Isolation of Functional Neuronal Terminals

Neurons with functional terminals were obtained by mechanical dissociation as described previously (Akaike and Moorhouse, 2003 (link); Yang et al., 2011 (link); Steffensen et al., 2018 (link)). In brief, one midbrain slice was transferred to a 35-mm culture dish (Falcon, Rutherford, NJ) filled with a standard external solution. The region of the slice containing the VTA was directly visualized through an inverted microscope (Nikon, Tokyo, Japan). A fire-polished glass pipette with a 50-μm diameter tip was mounted to a custom-constructed mechanoelectrical device for cellular dissociation. Using a manipulator, the pipette was then positioned just below the liquid-tissue interface of the VTA region. Neurons close to the surface of the slice were dissociated by horizontal vibration at a frequency of 15–20 Hz with a range from 0.1 to 0.3 mm for 2 min. The slice was then removed from the solution containing the dissociated cells. Within 20 min, the isolated neurons adhered to the bottom of the dish and were ready for electrophysiological recordings. These mechanically dissociated neurons differ from neurons dissociated using enzymatic techniques, with the latter losing most, if not all, presynaptic terminals during the dissociation process, the former can, in contrast, retain functional nerve terminals following this process.