Journal: Frontiers in Aging Neuroscience
Article Title: Detrimental effects of soluble α-synuclein oligomers at excitatory glutamatergic synapses
doi: 10.3389/fnagi.2023.1152065
Figure Lengend Snippet: Molecular effects induced by in vivo striatal injection of αSyn soluble oligomers in mice at the corticostriatal synapse. (A) Post-synaptic levels of AMPAR (GluA1, GluA2, and GluA3) and NMDAR (GluN2A, GluN2B, and GluN2D) subunits and scaffolding proteins (Rph3A and PSD95) were evaluated by Western blot in striatal TIF of sOligo- and PBS-injected mice 42 dpi. Protein levels normalized on tubulin were reported as OD% of PBS-mice. n = 5–7 mice. Post-synaptic levels of (B) AMPAR (GluA1, GluA2, and GluA3) subunits, (C) NMDAR (GluN2A, GluN2B, and GluN2D) subunits and (E) scaffolding proteins (Rph3A and PSD95) were evaluated by Western blot in striatal TIF of sOligo- and PBS-injected mice 84 dpi. Protein levels normalized on tubulin were reported as OD% of PBS-mice. n = 5–7 mice. (D) Expression of the dopaminergic marker Tyrosine hydroxylase was evaluated by Western blot in striatal homogenates of sOligo- and PBS-injected mice 84 dpi. Protein levels normalized on GAPDH were reported as OD% of PBS-mice. n = 7 mice. (F) Representative confocal images and quantification of spine morphology analyses (spine density, spine length and spine width) of SPNs of sOligo- and PBS-injected mice 84 dpi. Scale bar: 3 μm. n = 19–22 neurons from 3 mice. Data are represented as mean ± SEM. * P < 0.05 (Student’s t -test; data with non-normal distribution were tested with Mann–Whitney test).
Article Snippet: The primary antibodies used were: mouse anti-tubulin (1:30,000, #T9026, Sigma), rabbit anti-GAPDH (1:5,000, #sc-25778, Santa Cruz), rabbit anti-GluN2A (1:1,000, #M264, Sigma), rabbit anti-GluN2B (1:1,000, #718600, Invitrogen), mouse anti-GluN2D (1:1,000, #MAB5578, Millipore), rabbit anti-GluA1 (1:1,000, #13185, Cell Signaling), mouse anti-GluA2 (1:1,000, #75–002, Neuromab), mouse anti-GluA3 (1:1,000, #MAB5416, Millipore), polyclonal anti-Rph3A (1:2,000, Protein Tech, #11396-1-AP), mouse anti-PSD-95 (1:1,000, #K28/43, Neuromab), rabbit anti-tyrosine hydroxylase (1:10,000, #AB152, Millipore), rabbit anti-phospho-extracellular signal-regulated kinase (ERK) 44/42 (1:1,000, Cell Signaling, #9101), rabbit anti-ERK 44/42 (1:1,000, #9102, Cell Signaling), rabbit anti-phospho-cAMP responsive element binding protein (CREB) (1:1,000, #9198, Cell Signaling), and rabbit anti-CREB (1:1,000, #9197, Cell Signaling).
Techniques: In Vivo, Injection, Scaffolding, Western Blot, Expressing, Marker, MANN-WHITNEY
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