br Experimental procedures br Results br Discussion

Experimental procedures
Results
Discussion The data herein confirm the well documented neuroinflammatory effect of TLR3 activation following systemic administration of the viral mimetic poly I:C and the associated acute sickness response which includes fever, reduced body weight and allodynia [8,10,11,13,14]. Sickness behaviour induced following viral infection and TLR3 activation are associated with glial activation and the release of type 1 interferons (IFN-α and IFN-β) and NFĸB-inducible pro-inflammatory cytokines (e.g. IL-1β, IL-6 and TNF-α) [[8], [9], [10],13,17]. For example, cytokines including IL-1β, IL-6, TNF-α [11,41] and the COX-PGE2 pathway have been shown to be pivotal mediators of the TLR3 febrile response [42,43]. In addition to the acute neuroinflammatory response, TLR3 activation has been shown to result in the increased expression of the microglia/macrophage activation marker CD11b [17,44], reduced BDNF and increased indoleamine 2, 3, dioxygenase (IDO) and kynurenine in the CNS [17], effects which may underlie long-term neuronal and behavioural changes. Accordingly, the acute sickness response to immune stimulation in rodents has been found to resolve after 24 hs, however, lasting behavioural effects of TLR3 activation have been reported beyond this period. Anxiety-like and anhedonic behaviour [17] and enhanced mechanical nociceptive thresholds [44,45] have been reported in rats 24 hs post poly I:C administration, effects which have been replicated and expanded upon in the current study. Accordingly, the present data demonstrated that TLR3 activation results in anxiety-like behaviour in the OFT and EPM, anhedonia in the SPT and persistent mechanical allodynia in the von Frey test at 24 hs post poly I:C administration. Thus, taken together, the current and published data highlight the short and longer-term physiological consequences of TLR3 stimulation. Increasing evidence supports a role for FAAH, but not MAGL, substrates in the modulation of TLR3-mediated acute neuroinflammation in several 4sc regions [8,9]. The current data demonstrate that the systemic administration of the FAAH inhibitor URB597 attenuates TLR3-associated increases in the microglia/macrophage activation marker CD11b and M1 pro-inflammatory microglia/macrophage marker CD68 (but not MRC2, the M2 anti-inflammatory/restorative glial activation marker) in the hypothalamus. While it remains to be determined if similar effects occur in other brain regions, URB597 has been shown to modulate the TLR3-mediated increase in expression of neuroinflammatory genes in the hypothalamus, hippocampus and frontal cortex [8,9], although the specific genes and magnitude of the response differs between regions. Differences in the effect of FAAH inhibition on TLR3-mediated neuroinflammatory genes between brain regions may be attributed to differences in the resting state of neurons, glia or endocannabinoid activity in these regions. In accordance with the current study, FAAH inhibition also attenuates M1 microglia/macrophage activation (CD11b and CD68) and the expression of an array of pro-inflammatory mediators following TLR4 activation [30]. Thus, taken together the data indicate that enhancing central FAAH substrate levels can attenuate both the TLR3- and TLR4-mediated activation of M1 microglia/macrophages and associated increases in inflammatory mediators in the brain. Although GFAP expression in the hypothalamus tended to be enhanced in URB597-poly I:C treated animals, analysis revealed no significant change in GFAP expression in response to poly I:C in the presence or absence of URB597. Although further histological and functional studies are required, this data suggest that the effects of URB597 on TLR3-mediated neuroinflammation and associated behavioural responding are unlikely due to the inhibition of astrocyte activity. The current data demonstrate that the physiological consequences of FAAH substrate-induced attenuation of TLR3-mediated neuroinflammation include the attenuation of the fever response, without altering other aspects of the sickness response, namely reduction in body weight. Although FAAH substrates including AEA, OEA and PEA have been shown to modulate TLR4-induced changes in temperature [[27], [28], [29]], this is the first study to demonstrate that enhancing FAAH substrate levels can also modulate TLR3 associated temperature changes. As the hypothalamus is a key brain region involved in TLR3-mediated fever, it is likely that FAAH substrate modulation of TLR3-mediated inflammatory processes in this region underlies the effects on temperature observed. Further studies are required in order to determine if these effects are mediated by one or a combination of FAAH substrates. However, it should be noted that CB1-/- mice exhibit a robust fever response to poly I:C [46], indicating that AEA activation of CB1 receptors is unlikely to be the primary mediator of the effects of URB597 on TLR3-mediated fever.