基因敲除与学习、记忆：现状、问题和展望

基因敲除技术的应用使学习、记忆分子机制的研究出现了新的突破.目前已报道了多种学习、记忆以及LTP、LTD有缺陷的基因敲除动物,发现多种基因在学习、记忆的形成过程中必不可少.然而,现有研究的一个较大问题是忽视了遗传背景基因在表型改变中的作用,被认为由突变靶基因造成的表型缺陷实际上可能是由背景基因而不是由突变基因造成的.要排除背景基因的作用,必须建立新的ES细胞,选择纯遗传背景的小鼠品系,并且在时间、范围和程度上对基因敲除进行精细的控制.     

Reduced expression of the vesicular acetylcholine transporter causes learning deficits in mice

Storage of acetylcholine in synaptic vesicles plays a key role in maintaining cholinergic function. Here we used mice with a targeted mutation in the vesicular acetylcholine transporter (VAChT) gene that reduces transporter expression by 40% to investigate cognitive processing under conditions of VAChT deficiency. Motor skill learning in the rotarod revealed that VAChT mutant mice were slower to learn this task, but once they reached maximum performance they were indistinguishable from wild-type mice. Interestingly, motor skill performance maintenance after 10 days was unaffected in these mutant mice. We also tested whether reduced VAChT levels affected learning in an object recognition memory task. We found that VAChT mutant mice presented a deficit in memory encoding necessary for the temporal order version of the object recognition memory, but showed no alteration in spatial working memory, or spatial memory in general when tested in the Morris water maze test. The memory deficit in object recognition memory observed in VAChT mutant mice could be reversed by cholinesterase inhibitors, suggesting that learning deficits caused by reduced VAChT expression can be ameliorated by restoring ACh levels in the synapse. These data indicate an important role for cholinergic tone in motor learning and object recognition memory.     

Tfm-AR modulates the effects of ApoE4 on cognition

Female mice are more susceptible to apolipoprotein E (apoE4)-induced cognitive deficits than male mice. These deficits can be antagonized by stimulating androgen receptors (ARs). To determine the role of AR in the cognitive effects of apoE4, we backcrossed mutant mice with a naturally occurring defect in the AR [testicular feminization mutant ( tfm )] onto the Apoe −/− background to eliminate mouse apoE gene resulting in non-functional AR, and crossed the tfm / Apoe −/− female mice with apoE4 transgenic male mice. We behaviorally compared Apoe −/−, apoE4, tfm, and tfm /apoE4 male mice. Apoe −/−, apoE4, and tfm mice showed hippocampus-dependent novel location recognition but tfm /apoE4 mice did not. In contrast, all groups showed hippocampus-independent novel object recognition. Hippocampus-dependent learning and memory were also assessed in the water maze. In the water maze probe trial following the second day of hidden platform training, Apoe−/− and apoE4 mice showed spatial memory retention, but tfm and tfm /ApoE4 mice did not. In the water maze, probe trial following the third day of hidden platform training, Apoe−/− , apoE4, and tfm /Apoe −/− mice showed spatial memory retention, but tfm mice did not. These data support an important role for AR in protecting against the detrimental effects of apoE4 on hippocampus-dependent learning and memory.     

Holeboard discrimination learning in mice

We have adapted to mice a holeboard-learning task, which allows simultaneous assessment of spatial working and reference-memory performance. The holeboard apparatus consists of an open-field chamber with a 16-hole floor insert. Across trials, animals have to learn that the same four holes of 16 are always baited. Here, we show that C57BL/6 mice readily acquire this task within 4 days when submitted to six trials per day or within 8 days when submitted to only four trials per day. We also show that C57BL/6, Swiss-Webster, CD-1 and DBA/2 mice acquire this task similarly, despite the fact that some differences could be observed in measures of exploratory activity during habituation and training. Moreover, the muscarinic antagonist scopolamine disrupts learning at doses of 0.1 and 1.0 mg/kg, although the highest dose appeared to have side-effects. Lastly, we found that amyloid precursor protein transgenic mice have a selective disruption in their working-memory performance only during reversal training (i.e. after a change in the configuration of the baited holes). Overall, our data indicate that this spatial learning task is well adapted to mice and will be useful to characterize spatial memory in various genetic or pharmacological mouse models.     

