WO2016188112A1 - 特异性兴奋伏隔核中的gaba能神经元的组合物及其在改善精神分裂症异样行为中的应用 - Google Patents

特异性兴奋伏隔核中的gaba能神经元的组合物及其在改善精神分裂症异样行为中的应用 Download PDF

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WO2016188112A1
WO2016188112A1 PCT/CN2015/099667 CN2015099667W WO2016188112A1 WO 2016188112 A1 WO2016188112 A1 WO 2016188112A1 CN 2015099667 W CN2015099667 W CN 2015099667W WO 2016188112 A1 WO2016188112 A1 WO 2016188112A1
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nucleus accumbens
schizophrenia
composition
blue light
gene
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屠洁
王立平
杨帆
刘运辉
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Shenzhen Institute of Advanced Technology of CAS
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/867—Retroviral vectors

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  • the present invention relates to a composition for specifically stimulating GABAergic neurons in the nucleus accumbens and its use in improving the abnormal behavior of schizophrenia.
  • Schizophrenia is one of the most serious mental illnesses in human beings. It is characterized by changes in basic personality, division of thought, perception, emotion, and behavior, and disharmony between mental activity and environment. Epidemiological survey data show that the world's patients with schizophrenia and neurological disorders account for about 2% of the total population. Schizophrenia has the characteristics of high disability rate, high recurrence rate, poor prognosis and heavy social burden, which seriously affects the population health and economic development of our country.
  • D2 type dopamine receptor D2 (DRD2) antagonists have shown significant therapeutic effects as therapeutic psychotropic drugs.
  • D2 type dopamine receptor D2 (DRD2) antagonists have shown significant therapeutic effects as therapeutic psychotropic drugs.
  • D2 type dopamine receptor D2 (DRD2) antagonists have shown significant therapeutic effects as therapeutic psychotropic drugs.
  • amphetamine-like neurolally active substances that increase dopamine release in healthy individuals can induce psychosis and can also exacerbate psychotic symptoms in patients with schizophrenia.
  • the use of the dopamine precursor levodopa in patients with Parkinson's disease can induce psychotic symptoms (usually hallucinations and delusions) in many patients.
  • GABA ⁇ -aminobutyric acid
  • the nucleus accumbens is located at the junction of the basal ganglia and the marginal system, the outer part of the compartment, the inner and lower parts of the caudate nucleus, the anterior and anterior nucleus, and the ventral nucleus, the ventral side of the ventral pallidus and the olfactory tubercle. It is a larger nucleus of the basal forebrain.
  • the basic cell type of the nucleus is a medium-sized spiny neuron.
  • the neurotransmitter produced by such neurons is gamma-aminobutyric acid (GABA).
  • GABA gamma-aminobutyric acid
  • the main object of the present invention is to provide a technique for specifically regulating a target and thereby effectively improving the abnormal behavior of schizophrenia.
  • the invention utilizes the photogenetic regulation technology to specifically regulate the cells carrying the light-sensitive channel protein in the nucleus accumbens of schizophrenia animals, and simultaneously monitors the behavioral changes, and finds that the nucleus accumbens brain region is specifically excited and can significantly improve its innate Abnormal behavior of fear weakening and/or anxiety, which suggests that the nucleus accumbens is likely to be a new target for interfering with schizophrenia.
  • the invention provides a composition of a GABAergic neuron in a specific excitatory nucleus accumbens, the composition comprising:
  • a viral vector carrying a photo-sensing gene for infecting GABAergic neurons in the nucleus accumbens;
  • the vector comprises a promoter, a photoreceptor gene, and a green fluorescent marker gene, wherein the promoter is a Syn promoter;
  • a light-emitting device capable of generating blue light for illuminating the infected nucleus accumbens to excite it.
  • a lentiviral vector carrying a light-sensitive gene is injected into a nucleus accumbens region, which will infect GABAergic neurons in the nucleus accumbens, express corresponding photoreceptors in GABAergic neurons, and pass the illumination system.
  • GABAergic neurons expressing photoreceptors undergo light stimulation to excite them, and GABAergic neurons are excited by light. Play a role in improving the behavior of schizophrenia.
