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Abstract
Introduction
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Neuroscience

青少年C57BL/6雄性和雌性小鼠的社交失败应激模型

Published: March 15, 2024
doi:
10.3791/66455
, , , ,
1Integrated Program in Neuroscience,McGill University, 2Douglas Mental Health University Institute,McGill University, 3Department of Psychiatry,McGill University, 4Department of Neurology and Neurosurgery, Faculty of Medicine,McGill University, 5Ludmer Centre for Neuroinformatics & Mental Health,McGill University

Summary

我们为青少年 C57BL/6 小鼠开发了一种加速社交失败压力模型，该模型对男性和女性都有效，并允许在离散的青春期进行暴露。暴露于这种模型会引起社会回避，但仅在被击败的雄性和雌性小鼠的子集中。

Abstract

青春期的社会逆境很普遍，会对心理健康轨迹产生负面影响。需要对青少年雄性和雌性啮齿动物的社会压力进行建模，以了解其对持续大脑发育和行为结果的影响。慢性社会失败应激范式 （CSDS） 已被广泛用于模拟成年 C57BL/6 雄性小鼠的社会压力，方法是利用成年雄性啮齿动物对入侵其领土的入侵者表现出的攻击行为。这种范式的一个优点是，它允许根据它们在最后一次失败后 24 小时的社会行为的个体差异，将被击败的小鼠分为有弹性的组和易感组。在青少年C57BL / 6小鼠中实施该模型一直具有挑战性，因为成年或青少年小鼠通常不会攻击早期的青春期雄性或雌性小鼠，并且因为青春期是生命的短暂时期，包括谨慎的脆弱性时间窗口。通过调整CSDS的加速版本以用于青少年雄性和雌性小鼠，克服了这一限制。这种为期 4 天的压力范式，每天进行 2 次物理攻击，使用 C57BL/6 雄性成年小鼠来激发 CD-1 小鼠的攻击性，使其很容易攻击雄性或雌性青少年小鼠。这种模型被称为青春期小鼠的加速社会失败压力（AcSD）。青少年暴露于AcSD在男性和女性24小时后都会诱导社交回避，但仅在被击败的小鼠子集中引起。尽管在有弹性的组和易受攻击的组之间的会话中，攻击数量是一致的，但仍会发生此漏洞。AcSD模型足够短，允许在青春期的离散时期进行暴露，允许根据社会回避行为的存在与否对小鼠进行隔离，并且是第一个可用于研究青少年C57BL / 6雌性小鼠社会失败压力的模型。

Introduction

慢性社会失败压力范式被广泛用于模拟成年产后日 （PND） >65 雄性啮齿动物的社会压力。这种范式是基于成年雄性啮齿动物在入侵者入侵其领土时的自然攻击行为。该模型用于多种啮齿动物物种，包括大鼠、仓鼠和小鼠 1,2,3,4,5,6,7,8,9，包括持续约 10 天的身体攻击和心理压力的组合，在此期间，入侵啮齿动物实验几分钟的身体攻击来自常驻啮齿动物。这两只啮齿动物留在居民的家中笼子里，由一个隔板隔开，允许感官接触，但不允许身体接触7.在小鼠实验中，最常用的常驻/侵略小鼠是退休的育种瑞士CD-1小鼠，它们对入侵小鼠表现出强大的领土行为6,7。对于入侵小鼠，最典型的品系是近交系C57BL / 6品系2,4,5。被打败的老鼠每天都会暴露在新的侵略者面前，以防止对侵略者产生习惯。对照小鼠每天饲养不同的同种小鼠。在最后一次失败会话后24小时，在社会互动测试（SIT）中测试实验小鼠，其中它们可以在没有（无目标）或存在新型CD-1小鼠（目标）的情况下探索开放领域。对照组小鼠在与目标的交互区花费更多时间，而不是在任务的非目标部分花费更多时间。根据社会互动比率（在侵略者在场的互动区花费的时间/在侵略者不存在的互动区花费的时间），被击败的小鼠被分类为易感（比率1）。该程序提供了一个有用的工具来研究对压力反应的个体差异。

直到最近几年，慢性社会失败压力模型主要用于成年雄性小鼠，因为强调的雄性优势等级涉及与雄性战斗，而不是与雌性战斗 6,7。此外，雄性啮齿动物通常不会攻击雌性;相反，它们从事交配行为10.尽管存在这些障碍，但已经开发出不同的策略来适应成年雌性小鼠的慢性社会失败压力范式。例如，加利福尼亚老鼠的社会失败模型是基于这种一夫一妻制物种在保卫自己的领土时来自两性的自然侵略性 9,11。其他方法侧重于通过刺激其腹内侧下丘脑具有一致的攻击行为10,12 或在实验成年雌性小鼠中应用雄性尿液以接受 CD-1 侵略者的攻击13 来诱导 CD-1 小鼠的攻击行为。CD-1 小鼠的这种高度和一致的攻击性对于实验入侵小鼠在相互作用期间表现出对侵略者的反复攻击的明显从属行为迹象至关重要6.

