The Effects And Mechanisms Of Wnt3a In The Contextual Fear Memory

Background:Wnts constitute a family of evolutionaily conserved secreted lipid-modified glycoproteins that are generally about39-46kD in size. Wnts are devided into canonical Wnts and noncanonical Wnts according to the different signaling pathways mediated. There are three Wnt signaling pathways well characterized, including the canonical Wnt/(3-catenin, the non-canonical Wnt/Ca2+and Wnt/planar cell polarity pathways.It is known that Wnts and components of the Wnt signaling pathways are widly expressed in the nervous synstem. Wnts were reported to play critical roles in the development of the nervous system, including hippocampal formation, axon pathwayfinding, dendritic morphogenesis and synapse formation. Recently, it is showed that Wnts also exert functions in the adult nervous system. Recently, increasing evidence has shown that Wnts also exert functions in the synaptic plasticity and memory in adults. It is found that Wnt1mRNA in the amygdala decreased immediately following cued fear conditioning, and the infusion of Wnt1protein prior to training impaired long-term memory (LTM) in cued fear conditioning. In contrast, in the hippocampal dependent memory paradigm, spatial learning in a hidden platform water maze induced the elevation of Wnt7and Wnt5a. In the object recognition task, the infusion of the canonical Wnt antagonist DKK1into the dorsal hippocampus (DH) immediately after training impaired the memory consolidation, which suggests canonical Wnt signaling is required for hippocampal-dependent memory consolidation. To date, there are few reports about the roles of Wnts in the memory formation. In these studies, learning models and brain regains studied were different. The contribution of Wnts in learning and memory formation in animals is not well understood to date. It remains puzzled that which Wnt molecule was involved in memory formation and which signaling pathways were involved in the memory formation.Contextual fear conditioning (CFC) is a well established model to study hippocampal dependent memory processes, where the subject associates an aversive stimulus, such as a footshock [termed the unconditioned stimulus (US)], with a distinctive context [termed the conditioned stimulus (CS)]. After CFC training, the freezing time was increased when animals were returned into the distinctive context (CS). Freezing, a kind of animal defensive behavior, is regarded as a reliable index to evaluate the CFC memory of rodents. It is well documented the involvement of the DH in CFC.In the present study, using the CFC paradigm and a variety of molecular and pharmacological approaches, we aimed to investigate which Wnts in the hippocampus played roles in CFC memory formation and its underlying mechanism. Our study will help further understanding of the precise regulation of Wnts in different memory phase depending on different neural circuit, which will privide new target for the intervention of CFC memory. Objective:1. To investigate which Wnts in the DH played roles in CFC memory formation. 2. To investigate the mechanism of Wnt signaling on CFC memory formation.Methods:1. CFC memory formationCFC procedure can be divided into training and test. In brief, Mice were put into the conditioning chamber and allowed to habit for120s without any stimulation (habituation), then they received three consecutive foot shocks (0.4mAor0.7mA,2s duration each) through a stainless steel grid floor. Each foot shock was separated by a60s time period. After an additional60s following the last shock, mice were placed back to their home cages. The foot shock US was generated by a programmable animal shocker and the CS was the experimental context.Short-term memory (STM) and LTM were tested1h and24h after training respectively. The animals were returned to the previous chamber in which training occurred and tested for5min without foot shock; memory was assessed by measuring freezing behavior.2. Quantitative RT-PCRAfter CFC training, mice of experimental group were sacrificed at different time points. Different brain regions from each mouse (amygdala and DH) were dissected out and then frozen in liquid nitrogen followed by RNA extraction. RNA isolation was carried out using TRNzol-A+RNA isolation reagent.1ug of total RNA was then reversely transcribed in a final reaction mix of20ul using the RevertAid First Strand cDNA Synthesis Kit. Each sample was assayed in duplicate and relative levels of mRNA were normalized for each with expression levels of the (3-actin mRNA using the2-ΔΔCT method.3. Western blotMice were sacrificed at specific time point after CFC training. Brains were removed immediately after the animals killed and homogenized. The concentration of protein was detected using the BCA protein assay.30ug of samples were loaded to detect immunoreactivity by western blot. Densitometry analysis on the bands was calculated using Quantity one.4. ImmunohistochemistryMice were deeply anesthetized with5%chloral hydrate and perfused with0.9%normal