Proteins linked to extinction in contextual fear conditioning in the C57BL/6J mouse

Studying fear extinction is a major topic in neuroscience. No information on systematic studies on the linkage of contextual fear conditioning (cFC) with hippocampal protein levels is available and we were therefore interested in protein differences between animals with poor and good extinction. cFC was carried out in C57BL/6J mice, hippocampi were taken and proteins were run on two‐dimensional gel electrophoresis with subsequent quantification of protein spots. In‐gel digestion with trypsin and identification by ion trap MS/MS (high‐capacity ion trap) was used for the identification of significantly different hippocampal proteins between mice with good and poor performance of extinction. Signaling protein ras‐related protein rab‐7A and septin 8 levels were significantly higher in hippocampus of poor extinguishers, whereas ubiquitin carboxyterminal hydrolase isozyme L1 showed higher levels in animals with good extinction performance. A series of additional proteins showed significantly different levels between groups but the abovementioned were confirmed by immunoblotting. The abovementioned proteins have never been reported to be linked to extinction, memory, or learning and herein evidence for the involvement of several proteins in extinction mechanism as well as probably representing pharmaceutical targets is provided. Moreover, it is intriguing to demonstrate the differences between good and poor extinction performance at the protein level.     

Developmental Changes in Brain Glucose, Glycogen, Phosphocreatine, and ATP Levels in DBA/2J and C57BL/6J Mice, and Audiogenic Seizures

Abstract. Brains of rodents are primarily dependent on ketone bodies as a source of hydrogen for NADH and of acetyl-CoA during the perinatal period characterized by suckling. A mouse dam begins to wean her pups at about 14 days of age (DOA), the same age at which the brain reaches near-adult size and begins to shift to dependence on carbohydrate-derived sources of acetyl-CoA for normal function. Also at this time, the ear canals open, and mice of some strains become susceptible to audiogenic seizures (AGS). There may be a genetically determined derangement in the orderly transition from one source of brain energy to the other in AGS-prone mice, with a concomitant brief (days) reduction in the in situ energy reserve during the transition. In mice with a decreased energy reserve, a large energy expenditure within a short period of time (s), such as that induced by a substantial acoustic stimulus to newly opened acoustic pathways, might briefly lead to CNS disorganization before body energy repletion processes may occur, resulting in the onset of an AGS. Since glucose, glycogen, ATP, and phosphocreatine provide the bulk of the brain energy reserve, a developmental study was performed to measure the concentrations of these metabolites in brain tissues of DBA/2J mice (genetically/developmentally susceptible to AGS: onset at 12–14 DOA, peak at 18–21 DOA, rapid decline until 30 DOA, essentially lost by 42 DOA) and in C57BL/6J mice (not developmentally susceptible to AGS). Samples of frontal, temporal, cerebellar, and diencephalic regions were taken from mice 0–44 DOA and assayed. With the exception of higher glycogen levels in both DBA and C57 mice in cerebellar and diencephalic samples 0–16 DOA, no regional differences were found. A decrease in glycogen in all regions was observed in DBA mice 16–30 DOA, which was the inverse of susceptibility to AGS in these mice. This dip was not found in C57 mice. ATP levels were elevated in DBA mice 14–18 DOA, and glucose levels were decreased in DBA mice 24–40 DOA. These data lend support to the hypothesis that lowered brain energy reserves, or lowered access to brain reserves, underlies susceptibility to AGS.     

Differential expression and regulation of myristoylated alanine-rich C kinase substrate (MARCKS) in the hippocampus of C57/BL6J and DBA/2J mice

