Name: training rats to voluntarily dive underwater: investigations of the mammalian diving response
Uploaded: 2014-11-12T15:00:00
Description: Watch this Scientific Journal Video about Training Rats to Voluntarily Dive Underwater: Investigations of the Mammalian Diving Response at JoVE.com

Research supported by funding from the Midwestern University Office of Research and Sponsored Programs. Thanks also to the Midwestern University Animal Facility and Erik Warren.

NOTE: Experimental protocols described herein conducted at Midwestern University were approved by the Midwestern University IACUC.
1. Room Requirements
<ol>
	<li>Secure a procedure room that has running hot and cold running water, and a way to remove the water from the tank, typically a drain in the floor.</li>
	<li>Use a study table on which to place the diving tank. Since rooms with floor drains are typically contoured for drainage, cut down rubber stoppers and place them under the table l...

<ol>
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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bilateral+sectioning+of+the+anterior+ethmoidal+nerves+does+not+eliminate+the+diving+response+in+voluntarily+diving+rats.">Bilateral sectioning of the anterior ethmoidal nerves does not eliminate the diving response in voluntarily diving rats.</a> Physiol. Reports. 1, (2013).</li><li>Panneton, W. M., Anch, A. M., Panneton, W. M., Gan, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parasympathetic+preganglionic+cardiac+motoneurons+labeled+after+voluntary+diving.">Parasympathetic preganglionic cardiac motoneurons labeled after voluntary diving.</a> Front. Physiol. 5, 1-10 (2014).</li><li>McCulloch, P. F., Ollenberger, G. P., Bekar, L. K., West, N. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+regional+cerebral+blood+flow+in+voluntarily+diving+rats.">Distribution of regional cerebral blood flow in voluntarily diving rats.</a> J. Exp. Biol. 201, 549-558 (1998).</li><li>McCulloch, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+the+trigeminal+medullary+dorsal+horn+during+voluntary+diving+in+rats.">Activation of the trigeminal medullary dorsal horn during voluntary diving in rats.</a> Brain Res. 1051, 194-198 (2005).</li><li>Panneton, W. 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Rats in their feral form can and do exploit semi-aquatic environments, and will often dive underwater while foraging for food 6. Thus it is not too surprising that rats can be very easily trained to voluntarily dive underwater. The described training procedures may last up to 6 weeks, which will bring newly weaned rats to a body size used in most adult rat brain atlases (~300 g). Thus the brains from these trained animals will be more readily comparable to the anatomical structures identified in these atlases....

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 &#8220;stress&#8221; 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.

<table border="0" cellpadding="0" cellspacing="0" width="676">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Name of Material/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:159px;">Catalog Number</td>
			<td style="width:200px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 in internal diameter tubing&#160;</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:159px;">14-169-63</td>
			<td>Used to fill or drain tank</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">N95 mask - Moldex #2300N Series</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:159px;">19-003-246D</td>
			<td style="width:200px;">Used to limit inhalation of rat allergens</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Plexiglas rodent restraint device (Economy flat bottomed restrainer)</td>
			<td style="width:71px;">Braintree</td>
			<td style="width:159px;">FB-M/L&#160;</td>
			<td>For forced dives</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Telemetric transmitters&#160;</td>
			<td style="width:71px;">DSI</td>
			<td style="width:159px;">Model PA-C40 (270-0040-008)</td>
			<td style="width:200px;">Used to transmit pulsatile arterial blood pressure</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;">Hand-held antenna wand</td>
			<td style="width:71px;">DSI</td>
			<td style="width:159px;">Model RLA 3000 (272-5007)</td>
			<td style="width:200px;">Used to ensure radio antenna is near to transmitter while rat is negotiating underwater maze</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Intramedic PE50, 0.023&#34; ID</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:159px;">14-170-12B</td>
			<td style="width:200px;">Used as trailing arterial cannula</td>
		</tr>
	</tbody>
</table>

training rats to voluntarily dive underwater: investigations of the mammalian diving response

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.

Successful completion of the described swimming and diving training procedures can decrease the stress experienced by rats when diving under water. Blood corticosterone levels indicate that repetitive daily training decreases the stressfulness associated with voluntary diving, and trained rats find diving no more stressful than being handled daily by a human (Figure 2; 17). Conversely, rats not trained in the diving protocol find voluntary diving stressful (Figure 2; 17</...

Watch this Scientific Journal Video about Training Rats to Voluntarily Dive Underwater: Investigations of the Mammalian Diving Response at JoVE.com

Training Rats to Voluntarily Dive Underwater: Investigations of the Mammalian Diving Response

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.

Underwater submergence produces autonomic changes that are observed in virtually all diving animals. This reflexly-induced response consists of apnea, a ...

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