Animals
Transgenic male and female mice (8 to 12 weeks of age) were used for behavioural and biochemical experiments. Trpv1-Cre mice (JAX: 017769), Aldh1l1-creERT2 mice (JAX: 031008), Advillin-Cre mice (JAX: 032536), Cx3cr1-creERT2 mice (JAX: 020940), Ai9 mice (JAX: 007909), GCaMP6f mice (JAX: 024105), MitoTag mice (JAX: 032675), and BKS.Cg-Dock7m +/+ Leprdb/J mice (JAX: 000642) were purchased from Jackson Laboratory. Male and female CD1 mice were purchased from Charles River Laboratories. Generation of Myo10 knockout mice (tm1d) and mouse genotyping were described previously31. Mice were maintained at the Duke animal facility. db/db mice aged 12–16 weeks were used. All mouse experiments were approved by the Duke University Institutional Animal Care and Use Committee (IACUC). Mice were housed in an AAALAC-accredited animal facility under a 12 h:12 h light:dark cycle, with food and water provided ad libitum. All animals were housed at 22 ± 1 °C and 30–70 % humidity.
Human DRG tissues
A total of 16 human DRG samples were obtained from donors with and without diabetes (Supplementary Table 1) through the NDRI, under exemption approval from the Duke Institutional Review Board (IRB, Pro00051508). Diabetes was identified on the basis of medical history provided in the NDRI reports.
Reagents
PTX (T7402), Tam (T5648), oligomycin (O4876), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (C2920), rotenone (R8875), antimycin A (A8674), myxothiazol (T5580), CBX (C4790), CytoB (C2743) and Y-27632 (688001) were obtained from Sigma. Pitstop2 (ab120687) was obtained from Abcam. CPZ was obtained from MCE (HY-15640). TTX (1078) was obtained from Tocris. siMyo10 (siRNA ID: 157002), siMYO10 (siRNA ID: 118391) and negative control siRNA (4459405) were obtained from Ambion Life Technology.
Primary cultures from mouse DRG
Mouse DRG neuron culture
Mouse primary DRG neuron cultures were prepared as described44. In brief, DRG were digested with collagenase (0.2 mg ml−1, Sigma, 10103578001) and Dispase-II (3 mg ml−1, Sigma, D4693) for 60 min at 37 °C. The cells were then mechanically dissociated with a pipette, filtered through a 70-µm nylon mesh, and centrifuged at 300g for 5 min. The cells were plated on glass coverslips coated with 0.1 mg ml−1 poly-d-lysine (Thermo, A3890401) and cultured in Neurobasal medium supplemented with 10% FBS, 2% B27 supplement, and 1% penicillin-streptomycin (Neurobasal-supplemented medium) at 37 °C with 5% CO 2 .
Mouse DRG SGC culture
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