  • the invention proves that the stimulation of the nucleus accumbens by the composition of the invention can significantly improve the abnormal behavior of schizophrenia, such as congenital weakening fear and/or anxiety, by an elevated labyrinth experiment, which can effectively improve the animal model of schizophrenia. The wrong judgment of factors improves their ability to avoid related adverse environments. Therefore, the present invention provides a novel therapeutic target for the anatomical abnormal behavior of schizophrenia patients lacking safety awareness, and improving their cognitive ability to avoid danger; and provides a new strategy for developing new drugs.
  • the composition of the GABAergic neurons in the specific excitatory nucleus accumbens described in the present invention may also be referred to as a "set” or “kit”, including the present invention.
  • kit Specific reagents, materials, instruments and other components used in GABAergic neurons in specific nucleus accumbens, in the form of a product such as a kit, each component can be placed in a large package according to a predetermined position or Completely packaged and used after packaging.
  • the composition of the GABAergic neurons in the specific excitatory nucleus accumbens of the present invention further comprises: infecting the GARNA energy in the nucleus accumbens by the viral gene carrying the photoreceptor gene Neuronal reagent material.
  • the stereotaxic instrument and the related sample introduction system can accurately inject the virus carrier carrying the light-sensitive gene into the nucleus accumbens region, thereby infecting the GABAergic neuron cells in the nucleus accumbens region.
  • a photo-inducing gene-borne viral vector for infecting a GABAergic neuron cell or a precursor cell thereof in a nucleus accumbens;
  • a promoter in the vector is a Syn promoter;
  • the gene is ChR2;
  • the green fluorescent marker gene is eYFP;
  • the virus is a lentivirus; the specific construction process can be carried out by referring to the prior art in the field.
  • the viral vector carrying the light-sensitive gene is a lentiviral vector containing a Syn plasmid.
  • the promoter Syn initiates transcription with a light-sensitive gene and expresses a corresponding photoreceptor in GABAergic neurons in the nucleus accumbens. After being stimulated by light, the nucleus accumbens brain region is excited, which plays a role in improving the abnormal behavior of schizophrenia.
  • the composition for improving the abnormal behavior of schizophrenia according to the present invention wherein the lentiviral vector carrying a light-sensitive gene is a lentiviral vector containing a plasmid carrying Syn-ChR2-eYFP
  • the Syn-ChR2-eYFP can be constructed by using a plasmid containing a light-sensitive gene, a green fluorescent marker gene, and a promoter for specific marker neurons, Synapsin (Syn). And mixed plasmids associated with lentiviral packaging Together, pCMV ⁇ R8.74 and pMD2.G. were transfected into 293FT cells together with liposomes, and ruptured to obtain a lentiviral vector containing the plasmid containing Syn-ChR2-eYFP.
  • the composition of the GABAergic neuron cell in the specific excitatory nucleus accumbens of the present invention wherein the blue light is blue light having a wavelength of 460 to 480 nm, preferably blue light having a wavelength of 470 nm.
  • the invention provides the use of a composition of a GABAergic neuron in a specific excitatory nucleus accumbens for the preparation of a system for ameliorating schizophrenia.
  • a composition of a GABAergic neuron in a specific excitatory nucleus accumbens is a composition of the present invention, the use of the composition of the present invention in the preparation of a tissue for improving schizophrenia caused by congenital fear weakening and / Or an application in an anxiety system.
  • the improved schizophrenia abnormal behavior system utilizes a viral vector carrying a photo-sensing gene in the composition to infect a nucleus accumbens region of the brain.
  • the GABAergic neurons are stimulated by blue light to the infected nucleus accumbens by means of a light device to improve the abnormal behavior of schizophrenia.
  • the system further comprises an optical fiber for conducting blue light to the infected nucleus accumbens.
  • 200 ⁇ m of optical fiber is used to introduce light generated by the illumination device into the nucleus accumbens of the brain for light regulation of the infected nucleus accumbens.
  • the blue light illumination condition is: a blue light wavelength of 470 to 480 nm, an optical fiber outlet power of about 12 to 20 mW, and an illumination frequency of 40 to 80 Hz;
  • the duty ratio of :1 lasts for more than 5 minutes.
  • the invention also provides a system for ameliorating schizophrenia abnormal behavior, the system comprising a composition of the invention.
  • the heterosexual behavior of schizophrenia is innate fear of weakening and/or anxiety.
