Synaptic Connectivity in Engineered Neuronal Networks

Abstract

We have developed a method to organize cells in dissociated cultures using engineered chemical clues on a culture surface and determined their connectivity patterns. Although almost all elements of the synaptic transmission machinery can be studied separately in single cell models in dissociated cultures, the complex physiological interactions between these elements are usually lost. Thus, factors affecting synaptic transmission are generally studied in organotypic cultures, brain slices, or in vivo where the cellular architecture generally remains intact. However, by utilizing engineered neuronal networks complex phenomenon such as synaptic transmission or synaptic plasticity can be studied in a simple, functional, cell culture-based system. We have utilized self-assembled monolayers and photolithography to create the surface templates. Embryonic hippocampal cells, plated on the resultant patterns in serum-free medium, followed the surface clues and formed the engineered neuronal networks. Basic whole-cell patch-clamp electrophysiology was applied to characterize the synaptic connectivity in these engineered two-cell networks. The same technology has been used to pattern other cell types such as cardiomyocytes or skeletal muscle fibers.

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

“…31 The design for dendritic morphology was a generic one, mimicking four neuritebranches originating from the cell body. 33 Adult hippocampal neurons followed both the axonal and dendritic cues, although the dendritic attachment area seemed to be too restrictive, as the neurites tended to overgrow that portion of the pattern. The neurons were found to be both electrically active and synaptically connected, and displayed synaptic connectivity characteristic of excitatory glutamatergic neurons.…”

“…Neurons expressed synaptophysin and the presynaptic vesicle glycoprotein was used to quantify the synapses. 37 The NMDA and AMPA channel subunits NR2A, NR2B, and GluR2/3, the distinctive postsynaptic ligand-gated ion channels that control EPSP, 33,35,36,38 were all present in the adult hippocampal neurons. The presence of both presynaptic and postsynaptic EPSP proteins and characteristic excitatory synaptic electrophysiological parameters confirmed the presence of excitatory synapses between neurons on the patterned coverslips.…”

“…The masks were derived from previous work that demonstrated geometric control of neuronal polarity 31 and network formation 32 that were demonstrated using different SAM combinations for embryonic neurons. 33 The bidirectional polarity patterns have two somal adhesion sites (SAS) of 30 μm diameter approximately 150 μm apart connected by 5 μm lines of DETA. The dimensions of the patterns promoted the attachment of neurons onto the SAS, regrowth of axons along the connecting lines, and dendritic branching along the dotted strips of DETA.…”