Diverse inflammatory threats modulate astrocytes Ca2+ signaling via connexin43 hemichannels in organotypic spinal slices

Giulia Panattoni,Roberta Amoriello,Clara Ballerini,Laura Ballerini,Agnes Thalhammer,Christian Memo

doi:10.1186/s13041-021-00868-6

Giulia Panattoni, Roberta Amoriello + Show 4 more

Open Access

https://doi.org/10.1186/s13041-021-00868-6

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Abstract

Neuroinflammation is an escalation factor shared by a vast range of central nervous system (CNS) pathologies, from neurodegenerative diseases to neuropsychiatric disorders. CNS immune status emerges by the integration of the responses of resident and not resident cells, leading to alterations in neural circuits functions. To explore spinal cord astrocyte reactivity to inflammatory threats we focused our study on the effects of local inflammation in a controlled micro-environment, the organotypic spinal slices, developed from the spinal cord of mouse embryos. These organ cultures represent a complex in vitro model where sensory-motor cytoarchitecture, synaptic properties and spinal cord resident cells, are retained in a 3D fashion and we recently exploit these cultures to model two diverse immune conditions in the CNS, involving different inflammatory networks and products. Here, we specifically focus on the tuning of calcium signaling in astrocytes by these diverse types of inflammation and we investigate the mechanisms which modulate intracellular calcium release and its spreading among astrocytes in the inflamed environment. Organotypic spinal cord slices are cultured for two or three weeks in vitro (WIV) and exposed for 6 h to a cocktail of cytokines (CKs), composed by tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1 β) and granulocyte macrophage-colony stimulating factor (GM-CSF), or to lipopolysaccharide (LPS). By live calcium imaging of the ventral horn, we document an increase in active astrocytes and in the occurrence of spontaneous calcium oscillations displayed by these cells when exposed to each inflammatory threat. Through several pharmacological treatments, we demonstrate that intracellular calcium sources and the activation of connexin 43 (Cx43) hemichannels have a pivotal role in increasing calcium intercellular communication in both CKs and LPS conditions, while the Cx43 gap junction communication is apparently reduced by the inflammatory treatments.

Highlights

Neuroinflammation is a characterizing feature occurring in and contributing to central nervous system (CNS) pathologies such as amyotrophic lateral sclerosis (ALS) and multiple sclerosis (MS)[1, 2]
Sulforhodamine‐positive glial cells display slow spontaneous Ca2+ activity The presence of GFAP-positive astrocytes has long been described in cultured spinal explants ventral horns [11,12,13], as confirmed by Fig. 1A, where numerous astrocytes are visualized within the ventral area of a spinal organotypic culture after 2 weeks in vitro (WIV)
We labeled by fluorescent dye Fluo-4 AM cells in organotypic spinal cord and dorsal root ganglia (DRG) co-cultures to simultaneously visualize within the sampled area of the ventral horn pre-motor circuit [11], neuronal and glial cells calcium signaling

Summary

Introduction

Neuroinflammation is a characterizing feature occurring in and contributing to CNS pathologies such as amyotrophic lateral sclerosis (ALS) and multiple sclerosis (MS)[1, 2]. The emergent activity of neural circuits may Panattoni et al Mol Brain (2021) 14:159 be altered both acutely and chronically by inflammatory milieus activating intricate signaling pathways, orchestrated by various cell phenotypes, responsible for intercellular communication and contributing to the propagation of the inflammatory damage in the CNS. In this picture, astrocytes, the key cellular partners to neurons, play both beneficial roles, such as recovery of extracellular ionic homeostasis limiting inflammation [6] and deleterious ones, such as hypertrophy with increased astrogliosis [7, 8].

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