  • the present invention also provides a method of improving the abnormal behavior of schizophrenia, the method comprising:
  • the lentiviral vector carrying the light-sensitive gene of the invention is injected into the nucleus accumbens region to infect GABAergic neurons in the nucleus accumbens, and the corresponding photoreceptor is expressed in the GABAergic neurons;
  • the GABAergic neurons expressing the photoreceptor were stimulated by the light system to excite them.
  • the GABAergic neurons function to improve the abnormal behavior of schizophrenia after being excited by light.
  • the infected nucleus accumbens is subjected to blue light illumination stimulation by an illumination device to improve the abnormal behavior of schizophrenia.
  • the blue light conditions are: blue light wavelength 470 ⁇ 480nm, fiber outlet power 12 ⁇ 20mW, light frequency 40 ⁇ 80Hz; duty cycle 1:1, lasting more than 5min.
  • the technology of the present invention can accurately and specifically excite GABAergic neurons in the nucleus accumbens, and can effectively improve the abnormal behavior of schizophrenia, and achieve specificity compared with the traditional dopamine antagonist drug treatment. , is of great significance.
  • Figure 1A is a schematic diagram of the Syn-ChR2-eYFP plasmid
  • 1B to 1C are schematic diagrams showing successful expression of Syn-ChR2-eYFP in GABAergic neurons in mouse nucleus accumbens, in which Acbc is a nucleus accumbens and Acbsh is a nucleus accumbens;
  • FIG. 2 is a schematic diagram of illumination of a nucleus accumbens region of a mouse by illumination, in which Acbc is a nucleus accumbens and Acbsh is a nucleus spacer;
  • Example 3 is a graph showing experimental results of improving the excitability of the nucleus accumbens brain region under illumination conditions in Example 1;
  • FIG. 4 is a schematic diagram showing the results of an elevated cross maze experiment of wild type, DISC1::ChR2, and DISC1::eYFP mice in Example 1;
  • Fig. 5 is a graph showing the statistical results of the results of the elevated plus maze test of the wild type, DISC1::ChR2 and DISC1::eYFP mice in Example 1.
  • a lentiviral vector for infecting a nucleus accumbens in an animal which carries a Syn-ChR2-eYFP plasmid: a plasmid containing a light-sensitive gene, a green fluorescent marker gene, and a promoter for specific marker nerve cells (0.5) Gg) (the fusion plasmid structure is shown in Figure 1A), together with the mixed plasmids pCMV ⁇ R8.74, pMD2.G.
  • the virus titer can reach 3 ⁇ 10 8 TU/mL or more, and the produced virus particles are resuspended in a volume ratio of 1:1000 in a sterilized phosphate buffer.
  • the obtained virus resuspension was mixed into serum-free DMEM at a volume ratio of 1:400 for infection of the target brain region of the animal model: nucleus accumbens.
  • the lentiviral vector carrying the Syn-ChR2-eYFP plasmid is injected into the regulatory region to infect the cells.
  • the above-prepared lentivirus carrying the light-sensitive gene was injected into the nucleus accumbens of the mouse by a conventional stereotactic technique in the field of neuroscience and a micro-injection system, and the injection amount was generally 0.4 ⁇ L. After the injection, it was waited for 3 weeks, and 1 was taken. ⁇ 3 mouse brain tissues were identified by immunofluorescence staining, and the results showed green fluorescence, indicating that Syn-ChR2-eYFP was successfully expressed in situ (see Figure 1B and Figure 1C), and then under light control. Behavioral testing of mice with schizophrenia, in which mice with schizophrenia are transgenic mice that induce expression of the DISC1 mutant protein using tamoxifen.
  • the commercially available fiber of 200 ⁇ m diameter was vertically inserted, and the blue light was stimulated at 470 nm, and the light intensity was 1.1 mW, and the illumination frequency was 60Hz, 50ms every 50ms, the duty ratio is 1:1, after 5min, the discharge characteristics of the neurons in the nucleus accumbens are extracted and analyzed by the method of body light-electricity stimulation and recording. The results are shown in the figure. As shown in Fig. 3, the results in Fig.
  • DISC1::ChR2 indicates that the excitability of the cerebral nucleus accumbens is enhanced after light stimulation, and then the mouse (numbered DISC1::ChR2) is subjected to an elevated plus maze test, and to wild type mice and DISC1.
  • the schizophrenic mice (numbered DISC1::eYFP) transfected with the eYFP gene alone were compared.
