Surgery, In Vivo Single-Unit Recordings, and Drug Administration

Single-unit extracellular recordings were conducted as previously described, with minor modifications (Valenti and Grace, 2010). Mice assigned to the acute drug experimental protocols were transferred to the recording room and anesthetized on the same day of the experiment. CPP mice were anesthetized within 30 minutes from the CPP test (day 7) and randomly assigned to 1 of the protocols described below. Anesthesia was induced with 3% isoflurane, followed by 8% chloral hydrate (400 mg/kg; i.p.), which was also used for maintenance (280 mg/kg; i.p.). Subjects were secured on a stereotactic device (David Kopf Instruments), heart rate was monitored, and core temperature was regulated by a heating pad (Harvard Apparatus). Coordinates of brain regions were calculated using the Mouse Brain Atlas (Franklin and Paxinos, 2008). Extracellular recordings were conducted from the VTA (from Bregma, in mm P: 3; L: 0.4). In some experiments, cannulas were implanted within VP (P: 0; L: 1.6; V: 3.8) or NAcc (A: 1.6; L: 0.7; V: 3.5).

Pipettes were fabricated from borosilicate glass capillaries (1.2-mm-outer-diameter filament glass; Harvard Apparatus) with a vertical puller (PE-22, Narashige) and filled with 2% Chicago Sky Blue in 0.5M NaCl (in situ resistance 10–15 MΩ). Electrodes were lowered into the region of interest using a micromanipulator (S-IVM-1000, Scientifica) and slowly advanced until spontaneously firing cells were encountered (Figure 1A1–2). Neurons were isolated, and baseline activity was recorded for up to 5 minutes (Figure 1A3). Following drug administration, electrodes were advanced further to reach the end of the first track (from brain surface; VTA: 4–5 mm). Thereafter, recordings continued for another 5 to 8 vertical tracks in a predetermined pattern across horizontal and vertical axes (Figure 1A2). Signals were amplified by a headstage pre-amplifier, fed into an ELC-03XS universal amplifier (1000× gain, 100- to 4000-Hz bandpass; NPI Electronics), and digitized at 20 kHz (Power1401 A/D board; CED).

Single-unit extracellular recordings from ventral tegmental area dopamine neurons and post-hoc histological analysis. (A) Schematic illustration of single-unit recordings of VTA DA neuron population activity. (1) Glass microelectrodes were lowered to reach the upper edge of VTA and then slowly advanced while scanning for spontaneously active neurons; a dorsal to ventral electrode trajectory through the VTA is termed track (from surface, −4 to −5mm). (2) Top: after completion of the first trajectory, VTA measurements continued for another 5 to 8 tracks in a predetermined pattern. Bottom: activity pattern of VTA DA neurons. Approximately 50% of all VTA neurons are quiescent (light-grey) under baseline conditions; when active, DA neurons exhibit either irregular firing (black) or burst firing (black surrounded by grey circles). (3) Original trace of a representative recording with 2 burst firing events being indicated. The inset shows an enlarged action potential waveform typical of a DA neuron that is characterized by a notch and a prominent after-hyperpolarization, both indicated by arrows. (B) (1) Shows a coronal section from a mouse brain with the position of the recording electrode marked by iontophoretically injected Chicago Sky Blue (indicated by the arrow); (2) schematic drawings with spots indicating electrode positions in representative experiments; numeric values at the bottom indicate posterior distances of the section from bregma in mm. (C) Examples of cannula placements for drug application into ventral pallidum (1a) and nucleus accumbens (2), respectively; for VP, a cannula position is also shown at higher magnification (1b). Scale bars reflect 500 µm (C1a and 2) and 100 µm (1b), respectively.

For systemic effects, agents were injected i.p.; we employed vehicle and the very same drugs as used during CPP training. To identify sites of actions, drugs or tetrodotoxin were applied to specific brain areas using metallic cannulas (Plastics One; 31 GA). The latter were left in place for at least 10 minutes after infusion. Recordings started 30 minutes after drug administration.

Open filter settings (low pass, 30 Hz; high pass, 16 kHz; DPA-2F filter, NPI Electronics) were used to allow for proper post-hoc classification of neurons. At the end of experiments, mice were killed with a lethal dose of anesthetic; spots of electrodes were marked via electrophoretic ejection of Chicago Sky Blue (Fintronics Inc., USA). Electrode and cannula positions were confirmed by post-hoc histology (Figure 1B–C).