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(1) The activated CaMKII moves toward the glutamate receptor and phosphorylates the receptor to further activate the glutamate receptor.
(2) Activated CaMKII phosphorylates ion channels, thereby altering neuronal excitability.
(3) Activated CaMKII phosphorylated intermediate fibers alter the morphology, synaptic number and structure of neurons. Sun Chenyou et al reported that swimming training allowed the removal of Mg2+ in the NMDA receptor channel to prevent ca2+ influx, such that the channel opened, ca2+ inflow, and then activated ca2+/CaM second messenger, Ca2+/CaM activated CaMKII gene expression, thereby passing CaMKII Autophosphorylation increases phosphorylation of glutamate receptor subunits, ensuring efficient synaptic signaling and improving learning and memory.
Advances in research on animal experiments influencing learning and memory
Shanghai Xinsoft Information Technology Co., Ltd.
Abstract: The main research methods and methods of physiological psychology are to study the overall level or local organs and tissues of experimental animals by establishing relevant animal models, applying certain influencing factors, and using special measurement methods and indicators. Animal experiments can overcome many limitations of human experiments and achieve the desired results. Along with the continuous development of sports biology science, the production of animal models of exercise and learning and memory, and the measurement of indicators of measurement will be continuously improved, and will play an important role in the prevention and treatment of Alzheimer's disease and brain health related problems.
Keywords: learning and memory; animal model; movement mode; maze
Alzheimer's disease (also known as Alzheimer's disease) is a disease characterized by progressive cognitive dysfunction and memory impairment. With the aging of the social population, the incidence of Alzheimer's disease has increased year by year, and it has become a worldwide health problem that has attracted more and more attention. It not only affects the quality of life of patients, but also imposes a heavy burden on families and society. According to relevant literature, appropriate exercise can achieve significant effects in improving cognitive, memory and behavioral abilities in patients with Alzheimer's disease or ischemic cerebral infarction. The research on exercise and learning and memory has been limited by non-invasive indicators such as biochemical markers of brain nerves and other behaviors due to limitations of experimental methods and detection methods. In the late 20th century, with the development of animal experiments and the application of brain molecular biology, biochemistry, and various maze methods, the study of exercise and learning memory entered a new stage.
1 Animals influence animal models in learning and memory research
Establishing an ideal experimental animal model of learning and memory is the basis for conducting research work on exercise and learning and memory. The preparation of animal models should try to simulate clinical pathogenic factors, in order to obtain scientific data on pathogenesis, exercise intervention and other aspects.
1.1 Alzheimer's disease (AD) model
The ideal animal model of AD should have the same basic characteristics as Alzheimer's disease: 1. The model animal has the main neuropathological features of AD, senile plaque (SP) and neurofibrillary tangles (NFT). Second, the important pathological changes of AD occur. Cerebral neuronal death, synaptic loss, and reactive glial cell proliferation; third, behavioral cognitive and memory dysfunction.
The research on animal models of AD has made great progress after decades of efforts. At present, it can be summarized as follows: 1. Natural aging model: SAMPs rats were successfully developed by Professor Yukio Takeda, Kyoto University, Japan, mainly to accelerate the ageing of learning and memory functions. In the recession, the central nervous system such as cortex and hippocampus are mainly pathological changes, which is an ideal model for studying brain aging and dementia. Second, the damage model: Nelson et al. injected D-galactose into the lateral ventricle of rats. After 6 weeks, immunohistochemical analysis showed NFT-like phosphorylation and Aβ amyloid deposition plaque, causing severe memory loss. H et al. used an intracerebroventricular injection of AlCl3 in mice, a long-term intraperitoneal continuous injection or oral aluminum solution to obtain an animal model of NFT pathological changes and the like. Transgenic animal model: The APP variant gene of AD is implanted into mice by transgenic technology, and its brain can produce β amyloid deposition, neuroinflammatory senile plaques, synapse reduction, astrocyte and microglia hyperplasia, neuronal degeneration. And AD symptoms such as neurological programmed necrosis.
