PROJECT SUMMARY Throughout the central auditory pathway, the nervous system increasingly “colors” sensory representations with non-sensory features, such as an animal’s arousal state. Fluctuations in arousal has been found to influence sensory information processing and behavior. The Yerkes-Dodson law describes the relationship between arousal and task performance as an inverted-U in that intermediate states of arousal coincide with peak performance. While this has been shown in several auditory studies, the neural correlates of this behavioral phenomenon are poorly understood. Correlations between pupil diameter, a well-established biomarker of arousal state, and neural activity have been reported in the auditory system. Within the auditory cortex (ACtx), arousal has been shown to modulate the response properties of layer (L) 2/3 cells. However, the ACtx is composed of different subpopulations of cells that each differ with respect to their physiology, anatomy, and projection patterns. Moreover, cells contained in deeper layers, such as L5, provide the primary means by which the ACtx sends auditory information to major subcortical stations critical for decision-making. Layer 5 comprises two distinct projection cells types: intratelencephalic (IT) and extratelencephalic (ET) cells. The distinct anatomy and connectivity profiles of IT and ET cells lead us to hypothesize that arousal states differentially modulate their sensory tuning properties, which then influences behavior. Our preliminary data suggests that L5 cells are uniquely modulated by arousal. Aim 1 will characterize the arousal-dependent changes to L5 IT and ET cell response properties in mice passively listening to pure tones. We will simultaneously use a combination of in vivo two-photon calcium imaging and pupillometry to track neural activity and pupil diameters, respectively. Aim 2 will discern the optimal arousal state in an auditory categorization task and will relate L5 IT and ET cell activity at this optimal state to an animal’s performance. As in Aim 1, pupillometry and two-photon imaging in awake mice will be used. Through a cell-type specific approach, this research proposal will unveil the intricate relationship between arousal, distinct L5 neural activity, and auditory-guided behaviors.