Engineered Neuronal Networks for Functional Drug Screening
(NIH K01 EB003465)

Peter Molnar: PI
Anupama Natarajan, Ph.D. student

Using functionalized self-assembled monolayers combined with advanced surface patterning methods the inherent differentiation and self-organizing program in the neurons can be controlled and guided to form directed networks. Using the appropriate extracellular clues and cell types, different functional pathways of the brain could be recreated in vitro and used for a better understanding of physiology and pathophysiology of the nervous system. Moreover, surface patterns can be registered with surface-embedded extracellular electrodes allowing chronic or high-throughput recordings of synaptic transmission and network dynamics.
 

Systematic pharmacological characterization of synaptic transmission in engineered embryonic hippocampal networks with special emphasis on AMPA receptor modulators and metabotropic glutamate receptor agonists and antagonists

Original design for a functional high-throughput drug screening method to study drugs acting on synaptic transmission.

Surface patterns were created using self-assembled monolayers and photolithography. Cell attachment and axonal growth was determined by the surface patterns.

Asymmetric interconnectivity pattern has been designed
Patterning of multielectrode arrays has been developed, MEAs can be refunctionalized at least 3 times without degradation of signal quality (Abstract MEA2006.pdf)

As a connected project, we have designed, implemented and characterized two-neuron networks (1.pdf, 2.pdf):

A: Phase contrast and confocal microscopy images of the neurons on the patterns. Cells were filled with a fluorescent dye through the patch pipette. B: Mask design for two-cell networks. The diameter of the soma-attachment area is 25 µm. C: Ionic currents recorded from the patterned cells in voltage-clamp mode. D: Action potentials were evoked by current injections

Action potentials were evoked by current injection in one of the cells whereas postsynaptic currents were recorded from the other cell. A: AP - lower trace, EPSC recorded at -70 mV holding – upper trace. B: AP - lower trace, IPSC recorded at -70 and -30 mV holding – upper trace. Note the reversal of the current.

Effect of inhibitory synaptic interaction on the action potential firing of two-cell networks

As a side project we are continuing the systematic study of the factors which are affecting pattern formation and stability (shape, line width, feature size, surface, medium). These studies can lead to applications to determine factors affecting cell migration during development or regeneration. We have developed a high throughput method to measure cell migration along lines and we are validating the method using time-lapse microscopy.

Also, we are developing a technique to stabilize patterns based on 3D hydrogels (3D.pdf).