Social isolation rearing-induced impairment of the hippocampal neurogenesis is associated with deficits in spatial memory and emotion-related behaviors in juvenile mice

Experiences during brain development may influence the pathogenesis of developmental disorders. Thus, social isolation (SI) rearing after weaning is a useful animal model for studying the pathological mechanisms of such psychiatric diseases. In this study, we examined the effect of SI on neurogenesis in the hippocampal dentate gyrus (DG) relating to memory and emotion-related behaviors. When newly divided cells were labeled with 5-bromo-2'-deoxyuridine (BrdU) before SI, the number of BrdU-positive cells and the rate of differentiation into neurons were significantly decreased after 4-week SI compared with those in group-housed mice. Repeated treatment of fluoxetine prevented the SI-induced impairment of survival of newly divided cells and ameliorated spatial memory impairment and part of aggression in SI mice. Furthermore, we investigated the changes in gene expression in the DG of SI mice by using DNA microarray and real-time PCR. We finally found that SI reduced the expression of development-related genes Nurr1 and Npas4 . These findings suggest that communication in juvenile is important in the survival and differentiation of newly divided cells, which may be associated with memory and aggression, and raise the possibility that the reduced expression of Nurr1 and/or Npas4 may contribute to the impairment of neurogenesis and memory and aggression induced by SI.     

Over‐expressed MST1 impaired spatial memory via disturbing neural oscillation patterns in mice

The activated mammalian Ste20‐like serine/threonine kinases 1 (MST1) was found in the central nervous system diseases, such as cerebral ischemia, stroke and ALS, which were related with cognitions. The aim of this study was to examine the effect of elevated MST1 on memory functions in C57BL/6J mice. We also explored the underlying mechanism about the pattern alteration of neural oscillations, closely associated with cognitive dysfunctions, at different physiological rhythms, which were related to a wide range of basic and higher‐level cognitive activities. A mouse model of the adeno‐associated virus (AAV)‐mediated overexpression of MST1 was established. The behavioral experiments showed that spatial memory was significantly damaged in MST1 mice. The distribution of either theta or gamma power was clearly disturbed in MST1 animals. Moreover, the synchronization in both theta and gamma rhythms, and theta‐gamma cross‐frequency coupling were significantly weakened in MST1 mice. In addition, the expressions of GABAA receptor, GAD67 and parvalbumin (PV) were obviously increased in MST1 mice. Meanwhile, blocking MST1 activity could inhibit the activation of FOXO3a and YAP. The above data suggest that MST1‐overexpression may induce memory impairments via disturbing the patterns of neural activities, which is possibly associated with the abnormal GABAergic expression level.     

大鼠学习记忆能力与nov基因表达的关系

采用主动回避法进行大鼠学习记忆训练 ,选出学习成绩好和差的大鼠 ,用原位杂交、免疫细胞化学结合图像分析方法观察nov基因表达的差异。结果显示 ,novmRNA和NOV蛋白阳性神经元主要分布于海马、扣带皮质和联合皮质锥体层、基底神经节和下丘脑等脑区。好成绩组NOV蛋白免疫反应最强 ,阳性细胞最多 ,差成绩组nov基因的表达比假性条件反射组的表达稍强。novmRNA的表达在各组之间无明显的差异。以上结果提示 ,nov基因可能参与学习记忆的调控过程 ,这种调控发生在NOV蛋白翻译水平。     

Temporal Gene Expression Profile in Hippocampus of Mice Treated with <Emphasis Type="SmallCaps">d</Emphasis>-galactose

(1) Rodent chronically treated with D-galactose (D-gal) is increasingly used in pharmacological studies on aging; however, its mechanism remains unclear. The present study investigated the alterations of gene expression in the hippocampus of D-gal-treated mice. (2) C57 mice were subcutaneously injected with D-gal for 2, 4, and 8 weeks or vehicle, and then were subjected to behavioral tests. Gene expression profiles in hippocampus of each group were finally examined with cDNA microarray. (3) Both 4- and 8-week D-gal treatment led to a decrease of discrimination index in object recognition test, and 8-week D-gal-treated mice showed significant spatial learning & memory impairment in Morris water maze test. In comparison with the vehicle control group, the 2-, 4-, and 8-week D-gal treatment repressed the expression of 10, 13, and 30 genes by 2-fold or more, respectively. The early pattern was mainly characterized by down regulation of genes involved in ion transport. The delayed pattern included genes involved in protein biosynthesis, transport and signal transduction, which were highly related to synaptic functions. (4) These findings will contribute to the understanding of the mechanism of learning and memory impairment in mice treated with D-galactose.     

