Development of a measurement system of autonomic nervous activity in genetic pathological-model mice

在过去几年中，制造具有靶向突变（“敲除”突变）的转基因生物或动物的技术已成为神经科学领域日益重要的工具。一如既往，新技术除了提供研究问题或检验假设的新工具外，也会产生不可预见的困难，这些困难需要时间来克服。到目前为止，大多数构建的遗传动物都是老鼠，它们的大脑很难操纵（例如，在自由运动的条件下，老鼠很难实现对离散区域的刺激和单位记录）。因此，我们设计了以下程序在小鼠中进行生理研究。(1)为小动物（如老鼠）建造一个代谢笼，通过个人电脑调节，使实验人员可以在不干扰动物运动或扭曲记录导线和血管导管的情况下记录心血管参数。对于涉及中枢神经系统的研究，通常需要记录、刺激和/或将药物输送到大脑的离散区域。虽然这在麻醉制剂中可以常规地进行，但长期以来人们认识到麻醉可能会混淆心血管功能和体液平衡中枢调节研究的解释。因此，有必要开发一种程序，在清醒的动物身上进行类似的实验。当动物移动时，塑料笼子的地板向上翻转，以释放电缆引线和导管的扭曲；这是通过惯性传感器检测信号来完成的。(2)构建了一种在监视器屏幕上同时显示可变扫描速度的多个参数的系统，通过动作电位的振幅和噪声水平提供记录电极和被研究神经元的相对位置的线索信息，来阐明信号的长期趋势。该系统可用于评价记录电极的先进性。此外，还构建了多变量分析和三维图解的计算机程序。(3)对自由运动动物单单位活动的记录方法进行了以下改进：(a)首先改进了记录电极，该电极由涂有Formvar的不锈钢微丝制成。我们发现高阻抗电极适合于记录具有高信噪比的单单元活动。因此，用薄环氧溶液对不锈钢微导线进行多次绝缘，(b)其次，构建一个微型颅骨微型驱动器，允许细线记录电极的离散背腹侧运动，以推进细线电极并隔离单个活动，(c)第三，使用头戴式电压跟踪器在传输或进一步信号处理之前调节和稳定神经元信号。采用这些方法是为了将这三个要素整合到自由活动的小动物的工作电生理记录程序中。少

Techniques to create transgenic organisms or animals with targeted mutations ('knockout' mutants) have become increasingly important tools in neuroscience over the past few years. As always, new techniques, besides providing new tools to investigate problems or to test hypothesis, also give rise to unforeseen difficulties which take time to overcome. So far, most genetic animals constructed have been mice, whose brains are quite hard to manipulate (e.g., stimulation of discrete areas and unit recordings are difficult to achieve with mice in the freely-moving condition). Therefore, we devised the following procedures to perform physiological studies in mice.(1) Construction of a metabolic cage for small animals such as mice that is regulated by means of a personal computer and allows the experimenter to record cardiovascular parameters without disturbing the animal's movement or twisting the recording lead wires and vascular catheters. For studies involving the central nervous system, i … More t is often desirable to record, stimulate, and/or deliver drugs to discrete regions of the brain. Although this can be carried out somewhat routinely in anesthetized preparations, it has long been recognized that anesthesia may confound the interpretation of studies of the central regulation of cardiovascular function and body fluid balance. Therefore, it was necessary to develop a procedure for carrying out similar experiments in animals that are awake. The floor of the plastic cage was upturned to release the twist of the cable leads and catheters as the animal moved ; this was performed by signals detected with an inertia sensor.(2) A system for simultaneously displaying multiple parameters on a monitor screen with variable sweep speed was constructed to elucidate the long-term trend of signals whereby the amplitude of the action potentials and the noise levels provide cue information about the relative location of the recording electrodes and neurons under study. This system is useful to evaluate the advancement of the recording electrodes. In addition, computer programs for multi-variable analysis and three-dimensional illustrations were constructed.(3) Recording methods for single-unit activity in freely-moving animals have been improved as follows : (a) First, we improved the recording electrodes, which are made of stainless micro wires coated with Formvar. We found that electrodes with high impedance are suitable to record single-unit activity with a high signal-to noise ratio efficiently. Therefore, stainless micro wires were insulated several times with a thin Epoxy solution, (b) Second, a miniature skull-mounted microdrive that permits discrete dorsoventral movement of the fine-wire recording electrodes was constructed to advance the fine-wire electrode as well as to isolate single-unit activities, (c) Third, a head-mounted voltage follower was employed to condition and stabilize the neuronal signals prior to transmission or for further signal processing. These methods were followed in order to integrate these three elements into a working electrophysiological recording procedure in freely-moving small animals. Less