神经精神药理学模型 z-bo 2020-12-11 371

　　Introduction: Although the prevalence of neuropsychiatric diseases is very high, the etiology and molecular mechanism are still unclear. Zebrafish is increasingly used as a powerful animal model in neuropharmacology research and drug screening in vivo. Overall, this makes zebrafish a useful tool for drug discovery and identification of molecular pathways. Here, we discuss the zebrafish model of human neuropsychiatric diseases and drug-induced phenotypes. In addition to covering a wide range of brain diseases (from anxiety and psychosis to neurodegeneration), we also summarize the latest advances in zebrafish genetics and small molecule screening, which significantly enhance the discovery of disease models and new drug targets.

　　Zebrafish as an emerging animal model: The mechanism of widespread and debilitating neuropsychiatric diseases is not well understood, and effective treatments are often lacking. The identification of clinically relevant biomarkers, the underlying neurobiological mechanism, and the genetic and environmental factors of psychopathology are key steps to discover effective treatments. Although rodent human brain disease models have been used for this work, they are often hindered by high costs and low experimental efficiency. Zebrafish has recently received attention as a powerful animal model for a wide range of human brain diseases. The zebrafish is a small, low-cost, genetically domesticated aquatic vertebrate with high morphological, physiological and genetic homology with humans. The gene expression database and zebrafish brain atlas can also be used to explore the genomics and neuroanatomy of brain regions related to neuropsychiatric diseases. Modeling the human condition of zebrafish can reveal potential therapeutic targets and their potential molecular interactions. For example, in a recent study, a mutant mtdp-43 zebrafish discovered the therapeutic potential of methylene blue (mb). This was discovered by analyzing the efficacy of various compounds to improve amyotrophic lateral sclerosis (ALS)-like phenotype. Similarly, mtdp-43 mutant zebrafish have short, abnormally branched motor axons, increase oxidative stress, and abnormal escape responses. The administration of MB (a neuroprotective agent) can correct swimming and axon phenotypes while reducing endoplasmic reticulum (ER) stress due to the accumulation of unfolded mutant proteins. In the G93A-mtsod1 transgenic mouse model, ER stress is used as a potential target for ALS drug therapy, prompting further testing of the efficacy of several related drugs, thereby identifying and relocating the approved hypertension drug gunabenz as the potential for ALS New treatment. The zebrafish mutation model plays a key role in determining new treatment options for ALS. Two modulators of hematopoietic stem cells (hsc), recently discovered in zebrafish, are now a treatment for patients. The initial screening of nearly 2500 small molecules in zebrafish identified 35 "clues" for up-regulating the important hsc genes runx1 and c-myb, 10 of which regulate the prostaglandin pathway, indicating that it is involved in hsc regulation. One of the effective candidates, 16,16-dimethyl pge2 (dmpge2), was then tested in a mouse model, and the results showed that it can increase the number of transplanted hsc. Subsequent preclinical testing using primate blood models has achieved successful results, allowing the drug to enter the approved phase I clinical trial. These studies have recently achieved positive results in leukemia patients and proved the safety of the treatment, making it enter the phase II clinical trial. Both larvae and adult zebrafish are pre-clinical in vivo models that are very suitable for experiments, pharmacology and genetic manipulation. Due to its high transparency and small size, zebrafish larvae are particularly suitable for optical manipulation and imaging of neural activity, as well as large-scale high-throughput screening of molecular drug targets and candidate genes. With the latest development of genome editing technology and automated 3D behavioral phenotype, zebrafish has become an ideal model for studying the relationship between genotype-phenotype and genotype-drug-phenotype. In addition, zebrafish develop outside of the mother's body, mature quickly (about 90 days), and survive for about 4-5 years in the laboratory, allowing direct and convenient analysis of their pathogenesis. As a complement to the larval model, adult zebrafish exhibit complex behaviors related to cognition, reward, social behavior, and effects. Rats and mice are currently the most commonly used animals to study normal and abnormal brain functions; nearly one-third of all neuroscience papers published in 2015 used rodent models, while the use rate of other animal models (including zebrafish) It is less than 11%. However, zebrafish publications are growing faster than any other model organism, and experimental tools and resources for this organism are becoming more available. As a new animal model that needs to be validated across multiple fields, zebrafish has increasingly greater utility in high-throughput phenotype, gene and drug screening, so it is becoming more and more useful in neuropsychopharmacology and drug discovery research .

