For over two decades, the cholinergic hypothesis of Alzheimer’s disease (AD) has been accepted based on evidence showing the degeneration of cholinergic neurons and the deficit of acetylcholine (ACh) in AD patients and models, a key neurotransmitter in cognitive and memory processes in the hippocampus. Recently, increasing evidence suggests that neuroinflammation may play an important role in the etiology of AD. Among many inflammatory mediators, an increase in interleukin (IL)-1β, the most potent proinflammatory cytokine produced by microglial cells, has been consistently reported in patients with AD (Griffin et al. 1989; Kornman 2006). IL-1β is well known to trigger microglial activation, increase the expression of amyloid precursor proteins, and stimulate the hypothalamic–pituitary–adrenal (HPA) axis to secrete glucocorticoids (Dunn 2000; Griffin et al. 2006; Godoy et al. 2008). On the one hand, inflammation-induced excessive production of glucocorticoids could induce synaptic loss and neuronal apoptosis, which may contribute to the deficit of learning and memory (Nichols et al. 2001; Crochemore et al. 2005). For example, central administrations of IL-1β could induce spatial or working memory deficit in rats, which can be attenuated by IL-1 receptor antagonist (RA) or mifepristone (RU 486), a glucocorticoid RA (Song et al. 2004). At the cellular level, an increase in IL-1β concentration was found to relate to impaired long-term potentiation (LTP) in aged and stressed rats (Murray and Lynch 1998).
On the other hand, inflammatory responses in the brain may change astrocyte function, and thereby cause the dysfunction of neurotrophin systems (Takuma et al. 2004). Indeed, increased and decreased neurotrophin concentrations and receptor functions have been reported in AD patients (Counts and Mufson 2005; Peng et al. 2005). Furthermore, neurotrophins, such as nerve growth factor (NGF) and brain-derived neurotrophic factor have been used to treat AD (Cattaneo et al. 2004; Nagahara et al. 2009). In experimental studies, NGF was reported to attenuate cholinergic deficits following traumatic brain injury in rats (Dixon et al. 1997), and NGF gene transfer into aged animals can increase the level of depolarization-induced ACh release from hippocampal synaptic terminals (Wu et al. 2004).
However, in the last 20 years, most studies have only focused on the effects of IL-1β on behavior, the endocrine system, and catecholamine functions. Only one study reported that systemic administration of IL-1β caused a decrease in ACh levels in the hippocampus (Rada et al. 1991). To date, the correlation between ACh release and memory, the relationship between glucocorticoid secretion and ACh release, and between inflammation and NGF functions in IL-1-induced memory impairment is still unknown. The possible pathways of both IL-1-glucocorticoid-ACh release and IL-1-NGF-ACh release are presented by Fig. 1. Thus, the first aim of this study was to demonstrate these pathways. Our hypotheses were that (i) the reduction of ACh release is correlated with memory deficits after IL-1 administration; (ii) a glucocorticoid RA or IL-1 RA can block ACh reduction; and (iii) IL-1 would decrease NGF expression but increase brain inflammation, which is correlated with the reduction of ACh release. To demonstrate these hypotheses, an in vivo microdialysis technique was employed to measure ACh release from the dentate gyrus (DG) of the hippocampus, following IL-1β and saline injections during animal training and testing in an eight-arm radial maze, an apparatus for testing working memory. The reasons to choose the DG for this study are based on that the DG is innervated by basal forebrain cholinergic neurons and the loss of neurogenesis and cholinergic innervations in the DG play an important role in AD (Tatebayashi et al. 2003; deToledo-Morrell et al. 2007). Several studies have demonstrated that lesion of the DG significantly impairs hippocampus-dependent memory (Mohapel et al. 2005). In addition, the DG plays an important role in suppressive control of the HPA axis in AD. Lesion of the cholinergic projection in the basal forebrain is correlated with a significant increase in glucocorticoid receptor expression in the DG (Yau et al. 1992).
IL-1RA and RU 486 were administered before IL-1β administrations to demonstrate IL-1β and glucocorticoid effects on ACh release. mRNA expressions of NGF and IL-1β were measured by quantitative PCR in the hippocampus after IL-1 and IL-1RA administrations. Furthermore, this study also compared the effect of acute (1 day) and subchronic (7 days) IL-1β administrations on ACh release and memory performance in the rat because an increase in IL-1β release in acute brain conditions has been reported to protect neurons (Kuhlow et al. 2003; Dietrich et al. 2004), while in chronic neurodegeneration, excessive produced IL-1β has been considered to contribute to neuron death (Cunningham et al. 2005; Ferrari et al. 2006).
