Project Summary/Abstract Activity-dependent associative Hebbian plasticity, is recognized as a core mechanism underlying learning and memory, cognitive function, and brain development. Hebbian plasticity is intrinsically unstable due to its positive feedback nature, and has to be balanced by homeostatic mechanisms, which maintain the stability of overall neuronal activity. A major gap in the field concerns the incomplete understanding of cellular pathways by which neurons maintain homeostasis and how these mechanisms interact with Hebbian plasticity, especially on fast time scales. Our preliminary data suggest that a recently discovered neuronal membrane proteasome (NMP) may be involved in the fast homeostatic mechanism in vivo. NMPs are expressed in the tadpole brain and degrade nascent proteins in vivo. Inhibition of NMP activity led to a rapid increase in spontaneous neuronal activity and abolished learning-induced behavioral improvement in a visuomotor behavior paradigm. Activity-induced de novo synthesis of proteins that are important for the expression of downstream plasticity mechanisms is a hallmark of Hebbian plasticity. We hypothesize that NMP-mediated degradation of activity-induced nascent proteins serves as a negative feedback mechanism for fast homeostatic regulation of neuronal activity in response to plasticity-inducing activities. We will test this hypothesis in a well-established visually driven experience-dependent plasticity paradigm in Xenopus laevis tadpoles, which allows the combination of biochemical, physiological, molecular genetics, and behavioral experiments in an intact neural circuit with physiologically relevant sensory stimulation. Most critically, this experimental system provides the fast temporal resolution that is pivotal for the investigation of the rapid degradation of nascent proteins by NMPs in vivo. Specifically, in Aim 1, we will use in vivo BONCAT labeling to characterize the proteolytic activity of NMPs under different activity regimens and use expansion microscopy to delineate the spatiotemporal expression profile of NMPs in the optic tectum over development. In Aim 2, we will combine in vivo Ca++ imaging with molecular genetic tools to examine how NMPs regulate spontaneous and visually-evoked activity in tectal neurons, and determine whether NMP-mediated regulation of neuronal activity is cell-autonomous. In Aim3, we will use time-lapse structural and functional imaging and the visual avoidance behavior to assess the functional role of NMPs in experience-dependent plasticity at both cellular and circuit levels in the visual system. The proposed experiments will generate data for in-depth understanding of the NMP function in vivo. These results will shed light on a novel proteostasis-based fast homeostatic mechanism and lay the groundwork for future studies to further elucidate downstream cellular and molecular pathways underlying the functional interplay between activity-dependent proteostasis of nasc...