However, each model only simulates the symptoms and pathological changes of AD to a certain extent or in some aspects. They have their own advantages and disadvantages. The appropriate model should be selected according to different experimental purposes. The overall trend ranges from single-factor damage models to composite damage models, from single-transgenic models to double-transformed or even triple-transgenic models, but there are still many problems, such as high cost, etc. At present, most laboratories in China choose less costly composite damage AD. Animal model. With the deepening of the understanding of the molecular mechanism of the pathogenesis of AD, new and more complete animal models will emerge, further promoting the understanding of the pathogenesis of AD and promoting the progress of prevention and treatment research.
1.2 vascular dementia (VD) model
The main pathogenic factor of VD is cerebral vascular ischemia. The animal model is made by vascular occlusion, embolization, middle cerebral artery infarction, photochemistry, decerebral cortex, cerebrovascular occlusion, and spontaneous VD. Models, such as: the bilateral hippocampal infarction model induced by photochemical method in China to observe the effect of post-hippocampal infarction training on learning and memory function in rats. In foreign countries, the model of right cerebral artery ischemic infarction was made by reference to the Xiaoquan line suppository method. The left front forelimb was contracted and flexed on the same side, the Horner's sign on the same side, and the left side was circled when crawling, and the left side was dumped when standing. VD is a multi-factor disease, and its development is the result of multiple pathogenic factors, multiple pathological interactions and accumulation. At present, the preparation of VD animal models is completed from the simulation of a certain aspect of human VD, and there is still a certain deviation from clinical practice.
1.3 Gene (GD) model
Shimizu et al. used the third generation gene knockout technique to develop specific NR1 knockout mice in the hippocampal CA1 region to study the role of NMDAR in the memory consolidation stage. After the NR1 gene knockout, the NMDAR excitatory postsynaptic potential disappeared in the CA1 region. There is no LTP production, and the escape latency in the hidden platform water maze test is significantly prolonged, indicating that the spatial learning and memory of the hippocampal CA1 area requires the participation of NMDAR [5]. Using transgenic technology, Tang et al. overexpressed NR2B in the forebrain of transgenic mice and found that NMDAR channels have longer open time and increased activity. These transgenic mice showed better learning and memory ability in various behavioral tests [6]. .
2 The movement of animals in the study of exercise and learning and memory
2.1 Swimming training
Swimming is one of the main means of exercise load in exercise and learning memory experiments. Rats or mice are usually used as research objects for swimming sports, and still use a still water swimming pool. Exercise intensity needs to take into account factors such as water temperature, weight, and time.
2.2 Running platform / running wheel movement
Duan's animal treadmill is mainly an experimental device designed according to the running motion of rats or mice. At present, there are many applications. The exercise intensity can be accurately controlled by adjusting the running time, running speed and running platform slope, which belongs to passive movement. The running wheel refers to the animal crawling autonomously in the cage for active exercise.
2.3 Roller mesh training
The drum type mesh trainer is a circular mesh instrument with a length of 100cm and a diameter of 60cm. The middle is divided into 4 compartments. It can train 4 rats at the same time. The base has a fixed frame. One end has a handle and can be manually pressed by 5r/. Min performs rotation training, which can train the rat's functions of grasping, rotating, walking and so on.
2.4 Balance beam training
The balance beam training uses a 170cm long and 2cm wide square wooden stick, which is placed 7cm away from the ground. It acts as a balance beam to let the rats walk on it. It mainly evaluates and trains the balance function.
2.5 screen training
The screen is 50cm × 40cm mesh belt, the mesh is 1cm × 1cm, and the left and right sides of the stencil are made of 25cm high wooden board frame. The height of the screen is 80cm from the ground. Place the screen horizontally and place the mouse on it. Then slowly raise one end, turn the screen into a vertical position within 2 s, keep it for 5s, observe whether the mouse will fall off the screen or hold the screen with the front paws, so as to objectively evaluate Fore paw grip ability and muscle strength.
2.6 Rotating Rod Training
The rod fatigue meter is trained to take a wooden rod with a length of 150 cm and a diameter of 415 cm. The point is fixed on a 3r/min rotator and alternately rotated to the left and right to evaluate and train the dynamic balance function.
