Recombinant human bdnf

Stereotaxic surgeries were performed as described previously^{39 (link)}. Mice were anesthetized with a mixture of ketamine (100 mg/kg/10 ml) and xylazine (10 mg/kg/10 ml) (Henry Schein) in sterile saline. HSVs (-Gadd45b miR or -GFP) or BDNF (0.25 µg/side, recombinant human BDNF, R&D Systems) were bilaterally infused into the NAc (AP = 1.5, ML = ±1.5, and DV = −4.4 mm; 10° angle), while an AAV2 vector expressing ChR2, fused with enhanced yellow fluorescent protein (AAV2-EYFP-ChR2, purchased from University of North Carolina Vector Core) or its control (AAV2-EYFP), was infused into the VTA (AP = −3.2, ML = ±1.0, and DV = −4.6 mm; 7° angle). An infusion volume of 0.5 µl was delivered using 5 μL Hamilton syringe (Hamilton Company) over the course of 5 min (at a rate of 0.1 μl/min). Mice were allowed to recover for 4 days following the HSV infusion or for 7 days following the BDNF infusion before going through behavioral assessment. For the optogenetic stimulation of the VTA-NAc pathway, optic fibers were bilaterally implanted into the NAc (AP = 1.5; ML = ±1.3; DV = −3.9; 0° angle), three weeks after AAV2-EYFP-ChR2 or AAV2-EYFP infusion into VTA. Mice were allowed to recover for seven days following the cannulation, and then stimulated in home cages.

ES cell-derived motor neurons were cultured as previously described^{17 (link)}. Primary motor neurons were isolated from spinal cords of E12.5–13.5 mouse embryos^{54 (link)}. Briefly, embryos were sacrificed, the spinal cords dissected and the ventral horns isolated and dissociated by incubation with trypsin, followed by incubation with DNase and centrifugation through a BSA cushion. The resulting cell pellet was resuspended in complete motor neuron medium [Neurobasal (ThermoFisher, Waltam, MA), 2% v/v B27 supplement (ThermoFisher), 2% heat inactivated horse serum, 1% GlutaMAX (ThermoFisher), 25 μM β-mercaptoethanol, 10 mg/ml recombinant rat CNTF (R&D Systems, Minneapolis, MN), 100 pg/ml recombinant rat GDNF (R&D Systems), 1 ng/ml recombinant human BDNF (R&D Systems) and Pen/Strep antibiotics]. Cells were immediately plated on poly-DL-ornithine/laminin coated plates and maintained in culture for 5–8 d (5–8 DIV).

All drugs and reagents were from Sigma unless otherwise indicated. The main reagents were as follows: Neurobasal medium (Gibco), B27 Supplement (Gibco), Sulfo-NHS-LC-Biotin (Thermo Scientific), NeutrAvidin Agarose Resins (Thermo Scientific), recombinant human BDNF (R&D System), and K252a (Tocris).
The antibodies used for western blotting were ASIC1a (1:500, sc-13905, Santa Cruz) and GAPDH (1:2000, KC-5G4, KangChen). The HRP-conjugated secondary antibodies used for western blotting were goat anti-rabbit IgG (1:2000, AP132P, Millipore), rabbit anti-mouse IgG (1:2000, AP160P, Millipore), and rabbit anti-goat IgG (1:2000, AP106P, Millipore). The primary antibodies used for immunostaining were GFP (1:1000, A10262, Invitrogen), HA (1:1000, 901501, BioLegend), DsRed (1:1000, 632496, Clontech), and PSD-95 (1:1000, ab13552, Abcam). The secondary antibodies used for immunostaining were donkey anti-human IgG DyLight 550 (1:1000, SA5-10127, ThermoFisher), donkey anti-human IgG DyLight 488 (1:1000, SA5-10126, ThermoFisher), donkey anti-mouse IgG Alexa 647 (1:1000, 715-605-150, Jackson ImmunoResearch), donkey anti-rabbit IgG Alexa 568 (1:1000, A10042, Invitrogen), and goat anti-chicken IgG Alexa 488 (1:1000, A11039, Invitrogen).

Culture medium was composed of Neurobasal-A (Thermo Fisher, cat. # 10888022), 2% B-27 supplement (Thermo Fisher, cat. # 17504044), 1% N2 supplement (Thermo Fisher, cat. # 17502048), 0.1% gentamicin (Millipore Sigma, cat. # G1397), 200 mM L-glutamine (Thermo Fisher, cat. # 25030081). With or without the addition of 50 ng/mL recombinant human BDNF (R&D Systems, Minneapolis, MN, USA, cat. # 248-BDB) and 5 μM Forskolin (Stemcell Technologies, Cambridge, MA, USA, cat. # 72114).