  • the results are shown in Fig. 4, and Fig. 5 is the statistical result.
  • DISC1::ChR2 indicates that GABAergic neurons in the nucleus accumbens of schizophrenia are transfected with ChR2 and express a light-sensitive channel protein in DISC1 transgenic mice.
  • DISC1::eYFP indicates mental illness
  • the GABAergic neuron cells in the nucleus accumbens of schizophrenia are only infected with eYFP and express the DISC1 transgenic mouse of the gene.
  • ON indicates that the light stimulation is turned on, and the light stimulation shown is as described above, and OFF means OFF. Light stimulation.
  • the high plus maze is the use of animals to explore the characteristics of new and different environments and The fear of high open arms creates conflicting behaviors to examine the anxiety state of animals.
  • the elevated cross maze has a pair of open arms and a pair of closed arms. Rodents tend to move in the closed arms due to their darkness, but they will move in the open arms out of curiosity and inquiry. When stimulated, the animal simultaneously generates the impulse and fear of inquiry, which causes the conflicting behavior of inquiry and avoidance, which leads to anxiety.
  • the anti-anxiety drugs can significantly increase the number and time of entering the open arms.
  • the cross maze is higher than the ground, which is equivalent to people standing on the cliffs, causing fear and anxiety in the subjects.
  • the labyrinth of this embodiment is 60 cm above the ground, the open arm is 25 x 5 cm (length x width), and the closing arm is 25 x 5 x 14.3 cm (length x width x height).
  • the elevated plus maze experiment showed that the DISC1 schizophrenic mice (DISC1::ChR2 and DISC1::eYFP) had lower congenital fear of the open arms under the OFF state before the regulation. Wild-type mice have a higher probability of entering the open arm than the wild type.
