Abstract
As with many G protein-coupled receptors (GPCRs), the signalling pathways regulated by the dopamine D1 receptor (D1R) are dynamic, cell type-specific, and can change in the face of disease or drug exposures. In striatal neurons, the D1R activates cAMP/protein kinase A (PKA) signalling. However, in Parkinson’s disease (PD), alterations in this pathway lead to functional upregulation of extracellular regulated kinases 1/2 (ERK1/2), contributing to l-DOPA-induced dyskinesia (LID). In order to detect D1R activation in vivo and to study the progressive dysregulation of D1R signalling in PD and LID, we developed ratiometric fiber-photometry with Förster resonance energy transfer (FRET) biosensors and optically detected PKA and ERK1/2 signalling in freely moving rats. We show that in Parkinsonian animals, D1R signalling through PKA and ERK1/2 is sensitized, but that following chronic treatment with l-DOPA, these pathways become partially desensitized while concurrently D1R activation leads to greater induction of dyskinesia.
Highlights
G protein-coupled receptors (GPCRs) play pivotal roles in mediating neuronal communication in the brain
Using a 6-OHDA model of Parkinson’s disease, we show that D1 receptor (D1R)-mediated activation of protein kinase A (PKA) and extracellular regulated kinases 1/2 (ERK1/2) is at first enhanced by dopamine depletion, and partially attenuated by prolonged l-DOPA treatment
We opted to use adeno-associated viral vectors (AAVs) to express previously published Förster resonance energy transfer (FRET)-based reporters for PKA and ERK1/2, referred to as AKAR and EKAR respectively[9]. Variants of these biosensors have previously been used in vivo in applications such as microendoscopy3,9, 2-photon microscopy[14], and fluorescence lifetime photometry[16]. These biosensors are scaffolded on substrates for protein kinase activity and contain phosphorylation-dependent interaction domains, such that the biosensor undergoes a conformational change upon phosphorylation, leading to an increase in resonance energy transfer (Fig. 1a)
Summary
G protein-coupled receptors (GPCRs) play pivotal roles in mediating neuronal communication in the brain. We report a ratiometric fiber-photometry approach for real-time recording of geneticallyencoded FRET biosensors in freely moving rodents We apply this approach to investigate alterations in striatal GPCR signalling in a rat model of Parkinson’s disease and l-DOPA induced dyskinesia. In response to diminished synaptic dopamine levels, D1R signaling in dMSNs becomes rewired through alterations in receptor trafficking, upregulation of D1R-associated effectors including Gαolf, and formation of disease-specific receptor heteromers[21,22,23,24,25] These adaptations effectively sensitize the D1R and increase its ability to activate multiple signaling pathways mediated by PKA and extracellular regulated kinases 1/2 (ERK1/2). Our approach allowed us to monitor signalling in the same animals over the course of dopamine depletion and l-DOPA treatment, and can provide novel insights into how signalling processes shape behavioral outcomes over time
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