Interrogating endogenous neuromodulatory GPCR signal processing by real‐time imaging of cAMP dynamics through intact neuronal circuits

Abstract

The majority of neuromodulators exert their effects by activating G protein coupled receptors (GPCRs) which shape neuronal properties through the regulation of second messengers. The mechanisms of signal decoding at GPCRs amidst diversity of the effects associated with their ctivation remains elusive. Furthermore, there is a paucity of understanding of how GPCRs signal to produce unique responses impacting cellular physiology while converging on a limited set of second messenger signaling cascades. Although significant progress has been made studying GPCRs in reconstituted systems, information decoding processes by GPCRs in an endogenous setting is understood less due to a lack of appropriate tools. Here, we developed a novel in vivo reagent that allows monitoring GPCR signaling in the endogenous setting from genetically defined populations of cells. The approach takes advantage of knock‐in mice engineered to express a genetically encoded FRET‐based cAMP biosensor in a Cre‐dependent fashion. We applied this reagent to study integration of GPCR signals in striatal medium spiny neurons both in culture and in brain slices where intact circuits were activated by optogenetics. Combined with CRISPR/Cas9 genomic editing, this approach enabled us to identify individual receptors and analyze their contributions to downstream signaling in the native environment. We identified key spatial differences in pharmacological responsiveness by comparing dendritic regions with the neuronal cell body as well as plasma membrane and internal cellular signaling compartments. We then conducted comparative analysis of GPCR signaling mediated by two opposing circuits modulated by the neurotransmitter dopamine, revealing the mechanisms of dopaminergic processing as well as major differences in the generation of discrete second messenger signatures. We further demonstrate how cAMP signal processing mechanisms contribute to allostatic adaptations that imbalance tuning between striatal output neurons that may underlie states of reward and aversion.Support or Funding InformationNIH 1R01DK110621, APS STRIDE Undergraduate Summer Research Fellowship 1R25HL115473‐01.This work was supported by NIH grants: DA041207 (to B.S.M.), DA036596 (to K.A.M.), and DA026405 (to K.A.M.).