  • optogenetics specifically regulates the NAc brain region of DISC1 mice (DISC1::ChR2) (injection contains the photogene ChR2 in the NAc brain region and directly stimulates the brain region with light), which is compared with the wild type.

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Abstract

提供了一种特异性兴奋伏隔核中的GABA能神经元的组合物及其在改善精神分裂症异样行为中的应用。该组合物包括用于感染伏隔核中的GABA能神经元的携带光感基因的病毒载体和能产生蓝光的光照装置，其中所述病毒载体包括Syn启动子、光感基因、绿色荧光标记基因。

Description

特异性兴奋伏隔核中的GABA能神经元的组合物及其在改善精神分裂症异样行为中的应用 技术领域
本发明是关于一种特异性兴奋伏隔核中的GABA能神经元的组合物及其在改善精神分裂症异样行为中的应用。
背景技术
中国十二个地区精神疾病流行病学调查显示，目前全国约有1亿人有心理疾病，严重精神疾病患者约1600万人，每年约有二十五万人死于自杀。中国十七岁以下的3.4亿儿童、青少年中，约有三千万人受到情绪障碍和心理行为问题的困扰。当前神经精神疾病在我国疾病总负担中排名第一，占全部疾病和外伤所致残疾及劳动力丧失的五分之一。据专家预测，21世纪将是精神疾患流行的世纪，随着我国经济建设和社会发展的步伐加快，生活节奏和竞争日益增强，精神疾病会继续增加,预计到2020年，精神疾病在我国疾病总负担中将占到25％。世界卫生组织把新世纪的第一年定为精神卫生年，所以不管我们愿意与否，我们正无情地进入到了“精神疾病时代”。
精神分裂症(schizophrenia)是人类最严重的精神性疾病之一，以基本个性改变，思维、知觉、情感、行为的分裂，精神活动与环境的不协调为主要特征。流行病学调查资料显示，目前全世界患有精神分裂症和神经两级错乱的病人约占总人口的2％。精神分裂症具有致残率高、复发率高、预后不佳、社会负担沉重等特点，严重影响了我国人口健康和经济发展。
目前主流认为精神分裂症患者出现的症状与大脑内部多巴胺功能的亢进有某种联系。临床上把D2型多巴胺受体(Dopamine Receptor D2，DRD2)拮抗剂作为治疗精神病药物表现出了明显的治疗效果。相反，在健康个体上反复使用苯丙胺这类增加多巴胺释放的神经活性物质可以诱导出精神病，也可以加剧精神分裂症患者的精神病性症状。同样，对帕金森病患者使用多巴胺的前体左旋多巴，可以在许多患者中诱发精神病性症状(通常是幻觉和妄想)。
现在针对精神分裂症多巴胺类药物已持续了30多年，正反两方面的依据都有，但都没有突破性的进展。而且，精神分裂症患者是否存在多巴胺水平异常也缺乏直接的 证据：与健康个体比，精神分裂症患者的神经元在刺激下是否向突触释放出更多的多巴胺，至今没有定论；也有研究表明，精神分裂症患者可能存在对多巴胺运输和功能的异常调节。因此，目前控制精神病的发病主要依靠药物治疗和家庭社会的支持，无法对异常神经回路中多巴胺系统进行特异性的调控而无法实现真正治疗的目的，只是“治标”而已。
近年来γ-氨基丁酸(GABA)能网络在精神分裂症、抑郁症、自闭症及焦虑症等疾病中的作用正逐步被人们所认知。最新国际上的研究证据表明，GABA神经元的异常在精神分裂症中扮演了非常重要的作用。伏隔核位于基底核与边缘系统交界处，隔区的外下方，尾壳核的内下方，前方与嗅前核相连，后续终纹床核，腹侧为腹侧苍白球和嗅结节，是基底前脑的一个较大的核团。该核团的基本细胞类型是中型多棘神经元。这类神经元产生的神经递质是γ-氨基丁酸(GABA)。但伏隔核是否可作为调控精神分裂症异常行为的靶点还未进行研究，现有技术也尚未提供一种特异性干预伏隔核进而改善精神分裂症异常行为的方法。
发明内容
本发明的主要目的在于提供一种特异性调控靶标进而有效改善精神分裂症异常行为的技术。
本发明利用光遗传调控技术，特异性地调控精神分裂症动物伏隔核携带光敏通道蛋白的细胞，同时监测行为学的改变，发现伏隔核脑区特异性地兴奋后，能显著改善其先天恐惧弱化和/或焦虑的异常行为，从而认为伏隔核很可能是干预精神分裂症异样行为的新靶点。
从而，一方面，本发明提供一种特异性兴奋伏隔核中的GABA能神经元的组合物，该组合物包括：
