The nanogold labeled neurons were postfixed and gold toned with 0.05% gold chloride. Neurons were scraped into 1% Tx-100 in Tris-buffered saline (TBS) (50 mM Tris, 150 mM NaCl, pH 7.4) and protease and phosphatase inhibitor cocktail at 4°C. Lysates were sonicated and centrifuged at 100,000 × g for 30 min. The pellet selleck kinase inhibitor was washed and suspended in 2% SDS in TBS. Samples were separated by SDS-PAGE and immunoblotting was performed using primary antibodies described in Table S1. All immunoblots were performed a minimum of 3–8 times. Microfluidic neuronal culture devices with 2 somal compartments connected

by a series of microgrooves were obtained from Xona Microfluidics (Temecula, CA). Glass coverslips (Corning Inc.) were coated with poly-d-lysine and affixed to neuronal devices as per the manufacturer’s instructions. A total of 10,000 dissociated hippocampal neurons were plated. A 50 μl difference in media volume was maintained between the two compartments to regulate the direction of flow. α-syn 1-120-myc pffs (2 μg) were added to the neuritic compartment (retrograde experiments) or the

somal compartment (anterograde experiments) and were fixed 7–12 days later. The retrograde experiments were repeated 4 times and anterograde experiments repeated 3 times, each in triplicates. Hippocampal neurons were plated on MatTek dishes at 300,000 cells/dish http://www.selleckchem.com/products/ON-01910.html and treated with PBS or 5 μg/mL α-syn-hWT pffs. Neurons were loaded with Fluo4-AM (1 μM, Invitrogen,Carlsbad, CA). Spontaneous calcium activity from ∼200 neurons was recorded for 5 min, Purple acid phosphatases at 10Hz acquisition. Synchronous oscillations were forced with bicuculline (100 μM, Tocris) and increasing doses of the AMPAR antagonist, NBQX. When synchronous oscillations stopped, this final concentration of NBQX was used as an indication of excitatory tone (Breskin et al., 2006). Custom-coded MATLAB

scripts were used to analyze the images. Kurt Brunden, James Soper, Linda Kwong, Eddie Lee, and Jing Guo are thanked for reading the manuscript and for helpful discussions, and Patrick O’Brien, Christine Schultheiss, Victoria Kehm, Christina Haas, and Jeffrey Yeh for technical assistance. This work was supported by National Institutes of Health Grants NS053488, the Picower Foundation, the Benaroya Foundation, the RJG Foundation, the Jeff and Anne Keefer fund for Parkinson’s Research, the Parkinson Council, the Stein-Bellet Family Fund, and National Institutes of Health Grant NS015202 and Army Research Office W911F-10-1-0526. “
“Mutations in MECP2 cause Rett syndrome (RTT), a human neurodevelopmental disorder that can lead to cognitive impairment, autistic features, motor disabilities, seizures, and anxiety ( Chahrour and Zoghbi, 2007). Experiments that disrupt MeCP2 expression in specific populations of cells in mice indicate that dysfunction of neurons throughout the central nervous system contributes to the symptoms associated with RTT ( Guy et al., 2010).