通过慢病毒表达GAD67和CCK启动子驱动的视蛋白，在体外和体内靶向中间神经元。

Lentiviral expression of GAD67 and CCK promoter-driven opsins to target interneurons in vitro and in vivo.

作者信息

Mantoan Ritter Laura, Macdonald Douglas C, Ritter Georg, Escors David, Chiara Francesca, Cariboni Anna, Schorge Stephanie, Kullmann Dimitri M, Collins Mary

机构信息

Department of Clinical and Experimental Epilepsy, Institute of Neurology, University College London, London, UK.

Clinical Neurosciences Department, King's College NHS Foundation Trust, Denmark Hill, London, UK.

出版信息

J Gene Med. 2016 Jan-Mar;18(1-3):27-37. doi: 10.1002/jgm.2873.

DOI:10.1002/jgm.2873

PMID:26824337

Abstract

BACKGROUND

The ability to manipulate the activity of interneurons with optogenetic tools offers the possibility of interfering with diseases caused by altered neuronal inhibition and synchrony, including epilepsy and schizophrenia. To develop vectors for therapeutic approaches, targeting optogenetic constructs to interneurons is therefore a key requirement. We investigated whether the interneuron-specific promoters glutamic acid decarboxylase (GAD)67 and cholecystokinin (CCK) allowed targeted lentiviral delivery of opsins to interneurons as a whole, or specifically CCK+ interneurons.

METHODS

We generated lentiviral (LV) plasmids encoding channelrhodopsin (ChR2) and halorhodopsin (NpHR) tagged with fluorophores and driven by GAD67 or CCK promoters. Adeno-associated virus (AAV) and LV vectors carrying opsins driven by pyramidal cell promoters were used as controls. We transduced neuronal cultures and rodent brain in vivo, immunostained specimens 6-8 weeks after in vivo injection and 7-14 days after in vitro transduction, and evaluated volume and specificity of expression by confocal microscopy.

RESULTS

In vitro, 90% (19/21) of LV-CCK-NpHR2.0-EYFP expressing neurons were CCK+. In vivo, LV-GAD67-ChR2-mCherry was expressed in 2.6% (5/193), LV-GAD67-NpHR2.0-EYFP in approximately 15% (43/279) and LV-CCK-NpHR2.0-EYFP in 47% (9/19) of hippocampal GABA+ interneurons. GAD67 vectors expressed in larger volumes than CCK-driven constructs. AAV vector controls achieved the largest expression volumes.

CONCLUSIONS

LV-CCK-NpHR2.0-EYFP may be useful for targeting CCK+ interneurons in culture. GAD67/CCK-driven lentiviral constructs are expressed in vivo, although expression is not specific for interneurons. Overall, expression levels are low compared to opsins driven by pyramidal cell promoters. A better understanding of GAD67 and CCK promoter structure or alternative techniques is required to reliably target opsins to interneurons using viral vectors.

摘要

背景

利用光遗传学工具操纵中间神经元的活性，为干预由神经元抑制和同步性改变引起的疾病提供了可能，这些疾病包括癫痫和精神分裂症。因此，为开发治疗方法的载体，将光遗传学构建体靶向中间神经元是一项关键要求。我们研究了中间神经元特异性启动子谷氨酸脱羧酶（GAD）67和胆囊收缩素（CCK）是否能使视蛋白通过慢病毒靶向传递至整个中间神经元，或特异性地传递至CCK⁺中间神经元。

方法

我们构建了编码带有荧光团且由GAD67或CCK启动子驱动的通道视紫红质（ChR2）和嗜盐视紫红质（NpHR）的慢病毒（LV）质粒。将携带由锥体细胞启动子驱动的视蛋白的腺相关病毒（AAV）和LV载体用作对照。我们对神经元培养物和活体啮齿动物脑进行转导，在活体注射后6 - 8周以及体外转导后7 - 14天对标本进行免疫染色，并通过共聚焦显微镜评估表达的体积和特异性。

结果

在体外，表达LV - CCK - NpHR2.0 - EYFP的神经元中有90%（19/21）为CCK⁺。在体内，LV - GAD67 - ChR2 - mCherry在2.6%（5/193）的海马GABA⁺中间神经元中表达，LV - GAD67 - NpHR2.0 - EYFP在约15%（43/279）的海马GABA⁺中间神经元中表达，LV - CCK - NpHR2.0 - EYFP在47%（9/19）的海马GABA⁺中间神经元中表达。GAD67载体的表达体积大于CCK驱动的构建体。AAV载体对照的表达体积最大。

结论

LV - CCK - NpHR2.0 - EYFP可能有助于在培养物中靶向CCK⁺中间神经元。由GAD67/CCK驱动的慢病毒构建体在体内有表达，尽管表达并非中间神经元特异性的。总体而言，与由锥体细胞启动子驱动的视蛋白相比，表达水平较低。需要更好地理解GAD67和CCK启动子结构或采用替代技术，以便使用病毒载体将视蛋白可靠地靶向中间神经元。