WO2020118760A1 - 三级神经环路操纵组合物及动物三级神经环路操纵方法 - Google Patents

三级神经环路操纵组合物及动物三级神经环路操纵方法 Download PDF

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WO2020118760A1
WO2020118760A1 PCT/CN2018/122938 CN2018122938W WO2020118760A1 WO 2020118760 A1 WO2020118760 A1 WO 2020118760A1 CN 2018122938 W CN2018122938 W CN 2018122938W WO 2020118760 A1 WO2020118760 A1 WO 2020118760A1
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贾香连
王立平
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Shenzhen Institute of Advanced Technology of CAS
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  • the invention relates to the technical field of neuron activity manipulation, in particular to a three-level neural circuit manipulation composition and an animal third-level neural circuit manipulation method.
  • vector viruses such as AAV carrying specific promoters, light-sensitive genes and reporter genes are first injected into specific brain regions of model animals.
  • the AAV virus is expressed in cells with a promoter and spreads as far as the end of the axon. At present, this method can realize the manipulation between two brain regions, that is, between two levels of neurons A-B.
  • the neurons in the brain are connected to each other into an intricate network. Information is transmitted from top to bottom, and the multi-level neural loop can be controlled to analyze the flow of information.
  • the two-level neurons manipulated above can only analyze AB projections for model animal behavior, The influence of physiology, etc. cannot be analyzed downstream of the information flow projected by AB.
  • the main purpose of the present invention is to provide a three-level neural circuit manipulation composition and a third-level neural circuit manipulation method, aiming to use optogenetics, pharmacogenetics, etc. to achieve the purpose of manipulating the third-level neural circuit.
  • the three-level neural circuit manipulation composition provided by the present invention includes AAV-FLEX loxP- ChR2-eYFP virus, AAV-FLEx FRT- hM4D(Gi)-mCherry virus, and CAV-FLEx loxP- FLP virus .
  • the composition further includes N-oxide clozapine.
  • the present invention further proposes to apply the above three-level neural circuit manipulation composition to animals to perform animal three-level neural circuit manipulation methods.
  • the step of administering the tertiary neural circuit manipulation composition to the animal subject includes:
  • the CAV-FLEx loxP- FLP virus is injected into the C brain area of the animal subject.
  • the method further includes:
  • CNO was injected into the animal subject.
  • the animal object is a Cre-line animal.
  • the above three-level neural circuit manipulation composition of the present invention based on the problems of manipulating the third-level neural circuit such as optogenetics and pharmacogenetics, utilizes the CAV virus to be retrograde after being absorbed by nerve endings, and the AAV virus anterograde characteristics.
  • the combined use of viruses achieves the purpose of simultaneously manipulating tertiary neurons, ABC.
  • FIG. 1 is an architecture diagram of three-level neural circuit manipulation composition of the present invention in which three viruses manipulate a third-level neural circuit;
  • FIG. 2 is a virus injection scheme in which the three-stage neural circuit manipulation composition of the present invention manipulates three-stage neural circuit by three viruses;
  • FIG. 3 is a graph of detection results of target protein expression in a neural circuit experiment in which Cre transgenic tool mice were injected with virus according to an embodiment of the invention.
  • the present invention provides a three-level neural circuit manipulation composition, including AAV-FLEX loxP- ChR2-eYFP virus, AAV-FLEx FRT- hM4D(Gi)-mCherry virus, and CAV-FLEx loxP- FLP virus.
  • the AAV-FLEX loxP- ChR2-eYFP virus and the AAV-FLEx FRT- hM4D (Gi)-mCherry virus are recombinant viruses that are recombined with the target gene by using the AAV adeno-associated virus as the viral vector, and can be used in AAV-FLEX loxP And AAV-FLEx FRT recombinant expression system to express the target photosensitizing proteins ChR2-eYFP and hM4D(Gi)-mCherry.
  • the CAV-FLEx loxP- FLP virus is also a recombinant virus that uses the CAV virus (canine adenovirus) as a vector to express the FLP protein.
