使用基于酶的生物传感器来衡量进补和阶段性谷氨酸在阿尔茨海默氏症小鼠模型
Summary
在这里，我们描述了用于测量补药和采用酶联微电极阵列（MEA） 在体内阶段性细胞外谷氨酸的变化的空间和时间上精确的方法的设置，软件导航，和数据分析。
Abstract
神经递质的破坏往往是中枢神经系统（CNS）疾病的一个重要组成部分，发挥着病理阿尔茨海默氏症，帕金森氏症，抑郁症和焦虑潜在的作用。传统上，微透析一直是最常见的（赞美）技术检测发生在这些疾病中神经递质的变化。但由于微透析具有测量跨组织的大面积慢1-20分钟变化的能力，它具有侵袭性的缺点，可能会破坏大脑和慢速采样能力范围内的内在联系。一个相对较新的技术中，微电极阵列（MEA），具有用于所发生的测量的离散的大脑区域中的特定神经递质的变化，使得用于空间和时间上精确的方法的许多优点。此外，使用的MEA是微创，允许体内的神经递质的改变的测量。在我们的实验室，我们哈去过在神经递质谷氨酸，与阿尔茨海默氏病的病理变化特别感兴趣。这样，这里所描述的方法已经被用于评估在阿尔茨海默病的转基因小鼠模型中的潜在谷氨酸海马中断。简言之，该方法涉及使用涂布用酶对感兴趣，并使用自参照位点以减去背景噪声和干扰神经递质非常有选择性的多站点微电极。电镀和校准之后，可以MEA用微量被构造和使用立体定位设备下降到感兴趣的大脑区域。在此，所描述的方法涉及麻醉RTG（TauP301L）4510只小鼠和使用立体定位装置精确地定位子区域海马（DG，CA1，CA3和）。
Introduction
在大脑中的神经递质测量改变为神经科学家研究中枢神经系统（CNS），其通常的特征在于神经递质失调的疾病的重要工具。虽然微透析在用高压液相色谱法（HPLC / EC）组合已经以测量细胞外神经递质水平1，2，变化的最广泛使用的方法3， 图4，微透析探针的空间和时间分辨率可能不是理想的神经递质，如谷氨酸，被在细胞外空间5,6紧密调节。因为在遗传学和成像的最新进展，存在可用于在体内映射谷氨酸的其他方法。使用基因编码谷氨酸荧光记者（iGluSnFR）的ð双光子成像，研究人员能够通过在体外和体内 7，8，9的神经元和星形胶质细胞可视化的谷氨酸释放。值得注意的是，这允许从更大的视场记录和不破坏大脑的内在联系。尽管这些新的光学技术允许谷氨酸动力学和感官诱发反应和神经元活性的测量可视化，它们缺乏量化离散脑区域外空间的谷氨酸的量的能力。
另一种方法是酶联微电极阵列（MEA），其可以选择性地测量细胞外神经递质水平，如谷氨酸盐，通过使用自参考记录方案。该MEA技术已用于研究创伤性脑细胞外谷氨酸的变化损伤10，11，12，老化13，14，应力15，16，癫痫17，18，阿尔茨海默氏病19，20，和病毒模拟物21的注射和代表了在微透析中固有的空间和时间限制的改进。而微透析限制测量突触22，23附近的能力，具有的MEA具有高的空间分辨率，其允许细胞外谷氨酸溢出的选择性措施附近突触24,25。第二，微透析的低时间分辨率（1 – 20分钟）限制了调查的能力谷氨酸释放和清除的快速动力学在毫秒发生的第二范围26。因为谷氨酸的释放或间隙差异补品的措施，休息谷氨酸水平可能并不明显，这可能是必要的谷氨酸释放和间隙直接测量。 MEA的允许这样的措施，由于其高的时间分辨率（2赫兹）和检测的下限（<1微米）。第三，允许的MEA用于特定脑区域内神经递质分区域变型中，如大鼠或小鼠海马的检查。例如，使用的MEA我们可以分别针对齿状脑回（DG），海马，它们经由trisynaptic电路27连接的大角ammonis 3（CA3）以及大角ammonis 1（CA1），以检查细胞外谷氨酸的分区域的差异。造成通过植入28 < – （4毫米的长度1）和损坏，因为微透析探针的大小的/ SUP>，29，分区域的差异是难以解决。此外，光学系统仅允许通过刺激的外部刺激，例如晶须刺激或光的闪烁，这不允许分区域的刺激7。比其他方法的多边环境协定的最后一个好处是研究在体内这些次区域没有打乱他们的外在和内在联系的能力。
在这里，我们描述了如何在组合的记录系统（ 例如 ，FAST16mkIII）用的MEA，其由基于陶瓷的多点微电极，可以有差别地涂覆在记录位点以允许干扰试剂要被检测和从分析物信号中除去。我们还证明这些阵列可用于麻醉RTG的CA1海马亚区的DG，CA3内体内谷氨酸调节基于电流分析法研究，和（TauP301L）4510只小鼠中，通常使用的米阿尔茨海默病的乌斯模型。此外，我们通过用利鲁唑小鼠提供MEA系统对谷氨酸释放和清除的快速动力学的敏感性的确认， 在体外所示的药物以减少谷氨酸释放和增加谷氨酸盐摄取30，31，32，33，并展示在 TauP301L小鼠模型中的体内这些相应的改变。
Protocol
1.涂层用酶或矩阵层的微电极阵列 制备蛋白质基质溶液 称出10毫克的牛血清白蛋白（BSA）和转移到1.5毫升离心管中。 添加去离子水985μL含有BSA的微量离心管中。混合通过人工搅拌溶液（使用1000μL吸管〜3次重新移液，直到BSA溶解）。 注：请不要使用涡的解决方案，因为这样做可能会导入空气的混合溶液。另外，吸液管设定为体积<1000μL（ 例如 700μL?…
Representative Results
虽然这种技术可用于测量谷氨酸的改变在许多类型的动物模型，如创伤性脑损伤，衰老，压力和癫痫的信号，在这里我们展示了MEA技术如何可以用于检查转基因小鼠模型的谷氨酸改变的人tau蛋白病19,20。 RTG的（TauP301L）4510小鼠表达与额颞叶痴呆和帕金森综合征与染色体17相关的tau蛋白突变P301L，和通常用来研究与阿尔茨海默氏病?…
Discussion
所述MEA技术允许在体外和体内神经递质的释放和摄取的快速动力学的测量。因此，该技术产生了各种各样的数据输出包括补品神经递质水平，诱发神经递质释放，和神经递质的间隙。然而，由于采用多边环境协定是一个相对复杂的过程，有可能需要为成功使用进行优化许多因素。例如，在校准期间，人们可能注意到，不存在的信号波形（示波器屏幕），或者对刺激的响?…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
（; U54GM104942 MNR），NIA（MNR; R15AG045812），阿尔茨海默氏症协会（MNR; NIRG-12-242187），西弗吉尼亚大学学院研究参议院拨款（MNR），西弗吉尼亚大学和格兰特PSCOR这项工作是由普通医学科学研究所的支持（MNR）。
Materials
| FAST-16mkIII-8 channel | Quanteon | 16mkIII | |
| Microelectrode arrays | CenMet | W4 or 8-TRK | |
| Bovine Serum Albumin (BSA) | Sigma-Aldrich | A-3059 | 10 g (expires after 1 month) |
