GABA能中间神经元在调整中枢神经系统的神经网络中起关键作用,它们的缺陷与神经精神疾病有关。目前,mDlx增强子仅用于腺相关病毒(AAV)载体介导的转基因递送到皮质中间神经元中。这里,我们开发了一种新的抑制性神经元特异性启动子(称为mGAD65启动子),长度为2.5kb,来自编码谷氨酸脱羧酶(GAD)的Gad2基因的外显子1上游的小鼠基因组65。静脉输注血脑屏障穿透AAV-PHP。在mGAD65启动子的控制下表达增强的绿色荧光蛋白的B以抑制性神经元特异性方式转导整个脑。mGAD65启动子对GABA能中间神经元的特异性和效率,在运动皮层评估,几乎与mDlx增强剂相同或稍高。免疫组织化学分析显示,mGAD65启动子优先转导表达小清蛋白(PV)的中间神经元。值得注意的是,mGAD65启动子比mDlx增强子更有效地转导枝形吊灯细胞,并强烈标记它们的突触结,叫做墨盒,靶向兴奋性锥体神经元的轴突初始片段。为了测试mGAD65启动子表达功能分子的能力,我们病毒表达G-CaMP,荧光Ca2+指示剂,在运动皮层,这使我们能够监测GABA能抑制性神经元中自发和药物诱导的Ca2活性。这些结果表明,mGAD65启动子可用于AAV介导的靶向和操纵具有皮质PV表达神经元优势的GABA能神经元,包括吊灯细胞.
GABAergic interneurons play a critical role in tuning neural networks in the central nervous system, and their defects are associated with neuropsychiatric disorders. Currently, the mDlx enhancer is solely used for adeno-associated virus (AAV) vector-mediated transgene delivery into cortical interneurons. Here, we developed a new inhibitory neuron-specific promoter (designated as the mGAD65 promoter), with a length of 2.5 kb, from a mouse genome upstream of exon 1 of the Gad2 gene encoding glutamic acid decarboxylase (GAD) 65. Intravenous infusion of blood-brain barrier-penetrating AAV-PHP.B expressing an enhanced green fluorescent protein under the control of the mGAD65 promoter transduced the whole brain in an inhibitory neuron-specific manner. The specificity and efficiency of the mGAD65 promoter for GABAergic interneurons, which was assessed at the motor cortex, were almost identical to or slightly higher than those of the mDlx enhancer. Immunohistochemical analysis revealed that the mGAD65 promoter preferentially transduced parvalbumin (PV)-expressing interneurons. Notably, the mGAD65 promoter transduced chandelier cells more efficiently than the mDlx enhancer and robustly labeled their synaptic boutons, called the cartridge, targeting the axon initial segments of excitatory pyramidal neurons. To test the ability of the mGAD65 promoter to express a functional molecule, we virally expressed G-CaMP, a fluorescent Ca2+ indicator, in the motor cortex, and this enabled us to monitor spontaneous and drug-induced Ca2+ activity in GABAergic inhibitory neurons. These results suggest that the mGAD65 promoter is useful for AAV-mediated targeting and manipulation of GABAergic neurons with the dominance of cortical PV-expressing neurons, including chandelier cells.