The myristoylated alanine-rich C kinase substrate (MARCKS) is a major protein kinase C (PKC) substrate in brain that binds the inner surface of the plasma membrane, calmodulin, and cross-links filamentous actin, all in a PKC phosphorylation-reversible manner. MARCKS has been implicated in hippocampal-dependent learning and long-term potentiation (LTP). Previous studies have shown DBA/2 mice to exhibit poor spatial/contextual learning, impaired hippocampal LTP, and hippocampal mossy fiber hypoplasia, as well as reduced hippocampal PKC activity and expression relative to C57BL/6 mice. In the present study, we assessed the expression (mRNA and protein) and subcellular distribution (membrane and cytolsol) of MARCKS in the hippocampus and frontal cortex of C57BL/6 and DBA/2 mice using quantitative western blotting. In the hippocampus, total MARCKS mRNA and protein levels in C57BL/6J mice were significantly lower ( approximately 45%) compared with DBA/2J mice, and MARCKS protein was observed predominantly in the cytosolic fraction. MARCKS expression in frontal cortex did not differ significantly between strains. To examine the dynamic regulation of MARCKS subcellular distribution, mice from each strain were subjected to 60 min restraint stress and MARCKS subcellular distribution was determined 24 h later. Restraint stress resulted in a significant reduction in membrane MARCKS expression in C57BL/6J hippocampus but not in the DBA/2J hippocampus despite similar stress-induced increases in serum corticosterone. Restraint stress did not affect cytosolic or total MARCKS levels in either strain. Similarly, restraint stress (30 min) in rats also induced a significant reduction in membrane MARCKS, but not total or cytosolic MARCKS, in the hippocampus but not in frontal cortex. In rats, chronic lithium treatment prior to stress exposure reduced hippocampal MARCKS expression but did not affect the stress-induced reduction in membrane MARCKS. Collectively these data demonstrate higher resting levels of MARCKS in the hippocampus of DBA/2J mice compared to C57BL/6J mice, and that acute stress leads to a long-term reduction in membrane MARCKS expression in C57BL/6J mice and rats but not in DBA/2J mice. These strain differences in hippocampal MARCKS expression and subcellular translocation following stress may contribute to the differences in behaviors requiring hippocampal plasticity observed between these strains.     

Anxiety and fear behaviors in adult male and female C57BL/6 mice are modulated by maternal separation

This study investigated the effects of maternal separation in C57BL/6 male and female mice during infancy on later adult fear and anxiety behaviors. Additionally, we observed the maternal behavior of the dams to examine aspects of maternal care that may be modulated by daily bouts of separation. In males, mice that experienced maternal separation during the neonatal period displayed significantly higher levels of anxiety and fear behavior, as measured by the open field test and elevated plus maze, compared to control, standard facility reared males. In females, however, maternal separation reduced anxiety and fear behavior in the open field test, but only when the females were in the diestrous phase of their estrous cycle. The 30-min daily observation of the dams revealed that the separation did not significantly alter the frequency of the maternal care provided by the dam at the time point measured. These results indicate that the emotionality of adult male and female mice can be modulated by maternal separation. However, this effect is dependent on the sex of the offspring and the phase of the estrous cycle of the female.     

A comparative phenotypic and genomic analysis of C57BL/6J and C57BL/6N mouse strains

Background

The mouse inbred line C57BL/6J is widely used in mouse genetics and its genome has been incorporated into many genetic reference populations. More recently large initiatives such as the International Knockout Mouse Consortium (IKMC) are using the C57BL/6N mouse strain to generate null alleles for all mouse genes. Hence both strains are now widely used in mouse genetics studies. Here we perform a comprehensive genomic and phenotypic analysis of the two strains to identify differences that may influence their underlying genetic mechanisms.

Results

We undertake genome sequence comparisons of C57BL/6J and C57BL/6N to identify SNPs, indels and structural variants, with a focus on identifying all coding variants. We annotate 34 SNPs and 2 indels that distinguish C57BL/6J and C57BL/6N coding sequences, as well as 15 structural variants that overlap a gene. In parallel we assess the comparative phenotypes of the two inbred lines utilizing the EMPReSSslim phenotyping pipeline, a broad based assessment encompassing diverse biological systems. We perform additional secondary phenotyping assessments to explore other phenotype domains and to elaborate phenotype differences identified in the primary assessment. We uncover significant phenotypic differences between the two lines, replicated across multiple centers, in a number of physiological, biochemical and behavioral systems.

Conclusions

Comparison of C57BL/6J and C57BL/6N demonstrates a range of phenotypic differences that have the potential to impact upon penetrance and expressivity of mutational effects in these strains. Moreover, the sequence variants we identify provide a set of candidate genes for the phenotypic differences observed between the two strains.     

