Project Summary Non-invasive measurement of electrical activity in the body has been used for over a century to assess biological function. For example, characteristics of the electrocardiogram (ECG) are widely used to assess very specific cardiac functions. This is possible because the mechanisms and sources of ECG waves are known. Similarly, stimulus evoked potentials are routinely used to assess sensory function. For example, auditory brainstem responses (ABRs) evoked by sound are used to assess function of one (monaural) or both (binaural) ears. Distinct peaks in ABR waveforms map roughly to specific nuclei or fiber tracts in the ascending auditory pathway and can be used to assess function at these different levels. While ABRs are widely used to assess monaural hearing, assessment of binaural hearing remains a major clinical challenge - no routine objective clinical measure currently exists to test for it. However, a derived component of the ABR, referred to as the binaural interaction component (BIC), has been shown over the past decades to correlate with binaural hearing capabilities in normal and hearing impaired listeners and thus represents a promising objective measure of binaural function. The most prominent BIC peak, termed DN1, has been shown to be reduced (or even absent) in populations with binaural hearing impairments including children who have experienced temporary conductive hearing loss or been diagnosed with central auditory processing or autism spectrum disorders, and the aged. Moreover, the amplitude and latency of DN1 vary systematically with binaural cues, interaural time and level differences, and can predict perceived laterality of an auditory stimulus. Despite the promise of BIC as a biomarker, in humans BIC DN1 is small and unreliably measured using typical clinical methodology. We posit that a better understanding of the brainstem source of DN1 may provide clues to why it is unreliably measured and also suggest avenues for more reliable measurement methods. Earlier attempts to resolve the circuitry of the BIC using pharmacological and lesioning approaches were inconclusive. However, several recent studies suggest that the lateral (LSO) and not the medial (MSO) superior olive of the brainstem as a likely candidate. However, these studies are correlational and thus do not prove the LSO is the source of the BIC. The LSO receives near-coincident excitatory and inhibitory input from the two ears which could theoretically underlie BIC DN1. The experiments comprising Aim 1 employ a novel combination of optogenetic and electrophysiological techniques to conclusively determine the brain region generating the BIC. Based in part on these results, Aim 2 will determine the optimal stimulus for evoking the BIC, with the goal of reducing variability in BIC measurements. These experiments will reveal the neural generator of the BIC DN1 and reduce sources of variability in BIC measurements by determining the optimal stimuli to elici...