　　Zebrafish central nervous system: The overall structure, neuroanatomical features and cell morphology of the zebrafish central nervous system are similar to those of mammals. The amygdala is pathologically over-activated in clinical anxiety, social anxiety and drug abuse. The zebrafish medial eyelid showed increased expression of fos protein, which is a measure of nerve activation during acute d-amphetamine and drug-seeking behavior in Conditional Position Preference (CPP) analysis, which together supports the role of the zebrafish medial cerebral cortex. The role of the homologous structure of the mammalian amygdala has evolutionarily conserved functions to regulate key behaviors. Observing the activity of the central nervous system through imaging methods is an important step in discerning how the brain promotes normal and abnormal behavior. The small size and optical transparency of zebrafish larvae make it possible to perform high-resolution live imaging and manipulate the neural activity of active animals. For example, imaging of neuronal activity in zebrafish larval behavior is achieved by expressing genetically encoded calcium indicators and recording the activity of the entire brain using a light sheet microscope. The optogenetic neuromodulation of zebrafish larvae is particularly useful for studying the neural circuits of underlying behaviors related to brain diseases. By expressing optogenetic actuators including grooverhodopsin-2 and halorhodopsin, it has successfully activated neuronal excitement in behavioral larval zebrafish and inhibited targeted neuronal populations. So far, zebrafish visual genetics research has focused on several simple behaviors, such as flight, movement, and sensory processing. However, the optogenetic neuromodulation of zebrafish will help future research establish powerful models of complex human neuropsychiatric diseases. In addition, the adult zebrafish’s brain is large and opaque, making imaging of its neural activity more challenging. Contrast-enhanced X-ray micro-computed tomography with iodine as the contrast agent has recently been applied to adult zebrafish, which provides a three-dimensional visualization of the zebrafish brain anatomy of intact animals. Optical coherence tomography has also recently been used in adult zebrafish to generate high-resolution real-time cross-sectional images in a non-invasive manner, and then reconstruct them in 3D. Neurochemistry is generally conserved in vertebrates because they share major neurotransmitters, receptors, and transporters. Zebrafish are sensitive to major drugs such as psychostimulants, opioids, ethanol, hallucinogens, anxiolytics, antidepressants, and antipsychotics. The temporal and spatial distribution of the main neurotransmitter system of zebrafish is also similar to that of mammals. Glutamate, GABA, acetylcholine, dopamine, serotonin, norepinephrine and histamine are well described in zebrafish. For example, in addition to the d5 receptor, the main pathways and receptor subtypes of the zebrafish dopamine system are all present in the zebrafish. Recently compared the amino acid sequences of zebrafish and human d1-d4 receptors, and found that the binding sites of d1 and d3 have 100% amino acid homology, and the d2 and d4 receptors have 85-95% amino acid homology. Drugs that act on the dopamine system produce similar phenotypes, and because dopamine antagonists or removers impair movement, agonists predictably increase zebrafish movement, similar to rodents. The dopamine agonist apomorphine has a U-shaped dose-response relationship on the swimming distance of zebrafish larvae. Low doses increase center residence time (anti-anxiety effect) and high doses increase chemotaxis (anti-anxiety effect). A growing body of evidence points to changes in the neuroendocrine system in various brain diseases, including depression, anxiety, addiction, and Alzheimer's disease (AD). Activate the neuroendocrine hypothalamus-pituitary-neuronal axis (hpi) of zebrafish, release cortisol, and act on the glucocorticoid receptor (gr), similar to the human hypothalamic-pituitary-adrenal (hpa) axis. The neuroendocrine system of zebrafish is easily regulated by experiment, pharmacology and genetic manipulation, and fish cortisol can be sampled by various invasive and non-invasive methods. The genetic mutation of the gr gene in the adult zebrafish GRS357 mutant disrupts negative feedback and cortisol signaling by canceling the transcriptional activity of gr binding to cortisol.

