Effect Of Neurosteriods On Learning And Memory Ability Of Rats And Its Mechanisms

Objective: Cognitive disorders occurred in the presenium and gerontism, particularly, Alzheimer's Disease, vascular dementia, are very common neuropsychiatric disorders. Their prominent clinical features including progressing hypofunction of learning and memory, behavior disorders, abnormal feelings, etc. Neurosteroids participate in the regulation mechanism of many kinds of emotional expression such as anxiety and depress when stress happened, and neurosteroids also control convulsions, sleep, neural regeneration, biological circadian rhythm and many other physiologic functions. Studies have shown that neurosteroids have a regulatory effect on learning and memory. The neurosteroids such as PREGS and DHEA, can promote the GABAA receptor functions, and inhibit the effect of MNDA receptor, thereby enhance neuronal excitability and synaptic plasticity, promote the wakefulness, memory acquisition and consolidation. A major decrease in spatial learning after i.v. administration of AP can observed in rats. Central cholinergic system was involved in a lot of physiologic activities such as learning, memory, convulsion and attention. The balance of central acetylcholine level plays a crucial role in many advanced functions of brain. Cholinergic dysfunction, the change of metabolic enzymes levels, as well as the decrease in the number of the cholinergic receptors in different brain regions are all related to the occurrence and development of many diseases with cognitive disorders. The behavioral appearance test Morris water maze has been used in the validation of rodents models of neurocognitive disorders. The model of M receptor antagonist scopolamine induced learning and memory impairment in rats is widely used to study the damage mechanism occurred in learning and memory impairment and the special pathological change happened in presenium, as well as screening for the powerful nootropics. In the present study, the change of neurosteroids levels and behaviors performances in morris water maze of scopolamine-induced learning and memory impairment rats model were detected, the behavior performances and changes of the acetylcholinesterase levels were detected after neurosteroid intervention, in order to explore the influence and possible mechanism of neurosteroids on learning and memory. The present study will provide a experimental basis for clarification of the mechanisms of disases with cognitive disorders and the clinical application of neurosteroids.Methods:1 Grouping and drug treatment programs1.1 The preparation of scopolamine-induecd learning and memory impairment rats model16 male Sprague Dawley (SD) rats weight between 200g and 220g were designated to scopolamine model group and normal saline control group randomly with 8 in each. Each rat in model group was injected i.p. with 2mg·kg-1 scopolamine 30 minutes before the test, while in the control group each rat was injected with a corresponding volume of saline. The learning and memory ability of the rats were evaluated by the morris water maze test. The test results of the model rats obviously lower than that of saline control group demonstrate that the model established successfully.1.2 The effect of PROG on scopolamine-induced learning and memory impairment rats model40 male SD rats were designated to 5 groups randomly with 8 in each, including scopolamine model group, saline control group, low-dose PROG group (PROG-L), middle-dose PROG group (PROG-M) , high-dose PROG group (PROG-H). The PROG groups received the injected i.H. with respectively 4, 8, 16mg·kg-1 PROG 60 minutes before the morris water maze test, rats in saline control and model group received the equal volume of blank oil at the same time. Each rat in model group and PROG groups was injected i.p. with 2mg·kg-1 scopolamine 30 minutes before the test, while each rat in saline control group received the equal volume of saline. 2 Index detection2.1 Morris water maze testThe place navigation trials were conducted during 1-5 day. The rats were put in to the tank facing the wall at the same point everyday. Record the escape latency when the rats finding and climbing the platform. If rats fail to find the platform in 180s, record the escape latency as 180s, and guide them to the platform. Each rat is put to the platform and stay for 10s, whether find the platform or not. The spatial probe trials were conducted at the 7th day, remove platform, and the rats were put into the water facing the wall at the same point. Record the time when rats reach the position of the original platform first time and the frequency of rats passing through the position of the platform.2.2 Extraction and quantification of neurosteroidsRats were rapidly enthanized by decapitation after the spatial probe trial, trunk blood and brain regions were stored in–70℃for neurosteroids detection. Samples were extracted with Ethyl Acetate/n-hexane(9/1,v/v) for disas- sociative neurosteroids and the extractions were further purified by solid phase extraction