Systems genetic analysis of hippocampal neuroanatomy and spatial learning in mice

Variation in hippocampal neuroanatomy correlates well with spatial learning ability in mice. Here, we have studied both hippocampal neuroanatomy and behavior in 53 isogenic BXD recombinant strains derived from C57BL/6J and DBA/2J parents. A combination of experimental, neuroinformatic and systems genetics methods was used to test the genetic bases of variation and covariation among traits. Data were collected on seven hippocampal subregions in CA3 and CA4 after testing spatial memory in an eight‐arm radial maze task. Quantitative trait loci were identified for hippocampal structure, including the areas of the intra‐ and infrapyramidal mossy fibers (IIPMFs), stratum radiatum and stratum pyramidale, and for a spatial learning parameter, error rate. We identified multiple loci and gene variants linked to either structural differences or behavior. Gpc4 and Tenm2 are strong candidate genes that may modulate IIPMF areas. Analysis of gene expression networks and trait correlations highlight several processes influencing morphometrical variation and spatial learning.     

What can we teach Drosophila? What can they teach us?

A number of single gene mutations dramatically reduce the ability of fruit flies to learn or to remember. Cloning of the affected genes implicated the adenylyl cyclase second-messenger system as key in learning and memory. The expression patterns of these genes, in combination with other data, indicates that brain structures called mushroom bodies are crucial for olfactory learning. However, the mushroom bodies are not dedicated solely to olfactory processing; they also mediate higher cognitive functions in the fly, such as visual context generalization. Molecular genetic manipulations, coupled with behavioral studies of the fly, will identify rudimentary neural circuits that underly multisensory learning and perhaps also the circuits that mediate more-complex brain functions, such as attention.     

What can we teach Drosophila? What can they teach us?

A number of single gene mutations dramatically reduce the ability of fruit flies to learn or to remember. Cloning of the affected genes implicated the adenylyl cyclase second-messenger system as key in learning and memory. The expression patterns of these genes, in combination with other data, indicates that brain structures called mushroom bodies are crucial for olfactory learning. However, the mushroom bodies are not dedicated solely to olfactory processing; they also mediate higher cognitive functions in the fly, such as visual context generalization. Molecular genetic manipulations, coupled with behavioral studies of the fly, will identify rudimentary neural circuits that underly multisensory learning and perhaps also the circuits that mediate more-complex brain functions, such as attention.     

糖尿病小鼠胚胎早期发育的差异表达基因

目的利用小鼠糖尿病模型,探讨母体糖尿病环境对早期胚胎基因表达的影响。方法ICR雌性小鼠腹腔注射150mg／kg剂量STZ诱发糖尿病,与正常雄鼠交配受孕,取14d胎龄的胚胎,提取胚胎的总RNA。将Cy3和Cy52种荧光分别标记到实验组和对照组的RNA上,制成RNA探针,并与包含24859个基因的表达谱芯片进行杂交及扫描,重复3次实验,采用Agilent扫描仪进行扫描软件读取数据。结果筛选出差异表达基因397个,其中有328个基因在实验组表达量比对照组大2倍,69个基因在实验组表达量比对照组小2倍。结论母体糖尿病环境能影响早期胎儿的基因表达,通过上调代谢相关基因和下调发育相关基因影响小鼠胚胎的早期发育。为深入探讨糖尿病胚胎病理和代谢疾病的分子机理提供了基本数据和研究的方向。     

衰老性记忆障碍与脑突触体内游离[Ca~(2 )]_i的相关性

采用行为观察和生化检测相结合的方法 ,在过去工作的基础上 ,研究了 12月龄和 18月龄小鼠学习记忆能力的变化和 18月龄小鼠四个脑区 (海马、大脑皮层、四叠体和小脑 )突触体内 [Ca2 ]i 的水平 ,同时还比较了老年记忆保持良好组与记忆障碍组小鼠的脑钙水平。结果表明 ,随着年龄的增长 ,小鼠的学习记忆能力显著下降 ,上述脑区 (除大脑皮层外 )突触内 [Ca2 ]i 均明显升高 ,其中老年记忆障碍小鼠脑钙水平升高最为显著。提示 ,小鼠衰老性记忆障碍可能与其脑突触体内 [Ca2 ]i 的超载有关。     

Candidate Agtr2 influenced genes and pathways identified by expression profiling in the developing brain of Agtr2 mice

Intellectual disability (ID) is a common developmental disability observed in 1 to 3% of the human population. A possible role for the Angiotensin II type 2 receptor (AGTR2) in brain function, affecting learning, memory, and behavior, has been suggested in humans and rodents. Mice lacking the Agtr2 gene (Agtr2^−/y) showed significant impairment in their spatial memory and exhibited abnormal dendritic spine morphology. To identify Agtr2 influenced genes and pathways, we performed whole genome microarray analysis on RNA isolated from brains of Agtr2^−/y and control male mice at embryonic day 15 (E15) and postnatal day one (P1). The gene expression profiles of the Agtr2^−/y brain samples were significantly different when compared to profiles of the age-matched control brains. We identified 62 differently expressed genes (p ≤ 0.005) at E15 and in P1 brains of the Agtr2^−/y mice. We verified the differential expression of several of these genes in brain samples using quantitative RT-PCR. Differentially expressed genes encode molecules involved in multiple cellular processes including microtubule functions associated with dendritic spine morphology. This study provides insight into Agtr2 influenced candidate genes and suggests that expression dysregulation of these genes may modulate Agtr2 actions in the brain that influences learning and memory.     

