All experiments were carried out in accordance with the National Institutes of Health guidelines for the care and use of laboratory animals and were approved by the Uniformed Services University Institutional Animal Care and Use Committee. All efforts were made to minimize animal suffering and to reduce the number of animals used.

Morphine treatment. Sprague-Dawley male rats (P14–P21) received either one intraperitoneal injection of morphine sulfate (10 mg/kg) dissolved in 0.9% saline or injection of comparable volumes of saline 24 h prior to death for electrophysiological recordings or immunohistochemical studies. Only one cell per animal (saline or morphine) was recorded; therefore all reported n values represent the number of animals recorded.

Immunofluorescence and image analysis. Morphine- and saline-treated rats were anesthetized with an intraperitoneal injection containing ketamine (85 mg/kg) and xylazine (10 mg/kg) and perfused through the aorta with heparinized 1× phosphate-buffered saline (PBS) followed by 4% paraformaldehyde (PFA; USB, Cleveland, OH). The brains were dissected and placed in 4% PFA for 24 h and then cryoprotected by submersion in 20% sucrose for 3 days, frozen on dry ice, and stored at −70°C until sectioned. Sections of the VTA were cut with a cryostat (Leica CM1900) and mounted on slides. Serial coronal sections (20 μm) of the midbrain containing the VTA (from −4.92 to −6.72 mm caudal to bregma; Paxinos and Watson 2007) were fixed in 4% PFA for 5 min, washed in 1× PBS, and then blocked in 10% normal horse serum (NHS) containing 0.3% Triton X-100 in 1× PBS for 1 h. Sections were incubated in rabbit anti-tyrosine hydroxylase (TH) (1:1,000; Calbiochem, San Diego, CA) and mouse anti-HDAC2 (1:1,000; Abcam, Cambridge, MA) in carrier solution (5% NHS in 0.1% Triton X-100 in 1× PBS) overnight at room temperature. After rinsing in 1× PBS, sections were incubated for 2 h in Alexa Fluor 488-labeled goat anti-rabbit IgG and Alexa Fluor 568-labeled goat anti-mouse IgG (both diluted 1:200). Finally, sections were rinsed in 1× PBS, dried, and coverslipped with ProLong mounting medium containing DAPI to permit visualization of nuclei. Background staining was assessed by omission of primary antibody in the immunolabeling procedure (negative control). VTA tissue sections of rats with previously established presence of TH/HDAC2-immunoreactive neurons were processed as positive control tissue. Images in Fig. 1 were captured with a Zeiss Confocal Inverted Microscope System (Carl Zeiss) ×40/1.4 NA oil immersion objective. At three AP locations (−5.4, −5.7, and −6.0 relative to bregma) six TH-positive neurons were identified within the VTA. From each TH-positive neuron two HDAC2 density readings (3 μm × 3 μm) were taken from the somatic region (clearly labeled with TH) and the nuclear region (clearly labeled with DAPI). Three background density readings were taken from an area clearly not labeled with HDAC2. All density readings were normalized to background.

Slice preparation for electrophysiology and Western blot. Saline- or morphine-treated rats were anesthetized with isoflurane and immediately decapitated. The brains were quickly dissected and placed into ice-cold artificial cerebrospinal fluid (ACSF) containing (in mM) 126 NaCl, 21.4 NaHCO 3 , 2.5 KCl, 1.2 NaH 2 PO 4 , 2.4 CaCl 2 , 1.00 MgSO 4 , 11.1 glucose, and 0.4 ascorbic acid, saturated with 95% O 2 -5% CO 2 . Horizontal midbrain slices containing the VTA were cut at 250 μm (for electrophysiology) and incubated in ACSF at 34°C for at least 1 h. Slices were then transferred to a recording chamber and perfused with ascorbic acid-free ACSF at 28°C. Midbrain slices were cut at 400 μm for Western blot experiments. For HDACi treatment, slices were incubated in the presence of CI-994 dissolved in DMSO and diluted to the final concentration in ACSF. The final concentration of CI-994 (20 μM) was selected for brain slice incubation because it is approximately the concentration measured in the brain after a systemic, intraperitoneal administration of 30 mg/kg CI-994 (Graff et al. 2014). Controls were incubated in the same dilution of DMSO (1:1,000), which did not affect synaptic transmission. These slices were allowed to incubate for 2–4 h before transfer to the recording chamber or dissection of the VTA for Western blots. In some of our control recordings, slices were only incubated in DMSO-free ACSF and the data for both DMSO and DMSO-free groups were pooled together as controls in the graphs. For chemically induced GABAergic LTD experiments, drugs were bath applied after 10 min of stable baseline recordings of evoked inhibitory postsynaptic currents (IPSCs) and remained in the bath throughout the experiment. The CB1 receptor agonist R-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)-pyrrolo-[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone (WIN 55212-2) was dissolved in DMSO (1:1,000 dilution) and diluted to 2 μM in ACSF. Stock solutions for the group I metabotropic glutamate receptor (mGluR) agonist dihydroxyphenylglycine (DHPG, 50 μM) was made by dissolving in deionized water. All drugs were purchased from Sigma, Tocris, or Calbiochem.

