训练大鼠自愿潜水水下：哺乳动物潜水响应调查

Summary

Detailed instructions are provided on how to train rats to voluntarily dive underwater through a 5 m long Plexiglas maze. Because the brains of rats have been very well characterized, voluntarily diving rats may help elucidate the central pathways of the mammalian diving response.

Abstract

Underwater submergence produces autonomic changes that are observed in virtually all diving animals. This reflexly-induced response consists of apnea, a parasympathetically-induced bradycardia and a sympathetically-induced alteration of vascular resistance that maintains blood flow to the heart, brain and exercising muscles. While many of the metabolic and cardiorespiratory aspects of the diving response have been studied in marine animals, investigations of the central integrative aspects of this brainstem reflex have been relatively lacking. Because the physiology and neuroanatomy of the rat are well characterized, the rat can be used to help ascertain the central pathways of the mammalian diving response. Detailed instructions are provided on how to train rats to swim and voluntarily dive underwater through a 5 m long Plexiglas maze. Considerations regarding tank design and procedure room requirements are also given. The behavioral training is conducted in such a way as to reduce the stressfulness that could otherwise be associated with forced underwater submergence, thus minimizing activation of central stress pathways. The training procedures are not technically difficult, but they can be time-consuming. Since behavioral training of animals can only provide a model to be used with other experimental techniques, examples of how voluntarily diving rats have been used in conjunction with other physiological and neuroanatomical research techniques, and how the basic training procedures may need to be modified to accommodate these techniques, are also provided. These experiments show that voluntarily diving rats exhibit the same cardiorespiratory changes typically seen in other diving animals. The ease with which rats can be trained to voluntarily dive underwater, and the already available data from rats collected in other neurophysiological studies, makes voluntarily diving rats a good behavioral model to be used in studies investigating the central aspects of the mammalian diving response.

Introduction

The diving response consists of a suite of autonomic reflexes seen in animals of all vertebrate classes ¹. In response to submersion under water, this reflexly-induced response consists of apnea, bradycardia and an alteration of blood flow that maintains flow to the heart, brain and exercising muscles while limiting flow to viscera and non-exercising muscles ². Many of the metabolic and cardiorespiratory aspects of the mammalian diving response have been well investigated ^2,3, including those in humans ^4,5. However, what has been relatively lacking, until recently, is investigation of the central integrative aspects of the diving response. What happens within the brainstem, and what is the neuronal step-by-step pathway, that connects afferent inputs to efferent outputs during this autonomic reflex? Answering these questions will require an appropriate animal model ⁶. An adage in comparative physiology, the Krogh principle ⁷, is that for every research question there is some animal of choice on which the problem can be most conveniently studied. A most appropriate animal for studying the central aspects of the diving response is the rat ^6,8. In large part this is due to the fact that the brains of rats have been very well characterized, both anatomically and functionally, and many rat brain atlases are available ⁶. Additionally the rat is particularly useful in cardiorespiratory research, because the physiology of the rat is well known across all major organ systems, and the rat is well regarded as an animal model in systems biology ⁶. Finally, the laboratory rat is the domesticated version of the wild Rattus norvegicus, an animal that routinely swims and dives underwater ⁶. Based on these considerations, the rat is a good choice for studies investigating the central aspects of the mammalian diving response. In comparison, using marine animals to investigate the central aspects of the mammalian diving response would be much more difficult. This is due in large part to marine animals having comparatively large and non-uniformly sized brains, and the relative difficulty and high cost of housing these animals.

Rats have previously been used to investigate many aspects of the mammalian diving response, primarily in situations involving forced underwater submergence ^9-12. However many studies in marine and aquatic animals have shown that there can be a differential response to diving based upon whether the submergence was forced or voluntary ^2,13. Diving animals may show an extremely intense bradycardia during forced diving but a much less intense bradycardia during voluntary diving. The “stress” of forced submergence can significantly change the cardiovascular responses of diving in many animals ¹⁴. Small rodents such as muskrats also show a more intense diving response during forced submergence than during voluntary diving ^15,16. Thus, if rats are to be used to investigate the central aspects of the mammalian diving response, investigators should be aware that a rat forcibly submerged underwater may produce a response different from that of a voluntarily diving rat.

The goal of this article is to provide detailed instructions on how to train rats to voluntarily dive underwater. These procedures are not technically difficult, but can be time-consuming. The training is conducted in such a way as to reduce the stressfulness that could otherwise be associated with forced underwater submergence. This voluntary diving technique should minimize activation of central stress pathways and thus better allow investigation of the central aspects of the diving response. By itself, training rats to voluntarily dive underwater generates no data that can be used to investigate the central aspects of the mammalian diving response. Therefore examples of how voluntarily diving rats have been used in conjunction with other physiological and neuroanatomical research techniques, and how the basic dive training may need to be modified to accommodate theses other techniques, are also provided.

Protocol

注：所描述的实验方案，在中西部的大学在此进行批准了中西部大学IACUC。 1.房间要求安全过程的房间已运行冷热水，一种从水箱中取出的水，通常是漏在地板上。 使用在其上放置潜水坦克的研究表。由于配备了地漏一般轮廓供排水，减少橡胶塞，并把它们放在桌子腿，保证了桌面，并在坦克，因此水面，处于水平状态。 2.潜水坦克…

Representative Results

圆满完成了所描述的游泳和潜水训练过程可以减少老鼠经历时，在水中潜水的压力。血液中皮质酮水平表明，重复的日常训练降低自愿跳水有关的心理压力，训练大鼠找到潜水不超过每天由一个人（ 图2; 17）正在处理压力。相反，只在跳水协议没有经过培训找到自愿跳水压力（ 图2; 17）。此外，无论是训练，训练组找到被迫跳水是最紧张（ 图2;</stro…

Discussion

在他们的野性表格可老鼠做利用半水生环境，而且会经常潜入水下，而觅食^6。因此，这是不是太奇怪，老鼠可以很容易训练自愿潜水水下。所描述的训练过程可以持续长达6周，这将带来新断奶的大鼠在大多数成年大鼠脑图谱中使用的主体大小（〜300克）。因而从这些受过训练的动物的大脑将更加容易地比得上在这些图谱确定的解剖结构。

之后被放置在起步区最老鼠?…

Materials

Name of Material/ Equipment	Company	Catalog Number	Comments/Description
1 in internal diameter tubing	Fisher	14-169-63	Used to fill or drain tank
N95 mask – Moldex #2300N Series	Fisher	19-003-246D	Used to limit inhalation of rat allergens
Plexiglas rodent restraint device (Economy flat bottomed restrainer)	Braintree	FB-M/L	For forced dives
Telemetric transmitters	DSI	Model PA-C40 (270-0040-008)	Used to transmit pulsatile arterial blood pressure
Hand-held antenna wand	DSI	Model RLA 3000 (272-5007)	Used to ensure radio antenna is near to transmitter while rat is negotiating underwater maze
Intramedic PE50, 0.023" ID	Fisher	14-170-12B	Used as trailing arterial cannula