activity via distinct mechanisms [13]. In most cells, GSK-3 exists in a constitutively active form as a part of a protein complex consisting of APC (adenomatous polyposis coli), axin, and casein kinase Ia that phosphorylates b-Catenin leading to its degradation (Figure 1A). Binding of Wnt to the Frizzled-LRP6 (lipoprotein- related protein 6) receptor complex causes translocation of GSK-3 to the plasma membrane, away from its cytoplasmic substrates, where it binds to and phosphorylates LRP6 (Figure 1B) [14]. This leads to reduced phosphorylation of ß-Catenin and decreased proteosomal degradation. In addition, GSK-3 activity can be regulated by phosphorylation in response to growth factor binding via Akt (Figure 1C). This phosphorylation and inactivation of GSK-3 is thought to play a role in the anti-apoptotic properties of Akt activation [15]. Although increases in Akt activity have been observed following TBI, this appears to be insufficient to prevent TBI-induced apoptotic cell death. We therefore hypothesized that augmentation of GSK-3 inhibition post TBI would attenuate cell death and improve neurocognitive outcome in brain injured animals. In the present study, we examined the consequences of TBI on GSK-3 activity and post-TBI administration of GSK-3 inhibitors on neurocognitive outcome. Treatment of injured animals with lithium, a multi-target drug that inhibits GSK-3, offered significant neuroprotection and improved learning and memory in brain injured animals. However, the GSK-3-selective small molecule inhibitor SB-216763 resulted in improved motor function, but only modest cognitive enhancement that occurred in the absence of visible neuroprotection. Materials and Methods Ethics Statement All experimental procedures were approved by the Animal Care and Use Committee of The University of Texas Health Science Center at Houston (protocol# AWC-08-046),and were conducted in accordance with the recommendations provided in the Guide for the Care and Use of Laboratory Animals. Protocols were designed to minimize pain and discomfort during the injury procedure and recovery period. Materials Male Sprague-Dawley rats (275–300 g) were purchased from Charles River Laboratories (Wilmington, MA). Lithium chloride and SB-216763 was purchased from Sigma Aldrich (St. Louis, MO) and Tocris Bioscience (Ellisville, MO), respectively. Anti- bodies to phospho ß-Catenin (Ser33/37), total ß-Catenin, phospho GSK-3b (Ser9), total GSK-3b, phospho-Erk1/2 (Thr202/Tyr204, Thr185/Tyr187), total Erk1/2antibodies, phospho-Akt (Ser473), total Akt, phospho-LRP6 (Ser1490), and total LRP6 were purchased from Cell Signaling Technology (Danvers, MA). Antibodies to NeuN were obtained from Millipore (Billerica, MA). Production of traumatic brain injury All experimental procedures were approved by the Institutional Animal Care and Use Committee and were conducted in accordance with the recommendations provided in the Guide for the Care and Use of Laboratory Animals. Protocols were designed to minimize pain and discomfort during the injury procedure and recovery period. An electromagnet-driven controlled cortical impact (CCI) device was used to cause brain injury as previously described [16–18]. Briefly, animals were anesthetized using 5% isofluorane with a 1:1 O2/N2O mixture, mounted a stereotaxic frame, and a midline incision was made. Bilateral 6-mm craniectomies were produced midway between the bregma and Glycogen Synthase Kinase-3 and TBI lambda with the medial edges of the craniectomies 1-mm lateral to the midline. Rats received a single impact (2.7 mm deformation) on the right parietal lobe with an impact velocity of 6 meters/sec. Core body temperature was maintained at 37-38uC by use of a heating pad. The animals were given time to recuperate in a warming chamber before being returned to their home cages. Animals were weighed daily after the injury for the first 3 days, then weekly thereafter. Drug preparation and administration Lithium chloride was dissolved in sterile saline to a final concentration of 25 mg/ml. For testing the influence of lithium chloride on cognitive function following TBI, animals were injured then injected (s.cu.) with 0.5 ml (1 mEq/kg lithium) or an equal volume of vehicle 30 minutes post-injury, and every 24 hours thereafter for the first 5 days post-injury (n = 10/group). For biochemical analysis, na¨ıve rats were given an s.cu. injection of lithium (0.1, 0.25, 0.5 or 1 mEq/kg) or vehicle once a day for 5 continuous days (n = 3/dose). Three hrs after the last injection, rats were killed by decapitation and hippocampi removed for the preparation of protein extracts. For single dose studies, 1.0 mEq/ Kg lithium was injected, and the animals killed 3 hr later. The selective GSK-3b inhibitor, SB-216763, was dissolved in DMSO to a stock concentration of 8.33 mg/mL and further diluted in 60% PEG400 and 30% distilled water for a final concentration of 0.83 mg/mL. For testing the influence of GSK-3b inhibition on cognitive function following TBI, animals were i.p. injected with 5 mg/kg SB-216763 or an equal volume of vehicle 30 min following injury and every 24 hours thereafter for the first 5 days after injury (n = 10/group). For biochemical analysis, uninjured rats were given a single i.p. injection with SB-216763 (2.5 or 5 mg/kg) and killed 1 hr later (n = 3/dose). Sample Preparation Hippocampal tissues were quickly removed while submerged in ice cold artificial cerebral spinal fluid (10 mM HEPES pH 7.2, 1.3 mM NaH2PO4, 3 mM KCl, 124 mM NaCl, 10 mM dextrose, 26 mM NaHCO3 and 2 mM MgCl2) containing phosphatase inhibitors (2 mM sodium fluoride, 2 mM sodium molybdate and 1 mM sodium orthovanadate). Tissues were homogenized (20 strokes) in 10 volumes of a buffer containing 10 mM Tris pH 7.4, 1 mM EGTA, 1 mM EDTA, 0.5 mM DTT, phosphatase inhibitors (0.1 mM okadaic acid and 1 mM sodium orthovanadate) and protease inhibitors (1 mM PMSF and 10 mg/ml leupeptin) using a motorized teflon-glass homogenizer. The samples were immediately aliquoted and frozen at 280uC. Western Blotting Hippocampal tissue extracts were sonicated (5 pulses of 1 second each) using a Sonics Vibracell sonicator (Sonics & Materials, Inc., Newtown, CT) and a 0.4 mm diameter probe. The amount of protein in each sample was determined by a Bradford assay using bovine serum albumin (BSA) as the standard. Samples were denatured at 95uC for three minutes in 1x NuPage SDS sample buffer (Invitrogen, Carlsbad, Calif.). Equal amounts of protein were loaded, electrophoresed, and transferred to Immobilon-P membranes (Millipore, Billerica, MA) using the NOVEX X-Cell II system (Invitrogen, Burlingame, CA) and the buffers provided by the vendor. Membranes were blocked overnight in 5%BSA in TBST, followed by a three-hour incubation in primary antibodies (0.1–0.5 mg/ml in TBST containing 2% BSA) at room temperature. Membranes were then washed in TBST and incubated at room temperature with alkaline phosphatase (AP)-conjugated secondary antibodies for one hour as PLoS ONE | www.plosone.org 2 September 2011 | Volume 6 | Issue 9 | e24648