| Glutaraldehyde | Sigma-Aldrich | G-6257 | 100 mL (expires after 6 months) |
| Glutamate Oxidase | US Biological or Sigma Aldrich | G4001-01 or 100646 | 50 UI (expires after 6 months) |
| Hamilton Syringes | Hamilton | #701 | 2 syringes |
| Methanol | BDH | UN1230 | 4 L |
| m-Phenylenediamine dihydrochloride (mPD) | ACROS Organics | 1330560250 | 25 g |
| Reference Electrodes (RE-5B) | BAS | MF-2079 | 3 electrodes |
| Magnetic stir plate | Cole-parmer | EW-04804-01 | Can purchase from different supplier |
| Glutamate | Sigma-Aldrich | G-1626 | 100 g |
| Ascorbic Acid | TCI | 50-81-7 | 500 g |
| Dopamine Hydrochloride | Alfa Aesar | 62-31-7 | 5 g |
| Perchloric acid | VWR | UN2920 | 500 mL |
| Postassium chloride | VWR | 7447-40-7 | 1 kg |
| Sodium chloride | VWR | 7647-40-7 | 1 kg |
| Calcium Chloride | MP | 153502 | 100 g |
| Sodium Hydroxide | BDH | 1310732 | 500 g |
| Glass pressure ejection pipettes | CenMet | ||
| Sticky wax | Kerrlab | 625 | Can purchase from different supplier |
| Microsyringe | World Precision Instruments | MF28G-5 | |
| Modeling clay | WalMart | Can purchase from different supplier | |
| Picospritzer III | Parker | ||
| Silver wire | AM systems | 782000 | |
| Hydrochloric acid | BDH | 7647010 | 2.5 L |
| Platinum wire | AM Systems | 778000 | |
| Solder gun | Lowes or Home Depot | Can purchase from different supplier | |
| Multimeter | WalMart | Can purchase from different supplier | |
| PhysioSuite | Kent Scientific | Can purchase from different supplier | |
| SomnoSuite | Kent Scientific | Can purchase from different supplier | |
| Stereotaxic device | Stoelting | Can purchase from different supplier | |
| Digital Lab Standard | Stoelting | Can purchase from different supplier | |
| Meiji EMZ microscope | Meiji | EMZ-5 | |
| Drill | Dremel | Micro | |
| Metricide | Metrex | 102800 | |
| Scalpel | VWR | Can purchase from different supplier | |
| Surgery scissors | VWR | Can purchase from different supplier | |
| Sterile cotton swabs | Puritan | 25806 | Can purchase from different supplier |
| Eye ointment | Puralube Vet Ointment | Obtain from the vet | |
| Iodine swabs | VWR | S48050 | Can purchase from different supplier |
| Alcohol swabs | Local drug store | Can purchase from different supplier | |
| Sterile surgery drape | Dynarex | 4410 | Can purchase from different supplier |
| Sterile saline | Teknova | S5815 | Can make own soltuion using filters |
| Hydrogen Peroxide (3%) | Local drug store | Can purchase from different supplier | |
| Heating Pad | WalMart | Can purchase from different supplier |
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Diesen Artikel zitieren
Hunsberger, H. C., Setti, S. E., Heslin, R. T., Quintero, J. E., Gerhardt, G. A., Reed, M. N. Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer’s Mouse Models. J. Vis. Exp. (123), e55418, doi:10.3791/55418 (2017).