Wykes Alexander D, Ma Sherie, Bathgate Ross A D, Gundlach Andrew L
The Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Victoria, Australia.
Florey Department of Neuroscience and Mental Health, The University of Melbourne, Victoria, Australia.
IBRO Rep. 2019 Dec 13;8:1-10. doi: 10.1016/j.ibror.2019.11.006. eCollection 2020 Jun.
Modern neuroscience utilizes transgenic techniques extensively to study the activity and function of brain neural networks. A key feature of this approach is its compatibility with molecular methods for selective transgene expression in neuronal circuits of interest. Until now, such targeted transgenic approaches have not been applied to the extensive circuitry involving the neuropeptide, relaxin-3. Pharmacological and gene knock-out studies have revealed relaxin-3 signalling modulates interrelated behaviours and cognitive processes, including stress and anxiety, food and alcohol consumption, and spatial and social memory, highlighting the potential of this system as a therapeutic target. In the present study, we aimed to identify a promoter sequence capable of regulating cell-type specific transgene expression from an adeno-associated viral (AAV) vector in relaxin-3 neurons of the rat (NI). In parallel to relaxin-3 promoter sequences, we also tested an AAV vector containing promoter elements for the tropomyosin receptor kinase A (TrkA) gene, as TrkA is co-expressed with relaxin-3 in rat NI neurons. Stereotaxic injection of an mCherry-expressing AAV vector revealed widespread non-specific TrkA promoter (880 bp) activity in and adjacent to the NI at 8 weeks post-treatment. In contrast, mCherry expression was successfully restricted to relaxin-3 NI neurons with 98% specificity using a 1736 bp relaxin-3 promoter. In addition to detailed anatomical mapping of NI relaxin-3 networks, illustrated here in association with GABAergic medial septum neurons, this method for targeted transgene delivery offers a versatile tool for ongoing preclinical studies of relaxin-3 circuitry.
现代神经科学广泛利用转基因技术来研究脑神经网络的活动和功能。这种方法的一个关键特性是它与分子方法兼容，可在感兴趣的神经元回路中进行选择性转基因表达。到目前为止，这种靶向转基因方法尚未应用于涉及神经肽松弛素-3的广泛神经回路。药理学和基因敲除研究表明，松弛素-3信号传导调节相互关联的行为和认知过程，包括应激和焦虑、食物和酒精消耗以及空间和社交记忆，突出了该系统作为治疗靶点的潜力。在本研究中，我们旨在从大鼠神经中间叶（NI）的松弛素-3神经元中鉴定出一种能够调节腺相关病毒（AAV）载体细胞类型特异性转基因表达的启动子序列。与松弛素-3启动子序列并行，我们还测试了一种含有原肌球蛋白受体激酶A（TrkA）基因启动子元件的AAV载体，因为TrkA在大鼠NI神经元中与松弛素-3共表达。立体定位注射表达mCherry的AAV载体显示，在治疗后8周，NI及其附近区域存在广泛的非特异性TrkA启动子（880 bp）活性。相比之下，使用1736 bp的松弛素-3启动子，mCherry表达成功地以98%的特异性限制在松弛素-3 NI神经元中。除了这里与GABA能内侧隔核神经元相关展示的NI松弛素-3网络的详细解剖图谱外，这种靶向转基因递送方法为正在进行的松弛素-3神经回路临床前研究提供了一种通用工具。