saline followed by4%paraformaldehyde (PFA). Then the brains were removed for postfixation in30%sucrose in PBS. The brains were sectioned at30μm thickness using freezing microtome. Slices were blocked with a blocking solution (0.3%Trition X-100and10%normal donkey serum in TBS) for1h, and incubated in primary antibodies Wnt3a or GFP overnight at4℃. Slices were incubated with Donkey anti-mouse Alexa488or Donkey anti-rabbit Alexa488for1h at room temperature. Microphotographs were captured by using a confocal fluorescence microscopy with a Carl Zeiss LSM-780microscope (Microstructural Platform of Shandong University). Images were analyzed using Imaging J.5. Surgical procedureMice were anesthetized and restrained in a stereotaxic apparatus (8001, RWD Life Science), and implanted bilaterally with guide cannulas (stainless steel,26gauge) to the DH or amygdala. For security, the cannulas were fixed in place with acrylic dental cement and a stylus was placed in the guide cannula to prevent clogging. Mice were allowed to recover for1week before training.6. Lentivirus production and transfection293T cells were seeded into10cm culture dishes at a density of4-5×106cell/ml and achieved～80%confluency24h later. Two hours before transfection, the medium of dishes was refreshed with2ml medium. Four plasmids are transfected into293T cells:one transfer vector, o two packaging vectors, and one envelope vector. After48-72h, supernatant containing the virus is removed and centrifuged to concentrate virus. Lentivirus carrying an active-3-catenin expressing cassette (referred to as ’lenti-active-β-catenin’) or a green-fluorescent protein (referred to as ’lenti-GFP’) with a final infectious unit titer of109TU/ml was used for microinfusion or stereotaxic injections.Results1. Selective induction of Wnt3a expression in the DH following CFC trainingTo investigate the role of Wnts in CFC memory, we initially examined various Wnts mRNA and protein changes at various time points DH after CFC training using Real-time PCR and western blot. Specific changes of Wnt3a mRNA and protein after CFC training were observed in DH. Compared with the time course of Wnt3a mRNA levels change, Wnt3a protein in the DH showed a delayed increase. In addition, these changes were specific to Wnt3a. Real-time PCR results showed no changes in other Wnts (Wnt1, Wnt3, Wnt4, Wnt5a and Wnt7a). Interestingly, Wnt3a mRNA did not change significantly in the amygdala, a brain region also involved in CFC memory formation. To further investigate whether CFC training-induced Wnt3a mRNA change was specific to associative fear learning rather than only exposure to either context or shock alone. Mice were separated into the following four groups:naive mice as control group, context exposed group, immediately shock group and contextual fear conditioning group. The results revealed that levels of Wnt3a mRNA were only elevated in the associative fear learning among the four groups.2. Wnt3a dependent P-catenin and Wnt/Ca2+signaling pathways activation after CFC trainingWnt3a is reported to activate both the canonical Wnt/β-catenin and noncanonical Wnt/Ca2+signaling pathways. Whether the Wnt/β-catenin and/or noncanonical Wnt/Ca2+pathways downstream of Wnt3a are activated during CFC memory formation are still unknown. Results showed that although the total levels of GSK3β were not changed, p-GSK3(3(Ser9) levels in the DH were increased starting at1h and peaked at3h after CFC training. Furthermore, we observed that although the total β-catenin levels in the DH after CFC training had no significant change, the levels of active β-catenin and nuclear β-catenin were elevated at2h and3h after training, suggesting that CFC training induced the activation of Wnt/β-catenin signaling pathway. Furthermore, Real-time PCR results showed that the mRNA levels of axin2and tcfe2a were significantly increased at2h after the CFC training, suggesting that CFC training lead to the activation of downstream targets of Wnt/β-catenin pathway.In addition to the canonical Wnt/β-catenin pathway, Wnt3a is also reported to activate noncanonical Wnt/Ca2+-CaMKll pathway. Our results showed that the synaptic p-CaMKII levels were significantly increased at15min after CFC training, whereas the levels of CaMKII in the SNS were not significantly changed. To be noted, the synaptic p-CaMKII levels were elevated at15min after CFC training when Wnt3a protein levels did not change. It is possible that CFC training may induce rapid activity-dependent Wnt3a release, which contributes to the increased synaptic p-CaMKII levels. To confirm the release of Wnt3a, we performed fluorescence immunostaining of Wnt3a in the DH immediately or15min after CFC training and measured the fluorescence signaling intensity for Wnt3a positive cells in the DH to detect the release of Wnt3a. Compared with naive group, the values of signaling intensity for Wnt3a-positive cells in the DH were significantly reduced, suggesting a rapid Wnt3a release following the CFC training.Furthermore, we found that the increased levels of