大理石埋藏和小窝切碎作为小鼠重复、强迫性行为的测试
2013年12月24日
疾病、头部受伤、基因改造和药物治疗小鼠会对行为产生深远的影响。利用特征明确和经过验证的方法，例如大理石掩埋和巢切碎，可以作为人类强迫症和自闭症谱系障碍的模型，在小鼠中准确记录
强迫行为。该程序的总体目标是展示大理石掩埋和嵌套碎碎的简单性，作为评估和展示重复和强迫性行为的可靠范例。在用于大理石埋葬测试的啮齿动物中，准备一个笼子，其中 20 颗弹珠均匀分布在床上用品表面。然后将小鼠引入笼子中，并与弹珠一起不受干扰 30 分钟。
在此之后，移走鼠标，并对埋藏的弹珠进行 Nestle 测试。在引入鼠标之前，将完整的雀巢放入笼子中。30 分钟后，取出鼠标，让雀巢干燥一夜，然后再计算雀巢切碎的百分比。
最终，敲除小鼠和野生型小鼠可以显示大理石、埋葬和雀巢切碎行为的差异。演示这些程序的是研究生 Denise Briggs、博士后兼 Francescutti 院长、我实验室的助理研究员。所有动物都保持 12 小时的光照黑暗循环，可自由获取食物和水。
以获得最佳效果。在测试前三到五天，每天轻轻处理每只小鼠一次。当动物准备好进行测试时，在开始第一次测试前 60 分钟将笼子运送到测试室。
对于大理石轴承测试，使用带有安装过滤器顶部的标准聚碳酸酯鼠笼。将新鲜无味的小鼠垫料添加到每个笼子中，深度为 5 厘米，并通过插入另一个相同尺寸的笼子来平整垫料表面。这还有一个额外的好处，就是在床上用品表面压印一条平行线的模板。
标准玻璃玩具弹珠在温和的实验室清洁剂中彻底冲洗，并蒸馏离子水，并在每次使用前晾干。准备好后，轻轻地将弹珠压入床上用品的表面。在五行中，每行四个弹珠准备秒表或计时器，用于为会话计时。
要开始测试，请将鼠标面向笼子的一角，尽可能远离弹珠。将过滤器顶部放在笼子上，让鼠标不受弹珠的干扰 30 分钟。在测试期结束时，取出鼠标并将其放回其主笼。
在将对象从笼子中取出的过程中，请格外小心，不要移动或移开弹珠。然后，两到三名对处理条件不知情的研究人员应该对埋藏的弹珠数量进行评分。弹珠被计为 buried。
如果其表面积的三分之二被垫料覆盖，则对每只老鼠埋藏的弹珠数的分数求平均值。最后，取回所有 20 个弹珠并处理掉床上用品。使用带有合适过滤器顶部的标准聚碳酸酯小鼠笼。
对于雀巢切碎测试，将新鲜无味的小鼠垫料添加到每个笼子中至 0.5 厘米的深度，然后如前所述包装垫料。接下来，称量市售的棉纤维燕窝，并将一个雀巢放在床上用品上。在每个测试笼中，准备秒表或计时器来计时。
准备好后，将鼠标放在笼子面向墙壁的一角。将过滤器顶部放在笼子上，让鼠标与 Nestle 一起不受干扰 30 分钟。在此之后，取出鼠标并将其放回其固定架。
从笼子中取出剩余完好无损的巢箱材料，并使其干燥过夜。第二天。称量未切碎的燕窝，然后将该重量除以起始重量，以计算燕窝点燃的切碎百分比。
最后，丢弃剩余的切碎的巢穴材料和垫料。这里显示的是大理石埋设试验前后的代表性笼子。在测试野生型小鼠时，很少有弹珠被掩埋或从其初始位置移位。
然而，TPH 两只敲除小鼠埋葬了 20 颗弹珠中的 17 颗。剩余未掩埋的大理石用箭头表示。此侧视图显示了垫层表面的变化。
测试后，将野生型笼子中相对未受干扰的表面与包含敲除动物的大槽和峰的不平整表面进行比较。这里总结了大理石埋设测试的典型性能。与野生型小鼠相比，TPH 两只基因敲除小鼠埋藏的弹珠明显更多。
这些照片显示了切碎后具有代表性的巢穴。在这里，与来自两只敲除小鼠 TPH 笼子的巢穴相比，野生型小鼠的巢穴显示出少量的碎裂。该图显示了野生型和 TPH 两种基因敲除小鼠在雀巢切碎试验中的典型性能。
TPH 两只基因敲除小鼠切碎的雀巢材料明显多于野生型小鼠。一旦掌握，如果执行得当，这些技术可以在两个小时内完成。在尝试此过程时，重要的是要记住要符合定义大理石被埋葬的标准，并从干燥和未切碎的剩余物中取出雀巢的碎块。
在此程序之后，可以向小鼠注射用于治疗人类强迫症的药物，例如 SSRIs，以确定这些药物是否能减少小鼠的强迫症行为。
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概述
本文展示了使用大理石埋藏和巢垫碎片作为可靠范式来评估小鼠的强迫行为。这些行为是理解人类强迫症和自闭症谱系障碍的相关模型。
关键研究组成部分
科学领域
- 神经科学
- 行为科学
- 动物模型
背景
- 疾病和基因改变可以影响行为。
- 研究小鼠的头部损伤和药物治疗对行为变化的影响。
- 强迫行为对于模拟人类精神状况很重要。
- 验证的方法对于准确记录这些行为至关重要。
研究目的
- 说明大理石埋藏和巢垫碎片测试的简单性。
- 提供可靠的方法来评估啮齿动物的重复行为。
- 为理解强迫行为与人类疾病的关系做出贡献。
使用的方法
- 为大理石埋藏测试准备一个装有大理石的笼子。
- 观察小鼠在30分钟内与大理石的互动。
- 测试后计算埋藏的大理石数量。
- 使用巢垫在类似的观察期后评估碎片行为。
主要结果
- 埋藏的大理石数量提供了强迫行为的定量测量。
- 巢垫碎片的百分比指示了重复行为的水平。
- 两种测试都能有效地展示小鼠的强迫行为。
- 结果可用于模拟人类精神状况。
结论
- 大理石埋藏和巢垫碎片是评估强迫行为的验证方法。
- 这些范式可以帮助理解人类疾病的潜在机制。
- 进一步的研究可以在这些发现基础上探索治疗效果。
常见问题
Executive Industry Relevance
Marble burying and nestlet shredding tests provide quantitative, ethologically relevant readouts for compulsive-like behaviors in mice, supporting target validation in neuropsychiatric drug discovery. These assays enable mechanistic de-risking by linking genetic or pharmacological manipulations to measurable changes in repetitive behavior, informing go/no-go decisions in early discovery. Their simplicity, reproducibility, and sensitivity to small behavioral shifts make them valuable tools for portfolio triage and translational continuity from target identification to preclinical validation.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Interrogate therapeutic hypotheses by quantifying compulsive-like behaviors as a readout of target engagement in genetic or disease models.
- Operational Value: Enable biological de-risking through sensitive detection of behavioral changes resulting from target modulation, supporting target confidence assessment.
- Predictive Value: Support predictive confidence and portfolio triage by providing translatable phenotypic readouts relevant to OCD and ASD pathophysiology.
Screening & Assay Development
- Scientific Value: Prepare validated behavioral systems for downstream screening by establishing baseline and compound-responsive phenotypes in compulsive-like behavior assays.
- Operational Value: Address assay standardization and reproducibility through blinded scoring, fixed duration trials, and clear operational definitions (e.g., two-thirds marble burial threshold).
