Brain Slice Preparation
All procedures were performed according to Vanderbilt University Institutional Animal Care and Use Committee-approved procedures. Male C57Bl/6J mice (6–8 weeks old, Jackson Laboratories) were decapitated under anesthsia (isoflurane). The brains were quickly removed and placed in ice-cold sucrose-artificial cerebrospinal fluid (ACSF): (in mM) 194 sucrose, 20 NaCl, 4.4 KCl, 2 CaCl2
, 1 MgCl2
, 1.2 NaH2
, 10.0 glucose, and 26.0 NaHCO3
saturated with 95% O2
. Slices 300 µm in thickness were prepared using a Tissue Slicer (Leica). Rostral slices containing anterior portions of BNST (bregma 0.26–0.02 mm) (Franklin and Paxinos, 1997
) were identified using the internal capsule, anterior commissure, fornix, and stria terminalis as landmarks. Slices were then stored in a heated (approximately 28°C), oxygenated (95% O2
) holding chamber containing ‘normal’ ACSF (ACSF: (in mM) 124 NaCl, 4.4 KCl, 2 CaCl2
, 1.2 MgSO4
, 1 NaH2
, 10.0 glucose, and 26.0 NaHCO3) or transferred to a submerged recording chamber where they were perfused with heated (26°C, unless otherwise noted), oxygenated ACSF at a rate of about 2 ml/min. Slices were allowed to equilibrate in normal ACSF for 1 h before experiments began.
Whole-Cell Voltage-Clamp Recordings
Slices were placed in a submerged chamber (Warner Instruments) and neurons of the vBNST were directly visualized with infrared video microscopy (Olympus). Recording electrodes (3–6 MΩ) were pulled on a Flaming–Brown Micropipette Puller (Sutter Instruments) using thin-walled borosilicate glass capillaries. NMDA-EPSCs were evoked by local fiber stimulation with bipolar nichrome electrodes. Stimulating electrodes were placed in the vBNST, 100–500 µm medial from the recorded neuron, and electrical stimuli (5–40 V with a 100–150 µs duration) were applied at 0.1 Hz while recording NMDA-EPSCs and 0.2 Hz while recording AMPA-EPSCs. NMDA-EPSCs were recorded from a holding potential of −70 mV and pharmacologically isolated by adding 25 µM picrotoxin and either 20 µM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) or 10 µM NBQX in a zero Mg2+ ACSF (in mM: 124 NaCl, 4.4 KCl, 3.7 CaCl2, 1 NaH2PO4, 10 glucose, and 26 NaHCO3, pH 7.2–7.4; 290–310 mOsmol). Recording electrodes were filled with (in mM) K+-gluconate (135), NaCl (5), HEPES (10), EGTA (0.6), ATP (4), GTP (0.4), and biocytin (0.1%) pH 7.2, 290–295 mOsmol. AMPA-EPSCs were isolated by adding 25 µM picrotoxin and recording at a holding potential of −70 mV in normal ACSF. At this holding potential in the presence of picrotoxin in normal ACSF, application of either CNQX or NBQX completely abolished the postsynaptic current. Signals were acquired via a Multiclamp 700B amplifier (Axon Instruments), digitized and analyzed via pClamp 9.2 software (Axon Instruments).
For experiments in which NMDA was exogenously applied, the following protocol was used. A 1.0 mM NMDA solution was prepared in fresh ACSF and loaded into a thin-walled borosilicate glass capillary pulled using a Flaming–Brown Micropipette Puller (Sutter Instruments). The capillary tube was then mounted in a pipette holder connected to a Picospritzer II (Parker) delivery system. This pipette was then placed within approximately 10 µm of a cell to be recorded using video microscopy guidance. At this point, an independent patch electrode was positioned next to the cell and a whole-cell voltage-clamp configuration was obtained. Following break-in, the cell was allowed to rest for 5 min, and then the NMDA was applied using pressure ejection with pressure settings of 30–40 p.s.i. and pulse length of 3–15 ms. During the course of the experiment, NMDA was applied every 10 s to allow complete return to baseline holding current. The ACSF in these experiments was identical to those used for evoked NMDA-EPSC experiments.
Input resistance, holding current, and series resistance were all monitored continuously throughout the duration of experiments. Experiments in which changes in series resistance were greater than 20% were not included in the data analysis. Experiments were analyzed by measuring the parameter of interest; peak amplitude or area of the synaptic response which was normalized to the baseline period. The baseline period is defined as the 5 min period immediately preceding application of the drug. NMDA-EPSC decay was fitted with two exponentials using Clampfit 9.2 for averages of several traces from baseline and ‘drug’ values as done previously (Fu et al, 2005
). To allow for direct comparison of decay times between experimental conditions, the two decay time components, τ1
, were combined into a weighted time constant, τw
, using the equation: τw
) + (τ2a2
), where a1
are the relative amplitudes of the two exponential components.