3 Determination of behavioral indicators in the study of sports influence and memory
3.1 Labyrinth
The role of the maze in the formation and development of experimental animal psychology is crucial. From the beginning of the 19th century to the beginning of the 20th century, the maze has become a commonly used instrument for psychologists to test animal abilities. The labyrinth task is especially useful for laboratory testing because the instrument itself can tell the animal what they need to do (similar to the instructions for human experiments) [7]. At present, the most commonly used in the study of learning and memory are the Morris water maze, the Y labyrinth, the T labyrinth, and the radial arm maze of more than 4 arms. The subjects involved are mainly rats. The indicators commonly used in maze experiments are: the number of learning required before the subject reaches a specified standard; the number of errors and the position of each round of experiment; the time required for each round of experiments and the behavior in the experiment. The most commonly used maze methods in domestic research are the Morris water maze and the Y maze. As a tool for examining and identifying learning, the animal's learning and memory characteristics and brain mechanisms and the effects of exercise on animal learning and memory are examined.
The Morris water maze is a device designed and used by British psychologist Morris in the early 1980s for the study of spatial learning and memory in rats. It is an ideal experimental model for measuring spatial learning and memory in animals. The Morris water maze is a circular pool with a diameter of 200 cm, a height of 50 cm, a water depth of 30 cm, and a water temperature of 22 to 24 °C. The pool wall indicates 4 water inlet points, thereby dividing the pool into 4 quadrants. One of the quadrants is selected, and a platform with a diameter of 11 cm and a height of 29 cm is placed in the middle. Including: First, the positioning navigation experiment: the experimental rats are placed into the pool according to the four water inlet points of the east, south, west and north, and the time for finding the platform within 120s is recorded. Second, the space exploration experiment: the platform was removed, and the number of times the rat crossed the original platform position within 120s.
XR-XM101 Morris Water Maze
The Y labyrinth is a three-armed labyrinth with a signal light at the top of each arm. After the signal light is on for 6 s, the arm is a dangerous zone with 36V AC, which stimulates the rat to run from the bright arm to the dark arm. There is always a safe zone in the training, and the safety zones are changed in a random order. If the rat ran from the bright arm to the other bright arm after the power was turned on as an error, it was correct to run to the dark arm. Nine of the 10 consecutive tests were correct to master the maze structure. The less the number of trainings, the stronger the learning ability of the rats, which was used as an indicator to judge the learning resolving ability of the rats.
3.2 Multi-function reaction box
Experimental method: The multi-function condition reaction box was placed flat on the table of 60 cm high, the springboard was completely suspended, and the mouse tail was placed on the springboard, and the mouse could quickly find the door hole and enter the reaction box. Step into the incubation period before the electric shock, close the door hole, and use the appropriate voltage to shock the front paw 5 to 10s. This was repeated three times, and the incubation period of the mouse staying on the springboard was repeated 24 hours later, and it was recorded as the incubation period after the electric shock, and the longest observation time was 300 s. Those who do not enter the reaction tank for 300 s are considered to have good memory.
3.3 Opening behavior observation
The opening behavior observation is also called the open field experiment. The mice are placed in the opening environment before and after the exercise, and the number of cells running in 1 minute is recorded. The number of running in 3 minutes and the standing on the hind legs (including the buttress standing) And the number of free standing, the number of grooming, the number of urination, and the number of stools were used to observe the response of mice before and after exercise.
