Antidepressant fluoxetine engages astrocytic cAMP via purinergic signalling

The use of selective serotonin reuptake inhibitors (SSRIs), the first-line treatment for depression, has increased by about 50% over the past decade, placing them amongst the top 10 most frequently prescribed drug classes globally. Overall, SSRIs are effective in reducing frequency, severity, and duration of depressive episodes for a majority of patients, yet the mechanisms underlying their therapeutic effects are not fully understood. While SSRIs elevate synaptic serotonin, this action alone cannot account for their therapeutic effects. Additionally, SSRIs engage astrocytes, enhancing cyclic adenosine monophosphate (cAMP) signalling which is reported to be downregulated in depression. However, the signalling mechanisms underlying SSRI-induced upregulation of the astrocytic cAMP pathway remain unclear. Here, we identify a cascade of events by which the SSRI fluoxetine elevates intracellular cAMP levels in astrocytes, a process that depends on astrocyte-microglia crosstalk and purinergic signalling. Using FRET-based sensors in primary rat astrocytes, we show that fluoxetine elevates intracellular cAMP by 28% without altering calcium dynamics. cAMP increase was blocked by both serotonin (5-HT) 2B and adenosine 2B (A2B) receptor antagonists. Using the GRAB-ATP1.0 sensor and luminescence assays, we revealed that fluoxetine enhances astrocytic ATP release by 10% in a 5-HT2B receptor-dependent manner. Consistent with microglia-driven conversion of extracellular ATP to adenosine, which engages astrocytic A2B receptors, depletion of microglia in astrocyte cultures diminished fluoxetine-induced cAMP elevations and increased extracellular ATP. Together, these findings reveal that fluoxetine requires glial crosstalk and coordinated purinergic signalling to enhance astrocytic cAMP, a process shown to contribute to the therapeutic effect of SSRIs.