We have devised a new substrate patterning method that overcomes the limitations associated with random cell binding on pre-patterned substrates. Our approach provides the ability to create patterns that restrict, contain, allow or direct growth after cells have been plated. The ability to pattern substrates after plating allows selection of specific subsets of cells (i.e., transfected or knockout cells) and modification of patterns in response to changes in outgrowth. When combined with pre-patterned substrates comprising more than one material, polarization of neurons can be induced and interactions oriented. Using this approach we are able to create neuronal circuits with varying complexity and known connectivity. Our patterning technique is similar to maskless photolithography and uses a high-energy pulsed IR laser to directly unbind substrate materials. The areas where protein is removed become non-permissive for neuronal growth. We will use the combination of substrate patterning, microfluidics and electrophysiology to create and test functional neuronal circuits of hippocampal neurons. Once we have verified function and optimized the approach, we will focus on addressing specific biological questions. This will include studying the influence and regulation of neural activity on dendritic spine emergence, development and dynamics. We will also study the influence of an applied exogenous factor on spine formation and maturation, as well as alterations in spine dynamics in the absence of myosin IIB using cells from myosin IIB knockout mice. Finally, we will study the role of Rab5 in presynaptic function of hippocampal neurons connected in simple circuits.

Public Health Relevance

We have developed a new method of patterning substrates in the presence of living cells that allows creation of neuronal circuits with known connectivity in cell culture. This will advance our understanding of communication between nerve cells and facilitate modeling of brain circuits. This is important for understanding normal brain function and the potential for recovery from brain or spinal cord injury.

Agency
National Institute of Health (NIH)
Institute
National Institute of Mental Health (NIMH)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21MH081260-02
Application #
7618124
Study Section
Special Emphasis Panel (ZRG1-MDCN-K (50))
Program Officer
Asanuma, Chiiko
Project Start
2008-05-01
Project End
2011-01-31
Budget Start
2009-02-17
Budget End
2011-01-31
Support Year
2
Fiscal Year
2009
Total Cost
$171,900
Indirect Cost
Name
Washington University
Department
Neurosciences
Type
Schools of Medicine
DUNS #
068552207
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
Turney, Stephen G; Ahmed, Mostafa; Chandrasekar, Indra et al. (2016) Nerve growth factor stimulates axon outgrowth through negative regulation of growth cone actomyosin restraint of microtubule advance. Mol Biol Cell 27:500-17
Chandrasekar, Indra; Goeckeler, Zoe M; Turney, Stephen G et al. (2014) Nonmuscle myosin II is a critical regulator of clathrin-mediated endocytosis. Traffic 15:418-32
Chandrasekar, Indra; Huettner, James E; Turney, Stephen G et al. (2013) Myosin II regulates activity dependent compensatory endocytosis at central synapses. J Neurosci 33:16131-45