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 legs to ensure that the table top, and hence the water surface in the tank, is level.</li>
</ol>
2. Diving Tank
<ol>
	<li>Diving Tank Construction
	<ol>
		<li>Construct a rectangular tank (100 x 60 x 15 cm) using 3/4 in thick Plexiglas for the bottom and 1/2 in Plexiglas for the side walls (Figure 1). Permanently attach the outside Plexiglas pieces of the tank together using cyanoacrylate cement or solvents such as trichloromethane.</li>
		<li>Divide the tank into 5 channels each approximately 100 cm in length, by using removable 1/2 inch thick Plexiglas cut into 85 x 15 cm pieces. Permanently place grooves in the bottom and edges of the tank into which these channel dividers can be slotted into position.</li>
		<li> Suspend horizontal Plexiglas pieces from the channel dividers to create a &#8220;roof&#8221; for an underwater dive &#8220;tunnel.</li>
		<li>In one corner of the tank place a removable raised platform, the &#8220;finish area&#8221;, for rats to haul out of the water and rest between swimming and diving trials. Rats usually groom themselves and remove water from their fur while in the finish area. Place tall sides around the edges of the finish area to insure the rats remain on the platform between training trials.</li>
		<li>At the opposite corner of the tank from the finish area place a chamber that only allows underwater access to the maze. This removable &#8220;start area&#8221; is only used for diving trials. 
		NOTE: At the beginning of a diving trial a rat is lowered into the water while sitting on a platform within the start chamber; hence this platform is called the &#8220;elevator&#8221;. 
		NOTE: Use a modular design, where the start area, finish area, and all channels dividers are removable from the tank. This is done for two reasons: 1) to facilitate filling and emptying the tank with water after removal of all channel dividers, and 2) if a rat gets disoriented, especially while underwater, the pieces can be quickly disassembled to rescue the rat from the water.</li>
	</ol>
	</li>
	<li>Filling and emptying the tank with water
	<ol>
		<li>Filling the Tank with water
		<ol>
			<li>At the beginning of each training session, fill the tank with fresh 32 &#176;C tap water to a depth of about 12 cm. Stir the water in the tank to minimize warm or cool pockets. 
			NOTE: Typically it takes about 2 hr to train 12 rats. During this time water temperature decreases by about 4 &#176;C. By the end of the training session the water cools to approximately 28 &#176;C. Since 30 &#176;C water is thermoneutral to rats, using this warm water, rather than room temperature water, will minimize heat loss during repetitive training.</li>
		</ol>
		</li>
		<li>Emptying the Tank with water
		<ol>
			<li>At the end of each training session, drain the water from the tank using a large hose, such as a 4 m long piece of 1 inch internal diameter tubing. Completely submerge the tubing in the tank, ensuring no air pockets are in the tubing. Quickly place one end of the tubing into the floor drain. The resulting siphoning effect will drain the water from the tank.</li>
			<li>While keeping the tank end of the tubing under the water surface to maintain the siphoning, tip the tank on edge to facilitate complete drainage of the water. 
			NOTE: Alternatively, and rather than siphoning, install a tap near the base of the tank, and connect a hose from the tank tap to the floor drain.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Rat Allergen Considerations
<ol>
	<li>Wear disposable gloves whenever handling and training the rats. The gloves will limit contact with the water in the tank, which invariably will contain rat urine. 
	NOTE: The rats will become wet during the training, and the room will be filled with a &#8220;wet-rat&#8221; odor. Consequently a prophylactic regime of anti-histamines may prevent or limit the effects of rat allergies. Trainers may also need to wear disposable dust masks, or N95 masks, should they become sensitive to rat allergens.</li>
</ol>
4. Swim Training
<ol>
	<li>Daily swim training 
	NOTE: Personal experience indicates that younger rats learn the maze better and faster, and thus starting the training with 35 g newly weaned rats is preferable to using adult rats.
