Oxytocin Receptor-Mediated Signaling in Astrocytes

Especially in higher vertebrates, astrocytes are an indispensible part of signal processing within the brain. Thus, the mode of action of a neuroactive peptide such as OXT cannot be fully understood without this integral part of the CNS. The effects of OXT on neuronal cells have been well characterized, while its effects on astrocytic cells, specifically on OXTR-coupled signaling and its resulting cellular consequences, are poorly understood and might very well differ. To characterize the effect of OXT on astrocytic gene expression, intracellular signaling, as well as astrocyte-specific proteins, synthetic OXT was either administered icv in male Wistar rats or applied to cultured rat primary cortical astrocytes. Due to the results of this analysis implying an acute OXT-induced cytoskeletal remodeling and alterations to gap-junction coupling, I next examined the underlying molecular mechanisms and cellbiological consequences of these alterations. Here I found that OXT led to rapid elongation and formation of astrocytic processes in vitro and in vivo, while simultaneously impairing astrocytic intercellular connectivity. Mechanistically, both of these effects were OXTR-specific, conveyed via PKC and, to a lesser extent, MEK1/2 signaling. Notably, OXT-induced cytoskeletal remodeling and impairment of gap-junctions were characteristic for OXT, since its closely related sister-peptide AVP did not affect the examined parameters. CLSM and STED-microscopy following icv or ex vivo administration of OXT furthermore revealed changes to astrocyte-neuron spatial relationships in two brain regions associated with high responsiveness of astrocytic markers to OXT, i.e. PVN and hippocampus. In depth in vitro studies identified the previously undescribed Sp1-Gem signaling axis to be at the base of these effects. A combination of knockdown, knockout and overexpression experiments revealed that OXT drives Gem expression via the transcription factor Sp1 and that Gem is required and sufficient for the effects of OXT on astrocytes. The Sp1-Gem axis was differentially regulated by OXT in neuronal cells, identifying it as key driver in the cell type-specific response of astroglial cells to OXT. Based on these findings, astrocyte-specific AAV-mediated Gem or Oxtr shRNA knockdown vectors were established as tools for a targeted manipulation of astrocytic OXTR signaling and future assessment of astrocytic contribution to the physiological and behavioral effects of OXT. To this end, shRNA oligonucleotides were screened for knockdown efficiency in vitro and subsequently packaged into viral vectors providing astrocyte-specific expression via transcriptional control of shRNA expression under the hGFAP promoter