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

摘要

In this study, we demonstrate the directed formation of small circuits of electrically active, synaptically connected neurons derived from the hippocampus of adult rats through the use of engineered chemically modified culture surfaces that orient the polarity of the neuronal processes. Although synaptogenesis, synaptic communication, synaptic plasticity, and brain disease pathophysiology can be studied using brain slice or dissociated embryonic neuronal culture systems, the complex elements found in neuronal synapses makes specific studies difficult in these random cultures. The study of synaptic transmission in mature adult neurons and factors affecting synaptic transmission are generally studied in organotypic cultures, in brain slices, or in vivo. However, engineered neuronal networks would allow these studies to be performed instead on simple functional neuronal circuits derived from adult brain tissue. Photolithographic patterned self-assembled monolayers (SAMs) were used to create the two-cell “bidirectional polarity” circuit patterns. This pattern consisted of a cellpermissive SAM, N-1[3-(trimethoxysilyl)propyl] diethylenetriamine(DETA), and was composed of two 25 μm somal adhesion sites connectedwith 5 μm lines acting as surface cues for guided axonal anddendritic regeneration. Surrounding the DETA pattern was a backgroundof a non-cell-permissive poly(ethylene glycol) (PEG) SAM. Adult hippocampalneurons were first cultured on coverslips coated with DETA monolayersand were later passaged onto the PEG-DETA bidirectional polarity patternsin serum-free medium. These neurons followed surface cues, attachingand regenerating only along the DETA substrate to form small engineeredneuronal circuits. These circuits were stable for more than 21 daysin vitro (DIV), during which synaptic connectivity was evaluated usingbasic electrophysiological methods.