@article {68484,
	title = {Single-axonal organelle analysis method reveals new protein-motor associations.},
	journal = {ACS Chem Neurosci},
	volume = {4},
	year = {2013},
	month = {02/2013},
	pages = {277-84},
	abstract = {<p>Axonal transport of synaptic vesicle proteins is required to maintain neurons{\textquoteright} ability to communicate via synaptic transmission. Neurotransmitter-containing synaptic vesicles are assembled at synaptic terminals via highly regulated endocytosis of membrane proteins. These synaptic vesicle membrane proteins are synthesized in the cell body and transported to synapses in carrier vesicles that make their way down axons via microtubule-based transport utilizing kinesin molecular motors. Identifying the cargos that each kinesin motor protein carries from the cell bodies to the synapse is key to understanding both diseases caused by motor protein dysfunction and how synaptic vesicles are assembled. However, obtaining a bulk sample of axonal transport complexes from central nervous system (CNS) neurons to use for identification of their contents has posed a challenge to researchers. To obtain axonal carrier vesicles from primary cultured neurons, we fabricated a microfluidic chip designed to physically isolate axons from dendrites and cell bodies and developed a method to remove bulk axonal samples and label their contents. Synaptic vesicle protein carrier vesicles in these samples were labeled with antibodies to the synaptic vesicle proteins p38, SV2A, and VAMP2, and the anterograde axonal transport motor KIF1A, after which antibody overlap was evaluated using single-organelle TIRF microscopy. This work confirms a previously discovered association between KIF1A and p38 and shows that KIF1A also transports SV2A- and VAMP2-containing carrier vesicles.</p>
},
	keywords = {Animals, Axonal Transport, Cells, Cultured, Fluorescent Antibody Technique, Hippocampus, Kinesins, Membrane Glycoproteins, Mice, Microfluidic Analytical Techniques, Microscopy, Confocal, Nerve Tissue Proteins, Neurons, Organelles, Synaptic Vesicles, Synaptophysin, Transport Vesicles, Vesicle-Associated Membrane Protein 2, Vesicular Transport Proteins},
	issn = {1948-7193},
	doi = {10.1021/cn300136y},
	author = {Sgro, Allyson E and Bajjalieh, Sandra M and Chiu, Daniel T}
}
@article {68486,
	title = {A high-throughput method for generating uniform microislands for autaptic neuronal cultures.},
	journal = {J Neurosci Methods},
	volume = {198},
	year = {2011},
	month = {06/2011},
	pages = {230-5},
	abstract = {<p>Generating microislands of culture substrate on coverslips by spray application of poly-d lysine is a commonly used method for culturing isolated neurons that form self (autaptic) synapses. This preparation has multiple advantages for studying synaptic transmission in isolation; however, generating microislands by spraying produces islands of non-uniform size and thus cultures vary widely in the number of islands containing single neurons. To address these problems, we developed a high-throughput method for reliably generating uniformly shaped microislands of culture substrate. Stamp molds formed of poly(dimethylsiloxane) (PDMS) were fabricated with arrays of circles and used to generate stamps made of 9.2\% agarose. The agarose stamps were capable of loading sufficient poly D-lysine and collagen dissolved in acetic acid to rapidly generate coverslips containing at least 64 microislands per coverslip. When hippocampal neurons were cultured on these coverslips, there were significantly more single-neuron islands per coverslip. We noted that single neurons tended to form one of three distinct neurite-arbor morphologies, which varied with island size and the location of the cell body on the island. To our surprise, the number of synapses per autaptic neuron did not correlate with arbor shape or island size, suggesting that other factors regulate the number of synapses formed by isolated neurons. The stamping method we report can be used to increase the number of single-neuron islands per culture and aid in the rapid visualization of microislands.</p>
},
	keywords = {Animals, Cell Communication, Cell Culture Techniques, Cells, Cultured, Dendrites, Hippocampus, Mice, Neurons, Synapses, Synaptic Transmission},
	issn = {1872-678X},
	doi = {10.1016/j.jneumeth.2011.04.012},
	author = {Sgro, Allyson E and Nowak, Amy L and Austin, Naola S and Custer, Kenneth L and Allen, Peter B and Chiu, Daniel T and Bajjalieh, Sandra M}
}