synaptic p-CaMKII, p-GSK3(3and nuclear β-catenin were reversed by Wnt3a antibodies injection before CFC training, suggesting that Wnt3a was responsible for the activation of Wnt/Ca2+-CaMKM and Wnt/p-catenin signaling pathways induced by CFC training.3. Microinfusion of Wnt3a antibody before training impaired the acquisition of CFC.To assess if Wnt3a is required for CFC memory formation, Wnt3a antibody was bilaterally infused into DH15min before CFC traing.Mice injected with Wnt3a antibodies showed a significant decrease in freezing time during fear training, suggesting that Wnt3a in the DH was required for the acquisition of CFC memory formation. Furthermore, we found that STM and LTM were also impaired by Wnt3a antibody microinjection in the DH. In addition, no effect on the STM and LTM was found when Wnt3a antibodies were injected into amygdala.4. Microinfusion of Wnt3a antibody immediately after training impaired the CFC consolidationWe next assessed whether Wnt3a was involved in other memory processes such as consolidation or expression by varying the time point of Wnt3a antibody microinfusion into DH. Our result showed the intact STM and impaired LTM after microinfusion of Wnt3a antibody immediately after CFC training compared with the vehicle group, suggesting that Wnt3a was also involved in the CFC consolidation. However, our results showed that Wnt3a was not necessary for the expression of CFC.5. Wnt-Ca2+/CaMKII and Wnt/p-catenin signaling pathways downstream of Wnt3a are respectively involved in the CFC acquisition and consolidationTo determine the signaling pathways coupled to Wnt3a mediated CFC consolidation, Wnt3a antibodies were microinjected immediately after CFC training. Results showed that Wnt/β-catenin but not Wnt/Ca2+-CaMKII signaling pathway might be involved in Wnt3a mediated consolidation of CFC.To further distinguish the roles of Wnt/β-catenin and Wnt-Ca2+/CaMKII signaling pathways in different CFC memory processes,DKK1and sFRP1were applied. Immunoblot analysis showed that DKK1treatment selectively blocked the activation of the canonical Wnt/p-catenin signaling pathway but had no effect on the noncanonical Wnt-Ca2+/CaMKII signaling pathway induced by CFC training.However, sFRP1could block both the canonical Wnt/β-catenin and noncanonical Wnt-Ca2+/CaMKII signaling pathways activated by CFC training.Furthermore, we found that DKKI selectively impaired the LTM but not the STM while sFRP1in the DH disrupted both the STM and LTM of CFC. These data suggested that the Wnt-Ca2+/CaMKII signaling pathway was involved in the acquisition, while the Wnt/β-catenin signaling was involved in the consolidation of CFC.6. Wnt3a is sufficient for CFC memory formationWe used a weak CFC training protocol in which mice showed significantly attenuated STM and LTM memory when compared with normal training group. We found that exogenous Wnt3a infusion could enhance the activation of the Wnt/Ca2+-CaMKII and Wnt/β-catenin signaling pathway. In the behavioral test, we found that exogenous Wnt3a infusion could enhance both the STM and LTM induced by weak training to normal levels compared with the vehicle control.7. P-catenin acts downstream of Wnt3a to enhance CFC memory consolidationIn the present study, we constructed a lentiviral vector that expressed constitutively active β-catenin in which the first90amino acids (AN90) containing the GSK3P phosphorylation site were deleted to mimic active Wnt signaling. Our results showed that overexpression of constitutively active β-catenin in the DH enhanced the LTM but not the STM of CFC, suggesting that constitutively active β-catenin enhanced the CFC memory consolidation.To confirm β-catenin acts downstream of Wnt3a to mediate CFC memory consolidation, four weeks after infusion of lenti-abc virus or lenti-GFP virus, Wnt3a antibodies or vehicles were bilaterally injected into DH15min before CFC training. Results showed that in either Wnt3a antibodies or vehicles injection group, lenti-abc virus injection could enhance LTM but have no effect on STM of CFC compared with lenti-GFP group, which further confirmed that β-catenin acted downstream of Wnt3a to mediate CFC memory consolidation.Conclusion:1. CFC training could specifically induce the increased Wnt3a expression.2. We found CFC training could induce rapid activity-dependent Wnt3a release in advance of the increased Wnt3a synthesis. Moreover, we found that CFC induced the activation of Wnt/β-catenin and the Wnt/Ca2+signaling pathway which were dependent on Wnt3a.3. Wnt3a in the DH was not only necessary but also sufficient for CFC memory.4. Wnt3a activated Wnt-Ca2+/CaMKII and Wnt/β-catenin signaling pathways were respectively involved in CFC memory acquisition and consolidation.5. Overexpression of constitutively active β-catenin in the DH could rescue the deficit in CFC memory consolidation but not acquisition induced by Wnt3a antibody injection.Significance:Our study will help us understanding the role of Wnt3a in CFC memory formation and provide new target for the intervention of CFC memory.