Clark S D, Alderson H L, Winn P, Latimer M P, Nothacker H-P, Civelli O
Laboratory of Molecular Neurobiology, Centre for Addiction and Mental Health, Toronto, Ontario, Canada.
J Neurochem. 2007 Jul;102(1):112-20. doi: 10.1111/j.1471-4159.2007.04529.x. Epub 2007 Apr 10.
Urotensin II is a neuropeptide first isolated from fish and later found in mammals: where it has potent cardiovascular, endocrine and behavioral effects. In rat brain the urotensin II receptor (UII-R) is predominately expressed in the cholinergic neurons of the pedunculopontine (PPTg) and laterodorsal tegmental nuclei. Typically, the function of the PPTg has been examined using excitotoxins, destroying both cholinergic and non-cholinergic neurons, which confounds interpretation. We took advantage of UII-R's unique expression profile, by combining UII with diphtheria toxin, to engineer a toxin specific for cholinergic neurons of the PPTg. In vitro, two different toxin constructs were shown to selectively activate UII-R (average EC50 approximately 30 nmol/L; calcium mobility assay) and to be 10,000-fold more toxic to UII-R expressing CHO cells, than wildtype cells (average LD50 approximately 2 nmol/L; cell viability). In vivo, pressure injection into the PPTg of rats, resulted in specific loss of choline transporter and NADPH diaphorase positive neurons known to express the UII-R. The lesions developed over time, resulting in the loss of over 80% of cholinergic neurons at 21 days, with little damage to surrounding neurons. This is the first highly selective molecular tool for the depletion of mesopontine cholinergic neurons. The toxin will help to functionally dissect the pedunculopontine and laterodorsal tegmental nuclei, and advance the understanding of the functions of these structures.
尾加压素II是一种神经肽，最初从鱼类中分离出来，后来在哺乳动物中也被发现，它在哺乳动物体内具有强大的心血管、内分泌和行为效应。在大鼠脑中，尾加压素II受体（UII-R）主要表达于脚桥核（PPTg）和外侧被盖核的胆碱能神经元中。通常，人们使用兴奋性毒素来研究脚桥核的功能，这种方法会破坏胆碱能和非胆碱能神经元，从而混淆了实验结果的解释。我们利用UII-R独特的表达谱，将UII与白喉毒素结合，设计出一种对脚桥核胆碱能神经元具有特异性的毒素。在体外实验中，两种不同的毒素构建体均显示能选择性激活UII-R（平均半数有效浓度约为30 nmol/L；钙流测定法），并且对表达UII-R的CHO细胞的毒性比对野生型细胞高10000倍（平均半数致死浓度约为2 nmol/L；细胞活力测定）。在体内实验中，向大鼠的脚桥核进行压力注射，导致已知表达UII-R的胆碱转运体和NADPH黄递酶阳性神经元特异性缺失。损伤随时间发展，在21天时导致超过80%的胆碱能神经元丧失，而对周围神经元的损伤很小。这是第一种用于耗尽中脑桥胆碱能神经元的高度选择性分子工具。这种毒素将有助于从功能上剖析脚桥核和外侧被盖核，并增进对这些结构功能的理解。
