The neural cell adhesion molecule (NCAM) mediates cell-cell and cell-matrix adhesion. It is broadly expressed in the nervous system and regulates neurite outgrowth, synaptogenesis, and synaptic plasticity. Previous in vitro studies revealed that palmitoylation of NCAM is required for fibroblast growth factor 2 (FGF2)-stimulated neurite outgrowth and identified the zinc finger DHHC (Asp-His-His-Cys)-containing proteins ZDHHC3 and ZDHHC7 as specific NCAM-palmitoylating enzymes. Here, we verified that FGF2 controlled NCAM palmitoylation in vivo and investigated molecular mechanisms regulating NCAM palmitoylation by ZDHHC3. Experiments with overexpression and pharmacological inhibition of FGF receptor (FGFR) and Src revealed that these kinases control tyrosine phosphorylation of ZDHHC3 and that ZDHHC3 is phosphorylated by endogenously expressed FGFR and Src proteins. By site-directed mutagenesis, we found that Tyr18 is an FGFR1-specific ZDHHC3 phosphorylation site, while Tyr295 and Tyr297 are specifically phosphorylated by Src kinase in cell-based and cell-free assays. Abrogation of tyrosine phosphorylation increased ZDHHC3 autopalmitoylation, enhanced interaction with NCAM, and upregulated NCAM palmitoylation. Expression of ZDHHC3 with tyrosine mutated in cultured hippocampal neurons promoted neurite outgrowth. Our findings for the first time highlight that FGFR-and Src-mediated tyrosine phosphorylation of ZDHHC3 modulates ZD-HHC3 enzymatic activity and plays a role in neuronal morphogenesis.

Heparan sulfates (HSs) are complex and highly active molecules that are required for synaptogenesis and long-term potentiation. A deficit in HSs leads to autistic phenotype in mice. Here, we investigated the long-term effect of heparinase I, which digests highly sulfated HSs, on the spontaneous bioelectrical activity of neuronal networks in developing primary hippocampal cultures. We found that chronic heparinase treatment led to a significant reduction of the mean firing rate of neurons, particularly during the period of maximal neuronal activity. Furthermore, firing pattern in heparinasetreated cultures often appeared as epileptiform bursts, with long periods of inactivity between them. These changes in network activity were accompanied by an increase in the frequency and amplitude of miniature postsynaptic excitatory currents, which could be described by a linear up-scaling of current amplitudes. Biochemically, we observed an upregulation in the expression of the glutamate receptor subunit GluA1, but not GluA2, and a strong increase in autophosphorylation of a and b Ca 2þ /calmodulin-dependent protein kinase II (CaMKII), without changes in the levels of kinase expression. These data suggest that a deficit in HSs triggers homeostatic synaptic plasticity and drastically affects functional maturation of neural network.

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BACKGROUND Brain visual circuits are often studied in vivo by imaging Ca2+ indicators with green shifted emission spectra. Polychromatic white visual stimuli have a spectrum that partially overlaps indicators emission spectra, resulting in significant contamination of calcium signals. NEW METHOD To overcome light contamination problems we choose blue visual stimuli, having a spectral composition not overlapping with Ca2+ indicator emission spectrum. To compare visual responsiveness to blue and white stimuli we used electrophysiology (visual evoked potentials VEPs) and 3D acousto optic two photon(2P) population Ca2+ imaging in mouse primary visual cortex (V1). RESULTS VEPs in response to blue and white stimuli had comparable peak amplitudes and latencies. Ca2+ imaging revealed that the populations of neurons responding to blue and white stimuli were largely overlapping, that their responses had similar amplitudes, and that functional response properties such as orientation and direction selectivities were also comparable. COMPARISON WITH EXISTING METHODS Masking or shielding the microscope are often used to minimize the contamination of Ca2+ signal by white light, but they are time consuming, bulky and thus can limit experimental design, particularly in the more and more frequently used awake set up. Blue stimuli not interfering with imaging allow to omit shielding without affecting V1 physiological responsiveness. CONCLUSIONS Our results show that the selected blue light stimuli evoke physiological responses comparable to those evoked by white stimuli in mouse V1. This will make complex designs of imaging experiments in behavioral set ups easier, and facilitate the combination of Ca2+ imaging with electrophysiology and optogenetics.