Control of Local Intracellular Calcium Concentration with Dynamic-Clamp Controlled 2-Photon Uncaging

摘要

The variations of the intracellular concentration of calcium ion ([Ca²⁺]i) are at the heart of intracellular signaling, and their imaging is therefore of enormous interest. However, passive [Ca²⁺]i imaging provides no control over these variations, meaning that a full exploration of the functional consequences of [Ca²⁺]i changes is difficult to attain. The tools designed so far to modify [Ca²⁺]i, even qualitatively, suffer drawbacks that undermine their widespread use. Here, we describe an electro-optical technique to quantitatively set [Ca²⁺]i, in real time and with sub-cellular resolution, using two-photon Ca²⁺ uncaging and dynamic-clamp. We experimentally demonstrate, on neurons from acute olfactory bulb slices of Long Evans rats, various capabilities of this technique previously difficult to achieve, such as the independent control of the membrane potential and [Ca²⁺]i variations, the functional knocking-in of user-defined virtual voltage-dependent Ca²⁺ channels, and the standardization of [Ca²⁺]i patterns across different cells. Our goal is to lay the groundwork for this technique and establish it as a new and versatile tool for the study of cell signaling.