　　Zebrafish model of major central nervous system diseases: As mentioned earlier, an obvious advantage of non-human animals (such as zebrafish) in simulating brain diseases is their adaptability to experimental, genetic and pharmacological operations. In addition, the behavioral phenotype, genetic factors, and pharmacological sensitivity of zebrafish are usually similar to those reported in rodent brain disease models and human clinical populations.

　　Depression and anxiety: Stress is a common risk factor for the development of affective disorders, including severe depression and anxiety. In mammals, the stress response is mainly mediated by the interaction between the hypothalamus, pituitary gland and adrenal glands, which together form the hpa axis. Long-term stress and excessive activation of the hpa axis may reduce the expression of gr, and ultimately reduce the ability to adapt and respond to stress events, thereby triggering depression. In rodents, depression has been extensively modeled using early life and adult stress, as well as pharmacological intervention, selective breeding, or genetic engineering. Some hallmark depression symptoms (such as low self-esteem and low mood) are difficult to evaluate in animals because they cannot clearly show self-awareness. In contrast, the assessment of other phenotypes, including annucleopenia, comorbid anxiety or sleep and neuroendocrine disorders, can be easily modeled in animals. In zebrafish, long-term use of unpredictable chronic mild stressors (UCMS) can induce depression-like states. Adult fish exposed to UCMS for 7-14 days showed reduced movement, changes in shoal behavior, and changes in body color. When applied to zebrafish grown for 5 months under social isolation, UCMS will increase anxiety-like behaviors in the new tank test. The effects of UCMS on exploratory and group/shallow water behaviors can be reversed by fluoxetine (an SSRI) and the benzodiazepine anxiolytic bromazepam. Several key pro-inflammatory molecules, such as TNFα, IL-6 and COX-2, are differently regulated after 7 days of zebrafish UCMS. COX-2 transcription is higher in patients with recurrent depression and is hypothesized to negatively affect cognitive function, affectivity, and synaptic homeostasis. Treatment with psychotropic drugs (fluoxetine, bromzepam, and nortriptyline) can reduce the expression of IL-6 and TNFα, highlighting the sensitivity of this model to established clinically active antidepressants. Other drug interventions, such as the use of reserpine, can produce depression-like reactions in zebrafish, including social withdrawal, motor retardation, and elevated cortisol, which are similar to the clinical symptoms of depression. Several genetic models have been used to study depression in zebrafish. For example, zebrafish larvae with the mutant gr (gr/s357) exhibit higher physiological responses (such as higher levels of cortisol throughout the body) and dysfunctional hpi axis, similar to those observed in humans. Anxiety disorder is a debilitating mental illness with a lifetime prevalence of about 30%, higher than any other mental illness. There are several types of anxiety disorders, including panic disorder, post-traumatic stress disorder, generalized anxiety disorder, and special phobias. The hallmark symptoms of anxiety disorders are overwhelming and exaggerated concerns about perceived threats, which significantly reduce the patient’s quality of life and work efficiency. The first treatment for anxiety disorders is usually SSRIs or cognitive behavioral therapy. Patients who do not respond to these treatments are then given selective norepinephrine reuptake inhibitors (SNRIs) or tricyclic antidepressants. However, snris or tricyclic drugs increase the risk of tolerance and dependence, thereby limiting their use. In addition, although there are many treatments for anxiety disorders, about 30% of patients do not improve. This requires identification and development of treatment methods that do not have these limitations in terms of efficacy and tolerability. One of the problems in developing new treatments is to determine the mechanism of action of biochemical targets, genetic variants, or disease pathogenesis, which indicates the need for animal models. The zebrafish model is particularly suitable for high-throughput anti-anxiety drug screening. Zebrafish larvae hatch within 3 days after fertilization, and 5 dpf can cause their bladder to inflate and produce a wide range of behaviors. For example, staying on the periphery of the arena (tentacles) reflects anxiety-like behavior, which increases after exposure to anxiety stimuli or drugs. In the light-dark test, the fish can freely explore the bright light and dark sandbanks, but when the zebrafish spend more time in the dark, this indicates an anxiety-like response that may be affected by anxiolytic or anxiolytic The two-way impact of treatment. Genetic models of zebrafish anxiety are also available, including the knockout of vesicle monoamine transporter 2 (vmat2), which produces an anxiety-like image in the case of reduced social withdrawal and exploration.