  • AAV-FLEX loxP- ChR2-eYFP virus is injected in the A brain area, which can mark neurons in the A brain area, and embedding fiber in the B brain area can manipulate the AB nerve ring Road;
  • CAV-FLEx loxP- FLP virus is used for injection in the C brain area, CAV virus is absorbed by the end of the neuron, along the axon to the brain area upstream of the C brain area, Cre cells in the brain area upstream of the C brain area FLP protein was also expressed in cells;
  • AAV-FLEx FRT- hM4D(Gi)-mCherry virus was injected in the B brain area and expressed in cells with FLP protein in the B brain area.
  • hM4D Gi
  • CNO N-clozapine, a metabolite with many cell targets, which can be bound to a customized receptor set
  • BC-specific neural circuits are manipulated . Therefore, when the three are used together, CAV virus and AAV virus are combined to achieve the purpose of manipulating the functional activities of tertiary neurons.
  • the dose of the AAV-FLEX loxP- ChR2-eYFP virus injected into the A brain region is injected according to the dose ratio of 500nl per experimental mouse; the dose of the AAV-FLEx FRT- hM4D (Gi)-mCherry injected into the B brain region Injection according to a dose ratio of 200nl per experimental mouse; the dose of the CAV-FLEx loxP- FLP virus injected into the C brain area is injected according to a dose ratio of 100nl per experimental mouse; each virus, that is, AAV-FLEX loxP-
  • the concentration of ChR2-eYFP virus, AAV-FLEx FRT- hM4D(Gi)-mCherry, CAV-FLEx loxP- FLP virus can be 10 12 vg/mL.
  • the injection dose can be adjusted according to the ratio of the brain volume of other animals to the brain volume of the experimental mouse, and according to the injection dose of each brain region of the experimental mouse Injection dose.
  • the brain can be divided into many large and small areas according to function and structure, and defined according to its position, such as the hypothalamus, striatum bed nucleus, and solitary tract nucleus.
  • these brain regions are connected to each other through nerve fibers, which we call neural circuits.
  • the A brain area in each of the above examples is the striatum bed nucleus
  • the B The brain area is the hypothalamus
  • the C brain area is the solitary nucleus.
  • the recombinant expression systems Flp/FRT system and Cre/loxP system in the above recombinant viruses are site-specific recombinase systems, which have developed into powerful tools for genetic manipulation in vivo and in vitro. It is composed of a recombinase and a special DNA sequence, and belongs to the homologous system in eukaryotic cells.
  • the recombinase Flp is a monomeric protein composed of 423 amino acids in yeast cells; similar to Cre, Flp does not require any cofactors to function, and has good stability under different conditions.
  • FIG. 1 is an architecture diagram of three viruses operating a three-level neural loop
  • FIG. 2 is a virus injection scheme operating a three-level neural loop.
  • AAV-FLEX loxP -ChR2-eYFP was injected into Cre-line animals (the Cre animal commonly used in research is a Cre transgenic tool rat, which is widely commercialized.
  • a brain area AAV-FLEx FRT -hM4D(Gi)-mCherry injection in the B brain area, CAV-FLEx loxP -FLP injection in the C brain area. among them,
  • AAV-FLEX loxP -ChR2-eYFP virus was injected into the A brain area of Cre-line animals.
  • the ChR2 photosensitive protein was expressed on the cell membrane of the A brain area.
  • the optical fiber was buried in the B brain area downstream of the A brain area. Neural circuit function.
  • the CAV-FLEx loxP- FLP virus was injected into the C brain area of Cre-line animals.
  • the CAV virus carrying Loxp and FLP genes was absorbed by the nerve endings in the C brain area, and the CAV virus retrogrades along the nerve terminals into the superior neuron cell body of the C brain area. ;
  • CAV expresses FLP protein in neurons that express Cre protein at the upper level of the C brain region.
  • AAV-FLEx FRT- hM4D(Gi)-mCherry virus was injected into the B brain area of Cre-line animals. In cells expressing FLP protein in the B brain area, FLP and FRT combined to initiate hM4D(Gi) expression.
  • hM4D(Gi) functions to inhibit nerve cell activity.
  • the A-B neural circuit is activated with light in the B brain area.