	<ol>
		<li>Fill tank with water. See 2.2.1.</li>
		<li>Insert finish area and channel dividers to create 5 swimming channels.</li>
		<li>During the first training session gently lower the rats by hand into the water about 3-5 cm from the finish area. 
		NOTE: Upon their introduction to water rats will appear unsteady as they first encounter the sensation of floating. The rats will paddle about in an uncoordinated fashion while trying to locate a way to exit the water, eventually reaching the nearby finish area. Due to their innate ability to swim, in subsequent trials the rats will swim in a much more coordinated fashion toward the finish area. 
		NOTE: Support the rats from underneath so their feet are on the investigators hands. Gently lower the rats into the water, and let them swim away from the hand, rather than dropping them into the water.</li>
		<li>Between trials let the rats remain in the finish area for at least 1 min. This is to allow the rats to groom and explore, so that they regard the finish area as a &#8220;safe&#8221; place to go between trials. This is in contrast to grabbing a rat that has just completed a trial and immediately positioning it back in the water to start the next trial. 
		NOTE: Do not use external reward (i.e., food) during the training, so as to prevent activation of reward circuits in the brain.</li>
		<li>After the finish area wait period, gently hold the rats for 1 min before initiating the next trial. Gently dry the rats with a towel during this pre-trial hold since repetitive entry into the water will cause rats to get very wet, and drying them will prevent hypothermia.</li>
		<li>Repeat 4.1.3. through 4.1.5. to complete 3 to 5 trials for each rat to be trained during a daily swim training session.</li>
		<li>Remove all channel dividers and finish area from the tank.</li>
		<li>Empty the water from the tank. See 2.2.2.</li>
	</ol>
	</li>
	<li>Weekly swim training schedule
	<ol>
		<li>Conduct daily training sessions (see 4.1.) 5 days per week, ideally at the same time each day.</li>
		<li>Begin the first trial from the distance successfully negotiated on the previous day. Begin the second and third trials from an increased distance.</li>
		<li>With each subsequent daily training session increase the distance between where the rats are placed in the water and the finish area. For the first few training sessions increase this distance by 5-10 cm. After the rats appear more comfortable with swimming, increase the distances by 30-50 cm.</li>
		<li>Use larger Increases in the swimming distances while the rats are learning to swim a straight portion of a swim channel. In contrast, learning to swim the hairpin turns between channels might take 2-3 training sessions. 
		NOTE: Often, while waiting in the finish area, rats will re-enter the water to swim, and/or submerge their head underwater while still sitting on the finish area platform.</li>
		<li>Repeat this daily training protocol over 3 weeks to ensure successful swimming of the entire 5 channels. This repetitious training protocol ensures successful completion of the maze, especially since the maze includes alternating left and right hairpin turns.</li>
	</ol>
	</li>
</ol>
5. Dive Training
NOTE: After the rats have learned to successfully negotiate swimming through the maze they are ready to start dive training.
<ol>
	<li>Initial dive training (first day)
	<ol>
		<li>Insert start chamber. Fill tank with water (see 2.2.1.). Ensure water level is 1 cm below the opening to the start area.</li>
		<li>Insert finish area and channel dividers to create 5 swimming channels.</li>
		<li>During the first diving session train the rats to be lowered on the elevator into the water within the start chamber. Access to the maze is from the bottom of the start chamber. During this first session the water level is low enough so the rat can easily swim from the start chamber into the maze. Allow the rat to continue swimming through the maze to the finish area.</li>
		<li>Between trials let the rats remain in the finish area for at least 1 min. 
