Fused silica capillary tubing

Splitless, nano-flow liquid chromatography separation was performed using a Waters nanoACQUITY (Waters Corp., Milford, MA) UPLC system configured for partial loop injection coupled online to the Q-Exactive mass spectrometer. Fused silica capillary tubing (75 μm I.D.; Polymicro Technologies) was pulled to a tip of ~ 5 μm at one end and packed with 15 cm of Jupiter Proteo 4 μm C12 beads (Phenomenex, Torrance, CA). A Kasil fritted trap was prepared using a 100-μm I.D. capillary tube packed with 2 cm of the identical C12 packing material, and 2 μL of sample was trapped at a flow rate of 1 μL/min for 8 min prior to going in-line with the packed tip. Peptides were separated over a 60-min linear reversed phase gradient ranging from 2 to 32% Buffer B (0.1% w/v formic acid in acetonitrile) mixed with Buffer A (0.1% w/v formic acid in water) with a flow rate of 300 nL/min. Quality control runs analyzed a six bovine protein digest (Bruker-Michrom, Auburn, CA) using a 30-min linear gradient.

FSCV was performed using carbon fiber microelectrodes encased in fused-silica capillary tubing (Polymicro Technologies) (Clark et al., 2010 (link)). The carbon fiber microelectrode was positioned in the dorsomedial striatum at stereotaxic coordinates: A-P = 1.2 mm, M-L = 1.2 mm, D-V = 3.5 below the dura and a waveform was applied at a frequency of 60 Hz. After 40 min, the waveform frequency was reduced to 10 Hz for 10 min or until baseline recordings stabilized. Next, the carbon fiber microelectrode was lowered into the nucleus accumbens in 0.05 mm increments until maximum dopamine release was observed (D-V coordinates ranged from 3.60–3.98 below bregma, average 3.80). An Ag/AgCl reference electrode was placed in the contralateral hemisphere. The stimulation electrode (Plastics One) was placed above the PPTg at stereotaxic coordinates: A-P: −4.6 mm, M-L: 0.8 mm and lowered in 0.1 mm increments from 2.5 mm below the dura until optimal dopamine release was observed after 1 s stimulations of 60 Hz (average D-V coordinate: 2.7 mm below the dura). For the MFB stimulations, the stereotaxic coordinates were A-P: 2.4 mm, M-L: 1.1 mm, DV: 0.1 increments from 4 mm.