Genetically encoded fluorescent reporters to visualize Lewy pathology in live brain

Lewy bodies, a pathological hallmark of Parkinson's disease, are a-synuclein-enriched cytoplasmic inclusions that drive progressive neurodegeneration these reporters provide an opportunity to directly visualize a-synuclein inclusions in live brains and support various applications for measuring their pathological effects on individual neurons. Overall design: After anesthesia with tribromoethanol (200 mg/kg), the mice were transcardially perfused with ice-cold oxygenated sucrose-based ACSF and decapitated. Their brains were removed and placed into ice-cold oxygenated sucrose-ACSF, then were cut into slices (300 µm) in ice-cold oxygenated sucrose-ACSF with a vibratome (Leica VT 1200S). The slices were subsequently incubated in oxygenated ACSF at 34 °C for 30 min, then at roomtemperature for 1 h. To pick the a-Syn-6H-EGFP inclusion+ neurons in the SNc, glass capillaries (2.0 mm OD, 1.16 mm ID, Sutter Instruments) were autoclaved before pulling patch-seq pipettes. All environmental surfaces were kept clean and RNase-free with DNA-OFF (Takara Cat# 9036) and RNase Zap (Life Technologies Cat# AM9780), and great care was taken to maintain an RNase-free environment during sample collection. The patch-seq pipettes were pulled using a micropipette puller (Sutter Instrument, MODEL P-97), and the resistance was 2–4 MO. Cells were absorbed into patch-seq pipettes and the tips of the pipettes were broken and left in the RNase-free PCR tube containing 4 µl of lysis buffer consisting of 0.1% Triton X-100, 5 mM (each) dNTPs, 2.5mM Oligo-dT30, 1 U/ml RNase inhibitor, and ERCC RNA Spike-In Mix (Life Technologies Cat# 4456740). The samples were transferred to -80 °C until further processing (Cadwell et al., 2016; Xie et al., 2021).