title: Photolithography-Based Substrate Microfabrication for Patterning Semaphorin 3A to Study Neuronal Development.
abstract: Protein micropatterning techniques, including microfluidic devices and protein micro-contact printing, enable the generation of highly controllable substrates for spatial manipulation of intracellular and extracellular signaling determinants to examine the development of cultured dissociated neurons in vitro. In particular, culture substrates coated with proteins of interest in defined stripes, including cell adhesion molecules and secreted proteins, have been successfully used to study neuronal polarization, a process in which the neuron establishes axon and dendrite identities, a critical architecture for the input/output functions of the neuron. We have recently used this methodology to pattern the extracellular protein Semaphorin 3A (Sema3A), a secreted factor known to control neuronal development in the mammalian embryonic cortex. We showed that stripe-patterned Sema3A regulates axon and dendrite formation during the early phase of neuronal polarization in cultured rat hippocampal neurons. Here, we describe microfabrication and substrate stripe micropatterning of Sema3A. We note that same methodologies can be applied to pattern other extracellular proteins that regulate neuronal development in the embryonic brain, as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and Netrin-1. We describe modifications of these methodologies for stripe micropatterning of membrane-permeable analog of the second messengers cyclic AMP (cAMP) and cyclic GMP (cGMP), intracellular regulators of neuronal polarization that might act downstream of Sema3A.
authors: Department of Neurobiology and Behavior, Stony Brook University, Stony Brook, NY, 11794-5230, USA. maya.shelly@stonybrook.edu.
authors: Maya
authors: M
authors: Shelly
authors: Department of Neurobiology and Behavior, Stony Brook University, Stony Brook, NY, 11794-5230, USA.
authors: Seong-Ii
authors: SI
authors: Lee
authors: Department of Materials Science and Engineering, Stony Brook University, Stony Brook, NY, 11794-2275, USA.
authors: Giulia
authors: G
authors: Suarato
authors: Department of Materials Science and Engineering, Stony Brook University, Stony Brook, NY, 11794-2275, USA.
authors: Yizhi
authors: Y
authors: Meng
authors: ITAV-CNRS USR 3505, Toulouse, 31106, France.
authors: Sophie
authors: S
authors: Pautot
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D004129:Dimethylpolysiloxanes; D006624:Hippocampus / Q000166:cytology; D044085:Microfluidics; D055616:Microtechnology; D009474:Neurons / Q000166:cytology*; D051381:Rats; D040121:Semaphorin-3A / Q000378:metabolism*; D014466:Ultraviolet Rays
chemical_list: D004129:Dimethylpolysiloxanes; D040121:Semaphorin-3A; C013830:baysilon
keywords: Axon and dendrite polarization; Micro-contact printing; Microfluidics; Nanofabrication; Neuronal development; Poly(dimethylsiloxane) (PDMS); Protein stripe micropatterning; Semaphorin 3A