Inhibitory neurons modulate spontaneous signaling in cultured cortical neurons: density-dependent regulation of excitatory neuronal signaling

Abstract

Cortical neuronal activity depends on a balance between excitatory and inhibitory influences. Culturing of neurons on multi-electrode arrays (MEAs) has provided insight into the development and maintenance of neuronal networks. Herein, we seeded MEAs with murine embryonic cortical/hippocampal neurons at different densities (<150 or >1000 cells mm(-2)) and monitored resultant spontaneous signaling. Sparsely seeded cultures displayed a large number of bipolar, rapid, high-amplitude individual signals with no apparent temporal regularity. By contrast, densely seeded cultures instead displayed clusters of signals at regular intervals. These patterns were observed even within thinner and thicker areas of the same culture. GABAergic neurons (25% of total neurons in our cultures) mediated the differential signal patterns observed above, since addition of the inhibitory antagonist bicuculline to dense cultures and hippocampal slice cultures induced the signal pattern characteristic of sparse cultures. Sparsely seeded cultures likely lacked sufficient inhibitory neurons to modulate excitatory activity. Differential seeding of MEAs can provide a unique model for analyses of perturbation in the interaction between excitatory and inhibitory function during aging and neuropathological conditions where dysregulation of GABAergic neurons is a significant component.

Stimulation with a Low-Amplitude, Digitized Synaptic Signal to Invoke Robust Activity within Neuronal Networks on Multielectrode Arrays

“…We observed stimulation or suppression of spontaneous signaling using native or inverted Biological Signal, indicating that neuronal subpopulations were opposingly affected depending upon signal orientation. Inhibition of spontaneous signaling suggests a predominant impact on inhibitory neurons, consistent with the prior demonstration that GABAergic neurons are responsible for synchronized bursts in mature signal patterns (19,22). …”

“…Images present representative signal profiles for all recorded channels. Prior studies using combinations of excitatory and inhibitory neuronal antagonists have confirmed synaptic origin of signal in these cultures (17,19,22). …”

“…Spontaneous neuronal signals were discernable within ten days of plating. However, cultures were maintained for 1 month prior to recording in order to allow maturation and stabilization of the neuronal network (1719). …”

Robot-Embodied Neuronal Networks as an Interactive Model of Learning

“…Other appropriate software is available [1,28]. Networks were stimulated using a 1sec digitized synaptic signal 1mV in amplitude as in our prior studies [3,4,27]. Output was graphed and analyzed in Excel.…”

“…After culturing for 1 month to allow maturation of networks (confirmed by development of spontaneous signal streams rich in complex bursts [27], networks were segmented into two equivalent halves by slicing across the petri dish with a scalpel. This segmentation was not essential, but served to generate two independent "hemispheres."…”