Ann.kennedy.05

The identification of active neurons and circuits in vivo is a fundamental challenge in understanding the neural basis of behavior. Genetically encoded calcium (Ca2+) indicators (GECIs) enable quantitative monitoring of cellular-resolution activity during behavior. However, such indicators require online monitoring within a limited field of view. Alternatively, post hoc staining of immediate early genes (IEGs) indicates highly active cells within the entire brain, albeit with poor temporal resolution. We designed a fluorescent sensor, CaMPARI, that combines the genetic targetability and quantitative link to neural activity of GECIs with the permanent, large-scale labeling of IEGs, allowing a temporally precise “activity snapshot” of a large tissue volume. CaMPARI undergoes efficient and irreversible green-to-red conversion only when elevated intracellular Ca2+ and experimenter-controlled illumination coincide. We demonstrate the utility of CaMPARI in freely moving larvae of zebrafish and flies, and in head-fixed mice and adult flies. Authors: Benjamin F. Fosque, Yi Sun, Hod Dana, Chao-Tsung Yang, Tomoko Ohyama, Michael R. Tadross, Ronak Patel, Marta Zlatic, Douglas S. Kim, Misha B. Ahrens, Vivek Jayaraman, Loren L. Looger, Eric R. Schreiter

The cell adhesion molecule close homolog of L1 (CHL1) plays important functional roles in the developing and adult nervous system. In search of the binding partners that mediate the diverse and sometimes opposing functions of CHL1, the extracellular matrix-associated proteins vitronectin and plasminogen activator inhibitor-2 (PAI-2) were identified as novel CHL1 interaction partners and tested for involvement in CHL1-dependent functions during mouse cerebellar development. CHL1-induced cerebellar neurite outgrowth and cell migration at postnatal days 6–8 were inhibited by a CHL1-derived peptide comprising the integrin binding RGD motif, and by antibodies against vitronectin or several integrins, indicating a vitronectin-dependent integrin-mediated pathway. A PAI-2-derived peptide, or antibodies against PAI-2, urokinase type plasminogen activator (uPA), uPA receptor, and several integrins reduced cell migration. CHL1 colocalized with vitronectin, PAI-2, and several integrins in cerebellar granule cells, suggesting an association among these proteins. Interestingly, at the slightly earlier age of 4–5 d, cerebellar neurons did not depend on CHL1 for neuritogenesis and cell migration. However, differentiation of progenitor cells into neurons at this stage was dependent on homophilic CHL1–CHL1 interactions. These observations indicate that homophilic CHL1 trans-interactions regulate differentiation of neuronal progenitor cells at early postnatal stages, while heterophilic trans-interactions of CHL1 with vitronectin, integrins, and the plasminogen activator system regulate neuritogenesis and neuronal cell migration at a later postnatal stage of cerebellar morphogenesis. Thus, within very narrow time windows in postnatal cerebellar development, distinct types of molecular interactions mediated by CHL1 underlie the diverse functions of this protein.