An astrocyte-microglia crosstalk mediating neuroinflammation under exposure to psychostimulants

Methamphetamine (Meth) is a widely abused powerful psychostimulant, characterized at molecular level by monoaminergic disruption, high oxidative stress and mitochondrial dysfunction. Recently, increasing attention has been given to the interaction between neuronal and glial cells in the building and maintenance of addiction. In particular, microglia and gliotransmission have been implicated in addiction-associated neuroinflammation. However, the overall contribution of such process to the buil-up of addiction remains unclear. In the present study, we postulated that the long-term adverse consequences occurring within the reward circuitry under Meth exposure could be due, at least in part, to neuroinflammation and that limiting such process could be relevant to control the addictive behaviour and consequences.

Using flow cytometry, expression of signature genes and immunohistochemistry, we have established that acute exposure to Meth leads to robust microgliosis and activation of a pro-inflammatory profile in wild type mice (under a binge administration of 4 x 5 mg/kg, 2h intervals, i.p.). However, our data also showed that differently from what is commonly believed, Meth does not activate microglia in a cell-autonomous manner. This led us to hypothesize that microglia activation could depended on a crosstalk with other brain cells. In accordance, exposing primary microglia cultures to the conditioned media (CM) of primary astrocytes treated with Meth resulted in a strong activation of microglia, significantly increasing ROS production, iNOS expression and phagocytic activity, and release of pro-inflammatory cytokines. In contrast, exposure to the CM of different types of neurons exposed to Meth, did not result in microglia activation. To identify the astrocytic released factor(s) responsible for microglia activation, we analyzed the production of pro-inflammatory factors (IL-1B; IL-6 and TNF) by qRT-PCR, and glutamate release using FRET-based nanosensors in primary astrocytes exposed to Meth. We observe a huge increase in glutamate release. To gain mechanistic insight into this astrocyte-mediated microglia activation, we explored the pathways involved in astrocytic glutamate release, verifying that Meth and TNF regulated astrocytic glutamate release via mobilization of Ca2+ from the endoplasmic reticulum to the cytosol in an IP3R-sensitive manner. The cytosolic Ca2+ rise triggered by Meth promoted glutamate release from astrocytes via SNARE-dependent exocytosis. Supporting these data, we confirmed that in TNF knockout mice, the behavioural expression of Meth was abolished and Meth-induced microgliosis prevented. In summary, we describe here that Meth activates microglia via glutamate release from astrocytes, which is mediated by TNF. In this scenario, full understanding the nature of the neuroinflammatory response to psychostimulants may be crucial to devise new valuable therapeutic approaches.