THE OXYTOCIN RECEPTOR AS A KEY REGULATOR OF ASTROCYTE SIGNALING IN THE CENTRAL NERVOUS SYSTEM

The neuropeptide oxytocin (OT) is known to regulate crucial endocrine functions underpinning different behavioral aspects such as pair bonding, sociability, and emotional behavior, and to serve as a neuromodulator within the central nervous system (CNS), interplaying with other neurotransmission pathways. Within the context of synapse modulation, in the last decades, research has principally focused on the action of OT on neurons. However, different studies have demonstrated that OT and its cognate receptor (the oxytocin receptor, OTR) may play an active role in the regulation of astrocyte function. Nonetheless, the role of OT in orchestrating astrocyte signaling has been largely neglected, although they play an important role in controlling synapse coverage and efficacy, and the homeostasis of the excitatory amino acid glutamate via specific membrane transporters and its Ca2+-dependent release. We here investigated whether and if so, how, the activation of the OTR may influence gliotransmission by focusing on two different aspects correlated with the modulation of glutamate release: i) the involvement of native astrocytic OTR in the formation of G-protein coupled receptor (GPCR) heterodimers, and ii) the putative intracellular mechanisms underlying the activation of astrocytic OTRs. In the first part of the study, the characterization of the expression and the functionality of A2A-OT receptors and OT-D2 receptors heterodimers was evaluated on brain slices and purified striatal astrocyte processes (gliosomes) from adult male rats. We observed that our ex vivo models, characterized by the presence of typical functional astrocytic markers (GFAP, ezrin, and VGLUT1), expressed native OTR, A2A, and D2, receptors. Moreover, OTR was found to co-localize with either A2A or D2. To assess the possible formation of the hypothesized heterodimers, we combined different approaches including biophysical, biochemical, and functional studies. At first, on striatal astrocytes, we were able to observe positive signals using an in-situ proximity ligation assay (PLA), suggesting that the OTR is found in close proximity with either A2A or D2 receptors. This evidence was corroborated by co-immunoprecipitation analysis, showing that the receptors of interest interact physically on striatal astrocytes, in particular on flotillin-1-enriched membranes. On the functional level, we first assessed the effect of OT stimulation on depolarization-evoked endogenous glutamate release from striatal gliosomes in superfusion, observing that only 3 nM OT was able to exert an effect, negatively modulating the above-mentioned release. Concurrent stimulation with the A2A receptor agonist CGS21680 (10 nM), per se ineffective, completely abolished the effect mediated by OT and restored the ability of gliosomes to react to the depolarizing stimulus. This observation was also confirmed by the analysis of Ca2+ transients evoked upon depolarization, which followed the same pattern as glutamate release. By contrast, when exposing gliosomes to the D2 agonist quinpirole (1 µM), which on its own exerted an inhibitory effect on the release of glutamate release upon depolarization, a stronger reduction was observed, suggesting the existence of a facilitatory receptor-receptor interaction. This was confirmed by a similar effect in the presence of a subthreshold dose of quinpirole (100 nM). Lastly, we obtained in silico models of A2A-OTR and D2-OTR heterodimers and highlighted that, in both cases, specific amino acid residues located on transmembrane domains 4 and 5 of the three receptors are most likely involved in the establishment of the heterodimer interface. Altogether, these results indicate that, in striatal astrocytes, the native OTR interact physically with either A2A or D2 receptors, generating functional heterodimers and establishing a new level of regulation of glutamatergic transmission involving astrocytes. Following the observation that 3 nM OT was able to inhibit glutamate release from astrocytes, we decided to investigate the intracellular pathways potentially leading to such an effect. For this purpose, we set up a protocol to prepare primary cultured astrocytes that could reflect the releasing properties observed with gliosomes ex vivo. Accordingly, astrocytes were exposed to 10 µM Forskolin (FSK) from in vitro day (DIV) 7 to DIV 20-22, to induce stellation and functional maturation. As a result, FSK-treated astrocytes displayed a stellate morphology and were able to release endogenous glutamate in response to a depolarizing stimulus. Taking advantage of this in vitro model, we observed that 3 nM OT was able to induce a decrease in endogenous glutamate release upon depolarization and that the observed effect may be due to the coupling of OTR with the Gi/o protein, as suggested by a reduction in intracellular cAMP levels after treatment with OT. However, these observations are to be considered as preliminary and experiments are in progress to validate our hypothesis. Overall, the results obtained with this study provide a new way of considering OT and OTR signaling within the CNS, highlighting the crucial role played by astrocytes in the context of regulating excitatory synaptic transmission.