  • the above three-level neural circuit manipulation composition of the present invention based on the problems of manipulating the third-level neural circuit such as optogenetics and pharmacogenetics, utilizes the CAV virus to be retrograde after being absorbed by nerve endings, and the AAV virus anterograde characteristics.
  • the combined use of viruses achieves the purpose of simultaneously manipulating tertiary neurons, ABC.
  • the tertiary nerve circuit manipulation composition in the above embodiments and the animal tertiary nerve circuit manipulation method implemented by using the tertiary nerve circuit manipulation composition are limited to animal experiments and realize the animal tertiary nerve circuit manipulation Manipulated experimental research cannot be used for specific human clinical applications.
  • Cre transgenic tool mice with Cre/loxP and Flp/FRT recombinase expression systems directly purchased by Jackson Lab as the recipient animals;
  • AAV-FLEX loxP- ChR2-eYFP virus obtained AAV-FLEx FRT- hM4D (Gi)-mCherry virus, and CAV-FLEx loxP- FLP virus; among them, AAV-FLEX loxP- ChR2-eYFP virus, AAV-FLEx FRT -hM4D(Gi)-mCherry virus was directly purchased from Wuhan Shumi Brain Science and Technology Co., Ltd.
  • the result verified in Figure 3 shows that after the virus AAV-FLEX loxP -ChR2-eYFP is hit in the A brain area of Gad-cre mice, the virus is expressed in the neurons in the A brain area, and the neurons in the A brain area can be projected to B brain area, so if the expression of green fluorescent protein is seen in the fibers of B brain area, this virus is effective. That is, the AB neural circuit can be manipulated with optogenetics technology.
  • CAV-FLEx loxP- FLP was injected in the C brain area, and CAV virus was absorbed by nerve endings and reached the B brain area along the nerve fibers.
