Developmental Neurobiology Implications from Fabrication and Analysis of Hippocampal Neuronal Networks on Patterned Silane-Modified Surfaces

Abstract

We have determined the parameters necessary to fabricate reproducible neuronal patterns which we are using to begin studying fundamental issues in developmental neurobiology. The addition of a beam homogenizer, as well as a new surface preparation, has enabled the routine production of reproducible, high-resolution (2−20 μm) organosilane patterns. The effects of surface preparation and beam dosage were monitored using X-ray photoelectron spectroscopy (XPS) and proof of patterning is provided by high-resolution imaging XPS. We also report the guidance of neuronal adhesion and neurite outgrowth and the creation of reproducibly defined circuits of embryonic (E18−19) rat hippocampal neurons using these patterned surfaces in vitro. We have achieved a >50% rate of pattern formation, and at times the rate approaches 90%. We are using these patterns to address the issue of how geometric pattern cues might be used to affect cell-to-cell communication and we report the preliminary results on the synaptic development of the hippocampal neurons using dual patch-clamp electrophysiology. We monitored neurite outgrowth and the emergence of both spontaneous and evoked synaptic activity for both patterned and unpatterned (control) hippocampal cultures. The results indicate the intriguing possibility that geometry itself may be a modulating or trophic factor for cell development.

Two Cell Circuits of Oriented Adult Hippocampal Neurons on Self-Assembled Monolayers for Use in the Study of Neuronal Communication in a Defined System

“…The quality of the surfaces was in agreement with previously reported results. 32,46,47 Palladium-Catalyzed Metallization of Patterned Silane Monolayers. Patterned samples were visualized using a palladium-catalyzed copper reduction reaction, modified from Kind et al 48 In this reaction, copper is deposited in regions containing the amine terminated silane DETA.…”

Primary Neuron/Astrocyte Co‐Culture on Polyelectrolyte Multilayer Films: A Template for Studying Astrocyte‐Mediated Oxidative Stress in Neurons

“…[53] It has also been demonstrated by Wheeler and co-workers that patterned neurons synapse with each other, release neurotransmitters and develop electrical activity. [54][55][56] Furthermore, restricting neurons to patterns has been shown to enhance the cellular activity such as glutamine secretion and electrical activity as compared to random monocultures of neurons. [57,58] …”

Differentiation of skeletal muscle and integration of myotubes with silicon microstructures using serum-free medium and a synthetic silane substrate

“…22| Use XPS to characterize the monolayer of DETA by monitoring the N 1s peak 9,13 . ' PAUSE POINT Samples should be stored in dessicator before use.…”

“…First, a unique chemically defined serum-free medium supplemented with specific growth factors was developed to study the muscle differentiation process. Second, a synthetic, nonbiological, patternable 8,9 , cell growth promoting substrate, N-1 [3(trimethoxysilyl)propyl] diethylenetriamine (DETA), coated on glass coverslips [10][11][12] , was used as a template to grow the skeletal muscle cells. Third, it was demonstrated that when the dissociated muscle cells were plated on fabricated microcantilevers, they aligned along the long axis of the cantilever to form aligned myotubes.…”