Astroglial Ca2+-Dependent Hyperexcitability Requires P2Y1 Purinergic Receptors and Pannexin-1 Channel Activation in a Chronic Model of Epilepsy.

Mario Wellmann,Christian Bonansco,Juan C Sáez,Carla Álvarez-Ferradas,Carola J Maturana

doi:10.3389/fncel.2018.00446

Abstract

Astrocytes from the hippocampus of chronic epileptic rats exhibit an abnormal pattern of intracellular calcium oscillations, characterized by an augmented frequency of long lasting spontaneous Ca2+ transients, which are sensitive to purinergic receptor antagonists but resistant to tetrodotoxin. The above suggests that alterations in astroglial Ca2+-dependent excitability observed in the epileptic tissue could arise from changes in astrocyte-to-astrocyte signaling, which is mainly mediated by purines in physiological and pathological conditions. In spite of that, how purinergic signaling contributes to astrocyte dysfunction in epilepsy remains unclear. Here, we assessed the possible contribution of P2Y1R as well as pannexin1 and connexin43 hemichannels—both candidates for non-vesicular ATP-release—by performing astroglial Ca2+ imaging and dye uptake experiments in hippocampal slices from control and fully kindled rats. P2Y1R blockade with MRS2179 decreased the mean duration of astroglial Ca2+ oscillations by reducing the frequency of slow Ca2+ transients, and thereby restoring the balance between slow (ST) and fast transients (FT) in the kindled group. The potential contribution of astroglial pannexin1 and connexin43 hemichannels as pathways for purine release (e.g., ATP) was assessed through dye uptake experiments. Astrocytes from kindled hippocampi exhibit three-fold more EtBr uptake than controls, whereby pannexin1 hemichannels (Panx1 HCs) accounts for almost all dye uptake with only a slight contribution from connexin43 hemichannels (Cx43 HCs). Confirming its functional involvement, Panx1 HCs inhibition decreased the mean duration of astroglial Ca2+ transients and the frequency of slow oscillations in kindled slices, but had no noticeable effects on the control group. As expected, Cx43 HCs blockade did not have any effects over the mean duration of astroglial Ca2+ oscillations. These findings suggest that P2Y1R and Panx1 HCs play a pivotal role in astroglial pathophysiology, which would explain the upregulation of glutamatergic neurotransmission in the epileptic brain and thus represents a new potential pharmacological target for the treatment of drug-refractory epilepsy.

Highlights

Under physiological conditions, besides providing metabolic support for neural tissue, astrocytes are essential for neural function and actively participate in the modulation of neuronal excitability and synaptic plasticity (Fellin et al, 2007; Perea and Araque, 2007; Shigetomi et al, 2008; Bonansco et al, 2011; Tan et al, 2017)
MRS application did not change the mean frequency in both CT and KD conditions (CT 2.90 ± 0.15 min−1; CT plus MRS 2.92 ± 0.11 min−1; KD 2.56 ± 0.09 min−1; KD plus MRS 2.88 ± 0.15 min−1; p = 0.076), suggesting that P2Y1R blockade restored the balance between FT and ST without modifying the total amount of astroglial activity. These results suggest that slow astroglial Ca2+ transients require P2Y1R activation, which is responsible for the FT/ST imbalance that characterizes abnormal excitability shown by astrocytes from the epileptic brain
The above results indicate that the abnormal pattern of astroglial Ca2+-mediated signals observed in the hippocampus of chronic epileptic rats is likely driven by purinergic astrocyte-astrocyte signaling, and requires the activity of Panx1 HCs and P2Y1R

Summary

Introduction

Besides providing metabolic support for neural tissue, astrocytes are essential for neural function and actively participate in the modulation of neuronal excitability and synaptic plasticity (Fellin et al, 2007; Perea and Araque, 2007; Shigetomi et al, 2008; Bonansco et al, 2011; Tan et al, 2017). Reactive astrocytes exhibit severe changes in the expression of a wide variety of proteins regulating purinergic signaling as well as extracellular concentration of ATP and its metabolites, including over-expression of Cx43, Panx, P2 receptors as well as ectonucleotidases and adenosine kinases (Bonan et al, 2000a,b; Gouder et al, 2004; Fedele et al, 2005; Aronica et al, 2011; Shen et al, 2014; Barros-Barbosa et al, 2016; Cieslak et al, 2017). The contributions of astroglial ATP to the epileptogenesis process and the generation of epileptiform activity remain unclear (Cieslak et al, 2017)

Methods

Results

Conclusion