using Oasis HLB cartridges. High performance liquid chromatography-mass spectrometry (HPLC-MS) was used to quantify the neurosteroids in brain tissue and plasma.2.3 Quantification of acetycholinesterase activity in the samplesRats were rapidly enthanized by decapitation after the spatial probe trial, separate the cortex and hippocampus in the cold saline. Tissue homogeneization were made by normal saline 10% (m/V) after weighting, centrifuged 3500r·min-1 for 10min at 4℃. A reaction determination was strictly operated by using supernatant referring to kit instructions. Determine protein content of each sample by ultraviolet absorption spectrophotometry at the same time in order to conduct statistics calculation.Results:1 The identification of neurosteroid assayIn the present study, the linear calibration curves of neurosteroid were obtained in the 0.015~4.5 ng·ml-1. The inter-day and intra-day coefficients of variations of the DHEA, PREG, AP and PROG in the brain were less than 7.6% and 7.2%, 7.7% and 7.9%, 8.0 and 5.7, 7.9 and 4.5 respectively (n=5); The inter-day and intra-day coefficients of variations of the DHEA, PREG, AP and PROG in plasma were less than 8.1% and 7.0%, 6.7% and 12.3, 8.0% and 6.4%, 8.1% and 6.6% respectively (n=5).2 Morris water maze testPlace navigation trials results showed that the escape latency rats in both model group and control group shortened gradually. The escape latency of rats in model group began to become longer than that of the control group at the 3rd day, the difference was significant (P<0.05). In the spatial probe trials, the time rats in model group reach the position of original platform first time was longer than that of the control group (P<0.05). The frequency of rats in model group passing through the position of the platform was significantly less than that of the control group. (P<0.05). All the above showed that the scopolamine-induced learning and memory impairment rats model was successfully prepared.In the morris water maze test with PROG pre-treated, the place navigation trials results showed that escape latency of rats in each group reduced with the extension of training time. The escape latency of rats in low, middle-dose PROG groups began to shorten significantly at the 3rd day compared with that of the model group (P<0.05). The escape latency of rats in high-dose PROG group started to shorten at the 2nd day compared with that of the model group (P<0.05). However, among the three dosage groups, there was no significant difference in comparison. In the spatial probe trials, the time of the PROG groups for first reaching the original position of the platform were shortened significantly than that of model group (P<0.05). The frequency of rats in high-dose PROG group passing through the position of the platform was more than that of the model group(P<0.05).3 Changes of neurosteroids levels in samplesWhen scopolamine-induced learning and memory impairment happened, the PREG levels in rats frontal cortex of the model group decreased significantly compared with that of the control group (P<0.05), the DHEA, AP, and PROG levels had no significant change; DHEA, PREG, AP and PROG levels in the rats hippocampus of the model group had no significantly changed compared with the saline control group; PROG levels in the rats plasma of model group was significantly higher than that of the control group (P<0.05), but the DHEA, PREG and AP levels had no significant defferent changes compared with control group.4 Changes of acetycholinesterase activity in the samplesThe AchE activity in rats frontal cortex and hippocampus of model group were significantly higher than that of the control group (P<0.05). The AchE activity in rats frontal cortex and hippocampus of each PROG group were all lower than that of model group (P<0.05), whereas there was no significant difference compared with control group.Conclusion:1 When M receptor antagonist scopolamine induced and memory impair- ment happened, the neurosteroids synthesis metabolism were changed, the PREG level in rats frontal cortex decreases significantly, it can be inferred that the synthesis metabolism of neurosteroid may participate in the regulation effect of M receptor on the learning and memory ability.2 When scopolamine-induced learning and memory impairment happened, the neurosteroids synthesis metabolism in periphery was changed , the PROG level in the rats plasma increased significantly, it can be inferred that scopolamine may rivalry the effect of M receptor and then influence the endocrine system, especially the effect of the HPA axis, which is related with the steroidogenesis, and then participate in the regulation of the learning and memory.3 Exogenous PROG pre-treated can inhibit the AchE activity in the frontal cortex and hippocampus, improve the appearance of the scopolamine-induced learning and memory impairment model rats in the Morris water maze test. It can be infer that PROG may has the protect effect on the learning and memory ability of the scopolamine-induced learning and memory impairment model rats.