探討感覺神經元中力學傳導經細胞外間質牽引引發之延展力活化電流

Abstract

在感覺神經系統的力學傳導中，延展力活化通道扮演了很重要的角色，早先的研究已經發現直接施力於神經細胞本體可以引發力學活化電流，但是這些研究都無法在近似生理環境以及分子的層級探討專一性的施力下會引發什麼樣的反應。因此我們團隊發展了一套可以探討在體外類似生理環境下偵測感覺神經系統力學傳導的方法。首先我們將小鼠的背根神經元分離並且培養在有細胞外間質塗層 (例如: 纖維網蛋白) 的聚二甲基矽氧烷 (PDMS) 彈性介質上，待其纖維生長；再施力於彈性介質上，藉由彈性介質的形變牽引神經纖維，引發延展力活化電流再以電生理之方式紀錄。另外，在膜片鉗的電生理紀錄中我們也仔細監測輸入阻抗是否因為力學刺激而造成紀錄誤差。藉此技術，我們發現了一群背根神經元細胞會對延展力反應並且表現延展力活化電流，經過一連串的藥理實驗後我們發現有一群細胞的張力活化電流對於Amiloride有反應會產生抑制效果，而另外一群細胞則不對Amiloride有任何反應。其後，我們更加深入的以單一細胞層級反轉錄多聚酶鏈鎖反應技術去探討這些細胞中對於Amiloride反應的酸敏感通道基因表現，發現部份酸敏感通道經常表現於這些對於Amiloride有反應的細胞中，這樣的結果可能表示小鼠的酸敏感通道可能在一部份背根神經節的感覺神經系統力學傳導中扮演著特殊的角色。

Stretch-activated channels play an important role in neuronsensory mechanotransduction. Previous studies have revealed that direct mechanical indentation on neuron soma and neurites or applying radial stretch on neural cells can produce stretch-activated currents in dissociated sensory neurons. However, none of these methods have concerned the mechanical specificity in micro-environment in stretching neurons. We have recently developed a novel platform to probe the localized neurosensory mechanotransduciton in a physiologically relevant condition. We grew neurite-bearing dorsal root ganglion (DRG) neurons on elastic matrix (PDMS) anchored with specific extracellular matrix (ECM) such as fibronectin. While applying indentation onto the elastic-matrix, we can deliver a mechanical stimulation to stretch single neurite via the ECM tethering molecules. To obtain a consistent stretch response, we placed the indentation pipette ~10μm away from the neurite, and 100μm away from the cell soma, then programmed the movement of micromanipulator, and monitored reproducibility of the indentation with flurosphere movement. We used whole-cell patch clamp recording to investigate subtypes of stretch-activated currents delivered by ECM tethering neurites in dissociated dorsal root ganglion neurons. Furthermore, by a series of pharmacological examination, we found an amiloride sensitive and an amiloride insensitive stretch-activated current expressed in different neurons. In addition, I performed the single cell RT-PCR to screening the ASIC family (a mammalian DEG/ENaC channels), and found the ASIC1b and ASIC3 expression in the amiloride sensitive neurons. These results suggest that members of DEG/ENaC/ASIC family may participate in the neurosensory mechanotransduction in a subset of dorsal root ganglion neurons.