Autoradiography of muscarinic cholinergic receptors in cortical and subcortical brain regions of C57BL/6 and DBA/2 mice

Mice of the inbred strains C57BL/6 and DBA/2 show strain-dependent behavioural differences which have been correlated with variations in brain cholinergic systems. In the present study, the density of muscarinic cholinergic receptors in both strains of mice was determined by autoradiographic methods using [³H]quinuclidinyl benzilate (QNB) and [³H]pirenzepine as ligands. C57BL/6 mice showed a significantly lower [³H]QNB binding level in the frontal cortex by one third as compared to DBA/2 mice. In the striatum and the cholinergic pontomesencephalic nucleus laterodorsalis tegmenti the [³H]QNB binding was lower in C57BL/6 by 28% and 31%, respectively. The [³H]pirenzepine binding level was found to be significantly higher in C57BL/6 temporal cortex (by 22%). These results are discussed in relation to interstrain differences in cholinergic cell density and in the activity of cholinergic enzymes.     

Substrains matter in phenotyping of C57BL/6 mice

The inbred mouse strain C57BL/6 has been widely used as a background strain for spontaneous and induced mutations. Developed in the 1930s, the C57BL/6 strain diverged into two major groups in the 1950s, namely, C57BL/6J and C57BL/6N, and more than 20 substrains have been established from them worldwide. We previously reported genetic differences among C57BL/6 substrains in 2009 and 2015. Since then, dozens of reports have been published on phenotypic differences in behavioral, neurological, cardiovascular, and metabolic traits. Substrains need to be chosen according to the purpose of the study because phenotypic differences might affect the experimental results. In this paper, we review recent reports of phenotypic and genetic differences among C57BL/6 substrains, focus our attention on the proper use of C57BL/6 and other inbred strains in the era of genome editing, and provide the life science research community wider knowledge about this subject.     

Delayed matching-to-position performance in C57BL/6N mice

Delayed matching-to-sample is one of the most frequently employed behavioral tasks for assessing spatial working memory in animals. Although the advantages of the task have been widely acknowledged and it is used in the study of a variety of species, its application to mice has been rare. In the present study, we reported the efficacy of a delayed matching-to-position task in C57BL mice lever-pressing in an operant-conditioning chamber. Each trial started with the press of a back lever, followed by the presentation of either a left or right front lever. When the ratio requirement for presses to the front lever (sample) was met, a delay interval started. Delay interval continued until the mice made the first response after the elapse of the programmed delay interval. This was followed by the presentation of a choice of left or right front levers. The choice of the same front lever as the sample was reinforced, whereas the other was not. The proportion of correct choices showed a delay-dependent decrement. A higher ratio of response requirement to the sample resulted in increased accuracy, but the duration of the intertrial interval had no effect. Preceding trials also influenced response accuracy, indicating proactive interference. Overall, the results replicated the effects of parametric manipulations reported in other species, and thus, our findings validate the efficacy of the task for assessing spatial working memory in laboratory mice.     

The influences of age on T lymphocyte subsets in C57BL/6 mice

The aim of this study is to evaluate the age related changes of T lymphocyte subsets in C57BL/6 mice and immune function. Multi-color immunofluorescence techniques that were used to analyse relative numbers of T lymphocyte subsets include CD4⁺, CD8⁺, naive and memory CD4⁺ and CD8⁺, CD8⁺CD28⁺ T cells in peripheral blood of C57BL/6 mice from different age groups (Group I: 2 months old; Group II: 7 months old; Group III: 21 months old); Splenocytes isolated from different group mice were stimulated with Con A to evaluate the proliferative ability. Compared with group I, group II had a significant reduction in the percentage of CD4⁺, naive CD4⁺ and CD8⁺ T cells and an increase in the percentage of CD8⁺ T cells, while group III had a significant reduction in the percentage of CD4⁺, naive CD4⁺ and CD8⁺ T cells and increase in the percentage of CD8⁺, memory CD4⁺ and CD8⁺ T cells in peripheral blood. Compared with group II, group III had a significant reduction in the percentage of naive CD8⁺ T cells and increase in the percentage of memory CD4⁺ and CD8⁺, CD8⁺CD28⁺ T cells in peripheral blood. The T lymphocyte proliferation in vitro showed that groups II and III had a lower proliferative capacity than group I, between groups II and III, there was not a significant difference. We provide relative values for the T lymphocyte subsets in the different age groups of C57BL/6 mice. The immune system began aging at 7 months old in C57BL/6 mice under a specific pathogen free environment.     

