| Alzheimer's disease (AD) is a chronic, primary and irreversible neurodegenerative disorder characterized by deterioration of cognitive and mental functions, including learning and memory. AD is characterized pathologically by the presence of extracellular senile amyloid plaques and intracellular neurofibrillary tangles. The major component of AD senile plaques is amyloid-βprotein (Aβ), a 39-43 residue peptide. At present, the neurotoxicity of Aβand its active peptide fragments Aβ25-35 has been widely reported. Therefore, the pathogenesis of AD is associated with deposits of Aβin brain and its neurotoxicity, and the AP hypothesis in AD has been accepted widely. However, the possible mechanisms by which Aβimpairs cognitive function are complicated and unclear so far, and the effective measures in the prevention and treatment of AD are still seriously absent.Arginine vasopressin (AVP), as a neurotransmitter, is synthesized mainly in the suprachiasmatic nucleus (SCN), the bed nucleus of the stria terminalis (BNST), the medial amygdaloid nucleus (MeA), and parvocellular neurons in PVN. AVP involves in the endocrine regulation of the water balance and blood pressure, and also plays an important role, as a central neurotransmitter, in the regulation of some central nervous functions including learning and memory. It is reported that the concentration of AVP in the brain and the blood of the AD patients decreased significantly compared with the normal elderly population, indicating that vasopressinergic systems in the brain may be one of the targets of Aβneurotoxicity, and the incidence of the AD, to a certain extent, has a relationship with the decrease of AVP level in central nervous system. Moreover, AVP application is able to improve learning and memory in aged people, which suggests that AVP probably plays an important role in the improvement of memory impairment and the prevention and treatment of AD. However, the effects of AVP on cerebral function in electrophysiology and in behavior are still controversial up to now. Although it has been shown that Aβcan impair learning and memory, while AVP can enhance it, but whether AVP can reverse Aβ-induced impairment of learning and memory after co-application of AVP and Aβhas not been reported so far. The cellular mechanisms and signal transduction pathways by which AVP protects against Aβ-induced impairment are also urgently to be clarified.Therefore, the purposes of the present study are as follows:(1) Examining the effects of intracerebroventricular (i.c.v.) injection of AVP, Aβ25-35, and co-application of AVP and Aβ25-35 on rat spatial learning and memory by using a Morris water maze test. (2) Investigating the effects of AVP, Aβ25-35 and co-application of AVP and Aβ25-35 on the expression of p-CaMKII in hippocampus tissue using western blot technique. (3) Observing the effects of AVP, Aβ25-35 and co-application AVP and Aβ25-35 on the discharge frequency and discharge patterns of spontaneous single unit spikes in the hippocampal CA1 area by using multi-channel extracellular recording and spike sorting technique. So, in the present study, we tried to explore the mechanism by which AVP protects against Aβ-induced neurotoxicity in the cellular, molecular and behavioral levels and tried to provide a new idea in the prevention and treatment of AD.Part I:Arginine Vasopressin Prevents Against Aβ25-35-Induced Impairment of Spatial Learning and Memory in RatsObjective:Investigating the neuroprotective effects of arginine vasopressin (AVP) on spatial learning and memory impairment induced by AP25-35 in rats by using Morris water maze test.Methods:Adult-male Wistar rats (230-250g) were divided randomly into 4 groups (10 animals in each group):control, Aβ25-35, different concentration of AVP (0.1nmol, 1nmol, 10nmol) and co-application of AVP and Aβ25-35 groups. All drugs/vehicle were delivered by intracerebroventricular (i.c.v.) injection under the guidance of a brain stereotaxic apparatus. Morris water maze tests (Hidden platform test, probe trials, Visible platform test) were performed 2 weeks after drugs injection to obtain the ability of rat spatial learning and memory.Results:(1) In control group, the average latencies and distances for searching for the platform under water are 73.7±6.5 s,55.4±10.1 s,28.9±7.5 s,11.2±2.3 s and 9.5±1.2 s, as well as 1799.9±242.6 cm,1723.7±143.8 cm,677.5±176.4 cm,273.6±53.7 cm and 223±29.3 cm at training dayl-5, respectively. The percentages of total time elapsed and distance swam in right quadrant were 49.3±0.3% and 49.6±0.2%, respectively. (2) ICV injection of 25 nmol Aβ25-35 resulted in a significant decline in spatial learning, with longer latencies and distances for searching for the platform under water (P＜0.01), In the probe trials after removing platform, the percentage of total time elapsed and distance swam in right quadrant decreased to 35.1±0.3%and 35.1±0.2%, respectively (P＜0.01). (3) ICV injection of 0.1 nmol AVP slightly improves the behavior of learning and memory, but no significant differences compared with control groups (P>0.05). After using higher concentration (1 nmol and 10 nmol) of AVP, the latencies and the distances spent in searching for the platform under water significantly decreased than those in control (P<0.01). In probe test, the percentages of total time elapsed and distance swam in right quadrant were 57.9±0.03%and 58.1±0.04%in 1 nmol AVP (P＜0.01), and 67.6±0.02%and 67.1±0.04%in 10 nmol AVP (P＜0.01), respectively. (4) Co-application