- Predictive Value: Highlight screening readiness and scalability due to minimal equipment needs, short test duration, and high-throughput compatibility across laboratories.
Translational & Preclinical Research
- Scientific Value: Discuss disease relevance by modeling compulsive symptomatology seen in OCD and ASD, enabling translational biomarker alignment through behavioral quantification.
- Operational Value: Describe continuity from discovery through preclinical validation by using the same assay to evaluate target modulation across genetic, environmental, and pharmacological interventions.
- Predictive Value: Address risk-adjusted advancement decisions by linking changes in marble burying or nestlet shredding to target mechanism, supporting go/no-go criteria based on behavioral de-risking.
Pipeline & Workflow Integration
The marble burying and nestlet shredding tests function as discovery-stage phenotypic assays that support hypothesis testing, pathway clarification, and biological de-risking in early neuroscience research, with direct utility in lead identification and preclinical evaluation of target modulators.
- Discovery Biology: Explain how the method supports hypothesis testing, pathway clarification, or biological de-risking by quantifying compulsive-like behaviors as a functional readout of serotonergic, dopaminergic, or glutamatergic target activity in genetic models.
- Screening: Describe assay readiness, reproducibility, or quantitative outputs when supported by the article through standardized 30-minute trials, blinded scoring, and percentage-based nestlet shredding or marble burial counts.
- Analytics: Highlight measurements, readouts, or statistical outputs that help teams compare conditions, including marble burial frequency and nestlet shredding percentage as continuous variables for group comparisons.
- Translational Research: Connect the method to preclinical continuity or biomarker alignment only when the source supports it by demonstrating how assay results reflect symptom-relevant phenotypes in models of OCD and ASD, supporting cross-species validity.
- Enterprise Reuse: Frame the method as a reusable capability rather than a single-use technique due to low cost, minimal training requirements, and applicability across compound screens, target validation studies, and mechanism-of-action studies.
Operational & Enterprise Impact
- Scientific Value: Predictive confidence, target validation, reduction of mechanistic ambiguity in neuropsychiatric target engagement.
- Operational Value: Standardization, reproducibility, and scalability across sites and studies through fixed protocols and blinded endpoint assessment.
- Strategic Value: Better go/no-go decisions, capital efficiency, and reduced late-stage biological risk by identifying ineffective target modulators early in discovery.
- Portfolio Impact: Risk-adjusted prioritization and advancement decisions based on target-mediated changes in compulsive-like behavior phenotypes.
Implementation Considerations
- Required scientific expertise in rodent handling, behavioral observation, and blinded scoring to minimize bias and ensure data integrity.
- Instrumentation and analytical infrastructure needs include standard cages, bedding, marbles or nestlets, timers, and weighing equipment for nestlet assays.
- Cross-team standardization requirements involve consistent definition of marble burial (two-thirds surface coverage), blinded scoring by multiple observers, and environmental controls (light/dark cycle, acclimation).
- Adaptation considerations across model systems include validating baseline behavior in different strains and ensuring bedding depth and marble/nestlet presentation are standardized.
- Practical limitations supported by source material include the need for habituation handling, potential variability in spontaneous behavior, and the requirement for overnight drying of nestlets prior to weighing.