As introduced above, if inflammation can significantly reduce ACh release and neurotrophin function, any drug or natural product that reduces inflammation and enhance neurotrophin function could be an effective treatment for AD. Recently omega (n)-3 fatty acids have been suggested to treat AD since a higher n-3 fatty acid intake was associated with a lower risk of AD onset (Otsuka 2000; Morris et al. 2003). In a pilot clinical trial, a treatment with eicosapentaenoic acid (EPA) and docosahexaenoic acid significantly improve cognitive function in patients with mild cognitive impairment (Chiu et al. 2008). In animal models of AD or aging, significantly reversed learning deficits and normalized the expression of proteins involved in neuronal plasticity (Hashimoto et al. 2008). The mechanism by which n-3 fatty acids protect neurons may be though anti-inflammatory (inhibit nuclear factor-kappaB and reduce n-6 fatty acids to produce inflammatory mediators), anti-stress (reduce glucocorticoids), and modulation of apoptosis genes (Song et al. 2003; Lynch et al. 2007; Singer et al. 2008). Furthermore, we have reported that EPA can up-regulate the brain-derived neurotrophic factor receptor tropomyosin-related protein in fully differentiated SH-SY5Y cells (Kou et al. 2008). However, the ACh and neurotrophin mechanisms by which EPA improves memory are still unknown. The possible target points of EPA are also presented in the Fig. 1. Our hypothesis was that EPA might improve memory by blocking IL-1β effects on ACh and NGF. To demonstrate this hypothesis, as the second aim of this study, EPA effects on ACh release, memory and mRNA expressions of NGF and IL-1 were evaluated after saline or IL-1β administrations.
In this study, EPA in ethyl ester form was used to feed animals. The main advantages of the ethyl ester appears to be that it can be concentrated to a greater degree and has greater bioavailability than when provided as triglycerides (Brunton and Collins 2007). It also has the potential to be FDA approved, which requires it to be manufactured by good manufacturing practice facilities requiring consistency, purity, and minimal levels of toxins as has been done for prescription omega-3-acid ester. However, it remains unclear whether the advantages gained by esterification are lost by the fact that people absorbed ethyl-EPA less than the unesterified EPA (Lawson and Hughes 1988; Beckermann et al. 1990).
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This study, for the first time, reported four important findings in the IL-1-induced model of memory deficits: (i) a clear correlation between the decrease in ACh release and memory deficit was found during rat memory retrieval phase; (ii) glucocorticoids are involved in the reduction of ACh release; (iii) a down-regulated NGF expression in the hippocampus is correlated with the reduction of ACh release and associated with memory impairment; and (iv) E-EPA improves memory by attenuating the reduction of ACh release and NGF expression. Thus, the results have demonstrated our hypothesis that inflammation may increase glucocorticoids but suppress NGF to reduce ACh release, which is responsible to the memory deficit, and EPA treatment significantly reversed IL-1-induced changes by targeting inflammation and gene expression.
Using a unique technique combining behavioral test with microdialysis, this study observed a dynamic correlation between hippocampal ACh release and memory performance after each IL-1β administration. The results demonstrated that the reduction of ACh release was directly related to the memory deficit during memory retrieval phase, and E-EPA blocked the ACh reduction after IL-1β administration, and improved the memory. Furthermore, we have previously reported that IL-1RA can reverse IL-1-induced memory impairment, and this study further demonstrated that the effect of IL-1RA on memory is through reversing IL-1-induced decreases in ACh release and NGF expressions. The section below will discuss each step of the two pathways (shown by Fig. 1), by which IL-1 may impair memory and E-EPA improves memory.
IL-1β is a well-known stimulator for the HPA axis (Dunn 2000), and administration of RU 486 can significantly reduce corticosterone elevation and attenuate the memory impairments induced by i.c.v. IL-1 β administration (Song et al. 2004). This study revealed the mechanism by which glucocorticoids may affect ACh function in this model. It has been reported that stress can increase or decrease ACh release from the hippocampus, which is causally related to an increase in glucocorticoid secretion (Mizuno and Kimura 1997; Mitsushima et al. 2003). In this study, saline or IL-1 injections, as a mild stressor, significantly increased ACh release (for 20 min). After RU 486 treatment, the short increase in ACh release was significantly lower than that observed in the saline group, suggesting that the stress-induced ACh release may be partially related to glucorcoticoid effects. On the other hand, a deficit in working memory and decreased acetylcholinesterase activity or ACh release were reported in the hippocampus of rats that received chronic stress or chronic corticosterone administrations (Masuda et al. 2005; Srikumar et al. 2006). In this study, we showed that IL-1β administrations induced a significant and longer reduction in ACh release, which is different from the effect of the injection. Since both changes in ACh release could be partially but significantly attenuated by RU 486, it is possible that amount and duration of glucocorticoid secretion in response to different stressors may exert different effects on ACh release. The result from this study demonstrated that the fact that RU 486 improved memory may be, at least partially, related to the glucocorticoid effect on hippocampal ACh release. Since RU 486, an antagonist of glucocorticoid II receptor, did not completely reversed injections- and IL-1-reduced ACh release, there could be other factors, such as other glucocorticoid receptors, involved in ACh increase.