Performance of BALB/c and C57BL/6 mice under an incremental repeated acquisition of behavioral chains procedure

BALB/c (n = 8) and C57BL/6 (n = 11) male mice were trained under an incremental repeated acquisition (IRA) procedure using two distinct training procedures: forward and backward chaining. A new metric for assessing progress on the IRA procedure, progress quotient (PQ), quantified progress as the product of chain length and number of reinforcers earned during a session divided by the total number of reinforcers earned. BALB/c mice progressed further, had higher overall responding, earned more reinforcers, and acquired the response sequences faster than the C57BL/6 mice on both training procedures. There were only minimal effects of training procedure for either strain. The strain differences found between BALB/c and C57BL/6 mice confirm the importance of genetic background to behavior. C57BL/6 mice may be deficient in learning as compared with BALB/c mice but other contributing factors probably include overall responding, motivation, and more rapid satiation or habituation to sucrose reinforcement by the C57BL/6 mice. PQ is a sensitive and valid measure of progress for use in studies of mastery-based incremental repeated acquisition and BALB/c mice perform this challenging learning task better than do C57BL/6 mice.     

Susceptibility to re-infection in C57BL/6 mice with recombinant strains of Toxoplasma gondii

This work reports results of re-infection of BALB/c and C57BL/6 mice with different recombinant strains of Toxoplasma gondii. Mice were prime-infected with the non-virulent D8 strain and challenged with virulent strains. PCR–RFLP of cS10-A6 genetic marker of T. gondii demonstrated that BALB/c mice were re-infected with the EGS strain, while C57BL/6 mice were re-infected with the EGS and CH3 strains. Levels of IFN-γ and IL-10 after D8 prime-infection were lower in C57BL/6 than in BALB/c mice. Brain inflammation after D8 prime-infection was more intense in C57BL/6 than in BALB/c mice. It was shown that re-infection depends on mice lineage and genotype of the strain used in the challenge.     

The neurosteroid allopregnanolone impairs object memory and contextual fear memory in male C57BL/6J mice

Allopregnanolone (ALLO, or 3α-hydroxy-5α-pregnan-20-one) is a steroid metabolite of progesterone and a potent endogenous positive allosteric modulator of GABA-A receptors. Systemic ALLO has been reported to impair spatial, but not nonspatial learning in the Morris water maze (MWM) and contextual memory in rodents. These cognitive effects suggest an influence of ALLO on hippocampal-dependent memory, although the specific nature of the neurosteroid's effects on learning, memory or performance is unclear. The present studies aimed to determine: (i) the memory process(es) affected by systemic ALLO using a nonspatial object memory task; and (ii) whether ALLO affects object memory via an influence within the dorsal hippocampus. Male C57BL/6J mice received systemic ALLO either before or immediately after the sample session of a novel object recognition (NOR) task. Results demonstrated that systemic ALLO impaired the encoding and consolidation of object memory. A subsequent study revealed that bilateral microinfusion of ALLO into the CA1 region of dorsal hippocampus immediately following the NOR sample session also impaired object memory consolidation. In light of debate over the hippocampal-dependence of object recognition memory, we also tested systemic ALLO-treated mice on a contextual and cued fear-conditioning task. Systemic ALLO impaired the encoding of contextual memory when administered prior to the context pre-exposure session. Together, these results indicate that ALLO exhibits primary effects on memory encoding and consolidation, and extend previous findings by demonstrating a sensitivity of nonspatial memory to ALLO, likely by disrupting dorsal hippocampal function.     