Western blot. The VTA was dissected bilaterally from horizontal midbrain slices (400 μM) of morphine- or saline-treated rats. For HDACi treatment, slices were incubated in ACSF in the presence of CI-994 dissolved in DMSO or the same dilution of vehicle (DMSO) for 2–4 h before being snap frozen in liquid nitrogen and stored at −80°C. Tissues were thawed, washed in ice-cold PBS, and lysed in RIPA buffer containing protease inhibitors (Sigma). Samples were then sonicated, incubated on ice for 30 min, and centrifuged at 10,000g for 20 min at 4°C. Protein concentration in the supernatant was determined with a Pierce BCA Protein Assay Kit (Life Technologies). Equal amounts of protein (20 μg) were combined with loading buffer, boiled for 5 min, and loaded onto 4–20% precast polyacrylamide gel (Bio-Rad Laboratories). Separated proteins were transferred onto nitrocellulose membranes, blocked with casein-based blocking reagent (I-Block, Life Technologies) for 60 min at room temperature, and then incubated overnight at 4°C with rabbit anti-acetyl-histone H3K9 (Ac-H3K9, 1:1,000; Cell Signaling, no. 9649), or mouse anti-β-actin (1:10,000; Abcam, no. ab6276). After incubation, the membranes were washed with PBS-T and exposed to the appropriate horseradish peroxidase-linked secondary antibody (Cell Signaling). Blots were developed with Clarity Western ECL Substrate (Bio-Rad Laboratories) and detected with a Fuji LAS-3000 image acquisition system (Fuji, Stamford, CT).

Electrophysiology. Whole cell recordings were performed on midbrain slices with a patch amplifier (Multiclamp 700B) under infrared-differential interference contrast microscopy. Data acquisition and analysis were carried out with Digidata 1440A, pCLAMP 10 (Molecular Devices, Union City, CA), and Mini Analysis 6.0.3 (Synaptosoft). Signals were filtered at 3 kHz and digitized at 10 kHz. The recording ACSF was the same as the cutting solution except that it was ascorbic acid free. The appearance of an I h current (≥50 pA) in response to stepping cells from −50 mV to −100 mV was used to identify VTA DA neurons as previously described (Dacher et al. 2013). Paired AMPA receptor (AMPAR)-mediated excitatory postsynaptic currents (EPSCs) were stimulated with a bipolar stainless steel stimulating electrode placed 200–500 mm rostral to the recording site in the VTA at 0.1 Hz (100 μs). Combined EPSCs were evoked and recorded in ACSF perfusion containing picrotoxin (100 μM) while the cell was voltage clamped at +40 mV. Patch pipettes were filled with (in mM) 117 Cs-gluconate, 2.8 NaCl, 5 MgCl 2 , 2 ATP-Na+, 0.3 GTP-Na+, 0.6 EGTA, and 20 mM HEPES, with intracellular spermine (10 μM) (pH adjusted to 7.28 with CsOH, osmolarity adjusted to 275–280 mosM). d-APV (50 μM) was added to the perfusion to block NMDA receptor (NMDAR)-mediated currents and isolate AMPAR-mediated currents. Subtraction of AMPAR-mediated currents from combined excitatory currents allowed us to measure AMPA-to-NMDA ratios. Pharmacologically isolated AMPAR-mediated evoked EPSCs were also recorded to measure AMPAR rectification. These recordings were performed in the presence of picrotoxin (100 μM), d-APV (50 μM), and intracellular spermine (10 μM) included in Cs-gluconate-based internal solution. AMPAR EPSCs were recorded at several holding potentials ranging from −65 to +40 mV. AMPAR rectification was determined by dividing peak AMPAR EPSC amplitudes recorded at −65 mV by those recorded at +40 mV. Paired GABA A receptor (GABA A R)-mediated IPSCs were similarly evoked (at 0.1 Hz, duration 100 μs, 50 ms interstimulation interval), isolated, and recorded in ACSF containing 6,7-dinitroquinoxaline-2,3-dione (DNQX; 10 μM) and strychnine (1 μM). The internal solution was similar to miniature IPSC (mIPSC) recordings as stated below. Cells were voltage clamped at −70 mV for these recordings. Whole cell recordings of GABA A R-mediated mIPSCs were performed in ACSF perfused with DNQX (10 μM), strychnine (1 μM), and tetrodotoxin (TTX, 1 μM). The patch pipettes (3–6 MΩ) were filled with (in mM) 125 KCl, 2.8 NaCl, 2 MgCl 2 , 2 ATP-Na+, 0.3 GTP-Na+, 0.6 EGTA, and 10 HEPES (pH adjusted to 7.28 with KOH, osmolarity adjusted to 275–280 mosM). Similarly, AMPAR-mediated miniature EPSCs (mEPSCs) were isolated in ACSF perfused with picrotoxin (100 μM), d-APV (50 μM), and TTX (1 μM). Patch pipettes for mEPSC recordings were filled with (in mM) 117 Cs-gluconate, 2.8 NaCl, 5 MgCl 2 , 2 ATP-Na+, 0.3 GTP-Na+, 0.6 EGTA, and 20 HEPES. VTA neurons were voltage-clamped at −70 mV and recorded over 10 sweeps, each lasting 50 s. The cell input resistance and series resistance were monitored through all experiments, and if these values changed by >10% data were not included.