Abstract Activity of the brain across structures is an orchestrated process that spans a broad range of time and space scales. Highly coordinated communication is what activate responses to stimuli, makes behavior possible, and generates memories. To observe exchanges of information at the cellular and synaptic level, neuroscientists have been increasingly using non-invasive imaging techniques that rely on genetically encoded indicators based on fluorescent proteins (FPs). Calcium sensors, such as GCaMPs, are routinely used to probe neuronal firing, and, more recently, indicators targeting small molecule neurotransmitters/modulators (e.g.: glutamate, GABA, dopamine) were developed and quickly gained popularity in the field. Small-molecules indicators allow direct visualization of chemical communication, providing a large amount of information on the type of inputs used in neuronal networks in association with stimuli. Sensors based on fluorescent proteins are suitable for imaging fast, transient synaptic responses, however they are not able to provide information on integrated signals from large scale areas in the brain. Here, we propose a new sensor design for probing small molecules in the brain. We take advantage of chemical fluorophore, which are brighter, more photostable and have broader color-spectrum compared to FPs. The development of various in-cell labeling strategies have put the chemical fluorophore under genetic control. We thus propose to develop chemigenetic sensors based on self-labeling proteins (SNAP-tag, Halo-tag), which is engineered to become dependent on the presence of a neurotransmitter/modulator. In a preliminary study, we coupled a split version of SNAP-tag to a glutamate-binding protein (GltI, from E. coli) and showed that labeling of the construct with a fluorescent dye occurs proportionally to the amount of glutamate in solution. Aim 1 will build upon our preliminary results to improve the design of the construct and optimize the dynamic range of the sensor. We will use rational engineering, as well as random mutagenesis combined with high- throughput screening to improve the current design. We will then proceed with characterization of the sensor in vitro, as well as ex vivo in HEK cells and dissociated neurons. Ultimately, we will perform testing in cultured and acute hippocampal slices with 2-photon microscopy. Aim 2 will expand the scope of the sensor by exploring a broader range of fluorescent dyes and color variants to probe into the multiplexing capabilities of the sensor. Furthermore, we will incorporate the modular design into binding proteins derived from sensors for other neurotransmitters/modulators, with particular attention to GABA, acetylcholine and serotonin. We will also explore the use of Halo-tag as a self-labeling protein, to increase the multiplexing ability of our approach to more than one type of input signal.