| Co-Investigator(Kenkyū-buntansha) |
KUWAKI Tomoyuki Chiba University, Graduate School of Medicine, Dept. Autonomic Physiology, Professor, 大学院・医学研究院, 教授 (80205260)
KANITAKE Takato Miyazaki Med. College, Dept. Physiol., Assistant, 医学部, 助手 (20234461)
HANAMORI Takamitsu Miyazaki Med. College, Dept. Physiol., Associate Professor, 医学部, 助教授 (20041858)
KATO Kazuo Miyazaki Med. College, Dept. Physiol., Assistant (80284834)
ISHIZUKA Yuta Miyazaki Med. College, Dept. Psychiatry, Lecturer (20264377)
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| Research Abstract |
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
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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
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