　　Epilepsy: Approximately 50 million people worldwide suffer from epilepsy, which is characterized by recurrent cramps/seizures, behavioral disorders, pathological nerve activity, and endocrine dysfunction. Epilepsy can be modeled in juvenile zebrafish and adult zebrafish (mainly through the use of convulsive drugs and genetic modification) and assessed through various behavioral and physiological endpoints. The characteristic behaviors of adult zebrafish epileptiform state are hyperactivity, unstable swimming, loss of body posture, spastic spiral swimming, and central nervous system discharge. Experimental convulsions in zebrafish can be induced by acute caffeine (250 mg/l), pentylenetetrazole (PTZ, 2.5 g/l?) and pirotoxin (100 mg/l), leading to hyperactivity, circular/spiral swimming, Spasms and elevated levels of cortisol throughout the body. These symptoms are suppressed by antiepileptic drugs in both larval and adult zebrafish, enabling people to find more effective treatments for epilepsy. For example, administration of ptz not only induces characteristic seizures, but is accompanied by the rapid transcription of c-fos and npas4, similar to the response observed in mammalian seizures.

　　Psychosis: Psychosis is manifested as disturbances in cognition, influence, motor activity and social behavior, often accompanied by abnormal glutamate signals. MK-801 is a potent NMDA antagonist for schizophrenia models in rodents, zebrafish and other animal models. Similarly, pre-pulse inhibition (ppi) refers to the attenuation of the startle response when a weak non-startle response occurs before the startle stimulus. PPI damage in patients with schizophrenia can be rescued by antipsychotic medication. ppi is reliably replicated in zebrafish larvae, including currently available genetic mutants with reduced ppi. In general, the similarity of zebrafish's neural pathways and startle response proves their utility as a platform for discovering antipsychotic drugs and regulating genes.

　　Alzheimer's disease: AD is a progressive neurodegenerative disease that causes cognitive deficits, delusions, hallucinations, and changes in mood and behavior. One of the hallmark symptoms of AD is the development of neurofibrillary tangles and amyloid beta plaques. There are two major types of AD: occasional AD (65 years old and Development) and familial AD (FAD). Sporadic AD accounts for more than 95% of all AD cases and is related to the apolipoprotein Eε4 allele. This gene wants to correspond to the zebrafish APOE. Early-onset FAD is hereditary and is related to homologous mutations in presenilin 1 (psen1), presenilin 2 (psen2) and amyloid βA4 precursor protein (app) genes, zebrafish pen1, pen2, appa, and appb genes . Zebrafish is also of great value for studying the etiology of AD, especially the role of hypoxia as a putative risk factor. Under hypoxic conditions, mitochondria may release free radicals that increase oxidative stress. In zebrafish, it is easy to reproduce the hypoxic state by reducing water oxygen levels or by chemical simulation of sodium azide. Similar to humans, hypoxic conditions in larval and adult zebrafish up-regulate several genes related to AD, including SEN1, PSEN2, AppA, AppB, and Bace1.

　　Amyotrophic Lateral Sclerosis: ALS is a progressive neurodegenerative disease that affects motor neurons in the brain and spinal cord. The zebrafish is a particularly attractive model for studying spinal cord circuit function and dysfunction, because vision is transparent in the early stages of life, and there is a high degree of functional and anatomical similarity between the zebrafish spinal cord and humans. Similar to AD, there are two broad types of ALS: familial and sporadic ALS. About 10% of ALS cases are inherited. The cause of ALS is unclear, and the gene mutations that cause ALS are highly variable. However, SOD1 is the most easily understood gene associated with ALS, and mutations in the SOD1 gene account for 20% of familial ALS cases. Zebrafish larvae overexpress mutant SOD1 with abnormal neuromuscular connections (NMJ), which deteriorate as the fish mature. The NMJ volume of the larval mutant fish gradually decreased, the performance of the forced swimming test was poor, and the response to repeated stimulation was reduced. In short, this indicates that there is a defect in the nerve input of the muscle, rather than a defect in the inherent characteristics of the muscle. Through the heat shock stress response (hsr), zebrafish can also recognize disease-causing processes early. The hsr mechanism can repair damaged proteins in stressed cells and is a useful tool for monitoring cell disturbances. In sod1 mutant zebrafish containing the hsr reporter gene (hsp70-dsred), fluorescence promotes disease location and spread throughout the brain. This method has also been used to identify neuroprotective compounds and biological targets to improve early disease processes that are not fully understood. In addition to the role of zebrafish in monitoring the progression of ALS symptoms, genetic mutants and pharmacological models also help to identify the molecular mechanisms of this disease. For example, the loss of function of the zebrafish C9ORF72 leads to the degeneration of motor neuron axons, which is accompanied by a decrease in swimming speed and motor ability of larval zebrafish. Sensitivity of zebrafish to central nervous system drugs: zebrafish and mammalian neurotransmitter systems have sufficient similarity, which helps zebrafish models to show similar pharmacology and sensitivity to various central nervous system drugs Sex. Taking specific types of neuroactive drugs as examples, we will further illustrate this aspect of the zebrafish model and its relevance to the search for new treatments.