Postnatal expression of the lysine methyltransferase SETD1B is essential for learning and the regulation of neuron‐enriched genes

In mammals, histone 3 lysine 4 methylation (H3K4me) is mediated by six different lysine methyltransferases. Among these enzymes, SETD1B (SET domain containing 1b) has been linked to syndromic intellectual disability in human subjects, but its role in the mammalian postnatal brain has not been studied yet. Here, we employ mice deficient for Setd1b in excitatory neurons of the postnatal forebrain, and combine neuron‐specific ChIP‐seq and RNA‐seq approaches to elucidate its role in neuronal gene expression. We observe that Setd1b controls the expression of a set of genes with a broad H3K4me3 peak at their promoters, enriched for neuron‐specific genes linked to learning and memory function. Comparative analyses in mice with conditional deletion of Kmt2a and Kmt2b histone methyltransferases show that SETD1B plays a more pronounced and potent role in regulating such genes. Moreover, postnatal loss of Setd1b leads to severe learning impairment, suggesting that SETD1B‐dependent regulation of H3K4me levels in postnatal neurons is critical for cognitive function.     

Gene-Expression Changes in Cerium Chloride-Induced Injury of Mouse Hippocampus

Cerium is widely used in many aspects of modern society, including agriculture, industry and medicine. It has been demonstrated to enter the ecological environment, is then transferred to humans through food chains, and causes toxic actions in several organs including the brain of animals. However, the neurotoxic molecular mechanisms are not clearly understood. In this study, mice were exposed to 0.5, 1, and 2 mg/kg BW cerium chloride (CeCl₃) for 90 consecutive days, and their learning and memory ability as well as hippocampal gene expression profile were investigated. Our findings suggested that exposure to CeCl₃ led to hippocampal lesions, apoptosis, oxidative stress and impairment of spatial recognition memory. Furthermore, microarray data showed marked alterations in the expression of 154 genes involved in learning and memory, immunity and inflammation, signal transduction, apoptosis and response to stress in the 2 mg/kg CeCl₃ exposed hippocampi. Specifically, the significant up-regulation of Axud1, Cdc37, and Ube2v1 caused severe apoptosis, and great suppression of Adcy8, Fos, and Slc5a7 expression led to impairment of mouse cognitive ability. Therefore, Axud1, Cdc37, Ube2v1, Adcy8, Fos, and Slc5a7 may be potential biomarkers of hippocampal toxicity caused by CeCl₃ exposure.     

负重爬梯与有氧跑台运动对糖尿病大鼠学习记忆能力的影响及其机制探讨

目的：研究负重爬梯与有氧跑台运动对糖尿病大鼠学习记忆能力的改善效果并探索其可能分子机制。方法：40只雄性大鼠,随机分为正常对照组（NC）、糖尿病对照组（DC）、糖尿病负重爬梯组（DL）和糖尿病有氧跑台组（DA）,以单次腹腔注射链脲佐菌素构建糖尿病大鼠模型。DL组在晚上进行负重爬梯训练,10次/组×3组/天,每次间歇2 min,6天/周×6周;DA组在同一时间进行20 m/min的跑台训练,30 min/d。于造模成功和运动干预结束后采用Morris水迷宫检测大鼠的学习记忆能力;第2次水迷宫测试结束后断颈处死大鼠,采用RT-QPCR法检测大鼠海马内脑源性神经营养因子（BDNF）、TRKB、CREB mRNA表达水平。结果：与NC组相比,DC组大鼠海马BDNF、CREB基因表达显著下降,学习记忆能力显著降低。与DC组相比,DL和DA组大鼠海马BDNF、CREB基因表达显著上调,学习能力显著提高;DL大鼠海马TrkB基因显著上调,大鼠空间记忆能力显著改善,而DA组大鼠海马TrkB基因无显著变化,大鼠空间记忆能力无改善,与DA组相比,DL组大鼠海马TRKB、CREB基因显著上调。结论：有氧跑台运动与负重爬梯运动介导BDNF/TrkB/CREB信号通路对糖尿病大鼠的学习能力均有促进作用,而负重爬梯运动对糖尿病大鼠记忆能力的改善优于有氧运动方式。