GABAB receptor-dependent regulation of cAMP dynamics in hippocampal neurons

Abstract

Dysregulation of inhibitory G protein-dependent signaling, including signaling controlled by the GABAB receptor (GABABR), is implicated in many neurological disorders. In neurons, GABABR exerts much of its inhibitory influence by activating G protein-gated Inwardly Rectifying K+ (GIRK/Kir3) channels and inhibiting Adenylyl Cyclase (AC). Inhibitory G protein-dependent signaling is mediated by the Gαi/o subfamily of G proteins, which includes 3 Gαi isoforms (Gαi1, Gαi2, Gαi3), Gαo and Gαz, 5 Gβ isoforms (Gβ1-5) and 12 Gγ isoforms (Gγ1-12). However, the specific Gα subunits and Gβγ complexes that mediate GABABR-dependent regulation of these effectors in neurons is poorly understood. Here, we present the development of an imaging platform that enables systematic genetic interrogation of G protein subtypes and regulatory components shaping GABABR-AC signaling dynamics in mouse hippocampal (HPC) neurons. The platform involves viral expression of a cAMP luminescence sensor (GloSensorTM) in cultured neurons. GloSensorTM reliably reports a dose-dependent increase in cAMP in response to forskolin induced AC simulation, as well as a dose-dependent suppression of cAMP levels induced by the GABABR agonist baclofen. To investigate molecular mechanisms underlying the GABABR-dependent suppression of cAMP levels in neurons, we employed a CRISPR/Cas9 strategy. HPC neuron cultures derived from a Cas9-expressing mouse line were infected with adeno-associated virus (AAV) vectors carrying guide RNA (gRNA) sequences targeting specific genes of interest. In neurons treated with vectors containing a GABABR-specific gRNA, baclofen no longer suppressed the forskolin-induced increase in cAMP. When neurons were treated with either pertussis toxin (which inhibits Gαi/o proteins) or a mixture of AAVs carrying gRNAs targeting Gαi1, Gαi2, Gαi3, and Gαo, baclofen-induced cAMP suppression was abolished. Using this validated system, we then found that simultaneous ablation of Gαi1 and Gαo was sufficient to eliminate the baclofen-evoked suppression of cAMP. Simultaneous ablation of Gβ1-4 similarly eliminated the baclofen-induced cAMP decrease, whereas deletion of individual Gβ isoforms was without effect. Interestingly, either Gβ1 or Gβ2 alone was sufficient to support maximal GABABR-dependent cAMP suppression. Similarly, combined ablation of Gγ isoforms expressed at the highest levels in the adult HPC (Gγ2,3,4,7,10,12), but not any single Gγ isoform, abolished most of the baclofen response, while leaving either Gγ2 or Gγ3 as the sole Gγ isoform enabled maximum baclofen-induced suppression of cAMP. Our findings provide key insights into the composition of GABABR-AC signalosome in neurons, thus advancing our understanding of GPCR-effector compartmentalization in the central nervous system.