　　Anti-epileptic drugs: PTZ is one of the most widely used convulsants in rodents and zebrafish. It can produce a large number of potent epilepsy phenotypes that are inhibited by known anti-epileptic drugs to varying degrees. Small molecule screening can be performed on zebrafish as early as 2 dpf, and the potential therapeutic effect is not only evaluated by behavioral testing, but also by monitoring neural responses. Exposure to PTZ will increase the expression of c-fos, and the expression of c-fos will be attenuated by typical anticonvulsants, anti-inflammatory drugs, natural and synthetic steroids, antioxidants, and vasodilators. However, although these drugs can reduce PTZ-induced epilepsy, their mechanism of action is still unclear. In addition to PTZ, other drugs can also induce epileptiform states in zebrafish. Kenic acid (KA) is a commonly used convulsant in rodents and has a similar effect on zebrafish. Glutamate receptor antagonists can reduce KA-induced seizures, emphasizing the role of zebrafish models in studying glutamate excitatory neurotransmission. For example, clomiazole (a histamine receptor antagonist) is effective against gene-induced epilepsy in SCN1lab zebrafish (a model of Dravet syndrome caused by SCN1a mutations), which has spontaneous insensitivity to major antiepileptic drugs epilepsy.

　　Antipsychotics: The first-generation (typical) antipsychotics are high-affinity antagonists of dopamine D2 receptors and are the most effective drugs for the treatment of psychosis. However, they can cause serious side effects, including tremor, paranoia, and anxiety. Compared with typical antipsychotic drugs, the second-generation "atypical" antipsychotic drugs have lower affinity for D2 receptors and fewer side effects. However, new treatments for psychosis are still needed, and the zebrafish model is very useful in this regard. For example, taking MK-801 can cause hyperkinesia [similar to psychomotor agitation, which is a characteristic symptom of schizophrenia and social and cognitive deficits. The exercise effects induced by MK-801 were reversed by both typical (haloperidol) and atypical (olanzapine and sulpiride) antipsychotics. However, fish exposed to MK-801 performed poorly in inhibitory avoidance tasks, and their social and cognitive deficits were restored with atypical antipsychotics.

