Summary

有条件的遗传跨突触标记在胚胎小鼠脑

Published: December 22, 2014
doi:

Summary

Capitalizing on a binary genetic strategy we provide a detailed protocol for neural circuit tracing in mice that express complementary transsynaptic tracers after Cre-mediated recombination. Because cell-specific tracer production is genetically encoded, our experimental approach is suitable to study the formation and maturation of neural circuitry during murine embryonic brain development at a single cell resolution.

Abstract

Anatomical path tracing is of pivotal importance to decipher the relationship between brain and behavior. Unraveling the formation of neural circuits during embryonic maturation of the brain however is technically challenging because most transsynaptic tracing methods developed to date depend on stereotaxic tracer injection. To overcome this problem, we developed a binary genetic strategy for conditional genetic transsynaptic tracing in the mouse brain. Towards this end we generated two complementary knock-in mouse strains to selectively express the bidirectional transsynaptic tracer barley lectin (BL) and the retrograde transsynaptic tracer Tetanus Toxin fragment C from the ROSA26 locus after Cre-mediated recombination. Cell-specific tracer production in these mice is genetically encoded and does not depend on mechanical tracer injection. Therefore our experimental approach is suitable to study neural circuit formation in the embryonic murine brain. Furthermore, because tracer transfer across synapses depends on synaptic activity, these mouse strains can be used to analyze the communication between genetically defined neuronal populations during brain development at a single cell resolution. Here we provide a detailed protocol for transsynaptic tracing in mouse embryos using the novel recombinant ROSA26 alleles. We have utilized this experimental technique in order to delineate the neural circuitry underlying maturation of the reproductive axis in the developing female mouse brain.

Introduction

解剖路径跟踪是最常用的工具,以破译大脑和行为1之间的关系之一。进步的神经回路追踪技术已经赋予与神经科学家从小鼠基因2确定神经元群追踪神经回路的功能。尽管这些技术上的进步仍然具有挑战性解开尤其是在胚胎成熟的神经回路的形成。这是因为大多数开发迄今跟踪方法是基于立体定位注射跨突触示踪剂的或遗传修饰的嗜神经病毒( 1)2,3。虽然这些技术实现空间和时间分辨率的连通,几个固有的局限性,如技术上具有挑战性示踪剂注射入发育中的大脑中,注射部位的再现性,潜在炎症在注射部位和最祁门功夫,功夫造成嗜神经病毒tantly毒性限制了其使用4。

另一种方法是要表达的跨突触示踪剂如遗传改造的小鼠转基因。我们最近修改了此技术,开发了二进制遗传跨突触跟踪系统,以地图的任何基因鉴定神经元群5的神经回路。我们的试验策略是基于两个新敲入小鼠品系,其表达或者双向示踪剂大麦凝集素(BL)的6或逆行示踪剂破伤风毒素片段C的ROSA 26轨迹融合到绿色荧光蛋白(GTT)7后的Cre介导的重组。在这里,我们用这些小鼠品系选择性表达BL和GTT的产生亲吻促动素的神经元,即牵连调节生殖轴8,9的成熟神经肽。我们表明,这种技术适用于可视吻的发育和成熟在雌性小鼠大脑5的胚胎发育过程中peptin神经电路。

育种策略

在R26-BL-IRES-τlacZ(BIZ)和R26-GFP-TTC(GTT)示踪线敲入5株携带重组ROSA26等位基因。所述R26-BIZR26-GTT等位基因是转录沉默由于强烈的转录终止信号,它是由两个loxP序列侧翼5的存在。的BIZ和GTT转基因的表达是通过Cre重组酶介导的切除的转录终止信号的激活。在R26-BIZR26-GTT等位基因可以独立通过简单地用酶Cre车手穿越中使用。用于分析动物杂合的各自的CreR 26等位基因都可以使用。同窝承载1 的Cre或1 R 26等位基因,分别应用作对照。可替代地,也有可能产生吨riple敲入动物携带的Cre,R26-BIZR26-GTT等位基因,然而这将需要一个额外的十字架。

Protocol

注:伦理学声明:涉及动物对象的程序批准了汉堡萨尔州的大学和大学的动物福利委员会。 1.制备和胚胎组织的固定安排所有解剖出胚胎和牺牲动物前准备组织随后固定解决方案所需的设备。 注:始终制备新鲜的4%多聚甲醛(PFA)溶液(4%PFA的0.1M磷酸盐缓冲盐水(PBS),调节pH至7.4)。 安乐死定时怀孕的雌性小鼠与道德批准的程序。 转移到小鼠?…

