Summary Imbalanced levels of neuromodulators and other chemical signals contribute to a host of neurological disorders. Yet, previous studies describing these effects often examine only one molecule at a time, and typically provide a static description of signal levels in the brain or in the cerebrospinal fluid (CSF) that bathes all neurons. In reality, dozens of signals exhibit dynamic changes across states such as quiet waking and social or non-social arousal, which are altered in disease. The tracking and manipulation of patterns of neural activity has been critical to recent neuroscience progress. We lack analogous tools for estimation and control of dynamic patterns of neuromodulatory signals, which could revolutionize the study of brain states and effectively restore healthy states across neurologic and psychiatric disorders. Moreover, we do not understand how any given neuropsychiatric drug dynamically influences the levels of endogenous neuromodulators and peptides in the CSF or brain, thus impeding the rational design of optimal drug delivery strategies to maximize efficacy and minimize side effects. These blind spots are due to technical limitations: while cellular imaging and optogenetics have enabled ever-increasing precision in tracking and manipulation of brain cells, we lack the ability to accurately (i) record or (ii) control multiple neuromodulatory signals simultaneously in real time. We are overcoming the first challenge by developing novel methods for multiplexed, quantitative imaging of a panel of green fluorescent protein-based optical sensors of disease-relevant neuromodulatory signals (Aim 1): vasopressin, oxytocin, somatostatin, dopamine, norepinephrine, serotonin, acetylcholine, histamine, melatonin, corticotropin-releasing factor, vasoactive intestinal peptide, and adenosine. Briefly, sets of cultured cells expressing individual sensors are combined in a 3D hydrogel sensor array applied to the front of a gradient refractive index (GRIN) lens, which is inserted into the CSF or brain tissue of an awake, head-fixed mouse via a chronic cannula. Estimates of signal concentration using 3D two-photon imaging of the sensor array are then calibrated via post-hoc robotic dipping of the same sensor array into varying concentrations of each neuromodulator ex vivo. Once we have established this approach to track neuromodulatory composition across hours or days and across behavioral states (Aim 1), we will use closed-loop delivery methods to control dynamic patterns of up to a dozen neuromodulatory signals in the brain in awake mice and evaluate which patterns drive behavioral preference or avoidance (Aim 2).These experiments benefit from the use of fluorescence lifetime and well as fluorescence intensity measurements, allowing quantitative assessment of fluid composition across extended periods of time (hours to days) with minimal effects of bleaching. Together, these tools offer a novel, holistic framework for the study and control of multiple neuromodulators in the brain. The sensitive, real-time, multiplexed readout of signals in small volumes complements microdialysis and enables closed-loop control with applications to most domains of basic and clinical neuroscience research.

Project Narrative This proposal will develop new tools for real-time sensing of absolute concentrations of multiple neuromodulatory signals in the brain, using advanced one-photon and two-photon imaging. A detailed understanding of the levels of many neuromodulators and how this neuromodulatory signature changes across behavioral states is sorely lacking. Improved understanding and technology will allow testing of more sophisticated brain neuromodulation methods for improving cognitive and correction of chemical imbalances in the brain across neurologic and psychiatric disorders.