During development, brain circuits go through phases of synapse formation, stabilization, or elimination. γ-aminobutyric acid–mediated (GABAergic) synapse formation depends mainly on cell adhesion molecules, such as neuroligins and leucine-rich repeat transmembrane proteins, that interact with presynaptic neurexins and Slit- and Trk-like family proteins that bind to presynaptic protein tyrosine phosphatases. GABA and GABA type A (GABA_A) receptors are involved in an activity-dependent manner in the maturation and pruning of synapses. Adenosine triphosphate (ATP) and adenosine can be coreleased with GABA at synapses to be perceived by adenosine A_2A receptors. We tested the role of adenosine signaling in the stabilization and elimination of GABAergic synapses.

A_2A receptors control migration speed, axonal elongation, and dendrite branching. Whether A_2A receptors control synapse formation, stabilization, or elimination in the brain is not known. In the adult brain, A_2A receptors are mostly expressed on presynaptic terminals, where they control the probability of synaptic vesicle release. The amount, location, and function of A_2A receptors at neural synapses during early brain development has been unclear.

During synaptogenesis in the developing mouse hippocampus, between postnatal days P5 and P16, the density of A_2A receptors increases transiently around the postsynaptic density. Activity-dependent release of its endogenous ligand, adenosine, increases as well. A_2A receptors control the fate of GABAergic synapses. Suppression of A_2A receptors, their pharmacological blockade, or the removal of adenosine results in the destabilization of pre- and postsynaptic sites in vivo, ex vivo, and in vitro. If A_2A receptors remain inactive for >20 min, synapse destabilization is irreversible. We found that A_2A receptor activation is necessary and sufficient for GABAergic synapse stabilization, whereas GABA_A receptor activation is not necessary as long as A_2A receptors remain activated. We studied the molecular mechanism at play. A_2A receptor and GABA_A receptor signaling pathways converge onto calcium-calmodulin–sensitive adenylyl cyclases to produce adenosine 3′,5′-monophosphate (cAMP). The resulting activation of protein kinase A leads to phosphorylation of the postsynaptic scaffolding molecule gephyrin at the protein kinase A–sensitive serine residue 303 site. Expression of the gephyrin mutant mimicking this phosphorylation state prevents synapse loss upon the removal of extracellular adenosine. Phosphorylated gephyrin can be coimmunoprecipitated with the postsynaptic transmembrane Slit- and Trk-like family protein 3 that binds in the synaptic cleft to presynaptic protein tyrosine phosphatase σ to organize inhibitory synapses. The contribution of Slit- and Trk-like family protein 3 in stabilizing GABAergic synapses through adenosine signaling is demonstrated with a short hairpin RNA (shRNA) approach or after the expression of a mutant. Finally, antagonizing A_2A receptors during synaptogenesis in vivo results in the loss of GABAergic synapses during development and cognitive deficits when animals reach adulthood.

A_2A receptors regulate the elimination of certain GABAergic synapses when they become inactive. A_2A receptors are poised to detect active presynaptic terminals and trigger synapse removal after a defined period of synaptic inactivity.

(Left) Active synapse: Corelease of adenosine, ATP, and GABA activates A_2A receptors and GABA_A receptors, whose signaling pathways converge …