of 0.1 nmol AVP and AP25-35 did not induce any significant effect on the escape latency, distance and the percentages of total time elapsed and distance swam in the target quadrant (P>0.05) compared with those of Aβ25-35 alone group. However, co-application lnmol or lOnmol AVP and Aβ25-35 reversed the spatial learning and memory impairment induced by Aβ25-35 The escape latencies and distances were significantly decreased (P<0.01) compared to those in Aβ25-35 only group. At the same time, the percentage of total time elapsed and distance swam in the target quadrant were increased in these higher concentrations of AVP groups (P<0.01) (5) The visible platform tests showed that all drugs, including Aβ25-35 and different concentrations of AVP, did not affect the vision and the swimming speeds of rats.Conclusion:ICV injection of Aβ25-35 resulted in a significant decline in spatial learning and memory, while application of AVP alone (1 nmol or 10 nmmol) improved spatial learning and memory. Most importantly, AVP protected the spatial learning and memory against AP25-35 induced impairment in a dose-dependent manner, suggesting that up-regulation of central AVP can counteract the neurotoxicity of Aβ25-35 and may be useful in the prevention and treatment of AD.PartⅡ:Effects of Arginine Vasopressin and Aβ25-35on Phosphorylated Level of CaMKII in Hippocampus of RatsObjective:In order to clarify the mechanism by which AVP protects against Aβ-induced behavior impairment, the present study, by using Western blot technique, examined the effects of Aβ25-35 and AVP as well as co-application of these two drugs on the expression of phosphorylated Ca2+/calmodulin-dependent protein kinaseⅡ(p-CaMKⅡ) of hippocampus in rats.Methods:The rats in four different groups (control, Aβ25-35, AVP, and co-application of AVP and Aβ25-35) were decapitated after finishing Morris water maze behavioral test. The brain and bilateral hippocampus were removed in ice. Then, hippocampal slices were put into extraction buffer by sonication. The protein was extracted. The expression of p-CaMKII in the hippocampus was evaluated by using Western blot.Results:(1) Mean grey value of p-CaMKⅡof hippocampus in control group is 0.607±0.01. (2) AVP up-regulated the expression of p-CaMKⅡof hippocampus, with a higher mean grey value, being 0.745±0.02, significantly higher (P<0.01) than 0.607±0.01 in control. (3) Aβ25-35 down-regulated the expression of p-CaMKⅡin hippocampus, with a lower mean grey value, being 0.48±0.03, significantly lower than the value of 0.607±0.01 in control (P＜0.01). (4) AVP prevents against Aβ25-35-induced decrease of expression of p-CaMKⅡin hippocampus. Mean grey value of p-CaMKⅡof hippocampus is 0.593±0.02 in co-application group.Conclusion:AVP and Aβ25-35 can differentially affected the expression of p-CaMKⅡin hippocampus, up-regulation by AVP and down-regulation by Aβ25-35.Most importantly, AVP, to some extent, can protect the expression of hippocampal p-CaMKII against Aβ25-35 induced decline, suggesting that the expression p-CaMKII of hippocampus is closely associated with performance of learning and memory.PartⅢ:Arginine Vasopressin Protect against AP25-35 Induced Suppression of Spontaneous Discharges in the Rat Hippocampal CA1 Region In VivoObjective:To understand the possible electrophysiological mechanism by which AVP protects spatial learning and memory, the present study, by utilizing multi-channel recording technique, observed the effects of Aβ25-35 and AVP respectively, as well as co-application of Aβ25-35 and AVP, on spontaneous discharges in the hippocampal CA1 region in vivo in urethane anesthetized rats.Methods:A self-made bound recording electrode was inserted into the hippocampal CA1 area (AP 3.0-3.8 mm posterior to bregma, L 2.5-3.0 mm from the midline, H 2.5-3.5 mm from the dura) of rats. The spontaneous discharges were observed and recorded by using TDT-RX7 workstation. The change in discharge pattern and discharge frequency of hippocampal CA1 firing units were analized after spike sorting, a technique with which single unit activity can be extracted from usual extracellular recording. Besides Matlab analysis system was used for off-line analysis.Results:(1) Three patterns of cellular discharge were found:rapid and regular firing, slow and irregular firing and burst firing. The average frequency of the spontaneous discharges in the CA1 area in control is 7.2±0.81 Hz (n=81). (2) The average frequency of the spontaneous discharges in the CA1 area in Aβ25-35 (25nmol/5ul) rats was significantly decreased to 4.6±0.72 Hz, (n=72, P<0.01). (3) After pretreatment with AVP (10nmol/5ul), the average frequency of the spontaneous discharges was significantly increased to 7.9±0.63 Hz (n=83, P<0.05). (4) Compared with AP25-35rats, the average frequency of the spontaneous discharges in CA1 area in co-application of AP25-35 and AVP group increased from 4.6±0.72 Hz to 5.3±0.61 Hz (n=75, P＜0.05).Conclusion:The results mentioned above indicates that Aβ25-35 suppressed the frequency of spontaneous discharges of hippocampus in the CA1 area, while AVP can improve and reverse to some extent Aβ25-35-induced decline of frequency of the spontaneous discharges in the hippocampal CA1 region, suggesting that the alteration of single unit discharge and neuronal circuit activity may be involved in AVP-or Aβ-induced change in the brain higher function, including animal spatial learning and memory.
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