寒冷对雌性C57BL/6小鼠动情周期的影响*

目的:研究寒冷对雌性C57BL/6小鼠动情周期的影响。方法:12只雌性小鼠随机分为对照组、低温组,每组6只;低温组每天4℃暴露4 h,每天阴道涂片法观察小鼠动情状况,对照组饲养于常温动物房;每2 d称量体重,2周后心脏取血、子宫和卵巢,检测小鼠血清E2、FSH、LH、Prl、P水平,进行子宫、卵巢的组织病理学检查。结果:与对照组比较,低温组小鼠体重无显著性差异(P＞0.05),小鼠子宫脏器系数明显较低、动情间期明显延长(P＜0.01),血清FSH显著升高、Prl显著降低(P＜0.01),小鼠子宫腺管扩张,卵巢卵泡数量明显减少。结论:寒冷可使雌性C57BL/6小鼠动情周期延长,进而可能影响生殖功能。     

小鼠动物实验方法系列专题(三) 三硝基苯磺酸诱导的C57/BL6小鼠结肠炎模型的建立方法

2,4,6-三硝基苯磺酸(TNBS)诱导的小鼠结肠炎模型是研究人类炎性肠病(inflammatory bowl disease,IBD)的主要手段之一,但在实际应用中,常用的C57/BL品系小鼠却对TNBS有较高耐受性,不易建模。本文主要介绍一种可以有效诱导C57/BL6小鼠TNBS结肠炎的方法,并对疾病评价指标进行了具体的描述。对基因工程小鼠IBD模型的研究具有重要意义。     

C57BL/6小鼠性别差异对髓鞘少突胶质糖蛋白诱导的实验性自身脑脊髓炎疾病的影响

多发性硬化是人类常见的中枢神经系统自身免疫性炎症致脱髓鞘疾病．流行病学研究发现,女性患者多于男性,其平均发病时间早于男性．实验性自身免疫性脑脊髓炎(EAE)与多发性硬化症有相似的临床症状和病理特征,是被广泛应用于人类疾病研究的动物模型．本实验利用髓鞘少突胶质糖蛋白MOG_33-35免疫C57BL/6小鼠建立EAE模型,观察29天．通过疾病评分发现雌雄小鼠在发病率、起病时间上均无明显差别,但雄鼠的发病症状明显比雌鼠严重．在其病理切片HE染色中观察到雄性小鼠中枢浸润的炎性细胞多于雌性小鼠,并且在LFB染色中同样观察到雄鼠脱髓鞘区域明显增大．对其发病高峰期中枢浸润细胞的染色分析时,可以发现雄性小鼠中浸润的CD4 T细胞及其亚群T_H-1和T_H-17细胞均有明显增加．这些都表明MOG_33-35免疫C57BL/6小鼠建立的EAE模型存在着性别差异的影响,这一发现为今后建立多发性硬化症的动物模型中动物性别的选择提供了一定的参考依据．     

三个近交系C57BL/6J小鼠群体微卫星遗传变异分析(英文）

应用微卫星遗传标记对近交系C57BL/6J(B6)小鼠遗传稳定性进行分析。用FAM标记的引物PCR扩增了来自北京和上海三个实验动物生产单位提供的三个B6小鼠群体共15个微卫星位点并进行分型。结果显示,所有位点均处于纯合状态,其中7个位点为多态位点。研究表明各B6群体虽然为高度近交群体,但不同生产单位维持的B6群体之间存在遗传分化。     

C57BL／6J小鼠ES细胞系的建立及其特性分析

本文报道从C57BL/6J个鼠的囊胚中,建立了三个ES细胞系MESPU17,MESPU18,MESPU19。这些细胞的细胞学特征和强AKP反应,表明这三个细胞系具有干细胞的特征。这三个细胞系均为XY型,正常二倍体核型分别占70％、88％和59％。体外分化可形成简单类胚,体内分化可形成瘤块。与国际上通用的CCE和来自129/ter的ES细胞MESPU13相比,这三个细胞系的ES细胞较大;在体外培养时,生长较慢;细胞也较粘,进行显微注射时,很容易粘在注射针壁上。MESPU17,MESPU18进行了嵌合体制作,以BALB/c和昆明鼠的囊胚为受体,采用囊胚注射法未获嵌合体,但使用昆阴鼠的8细胞胚注射法和共培养法得到嵌合体。