| The central nervous system (CNS) has long been viewed as an immune privileged site; recent researches suggested that immune cell can enter into CNS and survey the healthy CNS. Accumulating evidence from recent studies suggests that immune function paly an important role in the maintenance neurogenesis and cognition. Long-term potentiation (LTP) is operationally defined as a long-lasting increase in synaptic efficacy in response to high-frequency stimulation of afferent fibers. LTP has been promoted as a putative neural mechanism of associative memory formation or storage in the mammalian brain. It has been widly used in physiology and pharmacology researches. Now there are little reports about the relationship between immune function and cognition, and there is no report about the relationship between immune function and LTP.In this study we established and improved the method of LTP in vivo in mice, and then different models of different immune state were used to study the relationship of immune function and LTP. Further more, the effect of Liuwei Dihuang decoction (LW) and CA4-3 (a polysaccharide derived from LW) on LTP and its mechanism were studied.1. Establishment and improvements of LTP in vivo in miceThe size of mouse hippocampus is much smaller than that of rat, and this makes it more difficulty to locating the dentate gyrus (DG) and perforant pathway (PP) of mouse hippocampus. Additionally the animal strain/age makes the location parameters various. Herein we described an improved and reliable method we developed for locating DG and PP. According to Rat/Mouse Brain in Stereotaxic Coordinates, we found the relative coordinates of DG and PP in rats and mice alike. We also observed the relation between the electrode depth and the response. Then we used relative coordinates to locate DG and PP, and the evoked response to confirm the depth of the electrode. This method was applied in KM mice, BALB/c mice, Nude mice and SAMP8 of different age in LTP experiment, and all animals were successfully evoked good popular spike (PS) and initiated LTP. Using relative coordinates avoids the difference caused by strain/age without confirming the location coordinates for each strain/age, and makes the performance of LTP experiment in vivo easy. Our results indicate that this method is reliable and useful for LTP in vivo in anesthetized mice, and it may be applied in in vivo study in rats and used for the electrode implantation for freely behaving mouse /rat model.2. The relationship between immune function and LTP2.1 Age-dependent changes of immune function and LTP in vivo in SAMP8In the 3[H]-TdR incorporation experiment, compared with SAMR1, the spleen lymphocytes'proliferation in SAMP8 was significantly decreased from 2-month old. These results suggested that the immune function of SAMP8 was significantly decreased at the early stage of its life. Then the subtypes of spleen lymphocytes were analysed, compared with SAMR1, CD4+ T cells and CD8+ T cells were significantly decreased, CD4-/CD8- double negative T cells were significantly increased, and the CD4+/CD8+ T cell ratio was significantly increased in 2-month-old SAMP8; CD4+ T cells and CD8+ T cells had no difference, CD4-/CD8- double negative T cells were slightly increased but not significantly, CD4+/CD8+ T cell ratio had no difference in 6-month-old SAMP8; CD4+ T cells CD8+ T cells were significantly decreased, CD4-/CD8- double negative T cells wre significantly increased, CD4+/CD8+ T cells had no difference in 12-month-old SAMP8. These results indicated that the decrease of T cell number was the main reason for SAMP8's immune deficiency.Then the subtypes of thymus lymphocytes were analysed, compared with SAMR1, CD4+/CD8+ double positive T cell was singnificantly decreased, CD4+ and CD8+ T cell were slightly increased in 2-month-old SAMP8; CD4+/CD8+ double positive T cell was singnificantly decreased, CD4+ and CD8+ T cell were significantly increased in 6 and 12-month-old SAMP8. These results indicated that the function of thymus was significantly decreased in SAMP8 from 2-month old.In LTP in vivo experiment, compared with SAMR1, LTP in 2-month-old SAMP8 was significantly increased, and significantly decreased in 6-month-old SAMP8, and there was no difference in 12-moth old SAMR1 and SAMP8. This result is similar to that of CFA model; it seems that increasing immune function to a certain level can decline the enhancement of LTP, and improve cognitive function. And then we observed the effect of immune function restoration on LTP in vivo in Nude mice. It is reported that immune function restoration could improve Nude mice's cognitive function. The result also indicated that increase of immune function could decline the enhancement of LTP.How could elevated immune function decline the enhancement of LTP and at the same time improve the cognitive function? LTP experiment could cause tissue damage, acute inflammation and cytokine release in situ, and cytokines can impair LTP. So the amount of IL-1β, IL-2, IL-6, IL-10 and TNF-αwere analysed in intact and LTP-used hippocampal tissue.The results in SAMP8 and SAMR1 also indicated that inflammatory cykokines was one of the reason for the increase of LTP in 2-month-old SAMP8.2.2 The effects of Freund's Adjuvant on LTP in vivo and learning-and-memory behaviors7 days after injection of IFA and CFA, BALB/c mice were used for experiments. In the 3[H]-TdR incorporation experiment, incomplete Freund's Adjuvant (IFA) had no