  • AAV-FLEx FRT- hM4D(Gi)-mCherry virus is injected into the B brain area, and the virus will be expressed only with the help of FLEx loxP and FLP components brought in by CAV virus. Therefore, seeing the yellow fluorescence in the B brain area proves that the viruses in the B and C brain areas are effective.

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Abstract

所述三级神经环路操纵组合物包括AAV-FLEX loxP-ChR2-eYFP病毒、AAV-FLEx FRT-hM4D（Gi）-mCherry病毒、以及CAV-FLEx loxP-FLP病毒。所述三级神经环路操纵组合物基于光遗传学、药物遗传学等操纵三级神经环路的问题，利用CAV病毒被神经末梢吸收后逆行、AAV病毒顺行特性，将三种病毒联合使用，实现同时操纵三级神经元即A-B-C的目的。

Description

三级神经环路操纵组合物及动物三级神经环路操纵方法 技术领域
本发明涉及神经元活动操纵技术领域，尤其涉及一种三级神经环路操纵组合物及动物三级神经环路操纵方法。
 背景技术
神经科学领域最难解决的问题是人为实时操纵神经元活动。在光遗传学被发明之后，才开始实现可以用光实时操纵神经元活动；后来逐渐药物遗传学、磁遗传学等也陆续被发明用来操纵神经元活动。采用以上技术操纵神经元的时候，先用携带特异性启动子、光敏感基因和报告基因的AAV等载体病毒注射入模式动物的特定脑区。AAV病毒在有启动子的细胞中表达，最远扩散至轴突末端。目前通过这种方法，最多可以实现两个脑区之间，即两级神经元A-B之间的操纵。
而大脑中的神经元相互连接成错综复杂的网络，信息自上而下传输，控制多级神经环路才能解析信息流向；以上操纵其中的两级神经元，只能解析A-B投射对于模式动物行为、生理等的影响，无法实现对A-B的投射的信息流下游进行解析。
 技术问题
本发明的主要目的在于提供一种三级神经环路操纵组合物及三级神经环路操纵方法，旨在用光遗传学、药物遗传学等实现操纵三级神经环路的目的。
 技术解决方案
为实现上述目的，本发明提供的三级神经环路操纵组合物，包括AAV-FLEX loxP-ChR2-eYFP病毒、AAV-FLEx FRT-hM4D（Gi）-mCherry病毒、以及CAV-FLEx loxP-FLP病毒。
优选地，所述组合物还包括N-氧化氯氮平。
本发明进一步提出将以上三级神经环路操纵组合物施用于动物进行动物三级神经环路操纵方法。
优选地，向所述动物对象施用所述三级神经环路操纵组合物的步骤包括：
将所述AAV-FLEX loxP-ChR2-eYFP病毒注射在动物对象的A脑区；
将所述AAV-FLEx FRT-hM4D(Gi)-mCherry病毒注射在动物对象的B脑区；
将所述CAV-FLEx loxP-FLP病毒注射在动物对象的C脑区。
进一步优选地，将所述CAV-FLEx loxP-FLP病毒注射在动物对象的C脑区步骤之后，还包括：
向所述动物对象注射CNO。
优选地，所述动物对象为Cre-line动物。
 有益效果
本发明的以上三级神经环路操纵组合物，基于光遗传学、药物遗传学等操纵三级神经环路的问题，利用CAV病毒被神经末梢吸收后逆行、AAV病毒顺行特性，将三种病毒联合使用，实现同时操纵三级神经元即A-B-C的目的。
 附图说明
图1为本发明三级神经环路操纵组合物三种病毒操纵三级神经环路的架构图；
图2是本发明三级神经环路操纵组合物三种病毒操纵三级神经环路的病毒注射方案；
图3是发明实施例以Cre转基因工具小鼠注射病毒后操作神经环路实验的目的蛋白表达的检测结果图。
 本发明的实施方式
本发明目的的实现、功能特点及优点将结合实施例，参照附图做进一步说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
本发明提供一种三级神经环路操纵组合物，包括AAV-FLEX loxP-ChR2-eYFP病毒、AAV-FLEx FRT-hM4D（Gi）-mCherry病毒、以及CAV-FLEx loxP-FLP病毒。
其中，AAV-FLEX loxP-ChR2-eYFP病毒和AAV-FLEx FRT-hM4D（Gi）-mCherry病毒均是以AAV腺相关病毒为病毒载体与目的基因重组后的重组病毒，分别可以在AAV-FLEX loxP和AAV-FLEx FRT重组表达系统中表达目的光敏蛋白ChR2-eYFP和hM4D（Gi）-mCherry。
CAV-FLEx loxP-FLP病毒同样也是以CAV病毒（犬腺状病毒）为载体表达FLP蛋白的重组病毒。
