ylation of the LRP6 receptor on Ser1490. This site has been shown to be phosphorylated in response to GSK-3 translocation to the LRP6 signalosome [14]. The summary data presented in Figure 1E show that the phosphorylation of LRP6 is significantly increased by 30 min post-injury (one-way ANOVA: F(5,24) = 4.297; P = 0.006). The total levels of LRP6 did not significantly change at any of the time points examined (one-way ANOVA: F(5,24) = 0.587; P = 0.710). As described above, GSK-3 is also inhibited following phosphorylation by Akt. Previous studies have reported that cortical impact injury results in increased Akt phosphorylation (on Thr308) and activity 3-day post-injury [24]. We therefore examined if GSK-3 phosphorylation is enhanced 3- day post-CCI. The representative western blots and the summary data shown in Figures 1F and 1G demonstrate that the phosphorylation of Ser9 of GSK-3ß increases after injury, reaching significance by the 3-day time point (one-way ANOVA: pGSK-3 F(5,24) = 7.335; p,0.001). This is consistent with a previous study which showed that GSK-3 phosphorylation is increased in the hippocampus for at least 24 hr after a weight drop model of TBI [25]. No change in the total levels of GSK-3ß was detected (Figure 1G; one-way ANOVA: GSK-3 F(5,24) = 0.622; p = 0.684). Upon inactivation by either the Wnt-LRP6 or Akt cascades, GSK-3-mediated phosphorylation of ß-Catenin on Ser33/37 is reduced, leading to its nuclear accumulation and enhanced gene expression. To assess if the TBI-associated decreases in GSK-3 activity are associated with changes in ß-Catenin phosphorylation and levels, western blots were again carried out. Figure 1H shows representative western blots illustrating phospho-ß-Catenin immu- noreactivity at different time points following injury. The summary data in Figure 1I demonstrates that TBI causes a significant, but transient, decrease in the phosphorylation of Ser33/37 of ß-Catenin acutely following injury (one-way ANOVA: F(5,24) = 4.948; p = 0.003). Although a significant decrease in ß-Catenin phosphor- ylation was observed by 3 hr post-injury, no concurrent increase in the total levels of this protein was observed (one-way ANOVA: F(5,24) = 1.208; p = 0.336). No changes in either the phosphorylation or total levels of ß-Catenin were observed at the time point at which GSK-3ß phosphorylation was observed. Lithium treatment improves hippocampal function and reduces neuronal loss following TBI Inhibition of GSK-3 has been shown to reduce neuronal death triggered by a number of stimuli [13]. Although GSK-3 activity appears to be modulated by both receptor- and Akt-mediated mechanisms following brain injury, these effects were transient and apparently insufficient to prevent TBI-associated hippocampal cell death. In order to determine the consequences of further inhibition of the GSK-3 pathway, post-injury administration of lithium was tested. A number of previous studies have shown that many of the protective effects of lithium are mediated by its influence on the GSK-3 cascade [26], although several other targets of its action have been reported. To confirm that systemic administration of lithium can inhibit the GSK-3 cascade in the brain, animals were s.cu. injected with 1 mEq/kg lithium (n = 3) or saline (n = 5) for five consecutive days. This dose was chosen based on a previous study that demonstrated that delayed administration of 1mEq lithium after cerebral ischemia reduced cortical infarct volume and facilitated neurological recovery [27]. In addition, we also tested the influence of lower doses of lithium, namely 0.10, 0.25, and 0.50 mEq/Kg (n = 3/dose). Three hours following the last injection, hippocampal protein extracts were prepared for western blots. Figure 2A shows representative western blots of phosphorylated (Ser9) GSK-3ß following different concentrations of lithium. Since the evaluation of the 1.0 mEq/Kg dose, and the Glycogen Synthase Kinase-3 and TBI dose-response study (0.1, 0.25, and 0.5 mEq/Kg) were carried out at two different times, an independent vehicle group was used for each analysis. The summary data show that when normalized across experiments, all doses tested effectively increased the phosphorylation of GSK-3ß (one–way ANOVA: F(4,12) = 5.326, p = 0.011). No significant difference was detected in the total levels of GSK-3ß (one–way ANOVA: F(4,12) = 1.770, p = 0.200) (Figure 2B). Although all doses increased GSK-3 phosphorylation, significant accumulation of ß-Catenin was only observed at the high doses (one-way ANOVA: F(4,12) = 49.283, p,0.001), with maximal accumulation observed at 1.0 mEq lithium (Figure 2C). Since maximal ß-Catenin accumulation was observed after 5 daily injections of the 1.0 mEq dose, we questioned if a single injection was sufficient to elicit this effect. To test this, animals received a single injection of either 1.0 mEq/Kg lithium or an equal volume of vehicle and killed 3 hr later. The western blot results shown in Figure 2D demonstrate that a single dose of lithium had no significant effect on GSK-3 phosphorylation (Student’s t-test: 0.956), total GSK-3 levels (Student’s t-test: 0.130) or ß-Catenin levels (Student’s t-test: 0.587). Based on these results, we tested the consequences of daily post-injury administration of 1.0 mEq lithium for the first 5 days after injury using a series of behavioral and cognitive tasks. The influence of post-injury lithium injection on vestibulomotor and motor function was tested on days 1–4 post-injury using the beam balance and foot fault tasks, respectively (Figure 3A). Rats were injured, and then injected s.cu. with 1mEq/kg (n = 10) or vehicle (n = 10) 30 min after the injury. Drug administration was continued every 24 hr for the first 5 days of injury. Figure 3B shows that post-injury administration of lithium did not signifi- cantly improve beam balance performance compared to simulta- neously tested vehicle controls (repeated measures two-way ANOVA: F(1,18) = 3.267, p = 0.087). In addition, neither ipsilateral (repeated measures two-way ANOVA: F(1,18) = 2.033, p = 0.170; Figure 3C) nor contralateral (repeated measures two-way ANOVA: F(1,18) = 1.589, p = 0.222; Figure 3D) foot faults were significantly different between the two groups. At the dose tested, however, lithium significantly exacerbated post-injury weight loss (two-way repeated measures ANOVA: F(1,18) = 14.324, p = 0.001). All animals had normal vestibulomotor and motor function by day 7 post-injury (data not shown), and had returned to their pre- injury weight. When these animals were tested for their ability to perform the Morris water maze task (beginning on day 14 post-injury), the rats treated with lithium had learning curves that were significantly different from their vehicle-injected counterparts (significant interaction by repeated measures two-way ANOVA: F(9,162) = 4.082, p,0.001; Figure 4A). When tested in a probe trial 24 hr following training, the lithium-treated injured animals crossed the original platform location significantly faster (vehicle: 48.5266.17 sec; lithium chloride: 26.5765.67 sec, p = 0.017) and more often (vehicle: 0.3060.15 crosses; lithium chloride: 2.0060.49 crosses, p = 0.003) than vehicle-infused animals, suggesting an improved memory for the platform location (Figure 4B). In support of this conclusion, the lithium chloride- injected rats demonstrated a preference for the quadrant in which the platform was located (repeated measures one-way ANOVA: F(3,27) = 11.50, p,0.001) while the saline-injected controls did not (repeated measures one-way ANOVA: F(3,27) = 0.62, p = 0.619) (Figure 4C). Comparison across groups revealed a significant interaction between treatment and quadrant (repeated measures two-way ANOVA: F(3,54) = 6.58, p,0.001) with a significant difference in the time each group spent searching in the target quadrant (quadrant I). Short-term memory was found to be PLoS ONE | www.plosone.org 5 September 2011 | Volume 6 | Issue 9 | e24648