The Study Of The Relationship Between Spatial Memory Impairment And The Hippocampal Insulin Signals In Ovariectomied-rats

Objective:Menopause sydrome caused by decreased estrrogen levels in perimemopausal women characterized by automomic nervous system disorder. Clinical data indicate perimemopausal women with memopausal syndrome usually accompanied memory decline. Animal studies had established surgical memopause model with ovariectomized rats; it was proved that estrogen deficiency following ovariectomy (OVX) negatively affects learning and memory using Morris water maze and fear conditioning test. Literatures have been showed that estrogen replacement therapy (ERT) can enhance the LTP and dendritic spine density in hippocampus of OVX rats. Although the accumulated evidence has been indicated estrogen as a mediator implicated in modification in learning and memory, but the mechanisms of estrogen underlying the regulated roles on memory formation was still unclear. Additionally, glucose is the main source of energy in the brain. Process of learning and memory is synchronized glucose utilization in specific brain regions. Hippocampus is a critical brain areas of learning and memory, and glucose uptaking in hippocampus increased under the task-based learning behavior patterns. Glucose uptaking by means of specific membrane glucose transporter, is determinged by the different glucose transporter isoforms and local glucose level in differnet tissue. GLUT4, as the one isform of glucose transporter, distributed in skeletal muscle and adipose tissue, and the efficacy of glucose transport is mediated by insulin. In hippocampus, GLUT4 was found distributed mainly in neurons, suggesting the glucose utilization in hippocampal neurons is insulin-dependent. Moreover, researchers have reported that estrogen can improve the insulin sensitivity, enhance insulin gene transcription and insulin release. Many evidence indicates cross-talking occures between insulin signal and estrogen signal in the brain.Therefore, we want to know whether insulin signaling is involved in the effect of estrogen on learning and memory.Methods:Experimental procedure:SD femal rats were randomly divided into four groups: non-ovariectomy (Normal), ovariectomy (OVX), non-ovariectomy+tamoxifen (N+TAM), and non-ovariectomy+anti-insulin antibody (N+anti). The OVX group were received ovariectomy surgery. The Normal, N+TAM and N+anti groups were performed the same procedure without removing the ovaries. Animals recovered for 7 days after surgery. Then stainless-steel guide cannulae were stereotactically implanted to bilaterally target the hippocampus according to the following coordinates:3.6 mm posterior to the bregma,2.00 mm lateral from the midline, and 3.5 mm ventral. The rats were returned to their cages and allowed to recover from surgery for 7 days. We used Morris water maze to analyze the change of memory. After training, the rats immediately received bilateral injections of drugs or saline. All hippocampal injections were performed in 2μL per side. The injection needle was left in place for 10 min to allow complete absorption. After behavioral tests, the rats were immediately sacrificed.Tissure preparation:All the animals were sacrificed before the collection of cerebrospinal fluid and blood serum. Part of the animals in each group (n= 6) were perfused, fixed and consecutive frozen sections. Another part of the animals in each group (n= 6) immediately decapitated, separated bilateral hippocampus and frozen in liquid nitrogen.Procedure of assay:Behavioral testing, use of Morris water maze to detect changes of spatial memory. Biochemical detection, insulin levels in serum, CSF and hippocampus were detected by ELISA; Brain slices in a continuous, select the hippocampus plane (Bregma-3.30mm,-3.45mm,-3.60mm,-3.75mm,-3.90mm,-4.05mm), using fluorescence microscopy to observe distribution of 2-NBDG positve cells in the hippocampus and count the number of 2-NBDG positive cells in hippocampal CA3 region; Hippocampal tissue total RNA was extracted using Q-RT-PCR technology to detect relative expression levels of insulin mRNA expression in hippocampus; The relative expression levels of ERβ and GLUT4 in hippocampus dectected by western blotting.Results:1. Estrogen deficit impaired the learning and memory in ovarietomied rats, we found that OVX impaired the spatial memory of rats; 2-NBDG positive cells in hippocampal CA3 and the relative expression of GLUT4 in hippocampus were reduced in OVX’s group compared with that in the normal group. In additon, the relative expression of insulin mRNA and protein in hippocampus were decreased.2. Impairment of learning and memory induced by blocking of estrogen signal is associated with the estrogen receptor p. we found that the relative expression of ERp in hippocampus was decreased in N+TAM group. When compared with the Normal group, N+TAM group impaired the spatial memory,2-NBDG positive cells in hippocampal CA3 and the relative expression of GLUT4 in hippocampus was reduced; the relative expression of insulin mRNA and protein in hippocampus were significant lower.3. Impairment of learning and memory induced by estrogen dificit is mediated by hippocampal insulin signals.we found that there was spatial memory dysfunction in N+anti group; 2-NBDG positive cells in hippocampal CA3 and the relative expression of GLUT4 in hippocampus was reduced in N+anti group compared with the Normal group.Conclusion:The effect of estrogen on memory via ERβ; Insulin signaling is involved in the effect of estrogen on learning and memory; During the process of learning and memory, deficient estrogen induced insulin level in hippocampus decreased, which reduced hippocampal glucose uptake.