In vivo extracellular dopamine neuron recordings

Rats were anesthetized with 8% chloral hydrate (400 mg/kg, i.p.) and placed in a stereotaxic apparatus. Chloral hydrate was used for all dopamine recordings to avoid significantly depressing dopamine neuron activity³⁷. Supplemental anesthesia was administered to maintain suppression of limb compression withdrawal reflex. Core body temperature of 37 °C was sustained using a thermostatically controlled heating pad (PhysioSuite, Kent Scientific Coorporation, Torrington, CT, USA). Extracellular glass microelectrodes (impedance ~6–10 MΩ) were lowered into the VTA (posterior 5.3 to 5.7, lateral 0.6 to 1.0 from bregma, and −6.5 to −9.0 mm ventral of the brain surface) using a hydraulic micro-positioner (Model 640, Kopf Instruments). Spontaneously active dopamine neurons were recorded using previously established electrophysiological criteria^38,39. Open filter settings (low-frequency cutoff: 30 Hz; high-frequency cutoff: 30 kHz) were used to identify and record dopamine neurons in the VTA. Various regions of the VTA were sampled by making multiple 6–9 vertical passes, separated by 200 µm, in a predetermined pattern. Three parameters of dopamine activity were measured and analyzed: the number of dopamine neurons firing spontaneously (population activity)¹¹, basal firing rate, and proportion of action potentials occurring in bursts. Electrophysiological analysis of dopamine neuron activity was performed using commercially available computer software (LabChart version 8; ADInstruments, Colorado Springs, CO, USA) and analyzed with Prism software (GraphPad Software, San Diego, CA, USA). All orexin antagonists were administered thirty minutes prior to extracellular recordings and single-cell extracellular recordings lasted no longer than two hours post injection.