用于感染伏隔核中的GABA能神经元的携带光感基因的病毒载体；该载体中包括启动子、光感基因、绿色荧光标记基因，其中，所述启动子为Syn启动子；
能产生蓝光的光照装置，用于对感染后的伏隔核进行光照调控以使其兴奋。
本发明中将携带光敏基因的慢病毒载体注入到伏隔核区，其将会感染伏隔核区中的GABA能神经元，在GABA能神经元内表达相应的光敏受体，通过光照系统对表达了光敏受体的GABA能神经元进行光刺激，使其兴奋，GABA能神经元受光兴奋后， 发挥改善精神分裂症异样行为的功能。本发明通过高架迷宫实验证明通过本发明的组合物刺激伏隔核兴奋后，能够明显的改善精神分裂症异样行为，例如先天性弱化恐惧和/或焦虑，可有效改善精神分裂症动物模型对危险因素的错误判断，提高其回避相关不利环境的能力。因此，本发明提供了针对精神分裂症患者缺乏安全意识的特征性异常行为，提高其回避危险的认知能力新的治疗靶点；同时为研制开发新型药物提供了新的策略。
根据本发明的具体实施方案，本发明中所述的特异性兴奋伏隔核中的GABA能神经元的组合物也可以称为一种“套组”或“套件”，其中包括了本发明中特异性兴奋伏隔核中的GABA能神经元所采用的各试剂、材料及仪器设备等组件，作为一种产品例如试剂盒的形式，各组件可按照预定的位置在大包装中分别放置或是独立包装后成套销售及使用。
根据本发明的具体实施方案，本发明中所述的特异性兴奋伏隔核中的GABA能神经元的组合物还包括：使所述携带光感基因的病毒载体感染伏隔核中的GABA能神经元的试剂材料。具体例如可包括：立体定位仪及相关进样系统，能够准确的将携带光敏基因的病毒载体注入到伏隔核区，从而使其感染伏隔核区中的GABA能神经元细胞。
在本发明的一具体实施方案中，构建了用于感染伏隔核中GABA能神经元细胞或其前体细胞的携带光感基因的病毒载体；该载体中的启动子为Syn启动子；光感基因为ChR2；绿色荧光标记基因为eYFP；所述病毒为慢病毒；具体的构建过程可以参照所属领域中的现有技术进行。
根据本发明的具体实施方式，其中所述的携带光敏基因的病毒载体为含带有Syn质粒的慢病毒载体。伏隔核中GABA能神经元细胞感染含带有Syn质粒的慢病毒载体后，启动子Syn启动携带光敏基因开始转录，在伏隔核区中GABA能神经元细胞内表达相应的光敏受体，在受到光刺激后从而使伏隔核脑区兴奋，起到改善精神分裂症异样行为的作用。根据本发明的具体实施方式，本发明中所述的改善精神分裂症异样行为的组合物，其中，所述的携带光敏基因的慢病毒载体为含带有Syn-ChR2-eYFP质粒的慢病毒载体，其结构如图1A所示。在本发明的一具体实施例中，所述的Syn-ChR2-eYFP可采用如下方法构建，将含有光敏基因、绿色荧光标记基因及用于特定性标记神经元的启动子Synapsin(Syn)的质粒和与慢病毒包装相关的混合质粒 pCMVΔR8.74、pMD2.G.一起，利用脂质体一起转染至293FT细胞，培养后破膜得到含带有Syn-ChR2-eYFP质粒的慢病毒载体。
根据本发明的具体实施方式，本发明所述的特异性兴奋伏隔核中GABA能神经元细胞的组合物中，其中，所述蓝光为波长460～480nm的蓝光，优选为波长470nm的蓝光。
另一方面，本发明提供特异性兴奋伏隔核中的GABA能神经元的组合物在制备用于改善精神分裂症异样行为系统中的应用。例如特异性兴奋伏隔核中的GABA能神经元的组合物为本发明所述的组合物，所述的应用为本发明的组合物在制备用于改善精神分裂症引起的先天恐惧弱化和/或焦虑系统中的应用。
根据本发明的具体实施方式，在本发明所述的应用中，其中，所述的改善精神分裂症异样行为系统是利用所述组合物中携带光感基因的病毒载体感染大脑伏隔核区中的GABA能神经元，并通过光照装置对感染后的伏隔核进行蓝光光照刺激，以改善精神分裂症异样行为。
根据本发明的具体实施方式，在本发明所述的应用中，其中，所述系统还包括用于将蓝光传导至感染后的伏隔核的光纤。在本发明的一具体实施例中，采用200μm的光纤将光照装置产生的光引入大脑伏隔核，用于对感染后的伏隔核进行光调控。
根据本发明的具体实施方式，在本发明所述的应用中，其中，所述蓝光光照条件为：蓝光波长470～480nm，光纤的出口功率12～20mW左右，光照频率40～80Hz的光；1：1的占空比，持续5min以上。
另一方面，本发明还提供一种改善精神分裂症异样行为系统，该系统包括本发明所述的组合物。例如，所述的精神分裂症异样行为是先天恐惧弱化和/或焦虑。
另一方面，本发明还提供了一种改善精神分裂症异样行为的方法，该方法包括：
