Three- to four-week-old Sprague Dawley rats were anesthetized and killed. The brain was rapidly dissected, and horizontal slices (230 μm thick) containing the VTA were prepared using a Vibratome. Slices were allowed to recover for at least 45 min in artificial CSF [aCSF; containing the following (in mm): 126 NaCl, 1.6 KCl, 2.4 CaCl2, 1.2 NaH2PO4, 1.2 MgCl2, 18 NaHCO3, and 11 glucose, saturated with 95% O2 and 5% CO2] before being transferred individually to the recording chamber and superfused with continuous flow (2 ml/min) of aCSF at 32°C containing picrotoxin (100 μm) to block GABAA receptor-mediated synaptic currents.
Cells were visualized using an upright microscope with infrared illumination. Whole-cell voltage-clamp recordings were made using an Axopatch 1D amplifier (Molecular Devices) with 3−5 MΩ glass electrodes containing the following (in mm
): 120 CsCH3
, 20 HEPES, 0.4 EGTA, 2.8 NaCl, 5 N(CH2
Cl, 2.5 Mg-ATP, and 0.25 Mg-GTP, pH 7.3. Putative DA cells were identified by the presence of a large hyperpolarization-activated potassium current, Ih
(Lacey et al., 1990
; Johnson and North, 1992
). According to previous studies, Ih
is present in 84% of VTA DA neurons (Sarti et al., 2007
), and there is general agreement that GABAergic neurons do not have Ih
(Margolis et al., 2006
). Although the presence of an Ih
current does not unequivocally identify DA cells, it is likely that the contribution of Ih
-positive tyrosine hydroxylase-negative cells to the observed effects is very small. A bipolar stimulating electrode was placed rostrally at a distance of 100−300 μ
m from the recording electrode. Afferents were stimulated at 0.1 Hz, and the evoked EPSCs were filtered at 2 kHz, digitized at 5−10 kHz, and recorded using Igor Pro software (WaveMetrics).
NMDAR or AMPAR traces were constructed by averaging 15 EPSCs elicited at +40 mV and −70 mV, respectively. NMDAR responses were calculated by subtracting the average response in the presence of 50 μm d
-APV) (AMPAR-mediated only) from that recorded in its absence. For LTP experiments, evoked EPSPs were recorded from neurons current clamped at −70 mV using 3−5 MΩ electrodes filled with a potassium methanesulfonate solution containing the following: 0.95% KOH (v/v), 0.76% methanesulfonic acid (v/v), 0.18% hydrochloric acid (v/v), 20 mm
HEPES, 0.2 mm
EGTA, 2.8 mm
NaCl, 2.5 mg/ml MgATP, and 0.25 mg/ml GTP, pH 7.3. LTP was induced by using a spike-timing-dependent (STD) protocol as previously described (Liu et al., 2005
): 20 bursts of EPSP–spike pairs were delivered, with each burst consisting of five paired stimuli delivered at 10 Hz (interburst interval of 5 s). The postsynaptic spikes were evoked ~5 ms after the onset of EPSPs by injecting depolarizing current pulses (1−2 nA, 3 ms). All recordings with a stable 10 min baseline were included. The magnitude of LTP was computed by averaging 30 consecutive EPSPs 5 min before and 30 min after the end of the induction protocol unless otherwise indicated.
In experiments involving in vivo drug exposure, rats were weighed, injected with cocaine (15 mg/kg, i.p.) or equal amount of 0.9% saline, and then returned to their home cage for 2 or ~23 h, after which VTA slices were prepared. Thus, whole-cell recordings started at 3−5 or ~24 h after cocaine or saline administration. In experiments involving in vitro drug exposure, VTA slices were normally prepared and after recovery were incubated with test substances (10−30 min as indicated). After this treatment, slices were transferred (twice) in a beaker containing regular aCSF for complete washout of the drugs and allowed to recover for 2−4 h. Thus, whole-cell recordings started 3−5 h after the end of drug exposure.
For experiments of rectification, spermine (100 μm
) was added to the intracellular solution. The rectification indices (RIs) were calculated by plotting the magnitude of the average EPSCs at −70, 0, and +40 mV and taking the ratio of the slope of the lines connecting values at 0−40 and at −70 to 0 mV as previously described (Adesnik and Nicoll, 2007
). Neurons were held at each potential for 2 min (12 sweeps) in the presence of d
-APV. Picrotoxin, cocaine, R
-3-benzazepine (SCH-23390), (±)-6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1 H
-3-benzazepine hydrobromide (SKF-81297), anisomycin, cycloheximide, spermine, and 1-naphthyl acetyl spermine (NASPM) were all purchased from Sigma. d
-APV was from Tocris Bioscience, and philantotoxin-74 (PhTx-74) was kindly provided by Dr. P. M. England (University of California, San Francisco, San Francisco, CA). Three- to four-week-old mice lacking functional D5
dopamine receptors were a generous gift from Dr. D. R. Sibley (National Institute of Neurological Disorders and Stroke/National Institutes of Health, Bethesda, MD). D5
knock-out mice (D5
−/−) and wild-type littermates (D5
+/+) were generated as previously described (Hollon et al., 2002