Interrogating the Spatiotemporal Landscape of Neuromodulatory GPCR Signaling by Real-Time Imaging of cAMP in Intact Neurons and Circuits

Abstract

Modulation of neuronal circuits is key to information processing in the brain. The majority of neuromodulators exert their effects by activating G protein coupled receptors (GPCR) that control the production of second messengers directly impacting cellular physiology. How numerous GPCRs integrate neuromodulatory inputs while accommodating diversity of incoming signals is poorly understood. In this study we develop an in vivo tool and analytical suite for analyzing GPCR responses by monitoring the dynamics of a key second messenger, cAMP with excellent quantitative and spatio-temporal resolution in various neurons. With this imaging approach in combination with CRISPR/Cas9 editing and optogenetics we interrogate neuromodulatory mechanisms of defined populations of neurons in an intact mesolimbic reward circuit and describe how individual inputs generate discrete second messenger signatures in a cell and receptor specific fashion. This offers a resource for studying native neuronal GPCR signaling in real time.

Molecular Deconvolution Platform to Establish Disease Mechanisms by Surveying GPCR Signaling

“…Finally, we investigated two representative mutants, V354A and S239N, in the endogenous setting of D1R-expressing striatal medium spiny neurons (D1R-MSNs) using CAMPER reporter mice specifically expressing cAMP sensor in D1R-MSNs ( Muntean et al., 2018 ). We observed that the V354A mutant exhibited increased basal cAMP levels compared with the wild-type; however, cAMP concentration in these neurons did not change in response to dopamine stimulation ( Figures 5 E–5H).…”

“…Mice (C57BL/6) were housed under standard conditions on a 12-hour light/dark cycle with continuous access to food and water in a pathogen-free facility. The CAMPER mouse was generated previously ( Muntean et al., 2018 ). Mice were appropriately genotyped and not subject to any prior procedures.…”

“…Primary striatal neurons were cultured as previously described ( Muntean et al., 2018 ). Brains from D1RCre-CAMPER pups (postnatal day 0) were rapidly excised followed by dissection of striata in ice cold Hanks’ Balanced Salt Solution (HBSS) (Thermo Fisher Scientific) supplemented with 20% FBS, 4.2 mM NaHCO 3 , and 1 mM HEPES.…”

“…Excitation of mTurquoise was achieved with a 442 nm diode laser paired with simultaneous 465-505 nm (mTurquoise) and 525-605 nm (Venus) bandpass emission filtration. Multiple Z stacks were captured at each time point and FRET ratios from neuronal cell bodies were calculated using ImageJ and converted to cAMP units from a calibration curve as previously described ( Muntean et al., 2018 ). Dopamine was administered at 100 μM in phasic 1 s pulses through an SF-77B perfusion apparatus (Warner Instruments, Hamden, CT).…”

Dopamine D1 receptor signalling in dyskinetic Parkinsonian rats revealed by fiber photometry using FRET-based biosensors

“…We set out to develop a platform for expressing and recording FRET-based biosensors that was both simple to use and readily generalizable to other biosensors and animal models. While transgenic mice have been generated that express a number of FRET biosensors 3,14,29 , we opted to use adeno-associated viral vectors (AAVs) to drive biosensor expression, since this would allow our tools to be exploited in both rats and mice without the need to maintain colonies of transgenic animals. In this work, we used previously published FRET-based reporters for PKA and ERK1/2 referred to as AKAR and EKAR respectively 13 .…”