New tools for targeted disruption of cholinergic synaptic transmission in Drosophila melanogaster.
New tools for targeted disruption of cholinergic synaptic transmission in Drosophila melanogaster.
复制标题
DOI：
10.1371/journal.pone.0064685
复制
发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Godenschwege TA
中科院分区：
文献类型：
--
作者：
Mejia M;Heghinian MD;Marí F;Godenschwege TA
烟碱乙酰胆碱受体（nAChR）是五聚体配体门控离子通道。nAChR的α7亚型涉及神经病理学，如帕金森病、阿尔茨海默病、成瘾、癫痫和自闭症谱系障碍。黑腹果蝇α7（Drosophilamelanogaster α7，Dα7）与脊椎动物α7亚基具有最接近的序列同源性，并能形成与脊椎动物相同的五聚体受体。Dα7亚基对巨纤维回路的功能至关重要，巨纤维回路介导果蝇的逃避反应。为了进一步表征受体功能，我们在Dα7 nAChR的配体结合结构域中产生了不同的错义突变。我们表征了在Dα7无效突变体和野生型背景中，两种携带单一突变D197 A和Y195 T的UAS构建体以及一种携带三重D77 T、L117 Q、I196 P突变的UAS构建体的靶向表达的效果。三重突变的表达能够恢复Dα7无效突变体中电路的功能，并且在野生型中表达时没有破坏性影响。与此相反，两个单突变严重破坏了Dα7依赖的突触传递，但在野生型背景下不破坏突触能或间隙连接依赖的突触传递，并且没有或仅部分挽救了无效突变体的突触缺陷。这些观察结果与由D197A或Y195T亚基和野生型Dα7亚基组成的杂合受体的形成一致，其中乙酰胆碱的结合或乙酰胆碱诱导的Dα7受体构象变化被改变，并引起胆碱能反应的抑制。因此，D197A或Y195T的靶向表达可用于选择性地破坏神经元回路中Dα7依赖性突触的突触传递。因此，这些构建体可以用作研究学习和记忆或成瘾相关行为的工具，通过允许操纵回路中的神经元加工而不影响其他细胞信号传导。
Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels. The α7 subtype of nAChRs is involved in neurological pathologies such as Parkinson’s disease, Alzheimer’s disease, addiction, epilepsy and autism spectrum disorders. The Drosophila melanogaster α7 (Dα7) has the closest sequence homology to the vertebrate α7 subunit and it can form homopentameric receptors just as the vertebrate counterpart. The Dα7 subunits are essential for the function of the Giant Fiber circuit, which mediates the escape response of the fly. To further characterize the receptor function, we generated different missense mutations in the Dα7 nAChR’s ligand binding domain. We characterized the effects of targeted expression of two UAS-constructs carrying a single mutation, D197A and Y195T, as well as a UAS-construct carrying a triple D77T, L117Q, I196P mutation in a Dα7 null mutant and in a wild type background. Expression of the triple mutation was able to restore the function of the circuit in Dα7 null mutants and had no disruptive effects when expressed in wild type. In contrast, both single mutations severely disrupted the synaptic transmission of Dα7-dependent but not glutamatergic or gap junction dependent synapses in wild type background, and did not or only partially rescued the synaptic defects of the null mutant. These observations are consistent with the formation of hybrid receptors, consisting of D197A or Y195T subunits and wild type Dα7 subunits, in which the binding of acetylcholine or acetylcholine-induced conformational changes of the Dα7 receptor are altered and causes inhibition of cholinergic responses. Thus targeted expression of D197A or Y195T can be used to selectively disrupt synaptic transmission of Dα7-dependent synapses in neuronal circuits. Hence, these constructs can be used as tools to study learning and memory or addiction associated behaviors by allowing the manipulation of neuronal processing in the circuits without affecting other cellular signaling.
登录
查看更多内容
影响因子：
4.8
作者：
Kasuya J;Ishimoto H;Kitamoto T
通讯作者：
Kitamoto T
影响因子：
9.8
作者：
Fayyazuddin A;Zaheer MA;Hiesinger PR;Bellen HJ
通讯作者：
Bellen HJ
DOI：
10.1111/j.1460-9568.2007.05686.x
发表时间：
2007-07
期刊：
The European journal of neuroscience
影响因子：
--
作者：
Allen MJ;Murphey RK
通讯作者：
Murphey RK
影响因子：
11.4
作者：
Hansen, SB;Sulzenbacher, G;Bourne, Y
通讯作者：
Bourne, Y
影响因子：
2.9
作者：
Ellison, M;Gao, F;Olivera, BM
通讯作者：
Olivera, BM