Ohnesorge Nils, Wilzopolski Jenny, Steinfath Matthias, Lewejohann Lars, Banneke Stefanie, Heinl Céline
German Centre for the Protection of Laboratory Animals (Bf3R), German Federal Institute for Risk Assessment (BfR), Berlin, Germany.
Institute of Animal Welfare, Animal Behavior and Laboratory Animal Science, Freie Universität Berlin, Berlin, Germany.
Front Neurosci. 2024 Sep 24;18:1456322. doi: 10.3389/fnins.2024.1456322. eCollection 2024.
Fast and effective anesthesia is the key for refining many invasive procedures in fish and gaining reliable data. For fish as for all vertebrates, it is also required by European law to reduce pain, suffering, and distress to the unavoidable minimum in husbandry and experiments. The most often used substance to induce anesthesia in zebrafish is tricaine (MS-222). When properly prepared and dosed, tricaine causes rapid loss of mobility, balance and reaction to touch. These signs are interpreted as a stage of deep anesthesia although its effects on the central nervous system have not convincingly been shown. Therefore, it might be possible that tricaine first acts only on the periphery, resulting in a paralyzed instead of an anesthetized fish. This has severe implications for animals undergoing procedures. To investigate the effects of tricaine on the central nervous system, we used zebrafish larvae [Tg(:H2B-GCaMP6s)] at 4 days post fertilization (dpf), expressing a calcium indicator (GCaMP6s) in all neurons, that allows monitoring and quantifying the neuronal activity. After treating larvae with 168 mg/L tricaine, a rapid loss of neuronal activity in the forebrain was observed in confocal microscopy. In contrast, only mild effects were seen in the midbrain and hindbrain. In conclusion, the different larval brain areas showed differences in the sensitivity to tricaine treatment. The effects on the central nervous system are indicative of tricaine's anesthetic function and are consistent with behavioral observations of inactivity and unresponsiveness to touch.
快速有效的麻醉是优化鱼类许多侵入性操作并获取可靠数据的关键。对于鱼类以及所有脊椎动物而言，欧洲法律还要求在养殖和实验中将疼痛、痛苦和不适降至不可避免的最低限度。斑马鱼中最常用的诱导麻醉的物质是三卡因（MS-222）。当正确配制和给药时，三卡因会导致活动能力、平衡能力迅速丧失以及对触摸无反应。这些迹象被解释为深度麻醉阶段，尽管其对中枢神经系统的影响尚未得到令人信服的证明。因此，三卡因可能首先仅作用于外周，导致鱼体麻痹而非麻醉。这对接受操作的动物具有严重影响。为了研究三卡因对中枢神经系统的影响，我们使用了受精后4天（dpf）的斑马鱼幼体[Tg(:H2B-GCaMP6s)]，其在所有神经元中表达钙指示剂（GCaMP6s），从而能够监测和量化神经元活动。在用168mg/L三卡因处理幼体后，共聚焦显微镜观察到前脑神经元活动迅速丧失。相比之下，中脑和后脑仅出现轻微影响。总之，不同的幼体脑区对三卡因处理的敏感性存在差异。对中枢神经系统的影响表明了三卡因的麻醉功能，并且与行为上的不活动和对触摸无反应的观察结果一致。