　　sedatives: sedatives are usually used to treat anxiety disorders, produce anxiety reduction, suppression and sedation, and mainly regulate histaminergic, GABAergic and adrenergic systems. Zebrafish have similarities with mammalian GABAA and GABAB receptor subunits and histamine H1 receptors, and are highly sensitive to many sedatives. High doses of chlordiazepoxide significantly reduce swimming speed, while diazepam has a two-way effect on anxiety. Low to moderate doses reduce bottom dwell, high doses can cause sedation. In zebrafish, long-term exposure to diazepam produces similar withdrawal symptoms for 2 weeks, including anxiety during light-dark preference tasks. Research progress in zebrafish small molecule and genetic screening: prospects for automated and high-throughput screening: zebrafish models are very suitable for behavioral, genomic and proteomic testing because they combine relatively simple nerves and a variety of behavioral processes from sleep to anxiety Complexity of behavior. Customized and commercial video tracking software can record various zebrafish behavior measurements, including speed, distance traveled, location preferences, and specific patterns. The automation of zebrafish video tracking can record multiple behavioral results at the same time, eliminating the need to repeat experiments and/or manually watch and re-watch the video each time a new result is measured. The social groups of zebrafish can also be recorded. The larval zebrafish allows recording more animals (for example, 96) while tracking their swimming patterns. In this rapidly growing field of zebrafish phenomenology, another advantage of technological advancement is the automation of drug management and the computerization of stimulation exposure. For example, in the paradigm of drug addiction or fear regulation, it jointly improves the standardization of testing procedures. And provides effective data collection, increased throughput and data reproducibility. Since multiple behavioral parameters of zebrafish can be monitored in 3D, zebrafish are easier to perform high-throughput in vivo screening. For example, X, Y, and Z swimming trajectories can be tracked by two cameras to generate two integrated two-dimensional trajectory files to generate three-dimensional tracking of swimming patterns, which helps to identify unique drug-induced phenotypic characteristics. Advances in behavior recognition allow for more detailed in vivo analysis of behavioral phenotypes. For example, software that can distinguish zebrafish's tail, mid-body, and head can quantify movement well and explain complex behaviors such as chasing or biting, chasing. These methods are particularly useful in multi-pharmacological studies using drugs that act on multiple targets. Since many psychiatric diseases are related to defects in multiple neurotransmitter systems and have polygenic causes, zebrafish screening becomes particularly important. Computational techniques, such as hierarchical clustering or similar ensemble methods, can also help identify target hit rates and predict the interaction of targets with psychoactive compounds. The combination of behavioral phenotypes and computing technology is helpful for the development and discovery of new medical targets, including in-vivo behavioral phenotyping of a single target compound in two-dimensional or three-dimensional tracking, to generate its unique swimming trajectory, and to identify the cause Compounds that require behavioral phenotypes, and use algorithms to predict their biological targets for subsequent in-depth analysis to generate hypotheses for target combinations.

　　Research progress in zebrafish genetic models: As mentioned above, genetic manipulation is crucial in animal research to identify candidate genes related to the cause of disease. Inject morpholine-modified antisense oligonucleotides (MOS) or small interfering RNA (siRNA) for functional loss studies to achieve short-term genetic regulation. MOS targets specific translation inhibitors and effectively reduces gene expression. RNA interference (RNAi) is the process by which RNA molecules inhibit the translation of targeted mRNA molecules. These methods have shown effectiveness in targeting specific genes and producing altered phenotypes, although the effectiveness of MOS has recently been questioned. The development of mutant zebrafish provides a more stable behavioral phenotype because it does not produce a knockout of a specific gene, but completely eliminates the target gene product. Mutants are produced by retroviral insertional mutation, in which DNA base pairs are integrated into the existing DNA of the organism or produced by chemical mutation. Chemical mutagenesis involves exposing male zebrafish to the methylating agent ethyl nitrosourea a few weeks before mating, in order to fix the mutation before the spermatogonia matures into sperm. Mutant zebrafish and deformed zebrafish are widely used in research and provide a deeper understanding of the role and importance of specific receptors and biological targets. For example, when developing a mutation model of autism, a group of highly active genes with large gene targets was discovered, which allowed for in-depth study of functional changes related to gene deletion and duplication. In addition, the size of previously unknown gene targets has been elucidated for targeted analysis in higher vertebrates and mammals.

　　Conclusion: Neuropsychiatric diseases afflict human beings all over the world and cause huge personal and social costs. For a long time, animal models have been used in neuropsychological research to better understand human disease states and play a key role in identifying biological and molecular targets, with the aim of developing safer and more effective treatments. The zebrafish is a promising new animal model that continues to provide important insights into the etiology of central nervous system diseases. The homology of key regions of the zebrafish and mammalian brains emphasizes the application of the zebrafish model in neurobehavioral and neuropsychological research. In addition, the conservation of neural pathways between zebrafish and mammals allows two-way translation of findings. Current genetic tools, tracking technologies, and statistical algorithms can help deepen understanding of molecular pathways, develop new compounds or reuse existing drugs. Combined with the high sensitivity of zebrafish to known anti-anxiety drugs, antipsychotics and other central nervous system drugs, this provides researchers with a comprehensive model organism that can identify and hypothesize molecular targets for drug treatments Conduct an empirical test.