Arcuate nucleus Neuropeptide Y/Agouti-related peptide (NPY/AgRP) neurons drive ingestive behavior in response to the internal and external environment of an organism. NPY/AgRP neurons are adjacent to the median eminence, a circumventricular organ, and circulating metabolic factors and hormones communicate the energy state of the animal via these neurons by altering the excitability of NPY/AgRP neurons, which produces an appropriate change in behavior to maintain homeostasis. One example of this plasticity is seen in the M-current, a subthreshold, non-inactivating K+ current that acts to modulate excitability. Fasting decreases while estradiol increases the M-current through regulation of subunit mRNA expression of Kcnq 2, 3, & 5. KCNQ2/3 heteromers are thought to mediate the majority of the M-current. Here we used a recently developed single adeno-associated viral (AAV) vector containing a recombinase-dependent Staphylococcus aureus Cas9 (SaCas9) and a single guide RNA against Kcnq3 to selectively delete Kcnq3 in NPY/AgRP neurons to produce a loss of function in the M-current. We found that this virus was effective at knocking down Kcnq3 but not Kcnq2 expression. With the reduced KCNQ3 channel expression NPY/AgRP neurons were more depolarized, exhibited a higher input resistance, and the rheobase current needed to induce firing was significantly reduced, indicative of increased excitability. Although the resulting decrease in the M-current did not overtly alter ingestive behavior, it did significantly reduce the locomotor activity as measured in open field testing. Therefore, the SaCas9-sgKcnq3 is efficient to knock down Kcnq3 expression thereby reducing the M-current and increasing the excitability of NPY/AgRP neurons.

中文翻译：

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CRISPR敲低Kcnq3减弱NPY / AgRP神经元的M电流

弓形核神经肽Y / Agouti相关肽（NPY / AgRP）神经元驱动食入行为，以响应生物体的内部和外部环境。NPY / AgRP神经元与中位隆突，室室器官相邻，循环代谢因子和激素通过改变NPY / AgRP神经元的兴奋性，通过这些神经元传达动物的能量状态，从而产生适当的行为改变以维持稳态。这种可塑性的一个例子是M电流，它是一个亚阈值，非灭活的K +电流，可调节兴奋性。空腹降低，而雌二醇通过调节Kcnq 2、3和5的亚基mRNA表达增加M-电流。KCNQ2/ 3异聚体被认为介导了大部分M-电流。在这里，我们使用了最近开发的包含重组酶依赖性金黄色葡萄球菌Cas9（SaCas9）和针对Kcnq3的单个指导RNA的单个腺相关病毒（AAV）载体，以选择性地删除NPY / AgRP神经元中的Kcnq3，从而在神经元中产生功能丧失。 M电流。我们发现该病毒可有效降低Kcnq3的表达，但不能有效降低Kcnq2的表达。随着减少的KCNQ3通道表达，NPY / AgRP神经元更去极化，表现出更高的输入电阻，并且诱导激发所需的流变碱电流显着降低，表明兴奋性增加。尽管由此导致的M电流降低并没有明显改变摄食行为，但确实降低了运动能力，如在野外试验中测得的那样。因此，