Fast wide-volume functional imaging of engineered in vitro brain tissues.

The need for in vitro models that mimic the human brain to replace animal testing and allow high-throughput screening has driven scientists to develop new tools that reproduce tissue-like features on a chip. Three-dimensional (3D) in vitro cultures are emerging as an unmatched platform that preserves the complexity of cell-to-cell connections within a tissue, improves cell survival, and boosts neuronal differentiation. In this context, new and flexible imaging approaches are required to monitor the functional states of 3D networks. Herein, we propose an experimental model based on 3D neuronal networks in an alginate hydrogel, a tunable wide-volume imaging approach, and an efficient denoising algorithm to resolve, down to single cell resolution, the 3D activity of hundreds of neurons expressing the calcium sensor GCaMP6s. Furthermore, we implemented a 3D co-culture system mimicking the contiguous interfaces of distinct brain tissues such as the cortical-hippocampal interface. The analysis of the network activity of single and layered neuronal co-cultures revealed cell-type-specific activities and an organization of neuronal subpopulations that changed in the two culture configurations. Overall, our experimental platform represents a simple, powerful and cost-effective platform for developing and monitoring living 3D layered brain tissue on chip structures with high resolution and high throughput.

Large-scale, high-resolution electrophysiological imaging of field potentials in brain slices with microelectronic multielectrode arrays.

Multielectrode arrays (MEAs) are extensively used for electrophysiological studies on brain slices, but the spatial resolution and field of recording of conventional arrays are limited by the low number of electrodes available. Here, we present a large-scale array recording simultaneously from 4096 electrodes used to study propagating spontaneous and evoked network activity in acute murine cortico-hippocampal brain slices at unprecedented spatial and temporal resolution. We demonstrate that multiple chemically induced epileptiform episodes in the mouse cortex and hippocampus can be classified according to their spatio-temporal dynamics. Additionally, the large-scale and high-density features of our recording system enable the topological localization and quantification of the effects of antiepileptic drugs in local neuronal microcircuits, based on the distinct field potential propagation patterns. This novel high-resolution approach paves the way to detailed electrophysiological studies in brain circuits spanning spatial scales from single neurons up to the entire slice network.

Low-frequency stimulation enhances burst activity in cortical cultures during development.

The intact brain is continuously targeted by a wealth of stimuli with distinct spatio-temporal patterns which modify, since the very beginning of development, the activity and the connectivity of neuronal networks. In this paper, we used dissociated neuronal cultures coupled to microelectrode arrays (MEAs) to study the response of cortical neuron assemblies to low-frequency stimuli constantly delivered over weeks in vitro. We monitored the spontaneous activity of the cultures before and after the stimulation sessions, as well as their evoked response to the stimulus. During in vitro development, the vast majority of the cultures responded to the stimulation by significantly increasing the bursting activity and a widespread stabilization of electrical activity was observed after the third week of age. A similar trend was present between the spontaneous activity of the networks observed over 30 min after the stimulus and the responses evoked by the stimulus itself, although no significant differences in spontaneous activity were detected between stimulated and non-stimulated cultures belonging to the same preparations. The data indicate that the stimulation had a delayed effect modulating responsiveness capability of the network without directly affecting its intrinsic in vitro development.

Role of calcium binding proteins in the control of cerebellar granule cell neuronal excitability: experimental and modeling studies.

Calcium binding proteins, such as calretinin, are abundantly expressed in distinctive patterns in the central nervous system but their physiological function remains poorly understood. Calretinin is expressed in cerebellar granule cells which provide the major excitatory input to Purkinje cells through parallel fibers. Calretinin deficient mice exhibit dramatic alterations in motor coordination and in Purkinje cell firing recorded in vivo through unknown mechanisms. In the present paper, we review the results obtained with the patch clamp recording techniques in acute slice preparation. This data allow us to investigate the effect of a null mutation of the calretinin gene on the intrinsic electroresponsiveness of cerebellar granule cells at a mature developmental stage. Calretinin deficient granule cells exhibit faster action potentials and generate repetitive spike discharge showing an enhanced frequency increase with injected currents. These alterations disappear when 0.15 mM of the exogenous fast calcium buffer BAPTA is infused in the cytosol to restore the calcium buffering capacity. Furthermore, we propose a mathematical model demonstrating that the observed alterations of granule cell excitability can be explained by a decreased cytosolic calcium buffering capacity due to the absence of calretinin. We suggest that calcium binding proteins modulate intrinsic neuronal excitability and may therefore play a role in the information processing in the central nervous system.

Theta-frequency bursting and resonance in cerebellar granule cells: experimental evidence and modeling of a slow k+-dependent mechanism.

Neurons process information in a highly nonlinear manner, generating oscillations, bursting, and resonance, enhancing responsiveness at preferential frequencies. It has been proposed that slow repolarizing currents could be responsible for both oscillation/burst termination and for high-pass filtering that causes resonance (Hutcheon and Yarom, 2000). However, different mechanisms, including electrotonic effects (Mainen and Sejinowski, 1996), the expression of resurgent currents (Raman and Bean, 1997), and network feedback, may also be important. In this study we report theta-frequency (3-12 Hz) bursting and resonance in rat cerebellar granule cells and show that these neurons express a previously unidentified slow repolarizing K(+) current (I(K-slow)). Our experimental and modeling results indicate that I(K-slow) was necessary for both bursting and resonance. A persistent (and potentially a resurgent) Na(+) current exerted complex amplifying actions on bursting and resonance, whereas electrotonic effects were excluded by the compact structure of the granule cell. Theta-frequency bursting and resonance in granule cells may play an important role in determining synchronization, rhythmicity, and learning in the cerebellum.