Depression has a substantial socioeconomic burden as the leading cause of disability globally, affecting 5% of the adult population. Although the use of antidepressants to treat depression and other mental health conditions has doubled in the last 20 years, a third of patients do not respond to antidepressant therapy.  Consequently, there is an unmet clinical need for improved treatment options for those living with depression.

There has been little development in this field, driven by our lack of understanding of the molecular and cellular mechanisms underpinning depression and other mental health conditions.  While selective serotonin reuptake inhibitors (SSRIs), including fluoxetine (FLX), are known to elevate serotonin levels by inhibiting its uptake into the presynaptic cell, evidence suggests that the therapeutic effects of antidepressants are independent of changes in monoamine levels. Therefore, the therapeutic effects of SSRIs cannot be entirely attributed to changes in serotonin levels. Thus, other signalling pathways are likely to be targeted by FLX.

Astrocytes, once thought to be solely supporting cells, are now widely recognised to play an essential role in emotional regulation. Both cAMP and Ca²⁺ signalling are essential for astrocyte function; however, their roles in emotional regulation or antidepressant mechanisms remain largely unknown.

Here, we aimed to determine the mechanism by which antidepressant FLX alters intracellular signalling in astrocytes.

Live cell imaging of primary rat astrocytes revealed that FLX application (10µM, 5 minutes) increases intracellular cAMP levels by 27%, but not Ca²⁺ levels (cAMP (Epac sensor): p<0.0001; Ca²⁺(Twtich2B sensor): p=0.1511). The FLX-driven increase in cAMP was dependent on adenylate cyclase (AC) signalling: application of AC inhibitor, NKY80, in combination with FLX attenuated FLX’s effect on cAMP (NKY80+FLX vs FLX: p=0.0044). In the presence of selective serotonin 2B (5-HT_2BR) and adenosine 2B receptor (A_2B) antagonists (PSB603 and LY266097), FLX was unable to increase cAMP (PSB+FLX p<0.0001 and LY+FLX p=0.0029 vs FLX). The FLX-driven increase in cAMP levels was 4x less than that of the direct action of adenosine on the A_2BR. We proposed that A_2BR activation is secondary to 5-HT_2BR activation. It has been previously demonstrated that ATP can be converted to adenosine by microglia. Although steps are taken to minimise microglial contamination, approximately 3% of microglia remain in our primary astrocyte cultures. Therefore, we used a real-time luminescence-based ATP release assay to demonstrate that FLX increases ATP release from astrocytes by 100% (p<0.0001), a process that is also dependent on 5-HT_2BR (LY+FLX vs FLX: p<0.0001). Finally, in microglia-depleted cultures (PLX5622, 10µM, 7 days), FLX was unable to increase cAMP (p=0.0118). All experiments included a minimum of 2 biological replicates, with 3 coverslips/wells and at least 4 cells/region of interests per coverslip. When two data sets were compared, an unpaired T-test was used; when more than two groups were compared, a one-way ANOVA with post-hoq Holm-Šídák’s multiple comparisons test was used.

To summarise, we have identified a novel antidepressant mechanism in which FLX potentiates ATP release via 5-HT_2BR. ATP is converted to ADO by microglia, which, in turn, activates the A2BR, thereby increasing intracellular cAMP levels.