调整慢性社会失败模型以用于青少年 C57BL/6 小鼠
青春期是一个以社会心理高度成熟为标志的时期，它与大脑微观和宏观结构的变化同时展开，尤其是前额叶皮层的变化。在人类和啮齿动物中，关于青春期的具体开始和结束几乎没有共识14,15。此外，在青春期存在关键的脆弱性窗口，即经验引起的对正在进行的大脑和认知发展的破坏16,17,18,19。青春期和青春期同时发生，但这些术语不是同义词。青春期标志着性成熟的开始，而青春期则代表着一个更广泛的阶段，其特征是从少年状态逐渐转变为实现独立20.不同的研究小组认为，小鼠的青春期从断奶（PND 21）到成年期（PND 60）21。特别是，青春期早期可称为断奶后第一周和第二周（PND 21-34），青春期中期称为PND 35-48期。这些范围包括离散的发育期，例如多巴胺系统的发育 22,23,24，对发育中的神经元网络的药物影响的脆弱性 17,25,26,27，以及不同的行为特征 16,20,28,29,30。

社交失败协议需要常驻老鼠的战斗行为。然而，与雌性小鼠一样，雄性小鼠通常不会与早期青春期小鼠进行攻击性互动，可能是因为它们不认为它们是威胁。大多数探索青少年C57BL / 6小鼠慢性社交失败影响的研究都是在青春期中期进行的31,32,33,34,35;其他人没有指定青少年暴露的产后日36,37，或将失败的日子延长到成年早期38 或不允许感官接触39;对青少年小鼠的其他研究使用不同的菌株40,41。表1总结了这些研究使用青春期雄性小鼠慢性社交失败压力的特征。

我们的研究小组对针对 C57BL/6 小鼠的特定青少年暴露窗口（包括青春期早期）感兴趣。由于不同青春期的持续时间较短，因此设计了慢性社会失败压力范式的加速版本的修改版本42。这种模型被称为青春期小鼠的加速社会失败压力（AcSD）。先前的研究表明，大鼠在青春期对社会压力的敏感性方面存在显着的性别差异 8,26,43,44,45，以及社会压力对人类心理健康轨迹的有害影响 46,47,48,49,50,51,52， 53,54,55,56.AcSD模型对青春期雌性小鼠也有效，使它们能够研究潜在的性别特异性后果，并探索神经生物学基础。

表1：在青春期雄性小鼠中使用社交失败压力范式的研究。 品系和物种：加利福尼亚小鼠： Peromyscus californicus。 C57BL/6： Mus musculus black 6 近交系小鼠品系。C57BL/6J：杰克逊实验室提供的M . musculus black 6近交小鼠模型。CD-1：来自瑞士近交白化小鼠品系的 肌肉分枝 杆菌。ICR： M. musculus 癌症研究所的白化小鼠品系优于近交系。OF1： M. musculus Oncins France 1 超过白化小鼠品系。
缩写：wk = 周;PND = 产后日;res = 弹性;SUS = 易感;UNSUS = 不易受影响。 请按此下载此表格。

Protocol

实验程序按照加拿大动物护理委员会的指导方针进行，并得到麦吉尔大学和道格拉斯医院动物护理委员会的批准（动物实验批准号：2005-5084）。所有小鼠被饲养在温度和湿度控制（21-22°C;60%）的菌落室中，并在道格拉斯心理健康大学研究所的神经表型中心进行12小时的明暗循环（8：00小时开光）。在整个实验过程中，小鼠可以 随意 获取食物和水。小鼠被随机分配到每个实验条件。实验动物…

Representative Results

在青少年 C57BL/6 雄性小鼠 （PND 21） 中使用慢性社交失败应激模型共进行了四个不同的实验。然而，该模型在早期青春期 C57BL/6 小鼠中的使用存在重要局限性。 青少年 C57BL/6 小鼠所需的设备修改第一个限制是用于社会失败装置的设备是为成年小鼠设计的。由于它们的大小，PND 21 的青少年 C57BL/6 小鼠能够穿过分隔器的穿孔到达侵略侧。这打断了感官接触的时期?…

