The Effects Of TRPC1on Environmental Enrichment-induced Neurogenesis And Cognition Enhancement And Its Underlying Mechanism In Hippocampus Of Adult Mice

BackgroundEnvironmental enrichment (EE) is associated with hippocampal plasticity, including increased neurogenesis and improved cognitive performance. Besides, EE has also been found to reduce the adverse effects on brain of stress, ageing and neurological disorders. The mechanisms of EE regulating hippocampal plasticity and improving brain function are complex, and are still not fully explained till now. Transient receptor potential1-canonical (TRPC1) channel is the Ca2+-permeable, nonselective cation membrane channels, which is selectively expressed by neurons in brain and is primarily expressed on cell somata and on dendrites of neurons. Evidences showed that TRPC1implicated in neural stem cell selfrenewal, neurite extension, promoted cell cycle process and cell proliferation, and so on. Moreover, a recent study found that TRPC1-mediated Ca2+influx via store-operated Ca2+entry (SOCE) is required for the proliferation of adult hippocampal neural progenitor cells in vitro. These results indicated that TRPC1might play important roles in the proliferation of neural progenitor cells (NPCs), hippocampal plasticity and learning and memory. However, whether TRPC1and related signaling pathways mediate EE-induced neurogenesis and cognition enhancement in adult mice has not been reported yet.Objective1. To explore the effects of environmental enrichment on TRPC1.2. To explore the roles of TRPC1on environmental enrichment-enhanced adult hippocampal neurogenesis and cognition.3. To explore the underlying mechanism of how TRPC1mediates the function of environmental enrichment.MethodsThe adult,8-10weeks old,129/SvEv (wild-type, WT) and TRPC1-/-male mice under the129/SvEv genetic background were randomly assigned to SC and EE, respectively. In each cage, the two kinds of mice were half-and-half. The effects of EE on TRPC1mRNA and protein levels were detected by using real-time PCR and Western blot. The newborn cells were labelled by intraperitoneal (ip) injection of BrdU, and then the proliferation, differentiation and survival of newborn NPCs were evaluated by immunofluorescence. Behavior tests were used to measure the cognition function and MF-CA3pathway long-term potentiation (LTP) was recorded to assess the synaptic plasticity. Moreover, to further confirm the role of TRPC1, we injected rAAV2-TRPC1or rAAV2-GFP (as control) into the hippocampus dentate gyrus (DG) of each group mice by brain stereotaxic technique, and evaluated the levels neurogenesis and cognitive memory of these mice after EE. Furthermore,to explore the underlying mechanism, the protein levels of MAPK family member and CREB in adult mice housed in SC or EE were detected by using Western blot.Results1. EE increased the expression of TRPC1in mice hippocampus.After the adult,8-10weeks old, WT mice were housed in SC and EE respectively for four weeks, the levels of hippocampal TRPC1mRNA and protein of EE mice were significantly higher than SC mice, which were detected by real time PCR and Western blot.2. Adult TRPC1-/-mice displayed impaired EE-induced neurogenesis and cognition enhancement.(1) The results of immunofluorescence showed that EE induced significantly increase of the proliferation of NPCs and the survival rate of newborn neurons in WT mice, whereas the proliferation of NPCs and the survival of newborn neurons were did not show any significant changes in TRPC1-/-after EE treatment.(2) The behavioral tests indicated that EE caused WT miceâ‘ the decrease of escape latencies in the hidden platform trial and the increase of time spent in the target quadrant in the probe trial in the Morris water maze;â‘¡a significant increase in freezing responses time of WT mice in the contextual fear conditioning;â‘¢a significant increased preference for the novel object of WT mice in the novel object recognition task. However, the performance of TRPC1-/-mice in the above mentioned behavior tests did not show any significant changes after EE.(3) The results of hippocampal slices LTP recording revealed that EE significantly increased the fEPSP slope of MF-CA3pathway in WT mice, whereas the fEPSP slope of MF-CAs pathway were did not show any significant changes in TRPC1-/-after EE.3. Overexpression of TRPC1by injection of rAAV2-TRPC1into hippocampus DG of TRPC1-/-mice rescued the impaired EE-induced neurogenesis and cognition enhancement.(1)The images of immunofluorescence showed that the proliferation of NPCs and the survival rate of newborn neurons in TRPC1-/-mice were significantly lower than that of WT mice after EE, whereas after overexpression of TRPC1by injection of rAAV2-TRPC1into hippocampus DG of TRPC1-/-mice, the proliferation of NPCs and the survival rate of newborn neurons in TRPC1-/-mice were almost restored to the level of WT mice.(2) The behavioral tests indicated:â‘ in the Morris water maze, the escape latencies in the hidden platform trial of TRPC1-/-mice were significantly higher than that of WT mice, and the time spent in the target quadrant in the probe trial of TRPC1-/-mice was significantly lower than that of WT mice;â‘¡the freezing responses time of TRPC1-/-mice was significantly less than that of WT mice in the contextual fear conditioning;â‘¢the recognition index of TRPC1-/-mice was significantly lower than that of WT mice in the novel object recognition task. However, after overexpression of TRPC1by injection of rAAV2-TRPC1into hippocampus DG of TRPC1-/-mice, the performance of TRPC1-/-in the above mentioned behavior tests were almost restored to the level of WT mice. (3) The results of hippocampal slices LTP recording revealed that the fEPSP slope of MF-CA3pathway in TRPC1-/-mice was significantly lower than that of WT mice after EE, whereas after overexpression of TRPC1by injection of rAAV2-TRPC1into hippocampus DG of TRPC1-/-mice, the fEPSP slope of TRPC1-/-mice was almost restored to the level of WT mice.4. TRPC1mediated the EE-induced adult neurogenesis and cognition enhancement might through ERK1/2-CREB pathway.To explore the underlying mechanism, we examine the level changes of proteins related with EE, adult hippocampus neurogenesis and learning and memory using Western blot, such as MAPK family members(ERK1/2, p38and JNK)and CREB protein. We found that the protein levels of phosphorylated ERK1/2, p38and CREB but not JNK were significantly increased in WT-EE mice compared with WT-SC mice; and the increased activity of ERK1/2and CREB rather than p38induced by EE was significantly suppressed in TRPC1-/-mice. Notably, overexpression of TRPC1restored the reduced pERK1/2and pCREB protein levels almost to WT levels after EE.ConclusionTRPC1mediated the enhancement effects of the environmental enrichment on neurogenesis and cognition might via ERK1/2-CREB pathway. Considering the beneficial effects of EE on a range of currently incurable brain disorders and the brain function of adult neurogenesis, TRPC1is a potentially novel therapeutic target to treat neurological diseases, such as neurodegeneration.