4 Evaluation indicators in sports impact learning and memory research
In the brain function localization zone, hippocampus is a classic brain region for studying animal and human learning and memory, emotion and behavioral functions, while synaptic plasticity of hippocampal nerve directly affects learning and memory function. In brain science research, the long-term potentiation (LTP) effect is considered to be a manifestation of synaptic plasticity and synaptic transmission, and is the neurocytological basis for learning and memory. Therefore, all indicators that can affect hippocampus and LTP can reflect the ability of learning and memory. Advances in research on animal experiments influencing learning and memory
4.1 Substances indirectly related to LTP
4.1.1 Apoptosis
Learning and memory are one of the main high-level neurological functions of the brain, and are the result of the interaction of different and closely related neurons. Therefore, maintaining the health of neurons and the plasticity of brain cells are prerequisites for learning and memory. It has been reported that impaired cognitive function in rats may be related to apoptosis of hippocampal neurons. Premature apoptosis of brain cells may cause brain atrophy, Alzheimer's disease, Parkinson's disease and other encephalopathy, thus affecting learning and memory. Bcl-2 and Bax are important regulatory genes involved in apoptosis. Bcl-2 is also called "survival gene", and the expression level is positively correlated with cell life. Bax is a pro-apoptotic gene that causes the organelles to release certain molecules, activates cysteine ​​proteases, and antagonizes the protective effect of Bcl-2, causing cells to become apoptotic, or by initiating mitochondrial permeability transition. The way to induce apoptosis. Wang Yang (2008) showed that long-term regular swimming can promote the expression of Bcl-2 in rats, decrease the expression of Bax, and inhibit the premature apoptosis of hippocampus cells.
4.1.2 Antioxidant substances
Free radicals have strong oxidative properties and can oxidize and damage many organs and tissues in the body, causing chronic diseases and aging effects. It can also react with unsaturated fatty acids in the bilayer structure of cell membrane to produce lipid peroxide. The latter is decomposed to form malondialdehyde. The malondialdehyde easily crosslinks with protein or lipid to form lipofuscin, which is difficult to be Lysosomal digestion, which accumulates with age, is considered to be a fundamental feature of cellular senescence. Lipofuscin mainly accumulates in the cerebral cortex and hippocampus, and the ability to learn and remember is negatively correlated with the amount of lipofuscin in the brain. Jin Hua et al (1995) reported that exercise training reduced the lipofuscin in the rat cerebral cortex. The mechanism may be that the appropriate amount of training reduces the lipid peroxidation level and weakens the cross-linking effect of malondialdehyde, thereby reducing the production of lipofuscin. , slows the accumulation in the cells and delays the aging process. The antioxidant system in the body also plays an important role in alleviating tissue damage caused by increased free radicals. One of the important antioxidant enzymes is superoxide dismutase (SOD), which catalyzes the conversion of O2- produced by biooxidation into H2O2 and O2+. To prevent damage to cells, Jenkins (1993) reported that aerobic exercise can increase SOD activity. There is also an important antioxidant in the antioxidant system, reduced glutathione (GSH), Kretzchmar (1990) reported that the concentration of GSH in the plasma of trained long-distance runners is higher than that of the average person. All of these indicate that moderate exercise can increase the antioxidant and anti-aging ability of brain tissue.
4.1.3 Synaptic plasticity
Synaptic plasticity includes synaptic transmission plasticity, developmental plasticity, and morphological plasticity. The cerebral cortex and hippocampus are brain regions related to learning and memory function. The structure and quantity of synaptic morphology in this region change during aging, including decreased synaptic density, decreased synaptic curvature, increased gap width, and thinning of postsynaptic dense material. Decreased fluidity of synaptic membranes and decreased synaptophysin content are closely related to learning and memory dysfunction. Changes in synaptic plasticity may also be the neurobiological basis of Alzheimer's disease. Ren Shanshan et al. (2010) showed that 9-week treadmill exercise can reverse the decline of synaptic number and synaptic membrane fluidity in aging mice, indicating that moderate exercise can promote synaptic plasticity in brain cognitive function zone during aging. Compensation, delay the occurrence of Alzheimer's disease.
4.1.4 Neurotransmitter in the brain
In 1958, Carlson first reported the presence of dopamine (DA) in the brain and elucidated the signaling function of DA and its role in human control of movement; Gingard discovered that dopamine and other similar transmitters stimulate nerve cells. On the basis of Greengard's theory, Kandel confirmed through the reef-sucking experiments of the sea rabbit that the memory function is caused by the direct changes of the synapses that constitute the indirect contacts of the nerve cells, and the neurotransmitter DA passes through the second. The messenger causes protein phosphorylation, which enters the nucleus and initiates new protein synthesis, which causes the organism to produce short-term and long-term memory. This shows that DA is closely related to learning and memory function. Xu Bo et al (2004) showed that long-term suitable swimming training can enhance DA in the hippocampus, nucleus accumbens and prefrontal cortex of rat brain, and then improve the learning and memory ability of rats through the positive phase regulation of neurotransmitters. .
