For VAMP7 knockdown, two constructs were used: VAMP7 KD3 5′-CTGAAGCATCACTCCGAGATTCAAGAGATCTCGGAGTGATGCTTCAG-3′ and VAMP7 KD4 5′-CTGAAAGGCATCATGGTCATTCAAGAGATGACCATGATGCCTTTCAG-3′.

In all cases, shRNA sequences were inserted into Xhol through Xbal cloning sites in the L307 lentiviral transfer vector, downstream of the human H1 promoter. Lentiviruses encoding pHluorin-tagged syb2, VAMP4, vti1a, VAMP7, mOrange-tagged syb2, and all shRNA constructs were prepared by transfection of human embryonic kidney (HEK) 293-T cells with FUGENE 6 and necessary viral coat and packaging protein constructs (pVSVG, pRsv-Rev, and pPRE). Three days selleck chemicals llc after transfection, virus www.selleckchem.com/products/chir-99021-ct99021-hcl.html was harvested from HEK293-T cell-conditioned media and added to neuronal media at 4 DIV. Lentiviral constructs to decrease expression of all four isoforms of the Doc2 protein family (Doc2A, Doc2B, Doc2G, and rabphilin) were a gift of Dr. Thomas C. Südhof (Stanford University) (Pang et al., 2011). Electrophysiological experiments were performed on dissociated hippocampal neurons as in Nosyreva

and Kavalali (2010) (see Supplemental Experimental Procedures for further details). Reelin was prepared and purified as described previously (Beffert et al., 2002) (see Supplemental Experimental Procedures for further details). Single-wavelength experiments were performed using an Andor iXon+ back-illuminated EMCCD camera using MetaFluor 7.6 software as described previously (Leitz and Kavalali, 2011) (see Supplemental Experimental Procedures for further details). Dual color experiments were performed using a Zeiss LSM510 confocal microscope using LSM5 software as described previously (Ramirez et al., 2012 and Raingo et al., 2012) (see Supplemental Experimental Procedures for further details). All error bars represent SEM and all statistical analyses were done using Microsoft Excel Software or

Sigma next Plot (see Supplemental Experimental Procedures for further details). We thank Drs. Megumi Adachi, Elena Nosyreva, and Catherine Wasser for discussions and comments on the manuscript. We also thank Brent Trauterman for his excellent technical assistance. We are grateful to Drs. Mikhail Khvotchev and Yildirim Sara for their insight at early stages of this project. This work was supported by National Institutes of Health grants MH066198 (E.T.K.), NS075499 (E.T.K.), T32 NS069562 (J.L.), MH070727 (L.M.M.) and HL063762 (J.H.) as well as funding from the from the Brain & Behavior Research Foundation (L.M.M. and E.T.K.) and International Mental Health Research Organization (L.M.M.). “
“At a synapse, neurotransmitters are released by Ca2+-triggered synaptic vesicle exocytosis (Katz, 1969) that is mediated by synaptotagmins (Südhof, 2012).