Development and Validation of an NPY-sensitive Microelectrode for Measuring NPY Release from Hippocampus

本研究方案旨在开发和验证对神经肽Y敏感的微电极 (NPY)用于测量海马区NPY的释放。通过这种方式，我们将能够找到关联 在神经肽Y水平与焦虑症之间。为了做到这一点，已经有两种电化学策略 旨在实时监测生物分子。大脑中的非电活性分子很难测量 具有高时间和空间分辨率的神经肽一直是一个挑战。电化学法 技术是强大的，可以用来测量表面的物理和化学性质，并 它们已被广泛用于检测具有非常低检测下限的分子。这两种技术的结合 具有快速扫描循环伏安和连续电化学阻抗的高选择性适配子 测量将提供两种新的策略来理解CA1区NPY的存在。 可能与NPY一起释放的不同分子将使用开发的 微电极，以证明选择性。转基因小鼠将受到和过度监管使用 四环素类药物改变NPY水平并使用开发的NPY敏感试剂确认测量 微电极。尺寸达25微米的铂微电极将提供适当的 用于吸附和解吸分子的底物以及过滤NPY的适体修饰 来自其他混杂分子的信号。相伴而生的电化学和电生理 将在CA1中进行测量，以便从测量的不同其他信号中滤除NPY。确认 NPY的作用将通过记录sc中fEPSP对低频电刺激的反应来测试 和TA途径。为了验证NPY水平，将使用ELISA法比较与 我们开发了海马区提取液中NPY敏感的微电极。 电化学阻抗谱和快速扫描循环伏安法已被证明是重要的 允许测量法拉第和非法拉第电流的技术，提供了 与电极表面相互作用的电活性物种以及非电活性物种。这个 将快速扫描循环伏安法与电化学阻抗测量相结合将使 研究团体使用微电极实时测量生物分子，如 神经递质和神经肽。

This research proposal aims to develop and validate microelectrode that is sensitive to neuropeptide Y (NPY) for measuring the release of NPY from hippocampus. In this way, we will be able to find correlations between NPY levels with anxiety disorders. In order to do this, two electrochemical strategies have been devised to monitor biomolecules in real-time. Non-electroactive molecules in the brain are difficult to measure with high temporal and spatial resolution and neuropeptides have been a challenge. Electrochemical-based techniques are powerful and can be used to measure the physical and chemical properties of the surface and they have been vastly used for the detection of molecules with very low detection limits. The combination of highly selective aptamers with fast scan cyclic voltammetry and continuous electrochemical impedance measurements will provide two novel strategies to understand the presence of NPY in the CA1 region. Different molecules that are potentially released together with NPY will be measured using the developed microelectrodes to prove selectivity. Genetically modified mice will be under and overregulated using tetracyclines to change NPY levels and confirm the measurement using the developed NPY-sensitive microelectrodes. Platinum microelectrodes measuring up to 25 micrometers will provide the appropriate substrate for the adsorption and desorption of molecules as well as the aptamer modification to filter NPY signals from other confounding molecules. Concomitant electrochemical and electrophysiological measurement in CA1 will be done to filter NPY from the different other signals measured. The confirmation of the effects of NPY will be tested recording fEPSPs in response to low-frequency electrical stimulation in the SC and TA pathway. In order to validate NPY levels, ELISA will be used to compare the measurements done with our developed NPY-sensitive microelectrodes in hippocampal extracts. Electrochemical impedance spectroscopy and fast scan cyclic voltammetry have shown to be important techniques that allow the measurement of faradaic as well as non-faradaic currents providing a picture of the electroactive species as well as non-electroactive species that interact with the electrode’s surfaces. The combination of fast scan cyclic voltammetry with electrochemical impedance measurements will empower the research community using microelectrodes for real-time measurement of biomolecules such as neurotransmitters and neuropeptides.