Human-Derived Cortical Neurospheroids Coupled to Passive, High-Density and 3D MEAs: A Valid Platform for Functional Tests.

With the advent of human-induced pluripotent stem cells (hiPSCs) and differentiation protocols, methods to create in-vitro human-derived neuronal networks have been proposed. Although monolayer cultures represent a valid model, adding three-dimensionality (3D) would make them more representative of an in-vivo environment. Thus, human-derived 3D structures are becoming increasingly used for in-vitro disease modeling. Achieving control over the final cell composition and investigating the exhibited electrophysiological activity is still a challenge. Thence, methodologies to create 3D structures with controlled cellular density and composition and platforms capable of measuring and characterizing the functional aspects of these samples are needed. Here, we propose a method to rapidly generate neurospheroids of human origin with control over cell composition that can be used for functional investigations. We show a characterization of the electrophysiological activity exhibited by the neurospheroids by using micro-electrode arrays (MEAs) with different types (i.e., passive, C-MOS, and 3D) and number of electrodes. Neurospheroids grown in free culture and transferred on MEAs exhibited functional activity that can be chemically and electrically modulated. Our results indicate that this model holds great potential for an in-depth study of signal transmission to drug screening and disease modeling and offers a platform for in-vitro functional testing.

Loss of Snord116 impacts lateral hypothalamus, sleep, and food-related behaviors.

Imprinted genes are highly expressed in the hypothalamus; however, whether specific imprinted genes affect hypothalamic neuromodulators and their functions is unknown. It has been suggested that Prader-Willi syndrome (PWS), a neurodevelopmental disorder caused by lack of paternal expression at chromosome 15q11-q13, is characterized by hypothalamic insufficiency. Here, we investigate the role of the paternally expressed Snord116 gene within the context of sleep and metabolic abnormalities of PWS, and we report a significant role of this imprinted gene in the function and organization of the 2 main neuromodulatory systems of the lateral hypothalamus (LH) - namely, the orexin (OX) and melanin concentrating hormone (MCH) - systems. We observed that the dynamics between neuronal discharge in the LH and the sleep-wake states of mice with paternal deletion of Snord116 (PWScrm+/p-) are compromised. This abnormal state-dependent neuronal activity is paralleled by a significant reduction in OX neurons in the LH of mutant mice. Therefore, we propose that an imbalance between OX- and MCH-expressing neurons in the LH of mutant mice reflects a series of deficits manifested in the PWS, such as dysregulation of rapid eye movement (REM) sleep, food intake, and temperature control.

Loss of Snord116 alters cortical neuronal activity in mice: a preclinical investigation of Prader-Willi syndrome.

Prader-Willi syndrome (PWS) is a neurodevelopmental disorder that is characterized by metabolic alteration and sleep abnormalities mostly related to rapid eye movement (REM) sleep disturbances. The disease is caused by genomic imprinting defects that are inherited through the paternal line. Among the genes located in the PWS region on chromosome 15 (15q11-q13), small nucleolar RNA 116 (Snord116) has been previously associated with intrusions of REM sleep into wakefulness in humans and mice. Here, we further explore sleep regulation of PWS by reporting a study with PWScrm+/p- mouse line, which carries a paternal deletion of Snord116. We focused our study on both macrostructural electrophysiological components of sleep, distributed among REMs and nonrapid eye movements. Of note, here, we study a novel electroencephalography (EEG) graphoelements of sleep for mouse studies, the well-known spindles. EEG biomarkers are often linked to the functional properties of cortical neurons and can be instrumental in translational studies. Thus, to better understand specific properties, we isolated and characterized the intrinsic activity of cortical neurons using in vitro microelectrode array. Our results confirm that the loss of Snord116 gene in mice influences specific properties of REM sleep, such as theta rhythms and, for the first time, the organization of REM episodes throughout sleep-wake cycles. Moreover, the analysis of sleep spindles present novel specific phenotype in PWS mice, indicating that a new catalog of sleep biomarkers can be informative in preclinical studies of PWS.

An approach to monitoring home-cage behavior in mice that facilitates data sharing.

Genetically modified mice are used as models for a variety of human behavioral conditions. However, behavioral phenotyping can be a major bottleneck in mouse genetics because many of the classic protocols are too long and/or are vulnerable to unaccountable sources of variance, leading to inconsistent results between centers. We developed a home-cage approach using a Chora feeder that is controlled by-and sends data to-software. In this approach, mice are tested in the standard cages in which they are held for husbandry, which removes confounding variables such as the stress induced by out-of-cage testing. This system increases the throughput of data gathering from individual animals and facilitates data mining by offering new opportunities for multimodal data comparisons. In this protocol, we use a simple work-for-food testing strategy as an example application, but the approach can be adapted for other experiments looking at, e.g., attention, decision-making or memory. The spontaneous behavioral activity of mice in performing the behavioral task can be monitored 24 h a day for several days, providing an integrated assessment of the circadian profiles of different behaviors. We developed a Python-based open-source analytical platform (Phenopy) that is accessible to scientists with no programming background and can be used to design and control such experiments, as well as to collect and share data. This approach is suitable for large-scale studies involving multiple laboratories.