrain to midbrain habenulo-interpeduncular nucleus (Hb-IPN) pathway consists of cholinergic and non-cholinergic neurons, which mediate different aversive behaviors. Simultaneous calcium imaging of neuronal cell bodies and of the population dynamics of their axon terminals reveals that signals in the cell bodies are not reflective of terminal activity. We find that axon terminals of cholinergic and non-cholinergic habenular neurons exhibit stereotypic patterns of spontaneous activity that are negatively correlated and localize to discrete subregions of the target IPN. Patch-clamp recordings show that calcium bursts in cholinergic terminals at the ventral IPN trigger excitatory currents in IPN neurons, which precede inhibition of non-cholinergic terminals at the adjacent dorsal IPN. Inhibition is mediated through presynaptic GABA B receptors activated in noncholinergic habenular neurons upon GABA release from the target IPN. Together, the results reveal a hardwired mode of competition at the terminals of two excitatory neuronal populations, providing a physiological framework to explore the relationship between different aversive responses.

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