Fig 2: Inhibition of calpain-2 rescues the hTau-induced inhibition of a4 nAChR currents in hippocampal neurons.(a) The cultured hippocampal neurons (7 DIV) were infected by lenti-mCherry-hTau virus or the vector. After 3 days, the neurons were treated with 10 µM mCalp-I for 48 h, and then the a4 nAChR agonist RJR2403 (1 mM, 50 ms) was puffed to elicit a4 current recorded by whole-cell patch clamp. (b) The amplitude of currents were analyzed (two-way ANOVA, factor infection: F1,24 = 6.492, p = 0.0177; factor drug: F1,24 = 4.380, p = 0.0471; infection × drug: F1,24 = 6.097, p = 0.0210; Tukey’s post hoc test). At least 7 neurons from each group were recorded and the data were expressed as mean ± SEM. **p < 0.01 vs Vec+Vehicle; #p < 0.05, vs hTau+Vehicle.

Fig 3: Effect of different frequencies of EAS of ST36-GB34 on expression of a4 nAChR and ß2 nAChR mRNA and proteins in the hippocampus in neuropathic pain rats (mean ± SD, n = 6 in each group); a a4 nAChR mRNA, b a4 nAChR protein; c ß2 nAChR mRNA, d ß2 nAChR protein; *P < 0.05, vs the normal group; #P < 0.05, vs the model group; \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$^\bullet$$\end{document}·P < 0.05, vs the 2/15 Hz EAS group

Fig 4: Overexpression of hTau reduces protein level of a4 nAChR with an increased cleavage of the receptor both in vitro and in vivo.(a) Representative images of the cultured hippocampus neurons, the lenti-mCherry-hTau or the vector was infected at 7 DIV and the neurons were cultured for another 5 day (left), or the one-month-old rat brain hippocampus after bilateral ventricular infusion of AAV-GFP-hTau (2 µl each side, 1.5 µl/min speed) at postnatal day 0–1 (P0-1) (right). (b–d) Western blotting data show that overexpression of hTau reduced a4 nAChR level in 12 DIV primary hippocampus neurons (left, from 3 independent cultures; two-sample unpaired t test, t4 = 4.949, p = 0.0078), or in rat hippocampal CA3 extracts (right, from at least 3 rats; two-sample unpaired t test, t4 = 5.862, p = 0.0042). The red dotted lines show the bands of cleaved a4 nAChRs fragment (two-sample unpaired t test, t4 = 6.634, p = 0.0027 in c; t4 = 3.982, p = 0.0164 in d). (e,f) Overexpressing hTau in primary neurons (two-sample unpaired t test, t4 = 0.5740, p = 0.5967) or rat hippocampus (two-sample unpaired t test, t4 = 0.4220, p = 0.6906) did not affect mRNA level of a4 nAChR measured by real-time fluorescent quantitative PCR. Data were expressed as mean ± SEM, *p < 0.05, **p < 0.01 vs control.

Fig 5: Effect of intra-hippocampal injection of M1mAChR selective antagonist (pirenzepine hydrochloride) and a4ß2 nAChR antagonist (Dihydro-beta-erythroidine, DHßE) on pain threshold (a mechanic, g; b thermal, sec) in neuropathic pain rats (mean ± SD, n = 4 in each group); *P < 0.05, vs the normal group; #P < 0.05, vs the model group