injury. Stereological cell counts revealed that these functional improvements were accompanied by a significant preservation in CA3 hippocampal neuron numbers. Although studies have shown that cell death after TBI can continue for weeks-to-months, the majority of hippocampal cell loss occurs within the first few days after the injury [8,61,62]. As inhibition of GSK-3 has been shown to offer neuroprotection in response to several toxic agents, it is likely that the protection observed was due to a reduction in hippocampal cell death within the initial 5-day period after injury (the time frame of lithium administration). However, we can rule out any lasting neuroprotective effects of lithium treatment that may have contributed to the behavioral and histological improvements we observed. Although oral lithium carbonate is an effective therapy of treating bipolar disorders, benefit is typically only seen following prolonged use. This delayed onset of action is in contrast to the relatively acute effects described herein. While the mechanism underlying lithium’s effect following TBI is not clear, a few studies have suggested that mechanistic differences exist between acute and chronic lithium treatment paradigms. For example, chronic, but not acute, lithium treatment increases BDNF expression in the rat hippocampus and temporal cortex [63], and BDNF expression has been observed following 28days of lithium treatment in persons with bipolar disorder [64]. In contrast, neuroprotection can be observed following acute lithium treatment, an effect thought to be due to a reduction of GSK-3-mediated apoptosis [65]. While we observed that GSK-3 activity could be inhibited using an acute administration routine (daily dosing for 5 days post- injury), a single dose of lithium did not significantly alter GSK-3 phosphorylation or result in significant accumulation of ß-Catenin. Thus, while the treatment window we observed in the present study is relatively shorter than that typically required to treat bipolar disorder, continuous dosing was still required to signifi- cantly alter GSK-3 activity. The therapeutic dose for lithium is in the range of 900 mg of lithium carbonate/day (24 mEq lithium). Usually, these patients References 1. Kochanek PM, Clark RS, Ruppel RA, Adelson PD, Bell MJ, et al. (2000) Biochemical, cellular, and molecular mechanisms in the evolution of secondary damage after severe traumatic brain injury in infants and children: Lessons learned from the bedside. Pediatr Crit Care Med 1: 4–19. 2. Yakovlev AG, Faden AI (1995) Molecular biology of CNS injury. J Neurotrauma 12: 767–777. 3. Thompson HJ, Lifshitz J, Marklund N, Grady MS, Graham DI, et al. (2005) Lateral fluid percussion brain injury: a 15-year review and evaluation. J Neurotrauma 22: 42–75. 4. Faden AI (2002) Neuroprotection and traumatic brain injury: theoretical option or realistic proposition. Curr Opin Neurol 15: 707–712. 5. Hayes RL, Wang KK, Kampfl A, Posmantur RM, Newcomb JK, et al. (1998) Potential contribution of proteases to neuronal damage. Drug News Perspect 11: 215–222. 6. Dixon CE, Kochanek PM, Yan HQ, Schiding JK, Griffith RG, et al. (1999) One-year study of spatial memory performance, brain morphology, and cholinergic markers after moderate controlled cortical impact in rats. J Neurotrauma 16: 109–122. 7. Colicos MA, Dixon CE, Dash PK (1996) Delayed, selective neuronal death following experimental cortical impact injury in rats: possible role in memory deficits. Brain Res 739: 111–119. 8. Yakovlev AG, Knoblach SM, Fan L, Fox GB, Goodnight R, et al. (1997) Activation of CPP32-like caspases contributes to neuronal apoptosis and neurological dysfunction after traumatic brain injury. J Neurosci 17: 7415–7424. 9. Grimes CA, Jope RS (2001) The multifaceted roles of glycogen synthase kinase 3beta in cellular signaling. Prog Neurobiol 65: 391–426. 10. Perez M, Rojo AI, Wandosell F, Diaz-Nido J, Avila J (2003) Prion peptide induces neuronal cell death through a pathway involving glycogen synthase kinase 3. Biochem J 372: 129–136. 