Stereotactic Delivery of Helper-dependent Adenoviral Viral Vectors at Distinct Developmental Time Points to Perform Age-dependent Molecular Manipulations of the Mouse Calyx of Held.
Stereotactic Delivery of Helper-dependent Adenoviral Viral Vectors at Distinct Developmental Time Points to Perform Age-dependent Molecular Manipulations of the Mouse Calyx of Held.
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DOI：
10.21769/bioprotoc.4793
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发表时间：
2023-08-20
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影响因子：
0.8
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突触是使神经元通信成为可能的专门结构，这对大脑功能和发育至关重要。突触蛋白的改变与各种神经和神经精神疾病有关。因此，在体内操纵突触蛋白可以深入了解这些疾病的分子机制，并有助于开发新的治疗策略。以前的方法如组成型敲除动物受到发育补偿和脱靶效应的限制。目前的方法概述了在不同的发育时间点使用辅助依赖性腺病毒载体（HdAd）在小鼠中进行年龄依赖性分子操作的程序。使用立体定向注射的HdAds在新生儿和幼年小鼠，我们证明了这种方法的通用性表达Cre重组酶的球状浓密的细胞幼年Rac 1fl/fl小鼠消融突触前Rac 1和研究其在突触传递中的作用。另外，我们在两个不同的发育时间点过表达CaV 2 α1亚基，以阐明决定突触前CaV 2通道丰度和偏好性的机制。这种方法提供了一种可靠、经济、微创的方法来控制小鼠大脑特定区域的基因表达，并将成为解读健康和疾病中大脑功能的有力工具。新生和年轻成年小鼠中病毒介导的遗传干扰。立体定位注射允许靶向不同发育阶段的大脑结构，以研究整个发育过程中遗传扰动的影响。
Synapses are specialized structures that enable neuronal communication, which is essential for brain function and development. Alterations in synaptic proteins have been linked to various neurological and neuropsychiatric disorders. Therefore, manipulating synaptic proteins in vivo can provide insight into the molecular mechanisms underlying these disorders and aid in developing new therapeutic strategies. Previous methods such as constitutive knock-out animals are limited by developmental compensation and off-target effects. The current approach outlines procedures for age-dependent molecular manipulations in mice using helper-dependent adenovirus viral vectors (HdAd) at distinct developmental time points. Using stereotactic injection of HdAds in both newborn and juvenile mice, we demonstrate the versatility of this method to express Cre recombinase in globular bushy cells of juvenile Rac1fl/fl mice to ablate presynaptic Rac1 and study its role in synaptic transmission. Separately, we overexpress CaV2 α1 subunits at two distinct developmental time points to elucidate the mechanisms that determine presynaptic CaV2 channel abundance and preference. This method presents a reliable, cost-effective, and minimally invasive approach for controlling gene expression in specific regions of the mouse brain and will be a powerful tool to decipher brain function in health and disease. Key features Virus-mediated genetic perturbation in neonatal and young adult mice. Stereotaxic injection allows targeting of brain structures at different developmental stages to study the impact of genetic perturbation throughout the development.