以上三种病毒作为神经元活动操纵使用时，AAV-FLEX loxP-ChR2-eYFP病毒用作注射在A脑区，可以标记A脑区的神经元，在B脑区埋置光纤可以操纵A-B神经环路；CAV-FLEx loxP-FLP病毒用作注射在C脑区，CAV病毒被神经元末端吸收，顺着轴突到C脑区上游的脑区中，在C脑区上游的脑区的Cre细胞中表达，同时细胞中FLP蛋白也被达；AAV-FLEx FRT-hM4D(Gi)-mCherry病毒注射在B脑区，在B脑区中有FLP蛋白的细胞中表达。hM4D（Gi）表达后，在CNO（N-氧化氯氮平，一种具有很多细胞靶标的代谢物，可以结合到所设定的定制受体上）药物作用下，操纵B-C特异性神经环路。从而三者共同结合使用时，通过CAV病毒、AAV病毒联合，实现操纵三级神经元功能活动的目的。
其中，所述AAV-FLEX loxP-ChR2-eYFP病毒注射A脑区的剂量按照每只实验小鼠500nl的剂量比例注射；所述AAV-FLEx FRT-hM4D（Gi）-mCherry注射B脑区的剂量按照每只实验小鼠200nl的剂量比例注射；所述CAV-FLEx loxP-FLP病毒注射C脑区的剂量按照每只实验小鼠100nl的剂量比例注射；各病毒，也即是AAV-FLEX loxP-ChR2-eYFP病毒、AAV-FLEx FRT-hM4D（Gi）-mCherry、CAV-FLEx loxP-FLP病毒的浓度可以为10 12 vg/mL。当然，当采用其他动物模型（也即是非小鼠动物模型）时，注射剂量可以根据其他动物脑容积与实验小鼠的脑容积比例，并按照上述实验小鼠各脑区注射剂量按比例进行调整注射剂量。
另外，大脑根据功能、结构可以区分成很多大大小小的区域，并且根据它的位置进行定义，比如下丘脑，纹状体床核，孤束核等。为了执行具体的功能比如，跑，吃等，这些脑区之间相互通过神经纤维连接，我们称之为神经环路。在本专利中，为了进一步清楚的阐明我们的系统在神经环路功能研究中的应用方法，我们举例说明，上述各实施例中的所述A脑区在为纹状体床核，所述B脑区为下丘脑，所述C脑区为孤束核。
其中，以上重组病毒中分别具有的重组表达系统Flp／FRT系统和Cre／loxP系统是位点特异性重组酶系统，已发展成为在体内、外进行遗传操作的有力工具。其都是由一个重组酶和一段特殊的DNA序列组成，属于在真核细胞内的同源系统。其中，重组酶Flp是酵母细胞内的一个由423个氨基酸组成的单体蛋白；与Cre相似，Flp发挥作用也不需要任何辅助因子，同时在不同的条件下具有良好的稳定性。
具体，其神经环路架构图和注射方案可以参见图1和图2所示，图1 是三种病毒操纵三级神经环路的架构图，图2是操纵三级神经环路的病毒注射方案示意图。实施中分别将AAV-FLEX loxP-ChR2-eYFP注射在Cre-line动物（现在研究中常用的Cre 动物，是Cre转基因工具鼠，商业化很广泛。可以从Jackson lab公司直接采购，或者从具有Cre小鼠资源库的相关机构或国家单位申请获取）的A脑区，AAV-FLEx FRT-hM4D(Gi)-mCherry注射在B脑区，CAV-FLEx loxP-FLP在注射C脑区。其中，
在Cre-line动物的A脑区注射AAV-FLEX loxP-ChR2-eYFP病毒，ChR2光敏蛋白表达在A脑区细胞膜上，将光纤埋在A脑区的下游B脑区中，可以利用光操纵A-B神经环路功能。
在Cre-line动物的C脑区注射CAV-FLEx loxP-FLP病毒，CAV病毒携带Loxp和FLP基因被C脑区中神经末梢吸收，CAV病毒顺着神经末梢逆行到C脑区上级神经元胞体中；CAV在C脑区上级表达Cre蛋白的神经元中，表达FLP蛋白。
在Cre-line动物的B脑区注射AAV-FLEx FRT-hM4D(Gi)-mCherry病毒，在B脑区中表达FLP蛋白的细胞中，FLP和FRT结合，启动hM4D(Gi)表达。
因此结合使用时具有CAV病毒被神经末梢吸收后逆行、AAV病毒顺行的特性；并且进一步将CNO注射入动物体内后，hM4D(Gi)发挥功能抑制神经细胞活动。同时，在B脑区用光激活A-B神经环路。
综上，用以上结合的方式即可以实现同时操纵三级神经环路A-B-C的功能。
本发明的以上三级神经环路操纵组合物，基于光遗传学、药物遗传学等操纵三级神经环路的问题，利用CAV病毒被神经末梢吸收后逆行、AAV病毒顺行特性，将三种病毒联合使用，实现同时操纵三级神经元即A-B-C的目的。
上述各实施例中的三级神经环路操纵组合物及利用所述三级神经环路操纵组合物实施的动物三级神经环路操纵方法均限于动物实验中，实现对动物三级神经环路操纵的实验研究，不能用于具体人体临床应用。
为使以上方法实施细节能被从事神经学技术的人员所能理解，以下通过具体的实验过程进行举例说明。
实施例1
