Neuromodulatory communication among various neurons and non-neuronal cells mediates myriad physiological and pathologic processes, yet defining regulatory and functional features of neuromodulatory transmission remains challenging because of limitations of available monitoring tools. Recently developed genetically encoded neuromodulatory transmitter sensors, when combined with superresolution and/or deconvolution microscopy, allow the first visualization of neuromodulatory transmission with nanoscale or microscale spatiotemporal resolution. In vitro and in vivo experiments have validated several high-performing sensors to have the qualities necessary for demarcating fundamental synaptic properties of neuromodulatory transmission, and initial analysis has unveiled unexpected fine control and precision of neuromodulation. These new findings underscore the importance of synaptic dynamics in synapse-, subcellular-, and circuit-specific neuromodulation, as well as the prospect of genetically encoded transmitter sensors in expanding our knowledge of various behaviors and diseases, including Alzheimer's disease, sleeping disorders, tumorigenesis, and many others.

During development, sensory hair cells (HCs) in the cochlea assemble a stereociliary hair bundle on their apical surface with planar polarized structure and orientation. We have recently identified a non-canonical, Wnt/G-protein/PI3K signaling pathway that promotes cochlear outgrowth and coordinates planar polarization of the HC apical cytoskeleton and alignment of HC orientation across the cochlear epithelium. Here, we determined the involvement of the kinase Gsk3β and the small GTPase Rac1 in non-canonical Wnt signaling and its regulation of the planar cell polarity (PCP) pathway in the cochlea. We provided the first in vivo evidence for Wnt regulation of Gsk3β activity via inhibitory Ser9 phosphorylation. Furthermore, we carried out genetic rescue experiments of cochlear defects caused by blocking Wnt secretion. We showed that cochlear outgrowth was partially rescued by genetic ablation of Gsk3β but not by expression of stabilized β-catenin; while PCP defects, including hair bundle polarity and junctional localization of the core PCP proteins Fzd6 and Dvl2, were partially rescued by either Gsk3β ablation or constitutive activation of Rac1. Our results identify Gsk3β and likely Rac1 as downstream components of non-canonical Wnt signaling and mediators of cochlear outgrowth, HC planar polarity, and localization of a subset of core PCP proteins in the cochlea.