11. Watcharasit P, Bijur GN, Zmijewski JW, Song L, Zmijewska A, et al. (2002) Direct, activating interaction between glycogen synthase kinase-3beta and p53 after DNA damage. Proc Natl Acad Sci U S A 99: 7951–7955. Glycogen Synthase Kinase-3 and TBI receive an initial dose of 300 mg (or 8mEq) to reduce the occurrence of adverse events such as nausea and kidney injury. The dose is then gradually increased to the recommended serum therapeutic dose (0.6–1.2mEq/L). Although we did not monitor the serum level of lithium, previous studies investigating its pharmacokinetics in rats have indicated that 5 mEq/kg lithium reaches a peak serum concentration of approximately 10 mEq/L with a half-life of 7 hr [66]. Thus, we anticipate that 1mEq/kg would give rise to a significantly lower serum concentration that is more comparable to the recommended dose. In addition to treatment of bipolar disorder with lithium alone, combined treatment with lithium and valproic acid has been used to treat patients resistant to monotherapy with either drug [67]. Further- more, a combination of 60 mg/kg lithium chloride (1.4 mEq) and 300 mg/kg valproic acid delayed disease onset, reduced neuro- logical deficits and prolonged survival in a mouse model of lateral sclerosis as compared to either drug alone [68]. As we have recently shown that valproic acid also offers neuroprotection and improves cognitive function following TBI [69], future studies will determine if a combination of lower doses of lithium (e.g. 0.25 mEq, Figure 2) and valproic acid can be used to reduce the consequences of TBI while minimizing adverse events associated with high doses of either drug [70,71]. Acknowledgments The authors would like to thank Dr. Jonathan Miller for his expert advice on the use of SB-216763. Author Contributions Conceived and designed the experiments: PKD SAO ANM SP. Performed the experiments: SAO MZ JC DJ RJG JZ ANM. Analyzed the data: SAO MZ JC DJ ANM PKD. Wrote the paper: PKD JC DJ SAO MZ JZ RJG ANM SP. 12. Hu S, Begum AN, Jones MR, Oh MS, Beech WK, et al. (2009) GSK3 inhibitors show benefits in an Alzheimer’s disease (AD) model of neurodegeneration but adverse effects in control animals. Neurobiol Dis 33: 193–206. 13. Jope RS, Johnson GV (2004) The glamour and gloom of glycogen synthase kinase-3. Trends Biochem Sci 29: 95–102. 14. Niehrs C, Shen J (2010) Regulation of Lrp6 phosphorylation. Cell Mol Life Sci 67: 2551–2562. 15. Duronio V (2008) The life of a cell: apoptosis regulation by the PI3K/PKB pathway. Biochem J 415: 333–344. 16. Dixon CE, Clifton GL, Lighthall JW, Yaghmai AA, Hayes RL (1991) A controlled cortical impact model of traumatic brain injury in the rat. J Neurosci Methods 39: 253–262. 17. Dash PK, Moore AN, Dixon CE (1995) Spatial memory deficits, increased phosphorylation of the transcription factor CREB, and induction of the AP-1 complex following experimental brain injury. J Neurosci 15: 2030–2039. 18. Hoskison MM, Moore AN, Hu B, Orsi S, Kobori N, et al. (2009) Persistent working memory dysfunction following traumatic brain injury: Evidence for a time-dependent mechanism. Neuroscience 159: 483–491. 19. Hamm RJ, Dixon CE, Gbadebo DM, Singha AK, Jenkins LW, et al. (1992) Cognitive deficits following traumatic brain injury produced by controlled cortical impact. J Neurotrauma 9: 11–20. 20. Dash PK, Mach SA, Moore AN (2002) The role of extracellular signal-regulated kinase in cognitive and motor deficits following experimental traumatic brain injury. Neuroscience 114: 755–767. 21. Royo NC, LeBold D, Magge SN, Chen I, Hauspurg A, et al. (2007) Neurotrophin-mediated neuroprotection of hippocampal neurons following traumatic brain injury is not associated with acute recovery of hippocampal function. Neuroscience 148: 359–370. 22. Sullivan PG, Bruce-Keller AJ, Rabchevsky AG, Christakos S, Clair DK, et al. (1999) Exacerbation of damage and altered NF-kappaB activation in mice lacking tumor necrosis factor receptors after traumatic brain injury. J Neurosci 19: 6248–6256. PLoS ONE | www.plosone.org 10 September 2011 | Volume 6 | Issue 9 | e24648