The mPFC was dissected from previously frozen brain tissue (= 3 young adult rats; 4C6 months). promoting activation of the NR2A-enriched synaptic pool of PFC NMDARs. These results implicate NR2A-NMDARs in normal working memory and suggest novel treatment strategies for improving working memory in cognitive disorders. SIGNIFICANCE STATEMENT Working memory, the ability to hold information in mind, requires prolonged activity of pyramidal neurons in prefrontal cortex (PFC) mediated by NMDA receptor (NMDAR) activation. NMDAR loss in PFC may account for working memory impairments in aging and psychiatric disease. Our studies demonstrate that NMDARs made up of the NR2A subunit, but not the NR2B subunit, are required for working memory and that loss of NR2A predicts severity of age-related working memory impairment. The importance of NR2A to working memory is likely due its abundant contribution to pyramidal neuron activity and location at synaptic sites in PFC. This information is useful in designing new therapies to treat working memory impairments by enhancing the function of NR2A-containing NMDARs. = 58) and aged (22C26 months aged, = 30) Fischer 344 rats were acquired from your National Institute on Aging Rodent Colony (housed at Charles River Laboratories). In Experiment 1, = 40 young rats were utilized for behavioral pharmacological experiments that assessed working memory overall performance after blockade of medial PFC (mPFC) NR2A or NR2B receptors, = 7 young rats were utilized for patch-clamp electrophysiology experiments that evaluated the relative contributions of NR2A and NR2B receptors to the overall NMDAR-mediated evoked EPSC in mPFC pyramidal neurons, and = 3 young rats were utilized for coimmunoprecipitation experiments to determine NR2ACPSD95 associations in mPFC. In Experiment 2, = 8 young and = 13 aged rats were used to evaluate age-related changes in mPFC expression of excitatory signaling proteins and their relationship with individual differences in working memory ability. In Experiment 3, = 11 aged rats were used to test the effects of modulation of NMDAR activity on working memory overall performance and = 6 aged rats were utilized for patch-clamp electrophysiology experiments to evaluate the effects of a d-amino acid oxidase inhibitor on evoked NR2A-NMDAR currents. Across experiments, rats were housed individually with Nalbuphine Hydrochloride access to food and water except during behavioral screening as explained below. All animal procedures were approved by the Institutional Animal Care and Use Committee of the University or college of Florida and conformed to the National Institutes of Health’s animal welfare guidelines. Experiment 1: Determining the role of NMDAR subtypes in working memory and mPFC neural physiology Surgical procedures. Rats were anesthetized with isofluorane gas Nalbuphine Hydrochloride and fixed into a stereotaxic frame (Kopf Devices) fitted with atraumatic ear bars. The incisor bar was set at ?3.3 mm relative to the interaural collection to provide a flat skull position. A midline incision was made and the skin and fascia over the skull were retracted. Burr holes were drilled in the skull over the mPFC for placement of three stainless steel screws. Bilateral guideline cannulae, consisting of a plastic body holding two 22-gauge stainless steel cannulae spaced 1.4 mm apart (Plastics One) were implanted to target mPFC at the coordinates (in mm) AP: +2.7 from bregma, ML: 0.7 from bregma, DV: ?3.8 from your skull surface. Cannulae were secured to the skull with stainless steel screws and dental acrylic and wire stylets were placed in the guideline cannulae to prevent contamination. Rats received injections of buprenorphine (1 mg/kg/d for 2 d postoperatively) and meloxicam (2 mg/kg/d for 3 d postoperatively) and Nalbuphine Hydrochloride topical triple antibiotic ointment (as needed) for analgesia and to prevent contamination. Rats were given a 2 week recovery period before beginning behavioral screening. Behavioral testing apparatus. Screening in the delayed response task (DRT) used to assess working memory was conducted in eight identical standard rat behavioral test chambers (30.5 25.4 30.5 cm; Coulbourn Devices) with metal front and back walls, transparent Plexiglas side walls, and a floor composed of steel rods (0.4 cm diameter) spaced 1.1 cm apart. Each test chamber was housed in a sound-attenuating cubicle and was equipped with a recessed food pellet delivery trough located 2 cm above the floor in the center of the front wall. The trough was fitted with a photobeam to detect head entries and a 1.12 W lamp for illumination. A single 45 mg grain-based food pellet (5TUM; TestDiet) was delivered to incentive correct responses. Two retractable levers were located to the left and right of the food trough (11 cm above the floor). An additional 1.12 W house light was mounted near the top of the CORO1A rear wall of the sound attenuating cubicle. Behavioral test chambers were connected to a computer running Graphic.