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

Abstract

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 cell permissive SAM, N-1[3-(trimethoxysilyl)propyl] diethylenetriamine (DETA), and was composed of two 25 μm somal adhesion sites connected with 5 μm lines acting as surface cues for guided axonal and dendritic regeneration. Surrounding the DETA pattern was a background of a non-cell-permissive poly(ethylene glycol) (PEG) SAM. Adult hippocampal neurons were first cultured on coverslips coated with DETA monolayers and were later passaged onto the PEG-DETA bidirectional polarity patterns in serum-free medium. These neurons followed surface cues, attaching and regenerating only along the DETA substrate to form small engineered neuronal circuits. These circuits were stable for more than 21 days in vitro (DIV), during which synaptic connectivity was evaluated using basic electrophysiological methods.

Morphological and functional characterization of human induced pluripotent stem cell‐derived neurons (iCell Neurons) in defined culture systems

“…Previous data obtained from these cells using culture parameters established by the manufacturer indicates their functional characterization over two weeks in vitro, including evidence for their ability to generate three major voltage‐dependent currents (Na v , Ca v , and K v ) . However, analysis of neuronal performance over a two week period in a single culture environment is insufficient if these cells are to be considered for incorporation into chronic drug evaluation studies in vitro . Careful assessment of maturation end points and cell survival over an extended time‐course is necessary to justify incorporation of CDI's hiPSC‐derived neurons into body‐on‐a‐chip and other devices for chronic drug toxicity and efficacy studies.…”

“…Previously, DETA has been used in conjunction with cytophobic surfaces to successfully culture as well as pattern cells into controlled configurations, providing the means to develop in vitro engineered cell networks for a variety of advanced culture applications . Furthermore, previous work has demonstrated that DETA is capable of promoting the survival and maturation of primary neurons in culture for extended periods . DETA, an analogue of spermine, has been shown to promote neuron survival in vitro through interactions with NMDA‐receptor complexes, and is also known to promote increased longevity in a number of model organisms by reducing the overproduction of harmful reactive oxygen species and autophagy regulation .…”

Presynaptic PICK1 facilitates trafficking of AMPA-receptors between active zone and synaptic vesicle pool

“…The anti-GluA2/3 antibody has been used in several recent immunocytochemical studies, which all have shown the expected characteristic staining pattern (Edwards et al, 2013;Sadeghi et al, 2014;Schuth et al, 2014;Peng et al, 2015).…”

Neural Engineering

“…Some of the more notable technological areas that have been employed include brain-device interfaces and surface technologies (Aregueta-Robles et al 2014 ;Helmke and Minerick 2006 ;El-Ali et al 2006 ;Cooke et al 2008 ;Edwards et al 2013 ); biomaterials such as synthetic extracellular matrices (Maclean et al 2016 ;Tibbitt and Anseth 2009 ;Theocharidis et al 2014 ); cell sources, particularly from stem cells (Merkle Florian and K. Eggan 2013 ); microfl uidic systems (Bhatia and Ingber 2014 ;Harink et al 2013 ;Barata et al 2015 ); and biomedical or biological microelectromechanical systems (Bio-MEMS). This chapter will focus on the in vitro modeling of the refl ex arc.…”

“…The demonstrated success of both methods indicates the usefulness of silane surface chemistry for creating patterned neuronal circuits and other groups have adopted this methodology . Examples of the application of this silane-based surface technology in neural engineering include two-cell circuit patterns of hippocampal neurons (Edwards et al 2013 ;Molnar et al 2007a ) (Fig. 9.2 ) and feed-forward patterns for mimicking signal propagation in the nervous system (Natarajan et al 2013 ) (Fig.…”