@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 = {

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.

}, 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} } @article {68488, title = {Single-synapse ablation and long-term imaging in live C. elegans.}, journal = {J Neurosci Methods}, volume = {173}, year = {2008}, month = {09/2008}, pages = {20-6}, abstract = {

Synapses are individually operated, computational units for neural communication. To manipulate physically individual synapses in a living organism, we have developed a laser ablation technique for removing single synapses in live neurons in C. elegans that operates without apparent damage to the axon. As a complementary technique, we applied microfluidic immobilization of C. elegans to facilitate long-term fluorescence imaging and observation of neuronal development. With this technique, we directly demonstrated the existence of competition between developing synapses in the HSNL motor neuron.

}, keywords = {Animals, Axons, Behavior, Animal, Caenorhabditis elegans, Diagnostic Imaging, Laser Therapy, Microfluidic Analytical Techniques, Movement, Neurons, Synapses, Time Factors}, issn = {0165-0270}, doi = {10.1016/j.jneumeth.2008.05.007}, author = {Allen, Peter B and Sgro, Allyson E and Chao, Daniel L and Doepker, Byron E and Scott Edgar, J and Shen, Kang and Chiu, Daniel T} } @article {68489, title = {Thermoelectric manipulation of aqueous droplets in microfluidic devices.}, journal = {Anal Chem}, volume = {79}, year = {2007}, month = {07/2007}, pages = {4845-51}, abstract = {

This article describes a method for manipulating the temperature inside aqueous droplets, utilizing a thermoelectric cooler to control the temperature of select portions of a microfluidic chip. To illustrate the adaptability of this approach, we have generated an "ice valve" to stop fluid flow in a microchannel. By taking advantage of the vastly different freezing points for aqueous solutions and immiscible oils, we froze a stream of aqueous droplets that were formed on-chip. By integrating this technique with cell encapsulation into aqueous droplets, we were also able to freeze single cells encased in flowing droplets. Using a live-dead stain, we confirmed the viability of cells was not adversely affected by the process of freezing in aqueous droplets provided cryoprotectants were utilized. When combined with current droplet methodologies, this technology has the potential to both selectively heat and cool portions of a chip for a variety of droplet-related applications, such as freezing, temperature cycling, sample archiving, and controlling reaction kinetics.

}, keywords = {Cell Survival, Freezing, Kinetics, Lymphoma, B-Cell, Microfluidic Analytical Techniques, Mineral Oil, Solutions, Water}, issn = {0003-2700}, doi = {10.1021/ac062458a}, author = {Sgro, Allyson E and Allen, Peter B and Chiu, Daniel T} }