NGF also plays an important role in learning and memory as result of its enhancement of ACh and LTP functions (Isacson et al. 2002). This study is the first to demonstrate that subchronic IL-1β administration significantly reduced hippocampal NGF expression, which is associated with reduced ACh release and the memory impairment. IL-1β may reduce NGF in two ways. First, glucocorticoids may up- or down-regulate NGF mRNA expression, depending on its amount and acting duration (Sapolsky 2000; Nichols et al. 2005). Excessive glucocorticoid secretion may suppress NGF functions (McLay et al. 1997). Down-regulated NGF functions were reported in animals exposed to psychological stress (Colangelo et al. 2004), and in an AD model (Salehi et al. 2006). However, once RU 486 administration in this study did not significantly attenuate the down-regulation of NGF mRNA expressions. This result may suggest that glucocorticoids were not involved in the down-regulation of NGF and mRNA expressions or longer treatment with RU 486 may be needed.
The other possible mechanism could be that IL-1β directly affects astrocyte functions, which changes NGF synthesis. In an AD model induced by aluminum, increased proinflammatory cytokines were associated with a decrease in NGF expression (Johnson and Sharma 2003). The result from this study supports that subchronic IL-1-induced inflammation down-regulates NGF expressions since IL-1RA significantly reversed the reduction of NGF and increased hippocampal IL-1 expression. In addition, we have previously reported that a treatment with anti-inflammation drug celecoxib inhibits glucocorticoid secretion, reverses NGF reduction, and improved memory in a rodent model of depression (Song et al. 2009). Therefore, both glucocorticoids and NGF may contribute to the reduction of ACh release and memory impairment in this model.
It should be emphasized that many studies have reported that inflammation can increase NGF expression and IL-1 has neuroprotective effects (Shaftel et al. 2007). We have recently found that acute IL-1β administration up-regulates, while subchronic IL-1 β administration down-regulates NGF mRNA expressions (Song et al. 2008). It is possible that during acute neuroinflammation and brain injury, the function of NGF and other neurotrophins may be enhanced to protect the brain. However, subchronic and chronic inflammation may cause astrocyte apoptosis, which reduces neurotrophin syntheses (Takuma et al. 2004).
Whether IL-1 is degenerative or protective in neurodegeneration and brain injuries also depend on its concentrations (Pinteaux et al. 2009). Low concentrations of IL-1, such 1-15 ng, were found to favorite microglia-initiate neuroinflammation, which causes cell death, while high concentration of IL-1 (500 ng) exerts neuroprotective effects (Pinteaux et al. 2009). In a model of IL-1β over-expression, increased IL-1 expressions (8–130 folds) was associated with up-regulation of astrocyte marker glial fibrillary acidic protein and a reduction in amyloid pathology (Shaftel et al. 2007).
Finally, this study revealed important mechanisms by which E-EPA benefits memory in this model. In our knowledge, this is the first report to show that E-EPA can modulate ACh release during learning and memory. As discussed above, glucocorticoids and NGF are important modulators for ACh release and memory. E-EPA effects on ACh and NGF may be related to its anti-inflammatory function (as shown by Fig. 1) because E-EPA significantly down-regulated IL-1β mRNA expression, and prevented IL-1-induced reduction in body weight, which are consistent with our previous findings that E-EPA can suppress inflammatory responses (Song et al. 2003, 2004; Su 2009). Previously, we have also reported that E-EPA can significantly reduce glucocorticoid secretion induced by IL-1 administration (Song et al. 2003, 2004). Therefore, anti-inflammation may reduce glucocorticoid production and restore astrocyte function. Furthermore, E-EPA may directly regulate ACh release because n-3 fatty acids can change neuron membrane fluidity and viscosity, especially in neuronal synapses (Puskas and Kitajka 2006). A decrease in n-3 fatty acids has been reported in AD patients or in Tg2576 AD mouse model of AD (Calon et al. 2004). Therefore, chronic feeding animals with n-3 fatty acids E-EPA may change membrane components and thereby benefit ACh release. Dr Lynch’s team has previously revealed the mechanism by which E-EPA improves memory through blocking IL-1-induced the inhibition on LTP (Lonergan et al. 2004; Lynch et al. 2007). In this study, our findings add further evidence that E-EPA may improve memory by the modulation of ACh and neurotrophin functions.
In conclusion, this study provides the first evidence that IL-1β can reduce ACh release, which is directly correlated to rat memory deficits in the radial maze. Furthermore, the results demonstrated that glucocorticoid and NGF may involve in IL-1-induced changes in ACh release. N-3 fatty acid E-EPA by suppressing inflammation blocked IL-1 effects on ACh release and NGF expression, and significantly improved memory.