N methyl d aspartate

Hydroethidine (HEt) was purchased from Assay Biotech (Sunnyvale, CA). Newport Green, FluoZin-3 AM, MitoTracker Green, Pluronic F-127, MEM, fetal bovine serum, glutamine, and horse serum were purchased from Life Technologies (Grand Island, NY). N-methyl-D-aspartate (NMDA), 2,2′-dithiodipyridine (DTDP), Rhodamine 123 (Rhod123), and N,N,N,N-tetrakis(2-pyridylmethyl)ethane-1,2-diamine (TPEN) were purchased from Sigma-Aldrich (St. Louis, MO). Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) was purchased from Tocris Bioscience (Ellisville, MO), apocynin obtained from Acros Organics (Morris Plains, NJ), and XF Base Medium (minimal Dulbecco’s Modified Eagle’s Medium) from Agilent Technologies (Santa Clara, CA). All other chemicals and reagents were purchased from common commercial sources.

After 1 week of feeding to facilitate recovery, mice were assigned to lesion or sham groups via matched-pair random assignment. Mice were anesthetized with isoflurane and fixed in a stereotaxic apparatus (1900 Stereotaxic Alignment System, David Kopf Instruments, Tujunga, CA) as previously described (Brigman et al., 2013 (link)). A 33-gauge infusion cannula (Plastics One, Roanoke, VA) attached with polyurethane tubing to a Hamilton syringe (Hamilton, Reno NV) was directed at 6 sites bilaterally targeting the hippocampus (−1.50, −1.80 and −2.25 mm AP, ±1.00, ±1.40 and ±1.75 mm ML, −2.00, −2.00 and −2.25 mm DV to Bregma). 0.2 μL N-methyl-D-aspartate (12.5 mg/mL, Sigma-Aldrich, St. Louis, MO) or saline vehicle was infused over 5 min using a pump (GenieTouch, Kent Scientific, Torrington, CT), with the cannula left in place for an additional 2.5 min to allow full diffusion. On completion of the last infusion, mice were sutured, given .05 mL Diazepam (.5 mg/mL), with an additional .025 ml as needed, to control seizures and returned to their home cages. Mice were given 1 week of recovery before being returned to food restriction. Mice began TUNL testing approximately 2 weeks after completion of surgery.

All experiments were performed in the presence of TTX (2 μM). The K⁺ channel blocker, 4-aminopyridine (4-AP; 5 mM; Sigma-Aldrich, St. Louis, MO) was applied with the ACSF in some experiments. . Agonists or antagonists of iGluRs, L-glutamate (50 μM), N-methyl-D-aspartate (NMDA; 15 μM), α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA; 3 μM), kainic acid (KA; 1 μM), the NMDA receptor antagonist, DL-5-aminophosphonovaleric acid (APV; 50 μM), the non-NMDA receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 μM; all from Sigma-Aldrich), and the AMPA receptor antagonist, GYKI-54266 (50 μM; Tocris Bioscience, Minneapolis, MN, United States) were bath applied for 5–15 min. The TRPV1 agonist, capsaicin (1 μM; Tocris Bioscience) was dissolved in ethanol and diluted in ACSF (final concentration of ethanol <0.01 % by volume) and was bath applied in the presence of glutamate receptor antagonists to identify receptors mediating the iGluR-dependent effects of TRPV1 binging on GABA release in the DMV (Derbenev et al., 2006 (link)).

Neurotoxic lesions of the lateral OFC were generated using injections of N-methyl-D-aspartate (Sigma Aldrich, St Louis, MO) dissolved in 0.9% saline (Sigma Aldrich) at a concentration of 20 mg/mL (Bissonette et al, 2008 (link)). For sham lesions, an equivalent volume of 0.9% saline was injected. Coordinates for bilateral OFC lesions were: anterior-posterior, +2.6 mm from bregma; medial-lateral, ±1.2 mm; dorsal-ventral, 2.2 from dura (Figure 1). At the injection site, 0.35 μL of N-methyl-D-aspartate or saline was injected at a rate of 0.1 μL/min, and the needle was left in place for 3 min. For viral expression studies, animals were injected at a rate of 0.1 μL/min with 0.3 μl of an AAV virus (UNC Viral Core) encoding the inhibitory (Syn-hM4Di-mCherry) DREADD receptor. Sham animals received an injection of a control AAV virus expressing green fluorescent protein (GFP). For both lesion and viral studies, mice were single housed after surgery and allowed 2 weeks to recover before beginning the drinking studies.