		NOTE: Do not use external reward (i.e., food) during the training, so as to prevent activation of reward circuits in the brain.</li>
		<li>After the finish area wait period, gently hold the rats for 1 min before initiating the next trial. Gently dry the rats with a towel during this pre-trial hold.</li>
		<li>Repeat 5.1.3. through 5.1.5. to complete 3 trials for each rat to be trained during this initial dive training session.</li>
		<li>Remove all channel dividers, start chamber and finish area from the tank.</li>
		<li>Empty the water from the tank. See 2.2.2.</li>
	</ol>
	</li>
	<li>Initial dive training (second day)
	<ol>
		<li>Insert start chamber. Fill tank with water (see 2.2.1.). Ensure water level is slightly above the opening to the start area.</li>
		<li>Insert finish area and channel dividers to create 5 swimming channels.</li>
		<li>During the second dive training session the water level has been raised so that to exit the start chamber the rat has to dip its head under the edge of the start chamber to enter the maze. Consider this as the rat&#8217;s first dive. Allow the rat to then continue swimming through the maze to the finish area.</li>
		<li>Let the rats remain in the finish area for at least 1 min between trials. 
		NOTE: Do not use external reward (i.e., food) during the training, so as to prevent activation of reward circuits in the brain.</li>
		<li>After the finish area wait period, gently hold the rats for 1 min before initiating the next trial. Gently dry the rats with a towel during this pre-trial hold.</li>
		<li>Repeat 5.2.3. through 5.2.5. to complete 3 trials for each rat to be trained during this dive training session.</li>
		<li>Remove all channel dividers, start chamber and finish area from the tank.</li>
		<li>Empty the water from the tank. See 2.2.2.</li>
	</ol>
	</li>
	<li>Initial dive training (third day)
	<ol>
		<li>Insert start chamber. Fill tank with water (see 2.2.1.), ensuring that the water level is above the opening to the start area.</li>
		<li>Insert finish area and channel dividers to create 5 swimming channels. Place a horizontal piece of Plexiglas immediately outside of the start area to create a 5 cm long diving tunnel.</li>
		<li>During the third dive training session the rat has to dip its head under the edge of the start chamber and swim 5 cm underwater to reach the open water of the swim channel. Allow the rat to then continue swimming through the maze to the finish area. 
		NOTE: Once placed in the start area, rats initiate their own underwater submergence, and thus these are considered to be &#8220;voluntary&#8221; dives. </li>
		<li>Let the rats remain in the finish area for at least 1 min between trials. 
		NOTE: Do not use external reward (i.e., food) during the training, so as to prevent activation of reward circuits in the brain.</li>
		<li>After the finish area wait period, gently hold the rats for 1 min before initiating the next trial. Gently dry the rats with a towel during this pre-trial hold.</li>
		<li>Repeat 5.3.3. through 5.3.5. to complete 3 to 5 trials for each rat to be trained during this dive training session.</li>
		<li>Remove all channel dividers, start chamber and finish area from the tank.</li>
		<li>Empty the water from the tank. See 2.2.2.</li>
	</ol>
	</li>
	<li>Weekly dive training schedule
	<ol>
		<li>Conduct daily training sessions (see 5.3.) 5 days per week, ideally at the same time each day.</li>
		<li>Begin the first trial from the distance successfully negotiated on the previous day. Begin the second and third trials with an increased diving distance.</li>
		<li>With each subsequent daily training session increase the length of the dive tunnel by adding additional horizontal dividers to extend the distance the rats have to swim underwater. For the first few training sessions increase this distance by 5-10 cm. After the rats appear more comfortable with diving, increase the distances by 30-50 cm. 
		 NOTE: Do not overextend the dive distance attempted during successive trials. If necessary, lift up the end of the horizontal channel cover to provide shorter dive distances. If during a dive trial a rat does not reach the end of the dive tunnel and starts to turn around underwater, quickly lift up the end of the channel cover and allow the rat to surface and continue his swim. This will enable the rat to successfully complete a longer dive distance. This positive reinforcement will keep the rat moving forward through the underwater tunnel with each dive trial.</li>
		<li>Use larger Increases in the diving distances while the rats are learning to dive a straight portion of a swim channel. In contrast, learning to dive around the hairpin turns between channels might take 2-3 training sessions. 
		NOTE: Often, while waiting in the finish area, rats will re-enter the water to swim, and/or submerge their head underwater while still sitting on the finish area platform.</li>
		<li>Repeat this daily training protocol over 3 weeks to ensure successful diving of the entire 5 channels. This repetitious training protocol ensures successful completion of the maze, especially since the maze includes alternating left and right hairpin turns. 
