Abstract Neurotransmitter specification is a crucial step in defining neural circuit identity and function because it helps establish which neurons communicate with each other. Surprisingly, neurotransmitter specification is neither mutually exclusive nor immutable. That is, some neurons release multiple types of neurotransmitters with different physiological functions, and the relative abundance of different neurotransmitters can be modified based on environmental conditions and neuronal firing patterns. These features endow neurons with a type of functional plasticity that is often overlooked but can have profound effects on neural circuit function and thus on behavior. Unfortunately the mechanisms responsible for neurotransmitter plasticity are poorly understood. This gap in knowledge makes it very difficult to determine the contribution of neurotransmitter plasticity to normal physiology and to psychiatric disorders involving neural circuit dysfunction. To overcome this obstacle using fruit flies as a model system we have pioneered a combination of (1) ribosome profiling to identify all neurotransmitter-associated transcripts in small groups of neurons and (2) RNAscope-based quantification of transcript distribution across those same neurons, which can be simultaneously targeted by reporters and thus identified by immunohistochemistry. In this proposal we will employ these exquisitely sensitive techniques to identify the different neurotransmitters that are used by various arousal-regulating neurons in the circadian clock network and by neurons that control sleep. We will also use RNAscope to measure how neurotransmitter identity is modified by changes in neuronal activity, such as those expected during the sleep/wake cycle, and the extent to which this plasticity is cell-autonomously regulated through synaptic transmission. Lastly, we will use RNAscope to measure the selectivity of commonly used drivers for labeling of neurotransmitter systems. Our studies will thus introduce a new, powerful combination of techniques for studying the central nervous system of the fly, and they will define for the first time the full complement of neurotransmitters used by each cell of a well-studied behavioral circuit. Our studies will also provide a foundation for determining how neurotransmitter identity can be modulated by physiological and pathophysiological events to alter neural circuit function and ultimately behavior. In the long-term such mechanisms are expected to contribute to our understanding of neural circuit dysfunctions underlying neurological disorders.