effect on spleen lymphocytes self-proliferation and ConA induced T cell proliferation, and significantly increased LPS induced B cell proliferation; Complete Freund's Adjuvant (CFA) significantly increased spleen lymphocytes self-proliferation and ConA induced T cell proliferation, and significantly decreased LPS induced B cell proliferation. Then the subtypes of spleen lymphocytes were analysed, both IFA and CFA had no effect on their amount.Then we observed the effect of IFA and CFA on cognitive behaviors. Results showed that, both IFA and CFA had no effect on locomotor activities and Morris Water Maze test, and they significantly increased active avoidance in Shuttle Box test which indicated that IFA and CFA could improve non-spatial cognition. In LTP in vivo experiment, IFA enhanced LTP slightly, and CFA decresed LTP significantly.These data showed that IFA and CFA increased immune function in a certain extent, and also increased non-spatial learning and memory in BALB/c mice, but had no effect on spatial learning and memory. It seems that non-spatial cognition is more susceptible to short-term immune change.How could elevated immune function decline the enhancement of LTP and at the same time improve the cognitive function? LTP experiment could cause tissue damage, acute inflammation and cytokine release in situ, and cytokines can impair LTP. So the amount of IL-1β, IL-2, IL-6, IL-10 and TNF-αwere analysed in intact and LTP-used hippocampal tissue. Both IFA and CFA had no effect on these cytokines in intact hippocampal tissue, except for the decease of IL-6 by IFA. In LTP-used hippocampal tissue, IFA increased anti-inflammatory cytokine IL-10, and decreased inflammatory cytokine IL-6, and had no effect on inflammatory cytokine IL-1β, IL-2 and TNF-α; CFA had no effect on anti-inflammatory cytokine IL-10 and inflammatory cytokine IL-6, and increased inflammatory cytokine IL-1β, IL-2 and TNF-αsignificantly. So the elevated inflammatory cytokines contributed to the decrease of LTP in CFA mouse model.2.3 The effect of immune reestablishment on LTP in vivo in Nude mice3 weeks after immune reestablishment, LTP in vivo was observed in nude mice. The results showed that the enhancement of PS after TS was significantly decreaded after immune reestablishment, and indicated that enhancement immune may decrease LTP due to increase of inflammatory cytokines in situ during LTP experiment.2.4 The effects of corticosterone on LTPThe results showed that corticosterone (Cort) could inhibit the proliferation and cytokines secretion of lymphocyte, and could also decreased the cytokine level in hippocampal tissue. The enhancement of LTP could also be decreased by cort in a dose-dependent manner. These results indicated that decreased the cytokines may be one of the cause for Cort's inhibitory effect on LTP.3. The mechanism of Liuwei Dihuang Decoction's cognitive enhancing effectIn LTP in vivo experiment, single time and long-term (7 days) administration of LW and CA4-3 had no effect on LTP in intact animals. In Cort model, single time and long-term (7 days) administration of LW had significantly protective effect; single time administration of CA4-3 had no protective effect, and long-term (7 days) administration of CA4-3 had significantly protective effect. These results indicated that immune modulation may be one of the mechanisms of protective effect on Cort model.7 days after administration of LW and CA4-3, BALB/c mice were used for the ~3[H]-TdR incorporation experiment, both LW and CA4-3 increased the self-proliferation and ConA induced T cell proliferation; and decreased the LPS induced B cell proliferation. Previous results indicated that CA4-3 could attach to the surface of B cell specially. The CA4-3 on the B cell surface could prevent LPS attching to B cell, so LW and CA4-3 decreased the LPS induced B cell proliferation. Then the subtypes of spleen lymphocytes were analysed, LW had no effect on their amount. CA4-3 decreased the CD8~+ T cell. Then we observed the effect of CA4-3 on spleen lymphoctes. In the 3[H]-TdR incorporation experiment, CA4-3 increased the spleen lymphoctes proliferation in a dose dependent manner, and it also had protective effect against Cort's damage on spleen lymphoctes. The cytokine in the culture supernatants were analysed, results showed that CA4-3 increased the amount of IL-2,IL-6 and IFN-γin intact spleen lymphoctes in a dose dependent manner, and it also increased the amount of IL-1β,IL-10,GM-CS,MCP-1,TNF-αwhich under detective limit without CA4-3. Incubation with Cort all cytokine were decreased, co-incubation with CA4-3 increased these cytokines. Previous studies in our lab showed that CA4-3 had no direct effect on neuron, and its molecular weight is about 30,000 Dalton, so it could hardly get through the blood brain barrier into the CNS. This study showed that CA4-3 could both improve the immune function and had protective effect against Cort's injury on LTP. These results indicated immune modulation was one of the mechanisms for cognition improvement of LW.Conclusion:1. Established an improved, standardized and stabled method of LTP in vivo in mice.2. The decline of immune function may be one of the primary causes for the decline of cognition of SAMP8. Immune function has an important effect on LTP due to the cytokine levels in the brain.3. Immune modulation may be one of the mechanisms for cognition improvement of LW and CA4-3.
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