S10，以Jackson lab公司直接采购的Cre转基因工具小鼠（具有Cre／loxP和Flp／FRT重组酶表达系统）作为受体动物；
S20，获取AAV-FLEX loxP-ChR2-eYFP病毒、AAV-FLEx FRT-hM4D（Gi）-mCherry病毒、以及CAV-FLEx loxP-FLP病毒；其中，AAV-FLEX loxP-ChR2-eYFP病毒、AAV-FLEx FRT-hM4D（Gi）-mCherry病毒直接采购至武汉枢密脑科学技术有限公司（http://www.brainvta.com/index.php?option=com_content&view=article&id=36&Itemid=248），以及CAV-FLEx loxP-FLP病毒采购自法国Plateforme de Vectorologie de Montpellier (PVM)公司（https://www.pvm.cnrs.fr/plateau-igmm/?vector=recombinase）。本实验步骤实施中，以上资源直接采用购买获得，在资源充分和设备条件充足时，技术人员也可以用各自的目的基因、表达载体系统与病毒宿主自行重组合成，筛选能正常在所描述的重组表达系统下表达目的蛋白的重组病毒。
S30，向A脑区纹状体床核注射AAV-FLEX loxP-ChR2-eYFP病毒500nl；B脑区下丘脑（ARC）注射AAV-FLEx FRT-hM4D（Gi）-mCherry病毒200nl；
C脑区孤束核注射CAV-FLEx loxP-FLP病毒注射100nl，各病毒的浓度为10 12 vg/mL；
S40，基于光实时操纵神经元的目的，在6周后，向受体动物A-B脑区埋置光纤；
S50，再1周后，CNO腹腔注射，进行实验；
S60，用免疫组化和原位杂交的方式进行（操作步骤按照购买的试剂盒的操作说明进行即可），验证病毒表达。结果参见附图3。
图3中验证的结果可知，将病毒AAV-FLEX loxP-ChR2-eYFP打在Gad-cre老鼠的A脑区之后，病毒在A脑区的神经元中表达，A脑区的神经元可以投射到B脑区，所以如果在B脑区纤维中看到绿色荧光蛋白的表达，说明此病毒有效。即可以用光遗传学技术操纵A-B神经环路。
将CAV-FLEx loxP-FLP注射在C脑区，CAV病毒被神经末梢吸收后顺着神经纤维达到B脑区。
将AAV-FLEx FRT-hM4D（Gi）-mCherry病毒注射在B脑区中，借助CAV病毒带过来的FLEx loxP和FLP组件，病毒才会表达。因此看到B脑区中黄色荧光证明，B和C脑区的病毒有效。
在B脑区中用原位杂交的方式，用Gad2探针标记红色荧光的细胞，共标记信号表明我们的确在B脑区Gad2神经元中的特异投射至C脑区中成功转染药物遗传学操纵组件hM4D（Gi）。
以上仅为本发明的优选实施例，并非因此限制本发明的专利范围，凡是利用本发明说明书及附图内容所作的等效结构或等效流程变换，或直接或间接运用在其他相关的技术领域，均同理包括在本发明的专利保护范围内。

Claims (6)

  1. 一种三级神经环路操纵组合物，其特征在于，包括AAV-FLEX loxP-ChR2-eYFP病毒、AAV-FLEx FRT-hM4D（Gi）-mCherry病毒、以及CAV-FLEx loxP-FLP病毒。
  2. 如权利要求1所述的三级神经环路操纵组合物，其特征在于，所述组合物还包括N-氧化氯氮平。
  3. 一种动物三级神经环路操纵方法，其特征在于，包括：
    向动物对象施用权利要求1或2所述的三级神经环路操纵组合物。
  4. 如权利要求3所述的动物三级神经环路操纵方法，其特征在于，向所述动物对象施用所述三级神经环路操纵组合物的步骤包括：
    将所述AAV-FLEX loxP-ChR2-eYFP病毒注射在动物对象的A脑区；
    将所述AAV-FLEx FRT-hM4D(Gi)-mCherry病毒注射在动物对象的B脑区；
    将所述CAV-FLEx loxP-FLP病毒注射在动物对象的C脑区。
  5. 如权利要求4所述的动物三级神经环路操纵方法，其特征在于，将所述CAV-FLEx loxP-FLP病毒注射在动物对象的C脑区步骤之后，还包括：
    向所述动物对象注射CNO。
  6. 如权利要求3至5任一项所述的动物三级神经环路操纵方法，其特征在于，所述动物对象为Cre-line动物。
PCT/CN2018/122938 2018-12-13 2018-12-22 三级神经环路操纵组合物及动物三级神经环路操纵方法 Ceased WO2020118760A1 (zh)

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