Spontaneous and Perturbational Complexity in Cortical Cultures

Abstract

Dissociated cortical neurons in vitro display spontaneously synchronized, low-frequency firing patterns, which can resemble the slow wave oscillations characterizing sleep in vivo. Experiments in humans, rodents, and cortical slices have shown that awakening or the administration of activating neuromodulators decrease slow waves, while increasing the spatio-temporal complexity of responses to perturbations. In this study, we attempted to replicate those findings using in vitro cortical cultures coupled with micro-electrode arrays and chemically treated with carbachol (CCh), to modulate sleep-like activity and suppress slow oscillations. We adapted metrics such as neural complexity (NC) and the perturbational complexity index (PCI), typically employed in animal and human brain studies, to quantify complexity in simplified, unstructured networks, both during resting state and in response to electrical stimulation. After CCh administration, we found a decrease in the amplitude of the initial response and a marked enhancement of the complexity during spontaneous activity. Crucially, unlike in cortical slices and intact brains, PCI in cortical cultures displayed only a moderate increase. This dissociation suggests that PCI, a measure of the complexity of causal interactions, requires more than activating neuromodulation and that additional factors, such as an appropriate circuit architecture, may be necessary. Exploring more structured in vitro networks, characterized by the presence of strong lateral connections, recurrent excitation, and feedback loops, may thus help to identify the features that are more relevant to support causal complexity.

In vitro cell models merging circadian rhythms and brain waves for personalized neuromedicine

“…On the other hand, in vitro studies on brain waves in 2D neuronal cell cultures may be limited by the difficulty of reproducing the properties of the underlying brain wave network and key differences may result comparing cultures of dissociated cortical neurons with in vivo models or with neurons cultured in vitro with more preserved architectures. 27 Indeed, 2D neuronal models are limited to side-by-side contact, lacking a 3D microenvironment and the relevant interactions between the cells and the surrounding extracellular matrix. However, pioneering studies carried out by the Tafti group 38 and by Colombi et al.…”

“…However, pioneering studies carried out by the Tafti group 38 and by Colombi et al. 12 , 27 on spontaneous and pharmacologically manipulated 2D neuronal culture activities, in terms of brain waves linked to the sleep/wake cycle, will be discussed in the following section. These authors claimed to artificially “drive” the connectivity of neurons within the cultured network, as cell cultures are not necessarily random networks, but their versatile neuronal growth can be manipulated to develop networks with different topologies, such as a 3D cytoarchitecture.…”

“…Along this line, Colombi et al. 27 proposed the addition of a cocktail of drugs, instead of carbachol alone, to their experimental design in order to better resemble the landscape of frequency oscillations observed in the awake human brain, together with a more intriguing possibility to artificially “drive” the connectivity of the cells within the cultured network. Rockstrom et al.…”

“…Development of the neural network involved neuromodulators, neurotransmitters, or electrical stimulation to induce sleep-like state switching wake-like state together with entrainment of co-cultured astrocytes to induce and modulate brain waves, such as cholinergically induced gamma oscillations. 6 , 26 , 27 Synchronized measurement of neuronal activity from multiple electrodes at different locations across the neural co-culture on 3D devices allows the analysis of the functional connectivity in the neuronal network. …”

The activity of deep cortical layers characterizes the complexity of brain responses during wakefulness following electrical stimulation

“…In vitro studies in cortical slices, found that application of noradrenaline and a cholinergic agonist increased the PCI (D'Andola, Rebollo et al 2018), whereas GABA antagonists reduced PCI (Barbero-Castillo, Mateos-Aparicio et al 2021). In contrast, rat cortical cell cultures show only moderate effects of cholinergic agonists on PCI values presumably due to a lack of appropriate circuit architecture (Colombi, Nieus et al 2021). However it is unclear which circuit elements, e.g.…”