The gut microbiota of environmentally enriched mice regulates visual cortical plasticity

ABSTRACT The complexity of brain circuits is sculpted both by innate genetic programs and environmental stimuli. Since the 1960s scientists have noticed that raising rodents in an enriched environment (EE) is able to improve all aspects of brain plasticity, from learning and memory to visual plasticity in adult and developing animals. Importantly, EE has also been shown to have beneficial effects on a variety of preclinical models of central nervous system diseases: Alzheimer’s and Parkinson’s disease, Rett syndrome, epilepsy etc, prompting intervention protocols in humans. However, the “enrichment derived key signals” through which this special environment performs its broad positive effects on brain health have not been completely elucidated yet. Here, we focused on signals coming from the body periphery and in particular on the gut microbiota. We found that the intestinal microbiota composition of EE mice is significantly different from the one of standard raised (ST) animals. Treatment of EE mice with an antibiotic cocktail completely prevented the EE-driven enhancement of OD plasticity. Strikingly, the fecal microbiota transplant from EE donors to adult ST mice was able to re-activate OD plasticity in the ST recipients. Thus, taken together our data suggest that experience-dependent changes in gut microbiota regulate brain plasticity. METHODS In the first dataset (Dataset1, files called zr2423) we report the raw data (.fastq) obtained from the sequencing of the fecal samples from C57BL/6J mice raised in EE or in ST from birth and collected at different time points during their lives. To analyze the composition of the microbiota of ST and EE mice at different ages, fresh faeces were collected longitudinally in the same subject at postnatal day (P)20 (n=6), P25 (n=6) and P90 (n=6). In the second dataset (Dataset2, files called zr2747) we report the raw data (.fastq) obtained from the sequencing of the fecal samples from C57BL/6J: adult donor mice living in EE (EE, n=8), adult recipient mice living in ST condition before the fecal transplantation (preFT, n=8) and 4 weeks after the fecal transplantation (postFT, n=8). For further details about the sample names see the “Explanation Table”. Bacterial DNA was extracted using a specific kit (QIAamp Powerfecal DNA kit, Qiagen) following the manufacturer's protocol. The 16S rRNA sequencing and analysis was performed by a service offered by Zymo Research (Irvine, CA, USA). <em>Targeted Library Preparation</em>: The DNA samples were prepared for targeted sequencing with the Quick-16S™ NGS Library Prep Kit (Zymo Research). The primer sets used were Quick-16S™ Primer Set V3-V4 (Zymo Research). The sequencing library was prepared using an innovative library preparation process in which PCR reactions were performed in real-time PCR machines to control cycles and therefore limit PCR chimera formation. The final PCR products were quantified with qPCR fluorescence readings and pooled together based on equal molarity. The final pooled library was cleaned up with the Select-a-Size DNA Clean &amp; Concentrator™, then quantified with TapeStation® (Agilent Technologies, Santa Clara, CA) and Qubit® (Thermo Fisher Scientific, Waltham, WA). <em>Sequencing:</em> The final library was sequenced on Illumina® MiSeq™ with a v3 reagent kit (600 cycles). The sequencing was performed with &gt;10% PhiX spike-in.摘要 脑回路的复杂性由先天遗传程序与环境刺激共同塑造。自20世纪60年代起，科学家便注意到，在富集环境（enriched environment，EE）中饲养啮齿类动物，可改善其脑可塑性的各个方面——从学习记忆能力，到成年及发育中动物的视觉可塑性。重要的是，研究还证实EE对多种中枢神经系统疾病的临床前模型具有有益作用，包括阿尔茨海默病、帕金森病、雷特综合征、癫痫等，这推动了人类相关干预方案的研发。然而，这种特殊环境通过何种“富集来源关键信号”对脑健康发挥广泛的积极作用，目前尚未完全阐明。本研究聚焦于来自机体外周的信号，尤其是肠道菌群（gut microbiota）。我们发现，富集环境饲养小鼠的肠道菌群组成与标准饲养（standard raised，ST）小鼠存在显著差异。使用抗生素混合制剂处理富集环境饲养小鼠，可完全阻断EE介导的眼优势可塑性（ocular dominance plasticity，OD plasticity）增强效应。令人惊喜的是，将富集环境饲养供体小鼠的粪便菌群移植至成年标准饲养小鼠体内，可重新激活受体小鼠的眼优势可塑性。综上，本研究数据表明，经验依赖的肠道菌群变化可调控脑可塑性。方法 在第一个数据集（Dataset1，文件名为zr2423）中，我们公开了从C57BL/6J小鼠粪便样本测序获得的原始数据（.fastq）：这些小鼠自出生起分别饲养于EE或ST环境，并在其生命过程中的不同时间点采集粪便样本。为分析不同年龄阶段ST与EE小鼠的菌群组成，我们对同一批小鼠进行纵向采样：分别在出生后第20天（P20，n=6）、第25天（P25，n=6）及第90天（P90，n=6）采集新鲜粪便。在第二个数据集（Dataset2，文件名为zr2747）中，我们公开了从以下C57BL/6J小鼠粪便样本测序获得的原始数据（.fastq）：饲养于EE环境的成年供体小鼠（EE组，n=8）、粪便移植前饲养于ST环境的成年受体小鼠（移植前组preFT，n=8），以及粪便移植4周后的成年受体小鼠（移植后组postFT，n=8）。关于样本命名的更多细节，请参见"Explanation Table"。细菌DNA提取采用特定试剂盒（QIAamp Powerfecal DNA kit，Qiagen），严格遵循厂商说明书操作。16S rRNA测序及分析由美国加利福尼亚州欧文市的Zymo Research公司提供的测序服务完成。靶向文库制备：使用Quick-16S™ NGS Library Prep Kit（Zymo Research）对DNA样本进行靶向测序文库构建。所用引物组为Quick-16S™ Primer Set V3-V4（Zymo Research）。本次文库构建采用创新流程：在实时荧光定量PCR仪中进行PCR反应，以精确控制循环次数，从而减少PCR嵌合体的形成。最终PCR产物通过qPCR荧光读数进行定量，并基于等摩尔浓度进行混合。混合后的最终文库通过Select-a-Size DNA Clean & Concentrator™进行纯化，随后使用TapeStation®（Agilent Technologies，美国加利福尼亚州圣克拉拉）与Qubit®（Thermo Fisher Scientific，美国华盛顿州沃尔瑟姆）进行定量。测序：最终文库在Illumina® MiSeq™平台上使用v3试剂试剂盒（600个循环）进行测序。测序时加入了超过10%的PhiX对照序列掺入。