Discussion

CD-1小鼠中一致的攻击性行为
在筛选阶段，注意 CD-1 显示的所有行为（追逐、安装行为、嗅探、梳理或咬合）并在为 AcSD 选择 CD-1 小鼠时密切遵循这些记录非常重要。CD-1小鼠与青春期小鼠相互作用而不攻击青春期小鼠，可能会在启动过程中对青春期小鼠产生攻击性。相比之下，攻击成年小鼠但不接近青春期小鼠的CD-1小鼠可能不会对青春期小鼠产生攻击性。

CD-1?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

这项工作由加拿大卫生研究院资助（CF资助号：MOP-74709;PJT 190045）、国家药物滥用研究所（CF 授权号：R01DA037911）、加拿大自然科学与工程研究委员会（CF 授权号：2982226）。Andrea Pantoja-Urban 得到了来自墨西哥的国家人文、科学和技术委员会/Consejo Nacional de Humanidades、Ciencias y Tecnologías （CONAHCYT） 和 FRQNT – 外国学生优异奖学金计划 （PBEEE） 的支持。塞缪尔·里彻（Samuel Richer）得到了麦吉尔大学神经科学综合项目的奖学金支持。插图是使用 BioRender.com 年的模板创建的。

Materials

C57BL/6 adolescent mice  In house breeding Mice were breeded at the Neurophenotyping Centre of the Douglas Mental Health University Institute.
C57BL/6 adult mice  Charles River Laboratories Strain Code: 027 Mice are ordered so as to arrive at PND>65 and are group housed (four mice per cage) in standard mice cages.
C57BL/6J adolescent mice  Jackson Labs Strain Code: 000664; RRID:IMSR_JAX:000664 Mice are ordered so as to arrive at PND 24 and are group housed (four mice per cage) in standard mice cages.
CD-1 mice  Charles River Laboratories Strain Code: 022 Mice retired breeders more than three months of age and singled housed throughout.
Cleaning solution  Virox Animal Health DIN 02537222 Prevail: Accelerated Hydrogen Peroxide. Desinfectant cleaner and deodorizer.
Clear perforated acrylic glass divider  Manufactured by Douglas Hospital, custom order 0.6 cm (w) × 45.7 cm (d) × 22.23 cm (h); perforations of 0.6 cm diameter. The dividers are perforated allowing sensory but no physical contact between the pair of mice.
Clear rectangular rat cages  Allentown 24 cm (w) × 48.3 cm (d) × 22.23 (h).
Cotton squares for bedding Inotiv Envigo T.6060 iso-BLOX 2.5 cm x 2.5 cm. Added to the social defeat apparatus.
Hard woodchip bedding Inotiv Envigo Teklad 7090, 7115 Sani-chip bedding.
Large binder clips to secure the steel-wire tops STAPLES Item #: 132429, Model #: 24178-CA 51 mm
Medium binder clips to secure the steel-wire tops Item #: 132367, Model #: 24172-CA 32 mm, in case the cover lids of the rat cages do not close with the large clips
Pain relief cream Polysporin Plus Pain Relief Cream (red format, NOT ointment), 2 Antibiotics plus lidocaine hydrochloride
Paired Steel-wire tops  24 cm (w) × 48 cm (d) with 0.6 cm (w) of separation between the grill
Removable wire-mesh enclosure  Johnston industrial plastics 11 cm (w) × 6.8 cm (d) × 42 cm (h) custom order; two per social interaction test arena secured in precut clear polycarbonate
Social interaction open-field arena PEXIGLAS 45 cm (w) × 45 cm (d) × 49 cm (h), custom-crafted from opaque acrylic glass (Plexiglas) 
Stopwatch  For timing defeat sessions
Video camera with infrared lights  Swann SRDVR-44580V  Swann Camera – 4 Channel 1080p Digital Video Recorder & 2 x PRO-T853
Video tracking software  Topscan 2.0 Clever Systems Inc.
DOWNLOAD MATERIALS LIST

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Social Defeat StressAdolescent MiceC57BL/6Male And FemaleSocial AvoidanceAccelerated Social Defeat Stress (AcSD) ModelBrain DevelopmentBehavioral Outcomes

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Pantoja-Urbán, A. H., Richer, S., Giroux, M., Nouel, D., Flores, C. Social Defeat Stress Model for Adolescent C57BL/6 Male and Female Mice. J. Vis. Exp. (205), e66455, doi:10.3791/66455 (2024).
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