Representative Results

本节将展示可以得到与R26-BIZ(B L- 我 RES-τlacZ)和R26-GTT(G FP-TT C)等位基因工作代表性的结果。在这里,我们使用了R26-BIZ和R26-GTT等位基因来分析神经回路调节生殖轴的成熟。再生在脊椎动物的中心由神经元的下丘脑,其分泌的促性腺激素释放激素(GnRH)的一小部分进行控制。亲吻促动素,GnRH神经元的一个有力的活化剂,已被牵涉到调节GnRH神经元的活?…

Discussion

相比于立体定位注射示踪剂或neurotopic病毒的表达跨突触示踪剂作为转基因跟踪基因定义神经元群体的神经回路具有几个优点。首先,将示踪剂是作为内源蛋白质,因此不引起任何免疫反应和选择性的神经通路可在不同的动物具有较高的再现性进行分析。第二,由于这是一种非侵入性的方法也可用于从神经元用于立体定位注射不容易进入跟踪电路,例如在子宫内 。限制包括在跨突触传递一?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

We thank Michael Candlish for critical comments on the manuscript. This project was supported by the Deutsche Forschungsgemeinschaft grants BO1743/6 and SFB/TRR 152 P11 and Z02 to Ulrich Boehm.

Materials

Name of Material/ Equipment Company Catalog Number Comments/Description
Bisbenzimide (Hoechst 33258 dye) Sigma 14530-100MG
Ethanol Sigma 32205-1L
Cryo mold (Peel-a-way) Polyscience Inc. 18646A-1 22mm x 22mm x 20mm
DMSO Sigma D8418-100ML
Dimethyl Formamide (DMF) VWR Chemicals 23470,293
EGTA ROTH 3054.3
Fluoromount G Southern Biotech 0100-01
Glutaraldehyde Sigma G5882-50ML
Hydrogen peroxide Sigma 34988-7
Isopentane (Methyl 2-butane) Sigma M32631-2.5L
Kaiser's Glycine gelatin Merck 1092420100
Methanol Sigma 494437-1L
MgCl2 Sigma M2670-100G
NaCl ROTH HN00.2
NBT Sigma 298-83-9
Nonidet P40 substitute Fluka 743.85
OCT Leica 14020108926
PAP pen Dako S2002
Parafarmaldehyde Sigma P6148-1KG
Sodium deoxycholate Sigma D6750-25G
Sucrose Sigma S7903-1KG
Superfrost slides Thermo Scientific FT4981GLPLUS
TSA kit PerkinElmer  NEL700
TSA plus kit PerkinElmer  NEL749A001KT
Tris ROTH AE15.2
Triton-X 100 ROTH 3051.2
Tween 20 ROTH 9127.1
X-gal ROTH 2315.1
Cryostat Leica na
Light microscope equipped with DIC imaging  Zeiss Axioskop2 equipped with Axio Vision software
Fluroscence microscope Zeiss Axioskop2 equipped with Axio Vision software
Photoshop Adobe PS6
Goat anti-WGA (recognizes BL) Vector Laboatories AS-2024
Biotinylayted horse anti-goat IgG Vector Laboatories BA-9500 
Biotinylated goat anti-rabbit IgG Vector Laboatories BA-1000 
Rabbit anti-GFP (recognizes GTT) Invitrogen A11122
Rabbit anti-GnRH Affinity Bio Reagent PA1-121
Dylight488-donkey anti-rabbit IgG Thermo Scientific SA5-10038
SA-Alexa Fluor 546 Life Technologies S-11225
Primers
BL Fwd (for BIZ genotyping) Eurofins MWG Operon  ATGAAGATGATGAGCACCAG
GGC 
BL Rev  (for BIZ genotyping) Eurofins MWG Operon  AGCCCTCGCCGCAGAACTC 
Cre Fwd  (for Cre genotyping) Eurofins MWG Operon GTCGATGCAACGAGTGATGAG
GTTCG
Cre Rev  (for Cre genotyping) Eurofins MWG Operon CCAGGCTAAGTGCCTTCTCTAC
ACCTGC
TTC Fwd  (for GTT genotyping) Eurofins MWG Operon AGCAAGGGCGAGGAGCTGTT
TTC Rev  (for GTT genotyping) Eurofins MWG Operon GTCTTGTAGTTGCCGTCGTCCT
TGAA
XY Fwd (for gender genotyping) Eurofins MWG Operon TGAAGCTTTTGGCTTTGA
XY Rev  (for gender genotyping) Eurofins MWG Operon CCGCTGCCAAATTCTTTG
ROSA26 Fwd Eurofins MWG Operon CGAAGTCGCTCTGAGTTGTTATC
ROSA26 Rev Eurofins MWG Operon GCAGATGGAGCGGGAGAAAT
SA Rev Eurofins MWG Operon CGAAGTCGCTCTGAGTTGTTATC

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Cite This Article
Kumar, D., Boehm, U. Conditional Genetic Transsynaptic Tracing in the Embryonic Mouse Brain. J. Vis. Exp. (94), e52487, doi:10.3791/52487 (2014).

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