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

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. G protein-coupled receptors (GPCRs) play pivotal roles in mediating neuronal communication in the brain. In fact, 90% of non-olfactory GPCRs are found in the brain 1 , where they regulate neuronal activity by engaging a variety of distal downstream effectors which include second messenger producing enzymes, ion channels, monomeric GTPases and protein kinases. Many GPCRs are pharmacologically targeted in the treatment of neurodegenerative and neuropsychiatric disease. Thus it is critical to understand how these receptors regulate intracellular signalling, and how this in turn regulates circuit function and ultimately, behavior. In cell culture models, these signalling pathways have been dissected extensively, through the use of genetically-encoded fluorescent and bioluminescent biosensors. Some of these biosensors have recently been used in the in vivo context, to link specific signalling patterns with behavioral outcomes 2-6. Many intracellular signalling biosensors utilize changes in Förster resonance energy transfer (FRET) between two fluorescent proteins 7 to report levels or activity of second messengers, protein kinases, GTPases, posttranslational modifications and protein-protein interactions 8-11. These tools have been widely used to dissect signaling pathways in cultured cells and have recently begun to be applied in vivo, aided by recent technological developments in intravital imaging 10,12 , microendoscopy 3 , 2-photon microscopy 13-15 and fluorescence lifetime measurement 16. Here 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. The dopamine D1 receptor (D1R) is a Gα s/olf coupled GPCR expressed throughout the forebrain. As for many GPCRs, several factors can impact D1R signalling, including the properties of specific ligands, the cellular context, interac...

Current Knowledge on the Background, Pathophysiology and Treatment of Levodopa-Induced Dyskinesia—Literature Review

“…The rate of D1R-dependent ERK1/2 phosphorylation is modulated by mGluR5, which alters the mGluR5/PLC/PKC pathway through the modulation of calcium/dependent responses. However, it does not influence the PKA activity [ 64 ]. The hypersensitivity of the D1R/cAMP/PKA pathway is associated with the elevation of the Gα olf protein level.…”

Current Knowledge on the Background, Pathophysiology, and Treatment of Levodopa-Induced Dyskinesia – Literature Review.

“…However, within 2 weeks after the treatment with L-DOPA, D1R were not able to activate PKA and ERK1/2 pathways. These observations indicate a remodelling of direct striatal pathways, that results from oversensitivity of dSPNs [49]. The rate of D1R-dependent ERK1/2 phosphorylation is modulated by mGluR5, which alters mGluR5/PLC/PKC pathway through modulation of calcium/dependent responses.…”

Single cell analysis of population-wide nuclear and cytosolic drug responses using high-content FRET imaging: measuring protein kinase activation in rat primary striatal neurons

“…Primary striatal neurons were prepared from postnatal day 1 pups as previously described 20 . One day in advance, 96-well optical bottom imaging plates (Nunc) were coated overnight with 0.1 mg/ml poly-D-lysine dissolved in phosphate buffered saline (PBS for cell culture - Sigma).…”