将本发明的携带光敏基因的慢病毒载体注入到伏隔核区，使其感染伏隔核区中的GABA能神经元，在GABA能神经元内表达相应的光敏受体；
通过光照系统对表达了光敏受体的GABA能神经元进行光刺激，使其兴奋。
本发明的改善精神分裂症异样行为的方法中，GABA能神经元受光兴奋后，发挥改善精神分裂症异样行为的功能。
根据本发明的具体实施方案，本发明的改善精神分裂症异样行为的方法中，是通过光照装置对感染后的伏隔核进行蓝光光照刺激，以改善精神分裂症异样行为。所述 蓝光光照条件为：蓝光波长470～480nm，光纤的出口功率12～20mW左右，光照频率40～80Hz；占空比1：1，持续5min以上。
本发明的有益技术效果：本发明的技术可以精确地特异性兴奋伏隔核中的GABA能神经元，可以有效改善精神分裂症异样行为，较传统的多巴胺拮抗剂药物治疗，真正实现了特异性，具有重要意义。
附图说明
图1A为Syn-ChR2-eYFP质粒示意图；
图1B～1C为小鼠伏隔核中GABA能神经元细胞成功表达Syn-ChR2-eYFP的示意图，图中Acbc为伏隔核，Acbsh为伏隔核壳体；
图2为通过光照对小鼠伏隔核区进行光照的示意图，图中Acbc为伏隔核，Acbsh为伏隔核壳体；
图3为实施例1中光照条件下提高了伏隔核脑区兴奋性的实验结果图；
图4为实施例1中野生型、DISC1::ChR2以及DISC1::eYFP型小鼠的高架十字迷宫实验结果示意图；
图5为实施例1中野生型、DISC1::ChR2以及DISC1::eYFP型小鼠的高架十字迷宫实验结果的统计结果图。
具体实施方式
为了对本发明的技术特征、目的和有益效果有更加清楚的理解，现结合具体实例对本发明的技术方案进行以下详细说明，应理解这些实例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照所属领域的常规条件或按照制造厂商所建议的条件。
以下实施例中，所用原始试剂和材料均可商购获得，或可按照现有技术的记载制备得到。
实施例1
(1)、携带Syn-ChR2-eYFP质粒的慢病毒载体的制备。
制备用于在动物活体感染伏隔核的慢病毒载体，其携带有Syn-ChR2-eYFP质粒：将含有光敏基因、绿色荧光标记基因及用于特定性标记神经细胞的启动子Syn的质粒 (0.5μg)(该融合质粒结构如图1A所示)，和与慢病毒包装相关的混合质粒pCMVΔR8.74、pMD2.G.(1.5μg)一起，利用脂质体(Invitrogen公司产品)一起转染至293FT细胞(ATCC公司产品，该细胞传代至25代后即需要更换新的一批细胞株)中(每200,000个细胞用6μL脂质体)。24小时后，培养293FT细胞的培养液换成无血清含有5mM丙酮酸钠的DMEM后继续孵育。16小时后，利用超速离心机在50,000g速度下破膜，将悬液通过20％的蔗糖滤柱，收集上清。该病毒滴度能达3×108TU/mL以上，产出的病毒颗粒以灭菌的磷酸盐缓冲液以1：1000的体积比进行重悬。将获取的病毒重悬液按1：400的体积比混入无血清DMEM中，用于感染动物模型的目标脑区：伏隔核。
(2)、将携带Syn-ChR2-eYFP质粒的慢病毒载体注入调控区使其感染细胞。
采用神经科学领域常规的立体定位技术以及微量进样系统将上述制备得到的携带光敏基因的慢病毒注入到小鼠的伏隔核，注入量一般为0.4μL，注入后，等待3周，取1～3只小鼠脑组织，进行免疫组织荧光染色鉴定，鉴定结果显示有绿色荧光，表明Syn-ChR2-eYFP在原位表达成功(可参见图1B及图1C)，即可开展光调控下的患精神分裂症的小鼠行为学检测，其中患精神分裂症的小鼠为采用他莫昔芬(tamoxifen)诱导DISC1突变蛋白表达的转基因小鼠。
(3)、光调控下的患精神分裂症的小鼠行为学检测。
如图2所示，在上述Syn-ChR2-eYFP原位表达成功的小鼠的伏隔核脑区垂直插入市售直径为200μm光纤，进行470nm的蓝光刺激，光强为1.1mW，光照频率为60Hz，每隔50ms照射50ms，占空比为1:1，持续5min后，采用在体光-电一体刺激和记录的方法对伏隔核脑区神经元放电特征进行提取分析，其结果如图3所示，图3结果表明光刺激后，提高了大脑伏隔核脑区的兴奋性，然后对小鼠(编号为DISC1::ChR2)进行高架十字迷宫实验，同时对野生型小鼠以及DISC1型及仅转染eYFP基因的精神分裂症小鼠(编号为DISC1::eYFP)进行对比实验，其结果如图4所示，图5为其统计结果。图4及图5中DISC1::ChR2表示患精神分裂症的伏隔核中的GABA能神经元细胞转染了ChR2并表达了光敏感通道蛋白的DISC1转基因小鼠，DISC1::eYFP表示患精神分裂症的伏隔核中的GABA能神经元细胞仅感染了eYFP并表达了该基因的DISC1转基因小鼠，图中ON表示打开光刺激，所示的光刺激的方式如上所述，OFF表示关闭光刺激。
