Surgeries for viral delivery and optic fiber and electrode implantation

Before the surgical procedures, the mice were anesthetized with 1% pentobarbital sodium (dissolved in saline; injection dose, 100 mg/kg, i.p.), the back of their necks were shaved for the delivery of pruritogens, and a magnet (1 mm in diameter, 3 mm in length) was implanted into the right hindpaw to record scratching behavior. Then, the mice were placed on a stereotaxic frame (Stoelting), and surgery was performed as previously described (Cetin et al., 2006). To specifically express the viral constructs in VTA DA neurons, recombinant adeno-associated viruses (AAVs) were injected into the VTA [relative to bregma: anteroposterior (AP), −3.35 mm; mediolateral (ML), 0.45 mm; dorsoventral (DV), −4.25 mm] of DAT-Cre mice. The virus was injected with a glass pipette (tip diameter, 20∼30 μm) at a rate of 0.05–0.1 μl/min (0.25–0.4 μl/site). The virus was infused unilaterally for the fiber photometry and identification of DA neurons in the optrode recording. For the slice electrophysiological recording and optogenetic inhibition of DA neurons during the behavioral test, the virus was injected into the VTA bilaterally.

For the photometry recording, Cre-dependent AAV vectors carrying fluorescent calcium indicator GCaMP6s [AAV-hSyn-DIO-GCaMP6s, 4.5 × 10¹² genome copies (gc)/ml] or control (AAV-Ef1α-DIO-EYFP, 5.54 × 10¹² gc/ml) were serotyped with AAV5 or AAV9 coat proteins and packaged by Shanghai Taitool Bioscience Co. Ltd. To identify the DA neurons in vivo, the transgene [AAV-Ef1α-DIO-ChR2 (H134R)-mCherry, 5.2 × 10¹² gc/ml; packaged by Vector Core at the University of North Carolina at Chapel Hill (UNC Vector Core), Chapel Hill, NC] was serotyped with AAV5 coat proteins. The transgenes used for the optogenetic inhibition (AAV-CAG-DIO-GtACR1-EGFP, 4.5 × 10¹² gc/ml; packaged by Shanghai Taitool Bioscience Co. Ltd) or control (AAV-Ef1α-DIO-EYFP, 4.4 × 10¹² gc/ml; packaged by UNC Vector Core) were serotyped with AAV5 coat proteins.

Following the AAV infusion, an optic fiber [diameter, 200 μm; numerical aperture (NA), 0.37] was immediately implanted into the VTA (AP, −3.35 mm; ML, 0.4 mm; DV, −4.20 mm), nucleus accumbens (NAc) lateral shell (LaSh; AP, 0.98 mm; ML, 1.75 mm; DV, −4.6 mm) or NAc medial shell (MeSh; AP, 1.54 mm; ML, 0.5 mm; DV, −4.5 mm) for the fiber photometry recording. To optogenetically inhibit VTA DA neurons during scratching, the optic fibers were implanted bilaterally above the VTA at the angle of 10 degrees (AP, −3.4 mm; ML, 1.25 mm; DV, −4.1 mm). A thin layer of tissue gel (Kwik-Cast Sealant) was added to the surgical hole to prevent contact between the dental cement and brain tissue. Then, gel (Vetbond Adhesive, 3M) was applied to the skull surface, and a thin layer of dental cement was immediately applied to the skull surface. A thick layer of dental cement was applied to build a head cap and cover the skull surface.

The electrode was homemade by using a screw-driven Microdrive, as previously described (Liu et al., 2014). Briefly, 12∼16 Ni-Chrome microwires (diameter, 33 μm/wire; California Fine Wire Co) were glued to a polyimide guide tube (inner diameter, 250 μm; outer diameter, 355 μm). To identify the DA neurons by light stimulation, an extra optic fiber was added (diameter, 200 μm; NA, 0.37) to the guide tube, and the end of the optic fiber was 200∼400 μm shorter than the ends of the microwires. The microwire array was inserted into the microdrive chamber and glued to a movable screw nut. The wire tips on one side were soldered to corresponding pins on a printed circuit board (PCB) connector. The wire tips on the other side were electroplated to a final impedance of ∼1.5 MΩ after the assembly of the electrode. The electrode with the optic fiber was referred to as an optrode. The optrode was implanted into the VTA (AP, −3.35 mm; ML, 0.4 mm; DV, −4.0 mm) of DAT-Cre animals, which were injected with AAV expressing channelrhodopsin 2 (ChR2) at least 3 weeks before the implantation. The ground and reference wires were separately soldered to the corresponding pins on the PCB on one side and to two skull-mounted cranial screws on the other side. Then, dental cement was carefully applied as described for the optic fiber implantation, except that the cement did not contact the microwires, allowing for the wires to be lowered after the surgery.