Statistical analyses were performed using Microsoft Excel, Graphpad Prism and Microcal Origin. Specifically, when determining if a compound had a significant effect (for example, 50 mM ethanol), a Student’s paired t-test was used, comparing the baseline value to the experimental value. For the ethanol experiments, the baseline value was the average value obtained from the recordings obtained in the 2 min immediately preceding application of ethanol, and the experimental value was determined by averaging the recordings obtained 4–6 min following removal of ethanol. In order to compare the effects of different concentrations of ethanol to one another, a one-way ANOVA was used, followed by a Tukey posttest to determine the significance of specific comparisons. When comparing the effects of multiple treatments on ethanol’s effect, a one-way ANOVA followed by Dunnet’s posttest was used to determine the significance of the differences between the groups. All values given for drug effects throughout the paper are presented as average±SEM. For results given in figures, significance is noted in the figure legend. For results not included in figures, significance is noted in the text.
Picrotoxin, CNQX, NMDA, Ro 25–6981, Ifenprodil, and dl-2-amino-5-phosphonopentanoic acid (dl-APV) were purchased from Sigma-Aldrich (St Louis, MO). NBQX was purchased from Ascent (England). Ethanol (95%) was purchased from Aaper Alcohol and Chemical (Shelbyville, KY). DMSO (0.02%) was used as a vehicle for picrotoxin. For experiments where the acute effects of a drug were examined on NMDA-EPSCs, only one cell per slice was recorded.
NR2A knockout (KO) breeder animals extensively backcrossed onto C57Bl6/j were obtained from Dr David Lovinger and were generated as previously reported (Kiyama et al, 1998
). Animals for this study were generated by the breeding of homozygous KO males and females.
Retrograde Labeling of BNST Neurons
C57Bl6/j male mice, 6–8 weeks old, were anesthetized with tribromoethanol (2.5% solution in saline, 0.19 ml/10 g, i.p.). Ophthalmic ointment (Puralube Vet) was applied to the eyes to prevent corneal damage. The fur on the top of the head was shaved off and the exposed skin was scrubbed sequentially with 70% ethanol, 10% povidone iodine, and 70% ethanol. The animal was mounted in a stereotaxic apparatus (Kopf Instruments) and placed on a warming pad kept at 39°C. The skin over the skull was cut and deflected enough to expose the bregma and lambda sutures. Local anesthetic cream containing benzocaine was applied to the wound. The head was leveled with respect to the intersections of bregma and lambda with the midline suture. Holes were drilled in the skull directly above the VTA according to the atlas of Franklin and Paxinos (3.3 mm posterior to bregma and 0.5 mm lateral to the midline). The 32-gauge needle of a 0.5 µl Hamilton zero dead volume syringe was heat sterilized immediately before backfilling with 30 nl of an undiluted suspension of fluorescent microspheres (Molecular Probes, 0.04 µm diameter, 488/560 µm excitation/emission). The dura was cut and the syringe needle lowered into the VTA (4.7 mm below surface of the skull). Tracer microspheres were slowly injected over 3–4 min and the needle left in place for an additional 10 min. Most animals received bilateral tracer injections. After withdrawing the needle, the wound was closed with sutures and the animal was hydrated with a saline injection (1 ml, s.c.) followed by freshly dissolved ampicillin (0.45 mg/0.1 ml per animal, s.c.). The animal was kept warm during recovery from anesthesia and was given an analgesic when ambulatory (buprenor-phen, 0.1 mg/kg, s.c.). Postsurgery, animals were housed singly and weighed and injected with analgesic twice per day for up to 6 days or until the animal gained weight for 2 consecutive days. Animals that lost more than 20% of their body weight or showed signs of uncontrolled pain, stress, or dehydration were euthanized. Healthy animals were used for electrophysiological experiments 5–14 days after surgery.
The hindbrain of all tracer-injected mice used for whole-cell recordings were immersion fixed in 4% paraformaldehyde/PBS, cryoprotected with 20% sucrose, and sectioned on a cryostat. Sections were mounted on slides and viewed with a fluorescence microscope. All animals with retrograde-labeled BNST neurons were confirmed to have tracer injection sites in the VTA.