4.2 Substances directly related to LTP
4.2.1 Brain-derived neurotrophic factor
BDNF was first reported by German neurobiologist Barde in 1982. It is mainly synthesized in brain tissue and is widely distributed in the central nervous system. The cerebral cortex and hippocampus are rich in content, which not only has a nutritional effect on the nervous system, but also Related to the regulation of synaptic function and plasticity. The main receptor for BDNF action is TrkB (trosine kinase B), which induces the formation of dimers and autophosphorylation of Trk tyrosine kinases on neuronal cell bodies through ligand receptor binding, thereby activating intracellular signal transduction pathways. Its function. Jovanovic et al. reported that BDNF facilitates the release of the neurotransmitter glutamate, which is closely related to the learning and memory process, by regulating the phosphorylation level of synaptic proteins through the BDNF/TrkB/MAPK signaling cascade. Xiong et al reported that BDNF can regulate the synthesis of NO in the central nervous system and contribute to the learning and memory process mediated by NO. Lin's research showed that BDNF phosphorylates NR1 and NR2 of postsynaptic NMDA receptors in the hippocampal CA1 region, producing LTP and improving learning and memory functions. Neeper et al. found that a week of voluntary revolving exercise significantly increased the levels of BDNF mRNA in rat hippocampus and neocortex, and then promoted learning and memory by mediating multiple signal transduction pathways in cells.
4.2.2 insulin-like growth factor-1
lGF-1 was first reported by Sallmon and Daugha-day in 1957. IGF-1 and its receptors are widely distributed in the brain, especially in hippocampus, which can promote the growth, differentiation and proliferation of nerve cells, reduce brain neuron damage caused by local hypoxia-ischemia, stimulate glial cell differentiation, and stimulate DNA synthesis. And axon growth, regulate the release of hippocampal acetylcholine, regulate the plasticity of hippocampal synapses, and have anti-apoptotic effects. In addition, IGF-1 is associated with the pathogenesis of age-related neurodegenerative diseases, such as Alzheimer's disease. Under exercise induction, IGF-1 levels in the hippocampus are elevated, and IGF-1 and BDNF in the hippocampus activate p-CAMKlI and p-MAPK II signaling cascades through their receptors, thereby increasing synaptophysin-1, synaptophysin One can promote Glu release, enhance information transmission between synapses, and enhance long-term memory. Therefore, exercise promoting IGF-1 expression may be one of the mechanisms of exercise-induced learning and memory.
4.2.3 MAPK/ERK
The MAPK/ERK signal transduction pathway is an important transmitter of a variety of extracellular signals from the cell surface to the cell. Mitogen-activated protein kinase (MAPK) is a type of serine/threonine protein kinase in cells. Extracellular signal-regulated kinase (EPK) is an important member of the MAPK family. Nuclear translocation after activation of ERK directly activates transcription factor Elk-1 and initiates transcription of immediate early genes; ERK also activates transcription factor cAMP response element binding protein (CREB) by activating ribosomal S6 kinase (Rsk), long term The formation of memory requires the initiation of gene expression. At the same time, ERK activated in the hippocampal CA1 region can increase the acetylation of histone H3. The histone is the main component of chromatin, and the DNA is tightly packed in chromatin, which contributes to gene expression. Shen reported appropriate exercise to increase the phosphorylation level of MAPK/ERK signaling pathway and improve learning and memory by inducing expression of target genes such as BDNF and other neurotrophic factors.
4.2.4 CREB
In the late 1980s, when Montrminy MRL and Yamamoto KK studied the regulation of gene transcription, they isolated and purified the cyclic adenosine monophosphate reaction from the nuclear extract of PEI2 cell line and rat brain tissue by affinity chromatography. Element binding protein (CREB), which acts as a nuclear transcription factor, plays an important role in learning and memory. The formation of long-term memory requires not only the synthesis of new proteins, but also the transcription of new genes, from the sea rabbit, fruit fly, small Numerous studies in rats and rats have shown that phosphorylated CREB is a switch for gene transcription in the hippocampus, and its gene expression enhances the formation of long-term memory in the hippocampus. Anderson BJ study showed that after 7 weeks of active training in rats, it can cause the increase of second messenger such as cAMP and Ca2+ in the central nervous system, and make CREB participate in memory formation. From the perspective of cell signal transduction, different pathways work together in CREB at the end, which activates the transcription of various memory-related target genes such as c-fos and BDNF by affecting the phosphorylation of CREB, ultimately leading to the improvement of learning and memory ability. .
