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  • Microfluidic local perfusion chambers for the visualization and manipulation of synapses.

Microfluidic local perfusion chambers for the visualization and manipulation of synapses.

Neuron (2010-04-20)
Anne M Taylor, Daniela C Dieterich, Hiroshi T Ito, Sally A Kim, Erin M Schuman
ABSTRACT

The polarized nature of neurons and the size and density of synapses complicates the manipulation and visualization of cell biological processes that control synaptic function. Here we developed a microfluidic local perfusion (microLP) chamber to access and manipulate synaptic regions and presynaptic and postsynaptic compartments in vitro. This chamber directs the formation of synapses in >100 parallel rows connecting separate neuron populations. A perfusion channel transects the parallel rows, allowing access with high spatial and temporal resolution to synaptic regions. We used this chamber to investigate synapse-to-nucleus signaling. Using the calcium indicator dye Fluo-4 NW, we measured changes in calcium at dendrites and somata, following local perfusion of glutamate. Exploiting the high temporal resolution of the chamber, we exposed synapses to "spaced" or "massed" application of glutamate and then examined levels of pCREB in somata. Lastly, we applied the metabotropic receptor agonist DHPG to dendrites and observed increases in Arc transcription and Arc transcript localization.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Anti-Microtubule-Associated Protein 2 (MAP2) Antibody, Chemicon®, from rabbit
Sigma-Aldrich
Anti-GAD67 Antibody, clone 1G10.2, clone 1G10.2, Chemicon®, from mouse
Sigma-Aldrich
Anti-phospho-CREB (Ser133) Antibody, clone 634-2, clone 634-2, Upstate®, from mouse
Sigma-Aldrich
Monoclonal Anti-MAP2 antibody produced in mouse, clone HM-2, ascites fluid