In Vivo Ca2+ and Voltage Imaging on The Urinary Bladder

描述（由申请人提供）：梗阻性尿路功能障碍是一种发病率极高、具有破坏性的社会和心理后果的疾病，可导致包括尿失禁、尿频增加和尿急感不适当在内的复杂症状，所有这些症状似乎都是由膀胱平滑肌异常收缩活动引起的。在本研究中，我们试图了解正常膀胱和梗阻后膀胱体内节律性或阶段性收缩活动的产生。尽管在体内研究相活动背后的细胞信号是非常困难的，但在神经和激素输入的背景下理解这种活动是很重要的，它们在收缩活动的调节中起着重要作用。我们最近开发了一种方法来监测细胞内游离Ca2+在体内通过发展转基因小鼠表达一种新的高信号噪声，遗传编码Ca2+指标。Ca2+指示剂GCaMP2在生理温度下是稳定的，接近GFP的亮度，能够持续、高分辨率地记录活小鼠的细胞Ca2+瞬态。这种分子在膀胱平滑肌中高度表达的小鼠已经被创造出来，并将用于观察体内细胞Ca2+信号。我们将使用这些小鼠来更全面地了解与尿路出口梗阻相关的功能障碍。我们将研究梗阻性小鼠自发性膀胱过度活动的异常收缩活动的基础，以了解膀胱异常收缩的细胞和分子基础。我们假设膀胱过度活动发生继发于异常起搏器活动和异常Ca2+波的发展，这些异常Ca2+波与平滑肌肌浆网（SR）或Cajal间质细胞（ICC） Ca2+释放失调有关。为了验证这一假设，我们将在正常情况下对平滑肌细胞中的相性Ca2+信号进行成像，试图了解自发活动的细胞和分子基础，确定这种模式被持续外流阻塞干扰的程度，并测试异常活动是否由于肌肉中SR Ca2+释放的改变或膀胱中起搏器细胞活动的变化而产生。这些发现应该提供更清晰的理解与尿路流出梗阻相关的平滑肌功能障碍。

DESCRIPTION (provided by applicant): Postobstructive urinary tract dysfunction is a disorder with extremely high morbidity and devastating social and psychological consequence, resulting in a complex of symptoms including incontinence, increased frequency of urination, and an inappropriate sense of urinary urgency, all of which appear to result from abnormal contractile activity of the smooth muscle lining the bladder. In this proposal we seek to understand the generation of rhythmic or phasic contractile activity in the normal and postobstructed urinary bladder in vivo. Although the study of the cellular signals underlying phasic activity is extraordinarily difficult in vivo, it is important to understand this activity in the context of neural and hormonal inputs that play an important role in the regulation of contractile activity. We have recently developed methods to monitor intracellular free Ca2+ in vivo through the development of transgenic mice expressing a novel high signal-noise, genetically encoded Ca2+ indicator. GCaMP2, the Ca2+ indicator, is stable at physiological temperatures and approaches the brightness of GFP, enabling sustained, high resolution recording of cellular Ca2+ transients in the living mouse. Mice in which this molecule is highly expressed in urinary bladder smooth muscle have been created and will be used to visualize cellular Ca2+ signals in vivo. We will use these mice to more fully understand the dysfunction associated with urinary tract outlet obstruction. The basis of abnormal contractile activity in postobstructive mice that develop spontaneous bladder overactivity will be studied to understand the cellular and molecular basis for abnormal bladder contractions. We hypothesize that bladder hyperactivity occurs secondary to the development of abnormal pacemaker activity and anomalous Ca2+ waves associated with dysregulated Ca2+ release from the sarcoplasmic reticulum (SR) of smooth muscle or Interstitial Cells of Cajal (ICC). To test this hypothesis, we will image phasic Ca2+ signals in smooth muscle cells under normal conditions, seek to understand the cellular and molecular basis for spontaneous activity, determine the extent to which this pattern is disturbed by sustained outflow obstruction, and test whether abnormal activity arises due to alterations in SR Ca2+ release in muscle or changes in the activity of pacemaker cells in the urinary bladder. These findings should provide a clearer understanding of smooth muscle dysfunction associated with urinary tract outflow obstruction.