Our 
results show a remarkable ability of two different Tat-interfering peptides to prevent the downstream actions of TLR4 receptor stimulation at the molecular, cellular and behavioral levels. Although similar peptides have previously been developed, we 22948146 have been the first to show a behavioral impact of blocking TLR4-MyD88 interaction, likely mediated by a rescue of microglia morphology changes and cytokine production that are normally induced by LPS. These peptides cross the BBB and enter cells where they disrupt the protein-protein interactions between TLR4 and MyD88. The peptides mimicked the key sequences necessary for dimerization and interaction of MyD88 and TLR4 TIR domains. Natural mutations of this key sequence in the TLR4 receptors were previously discovered to explain the unresponsiveness of a specific strain of rat to LPS [29]. We found that interfering peptides that mimic either the sequence of TLR4 receptors or the sequence on the recognition site on MyD88 prevented the co-immunoprecipitation of these proteins and the ability of LPS to activate second messengers and increase cytokine formation in intact tissue in brain slices and in vivo. Using two photon imaging we have further shown the dynamic morphological changes that microglia can undergo in responses to LPS and that these changes can be mediated by TLR4 signaling. In addition these Tat-interfering peptides were remarkably effective at preventing the behavioral syndrome that accompanies sickness caused by LPS. When mice were administered LPS by peripheral i.p. injection, a SRIF-14 series of behavioral changes AN 3199 biological activity occurred within a one hour period, as previously reported [2]. Following treatment with either Tat-MyD88 or Tat-TLR4, but not a Tat-scrambled peptide, there was a complete absence of motoric (behavioral screen) and motivational (behavioral screen and titrated ICSS) effects of LPS-induced sickness which we attribute to the direct action of the peptide on brain function.Microglia and Sickness BehaviorFigure 4. LPS induced sickness behavior was blocked by Tat-MyD88 and Tat-TLR4 as assessed in mice using a modified SHIRPA screen and in the novel home cage following various treatments. A. Cumulative score of sickness obtained using the SHIRPA screen. B. Representative paths of mice over 30 min in the home cage showing decreased exploration induced by LPS, and effective block by Tat-MyD88 and Tat-TLR4 but not Tat-scram. Average speed travelled (C), cumulative distance travelled (D), number of 15755315 rears in the home cage (E). F, G. Assessment of sickness behavior in rats using intracranial self stimulation. F. Number of responses per minute during baseline, LPS treatment, and following LPS treatment. G. Number of responses per minute during baseline, LPS plus Tat-MyD88 treatment, and following LPS plus Tat-MyD88. doi:10.1371/journal.pone.0060388.gThe sickness behavior of animals, triggered by the inflammatory release of cytokines, mirrors the well known symptoms of sickness in humans which include fatigue, loss of appetite and cognitive changes. It is also becoming evident that sickness and inflammation are important contributors to the occurrence of depressive episodes [2]. Therefore, these peptides represent a novel means of blocking the behavioral impact of sickness, and potentially an effective strategy for alleviating symptoms of depression induced by chronic inflammation and sickness. Our detailed description of a molecular target linking inflammation and sickness to motiva-.Our results show a remarkable ability of two different Tat-interfering peptides to prevent the downstream actions of TLR4 receptor stimulation at the molecular, cellular and behavioral levels. Although similar peptides have previously been developed, we 22948146 have been the first to show a behavioral impact of blocking TLR4-MyD88 interaction, likely mediated by a rescue of microglia morphology changes and cytokine production that are normally induced by LPS. These peptides cross the BBB and enter cells where they disrupt the protein-protein interactions between TLR4 and MyD88. The peptides mimicked the key sequences necessary for dimerization and interaction of MyD88 and TLR4 TIR domains. Natural mutations of this key sequence in the TLR4 receptors were previously discovered to explain the unresponsiveness of a specific strain of rat to LPS [29]. We found that interfering peptides that mimic either the sequence of TLR4 receptors or the sequence on the recognition site on MyD88 prevented the co-immunoprecipitation of these proteins and the ability of LPS to activate second messengers and increase cytokine formation in intact tissue in brain slices and in vivo. Using two photon imaging we have further shown the dynamic morphological changes that microglia can undergo in responses to LPS and that these changes can be mediated by TLR4 signaling. In addition these Tat-interfering peptides were remarkably effective at preventing the behavioral syndrome that accompanies sickness caused by LPS. When mice were administered LPS by peripheral i.p. injection, a series of behavioral changes occurred within a one hour period, as previously reported [2]. Following treatment with either Tat-MyD88 or Tat-TLR4, but not a Tat-scrambled peptide, there was a complete absence of motoric (behavioral screen) and motivational (behavioral screen and titrated ICSS) effects of LPS-induced sickness which we attribute to the direct action of the peptide on brain function.Microglia and Sickness BehaviorFigure 4. LPS induced sickness behavior was blocked by Tat-MyD88 and Tat-TLR4 as assessed in mice using a modified SHIRPA screen and in the novel home cage following various treatments. A. Cumulative score of sickness obtained using the SHIRPA screen. B. Representative paths of mice over 30 min in the home cage showing decreased exploration induced by LPS, and effective block by Tat-MyD88 and Tat-TLR4 but not Tat-scram. Average speed travelled (C), cumulative distance travelled (D), number of 15755315 rears in the home cage (E). F, G. Assessment of sickness behavior in rats using intracranial self stimulation. F. Number of responses per minute during baseline, LPS treatment, and following LPS treatment. G. Number of responses per minute during baseline, LPS plus Tat-MyD88 treatment, and following LPS plus Tat-MyD88. doi:10.1371/journal.pone.0060388.gThe sickness behavior of animals, triggered by the inflammatory release of cytokines, mirrors the well known 
symptoms of sickness in humans which include fatigue, loss of appetite and cognitive changes. It is also becoming evident that sickness and inflammation are important contributors to the occurrence of depressive episodes [2]. Therefore, these peptides represent a novel means of blocking the behavioral impact of sickness, and potentially an effective strategy for alleviating symptoms of depression induced by chronic inflammation and sickness. Our detailed description of a molecular target linking inflammation and sickness to motiva-.