Figure 8. Post-injury administration of SB-216763 partially improves spatial memory, but does not offer neuroprotection. A) SB-216763-treated rats performed similarly to rats treated with vehicle in the hidden platform version of the Morris water maze task. No significant influence of SB-216763 administration was seen in either the B) latency to the first platform crossing or number of platform crossings, nor C) quadrant preference in a probe trial given 24 hr following training. *, P,0.05 by one-way repeated measures ANOVA. D) When given a probe trial 30 min after training, SB-216763-treated rats crossed the hidden platform location significantly more times than did the vehicle treated controls. *, P,0.05 by Student’s t-test. E) Representative photomicrographs of NeuN immunoreactivity from the dorsal, ipsilateral hippocampi of a vehicle- and a SB-216763-treated animal. Data are presented as the mean 6 SEM. DG: dentate gyrus. Scale bar = 500 mm. doi:10.1371/journal.pone.0024648.g008 strongly inhibit GSK-3 [31,32]. Consistent with increased Wnt signaling after TBI, the enhanced LRP6 phosphorylation we observed was followed by a significant decrease in GSK-3- dependent ß-Catenin phosphorylation. In addition to Wnt signaling, GSK-3 has been shown to be inhibited by direct phosphorylation by a number of kinases including protein kinase A (PKA) [33], Akt [34], PKC [35], p90 ribosomal S6 kinase (p90RSK) [36] and p70 ribosomal S6 kinase (p70S6K) [37]. Our results indicate that TBI causes a delayed increase in GSK-3ß phosphorylation on Ser9. Although the kinase responsible for this phosphorylation has not been identified, the time point at which we observed the increase (3 days post-injury), is consistent with that seen for activation of Akt. Using a cortical impact model of injury, Zhang et al., reported that TBI causes a delayed phosphorylation of Akt on Thr308 that occurs 72 hr post- injury [38]. Although we observed a significant increase in GSK- 3ß phosphorylation, no corresponding decrease in ß-Catenin phosphorylation was seen at this time point. While the reason for this is not clear at present, it is possible that the activity of GSK- 3a, which can also phosphorylate ß-Catenin, was unaffected by TBI [39] and could have maintained ß-Catenin phosphorylation. Glycogen Synthase Kinase-3 and TBI Accumulating evidence suggests that GSK-3 may play an important role in apoptosis with its overexpression inducing, and its inhibition preventing, apoptosis [9,40,41]. Although we observed that GSK-3 is negatively regulated as a result of TBI, the continued presence of neuron death suggests that this inhibition is not sufficient to protect the hippocampus from TBI- associated apoptosis [7,8]. As several studies have indicated that administration of the GSK-3-selective inhibitor SB-216763 can protect cultured neurons against excitotoxicity and toxin-induced apoptosis [42–44], and it has been shown to reduce the presence of pyknotic neurons in a model of Alzheimer’s disease [12], we questioned if similar benefits could be observed following TBI. In contrast to our anticipation, animals treated with the selective GSK-3 inhibitor SB-216763 had comparable hippocampal cell loss to injured rats treated with vehicle, and only showed a modest improvement in short-term memory. Our biochemical assessment of the consequences of SB-216763 demonstrated that the dose employed in our behavioral studies (5.0 mg/kg) effectively reduced GSK-3 activity in the hippocampus, and this dose has been used by other investigators to examine the consequences of GSK-3 inhibition in vivo [28]. While these results suggest that SB-216763 is only partially effective at reducing TBI pathology, it is possible that a higher concentration of the drug would have been more effective in reducing TBI-associated behavioral dysfunction. Since multiple mechanisms contribute to TBI pathophysiology, it is thought that a drug specific for a single target may not give rise to meaningful improvements in neurobehavioral outcome. There is a general consensus among TBI researchers that either a combination treatment, or a drug that targets multiple TBI- associated pathological cascades, is likely to be effective as a treatment [45]. Based on this premise, we tested the efficacy of lithium, a relatively non-selective inhibitor of GSK-3. Lithium inhibits GSK-3 both directly [36,46], and indirectly by reducing the activity of protein phosphatase-1 [47] and by increasing the phosphorylation of GSK-3 via activation of Akt [48]. Lithium has been shown to attenuate multiple pathological processes that have been reported to contribute to TBI pathophysiology such as apoptosis, oxidative stress, and mitochondrial and endoplasmic dysfunction. For example, pre-treatment with lithium protects cultured neurons from glutamate-induced cell death [49,50], and reduces the oxidative stress associated with neuropeptide S [51]. Bipolar patients on lithium have reduced levels of N-acetyl aspartate (NAA), a marker for neural injury, and have more gray matter volume as compared to control patients that are not on lithium therapy [52–55]. Although many of these neuroprotective properties of lithium are thought to be due to its influence on GSK-3 [56], lithium has been demonstrated to alter the activity of several other pathways. For example, lithium can indirectly block PKC activity by inhibiting inositol monophosphatase (IMPase) activity [57], thereby reducing the generation of diacylglycerol and inositol triphosphate. Nucleotide bisphosphate 39-nucleotidase (BPntase, also called RnPIP) is noncompetitively inhibited by lithium [58], whereas lithium has been shown to activate the ERK/MAPK cascade [59]. Although we observed that lithium effectively inhibited GSK-3, we cannot rule out the contribution of these other targets of lithium on the results we observed. Recently, it has been shown that pretreatment of mice with lithium prior to brain injury reduces inflammation, prevents TBI- induced depression, improves spatial learning and memory, and protects against hippocampal cell loss [25,60]. Consistent with this, we observed that lithium, when administered post-TBI and continued for an additional 5 days, significantly improved hippocampal function as indicated by improved learning and memory in the Morris water maze task tested days 14–25 post- PLoS ONE | www.plosone.org 9 September 2011 | Volume 6 | Issue 9 | e24648