One challenge in biology is signal transduction monitoring in a physiological context. Intravital imaging techniques are revolutionizing our understanding of tumor and host cell behaviors in the tumor environment. However, these deep tissue imaging techniques have not yet been adopted to investigate the second messenger calcium (Ca2+). In the present study, we established conditions that allow the in vivo detection of Ca2+ signaling in three-dimensional tumor masses in mouse models. By combining intravital imaging and a skinfold chamber technique, we determined the ability of photodynamic cancer therapy to induce an increase in intracellular Ca2+ concentrations and, consequently, an increase in cell death in a p53-dependent pathway.

The deafness locus DFNB1 contains GJB2, the gene encoding connexin26 and GJB6, encoding connexin30, which appear to be coordinately regulated in the inner ear. In this work, we investigated the expression and function of connexin26 and connexin30 from postnatal day 5 to adult age in double transgenic Cx26^Sox10Cre mice, which we obtained by crossing connexin26 floxed mice with a deleter Sox10–Cre line. Cx26^Sox10Cre mice presented with complete connexin26 ablation in the epithelial gap junction network of the cochlea, whereas connexin30 expression was developmentally delayed; immunolabeling patterns for both connexins were normal in the cochlear lateral wall. In vivo electrophysiological measurements in Cx26^Sox10Cre mice revealed profound hearing loss accompanied by reduction of endocochlear potential, and functional experiments performed in postnatal cochlear organotypic cultures showed impaired gap junction coupling. Transduction of these cultures with a bovine adeno associated virus vector restored connexin26 protein expression and rescued gap junction coupling. These results suggest that restoration of normal connexin levels by gene delivery via recombinant adeno associated virus could be a way to rescue hearing function in DFNB1 mouse models and, in future, lead to the development of therapeutic interventions in humans.

Phosphatidylinositol phosphate kinase type 1γ (PIPKIγ) is a key enzyme in the generation of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P ₂ ] and is expressed at high levels in the nervous system. Homozygous knockout mice lacking this enzyme die postnatally within 24 h, whereas PIPKIγ ^+/− siblings breed normally and have no reported phenotype. Here we show that adult PIPKIγ ^+/− mice have dramatically elevated hearing thresholds for high-frequency sounds. During the first postnatal week we observed a reduction of ATP-dependent Ca ²⁺ signaling activity in cochlear nonsensory cells. Because Ca ²⁺ signaling under these conditions depends on inositol-1,4,5-trisphosphate generation from phospholipase C (PLC)-dependent hydrolysis of PI(4,5)P ₂ , we conclude that ( i ) PIPKIγ is primarily responsible for the synthesis of the receptor-regulated PLC-sensitive PI(4,5)P ₂ pool in the cell syncytia that supports auditory hair cells; ( ii ) spatially graded impairment of this signaling pathway in cochlear nonsensory cells causes a selective alteration in the acquisition of hearing in PIPKIγ ^+/− mice. This mouse model also suggests that PIPKIγ may determine the level of gap junction contribution to cochlear development.

We have previously shown that in vitro transduction with bovine adeno–associated viral (BAAV) vectors restores connexin expression and rescues gap junction coupling in cochlear organotypic cultures from connexin–deficient mice that are models DFNB1 nonsyndromic hearing loss and deafness. The aims of this study were to manipulate inner ear connexin expression in vivo using BAAV vectors, and to identify the optimal route of vector delivery. Injection of a BAAV vector encoding a bacterial Cre recombinase via canalostomy in adult mice with floxed connexin 26 (Cx26) alleles promoted Cre/LoxP recombination, resulting in decreased Cx26 expression, decreased endocochlear potential, increased hearing thresholds, and extensive loss of outer hair cells. Injection of a BAAV vector encoding GFP-tagged Cx30 via canalostomy in P4 mice lacking connexin 30 (Cx30) promoted formation of Cx30 gap junctions at points of contacts between adjacent non-sensory cells of the cochlear sensory epithelium. Levels of exogenous Cx30 decayed over time, but were still detectable four weeks after canalostomy. Our results suggest that persistence of BAAV-mediated gene replacement in the cochlea is limited by the extensive remodeling of the organ of Corti throughout postnatal development and associated loss of non-sensory cells.