所述的高架十字迷宫(High plus maze)是利用动物对新异环境的探究特性和对 高悬敞开臂的恐惧形成矛盾冲突行为来考察动物的焦虑状态。高架十字迷宫具有一对开臂和一对闭臂，啮齿类动物由于嗜暗性会倾向于在闭臂中活动，但出于好奇心和探究性又会在开臂中活动，在面对新奇刺激时，动物同时产生探究的冲动与恐惧，这就造成了探究与回避的冲突行为，从而产生焦虑心理。而抗焦虑药物能明显增加进入开臂的次数与时间，十字迷宫距离地面较高，相当于人站在峭壁上，使实验对象产生恐惧和不安心理。本实施例的迷宫离地高60cm，开放臂为25×5cm(长×宽)，闭合臂25×5×14.3cm(长×宽×高)。
从图4及图5可以看出，高架十字迷宫实验表明，在调控前的OFF状态下，DISC1精神分裂症型小鼠(DISC1::ChR2及DISC1::eYFP)对开放臂的先天恐惧低于野生型小鼠，进入开放臂的概率大于野生型。但是在ON状态下，光遗传技术特异性调控DISC1小鼠的NAc脑区(DISC1：：ChR2)后(注射含有光基因ChR2在NAc脑区，并用光直接刺激该脑区)，其对比野生型对照组，其进入开放臂的概率就没有显著差异，提示调控起效；另外，对比打了空壳病毒(DISC1：：eYFP)的DISC1小鼠，其显著降低了进入开放臂的概率。

Claims (13)

  1. 一种特异性兴奋伏隔核中的GABA能神经元的组合物，该组合物包括：
    用于感染伏隔核中的GABA能神经元的携带光感基因的病毒载体；该载体中包括启动子、光感基因、绿色荧光标记基因，其中，所述启动子为Syn启动子；
    能产生蓝光的光照装置，用于对感染后的伏隔核进行光照调控以使其兴奋。
  2. 根据权利要求1所述的特异性兴奋伏隔核中的GABA能神经元的组合物，该组合物还包括：使所述携带光感基因的病毒载体感染伏隔核中的GABA能神经元的试剂材料。
  3. 根据权利要求1所述的特异性兴奋伏隔核中的GABA能神经元的组合物，其中，所述蓝光为波长460～480nm的蓝光，优选为波长470nm的蓝光。
  4. 特异性兴奋伏隔核中的GABA能神经元的组合物在制备用于改善精神分裂症异样行为的系统中的应用，其中，所述特异性兴奋伏隔核中的GABA能神经元的组合物优选为权利要求1～3中任一项所述的组合物。
  5. 根据权利要求4所述的应用，其中，所述的改善精神分裂症异样行为的系统是利用所述组合物中携带光感基因的病毒载体感染伏隔核中的GABA能神经元，并通过光照装置对感染后的伏隔核进行蓝光光照刺激，以改善精神分裂症异样行为。
  6. 根据权利要求4所述的应用，其中，所述的精神分裂症异样行为是先天恐惧弱化和/或焦虑。
  7. 根据权利要求5所述的应用，其中，所述改善精神分裂症异样行为的系统还包括用于将蓝光传导至感染后的伏隔核的光纤。
  8. 根据权利要求7所述的应用，其中，所述蓝光光照条件为：蓝光波长470～480nm，光纤的出口功率12～20mW，光照频率40～80Hz的光；1：1的占空比，持续5min以上。
  9. 一种改善精神分裂症异样行为的系统，该系统包括权利要求1～3中任一项所述的组合物。
  10. 根据权利要求9所述的改善精神分裂症异样行为的系统，其中，所述的精神分裂症异样行为是先天恐惧弱化行为和/或焦虑。
  11. 一种改善精神分裂症异样行为的方法，该方法包括：
    将一种携带光敏基因的慢病毒载体注入到伏隔核区，使其感染伏隔核区中的GABA能神经元，在GABA能神经元内表达相应的光敏受体；其中所述携带光感基因的病毒载体包括启动子、光感基因、绿色荧光标记基因，其中，所述启动子为Syn启动子；
    通过光照系统对表达了光敏受体的GABA能神经元进行光刺激，使其兴奋。
  12. 根据权利要求11所述的方法，其中是通过光照装置对感染后的伏隔核进行蓝光光照刺激，以改善精神分裂症异样行为。
  13. 根据权利要求12所述的方法，其中，所述蓝光光照条件为：蓝光波长470～480nm，光纤的出口功率12～20mW左右，光照频率40～80Hz；占空比1：1，持续5min以上。
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