4.2.5 GLU/NMDA
Glutamate is the most important excitatory neurotransmitter in the central nervous system. It is closely related to the learning and memory process by activating glutamate receptors to mediate rapid excitatory synaptic transmission. The glutamate ionotropic receptor NMDA (N-methyl-D-aspartate) is a very important post-synaptic component that not only induces excitatory-dependent synaptic plasticity, but also transmits information between cells. China also plays an important role. The formation and maintenance of LTP requires the involvement of NMDA receptors. Therefore, NMDA receptors are considered to be the main regulators of synaptic plasticity and long-term potentiation effects of cortical and hippocampal neurons, which constitute important functions and learning and memory of the central nervous system. The molecular basis, which consists mainly of two subunits, NR1 and NR2. Zhang Hui et al. (2007) reported that appropriate exercise training, such as roller mesh training and balance beam training, can promote the expression of NR1 and NR2, and then increase the density of NMDA, increase the production of LTP, improve the transmission efficiency of synapses, and facilitate learning and memory. Increased capabilities.
4.2.6 NO/NOS
Hippocampal NO is mainly derived from neuronal NOS (nitric oxide synthase), and NO has been identified as a retrograde transmitter involved in the formation of LTP. The mechanism is: post-synaptic NMDA receptor and non-NMDA receptor activation, Ca2+ influx Activates NOS with calmodulin to catalyze the production of NO; ​​NO can freely and rapidly penetrate the cell membrane, enter presynaptic components, activate soluble guanylate cyclase (sGc), and cause sGC to activate and cause cells Increased cGMP levels further promote glutamate synthesis and transmitter release; glutamate re-acts on postsynaptic NMDA and non-NMDA receptors to induce LTP. Sun Guoxin et al (2006) reported that moderate-intensity exercise can increase the expression of NOS and its subtypes in the hippocampal CA1 region, and increase the amount of NO production, thus improving learning and memory. Liu Hongyu et al (2006) reported that high-intensity training stimulated overexpression of hippocampal N0/N 0S, and excessive NO in the central nervous system has toxic effects, affecting learning and memory.
4.2.7 C-FOS
C-fos and c-jun belong to the early-inscribed gene family and are related to learning and memory. When it is rapidly induced to transcribe, the amount of mRNA in the cytoplasm increases rapidly, and the translated Fos and Jun enter the nucleus and form a heterodimeric Fos-Jun complex, which binds to the regulatory region of the target gene. It becomes a 7 bp enhancer unit of the TPA response element (TRE), thereby further affecting the expression of the target gene. It has been found that many genes closely related to memory, such as collagenase, nerve growth factor and globin promoter, have a TRE region, and the Fos-Jun complex can bind to the TRE region of these genes to induce their expression, thus, c The -fos gene affects learning and memory by affecting transcription and translational control. Yang Yifei et al (2005) reported that long-term appropriate exercise training can cause a moderate increase in cAMP, ca2+ and other second messengers in the central nervous system, thereby inducing c-fos gene expression, controlling transcription of downstream target genes, and synthesizing new proteins. Conducive to the memory function of the brain, which will provide a strong evidence for the promotion of learning and memory at the molecular level.
4.2.8 CaM/CaMK
Long-term potentiation of LTP is based on a post-synaptic dense structure (PSD), a calcium-dependent protein kinase that accounts for approximately 30% to 50% of the total PSD protein. %, the molecular switch function of memory at the synaptic site. CaMKII autophosphorylates into a Ca2+-independent activation state, and activated CaMKII has the following effects on learning and memory:
The above indicators reflect the changes and extent of learning and memory of model animals caused by exercise from different angles, but these indicators have limitations and need to integrate a variety of information to accurately judge.