		NOTE: The full 6 week swim and dive training schedule coincides with the growth of 35 g newly weaned rats to reach a body weight of 300 g.</li>
		<li>After daily training sessions are complete, and the rats have been returned to their home cage, ensure rats quickly dry their fur through grooming, or if necessary, place a heating pad under their cage to keep the rats warm until their fur is dry.</li>
	</ol>
	</li>
</ol>
6. Experimental Variations
NOTE: The basic experimental set-up and animal training have been described above. However, behavioral training only provides a model to be used with other experimental techniques to collect data of interest. Basic protocols are modified to investigate specific aspects of the diving response. Examples of these modifications, and some considerations for the collection of data using these physiological and neuroanatomical techniques, are given below.
<ol>
	<li>Implantable Telemetric Transmitters
	<ol>
		<li>After completion of the training, use commercially available telemetric transmitters to transmit pulsatile arterial blood pressure from swimming and diving rats. Obtain local IACUC approval for procedures concerning surgery and post-surgical recovery. Follow the implantation procedures suggested by the company for their transmitter, and ensure complete recovery from the surgery before returning the rat into the water.</li>
		<li>Ensure the antenna receiving the radio signal is nearby when the rat is in the water. 
		NOTE: Water attenuates radio signals, and so the distance the radio signal needs to travel through the water becomes a limiting factor of the tank dimensions.</li>
		<li>Use a hand-held antenna wand, rather than a rat-cage sized antenna, to follow the rat as it progresses through the maze. Keep the wand antenna within 30 cm of the rat to ensure that the radio signal is not lost while the rat is underwater.</li>
	</ol>
	</li>
	<li>Trailing Cannulae
	<ol>
		<li>Redesign the channel dividers and horizontal pieces creating the roof of the diving tunnel so that the cannula trails along behind the rat during its progression through the maze. 
		NOTE: The modular design of the channels is of utmost importance and must also allow quick recover of the rat from the underwater maze. If a cannula snags and became dislodged from the rat, the rat could soon bleed out while underwater if the cannula is not quickly reattached.</li>
		<li>Use trailing arterial cannulae to record arterial blood pressure and heart rate in voluntarily diving rats. Use a 90 cm piece of PE50 as a trailing cannula. 
		NOTE: This cannula length is long enough to connect the rat to the pressure transducer while still allowing the rat to progress through the maze, yet is short enough to minimize cannula dead space and is close enough to the pressure transducer to allow sufficient fidelity of the arterial pressure signal.</li>
		<li>Use trailing venous (or arterial) cannulae to inject pharmaceutical agents such as parasympathetic and sympathetic agonists and antagonists, or to inject tracers or dyes that determine the distribution of cardiac output.</li>
		<li>Withdraw blood samples from the rats using the venous (or arterial) cannulae while the rats are underwater to determine underwater catecholamine levels or blood chemistry. 
		NOTE: Minimize the length of the trailing cannula and account for the cannula dead space during blood draws.</li>
	</ol>
	</li>
	<li>Detection of Activated Brainstem Neurons
	<ol>
		<li>Use immunological detection of the Fos protein to identify specific areas of the brainstem that are part of the diving response. 
		NOTE: During repetitive initiation of a cardiorespiratory reflex, brainstem neurons that are part of that reflex circuit can become activated and produce a protein called Fos.</li>
		<li>Repetitively dive rats through the maze every 5 min for 2 hr for a total of 24 dives. 
		NOTE: Other protocols that also induce neuronal Fos production can also be used. To avoid activation of brainstem neurons involved in the response to stress, the behavior that is repeated should be included as part of the behavioral training.</li>
	</ol>
	</li>
	<li>Blood Corticosterone Levels during Diving
	<ol>
		<li>Use corticosterone as an indicator of the level of stress rats experience during swimming and diving.</li>
		<li>To obtain blood for corticosterone analysis, draw 0.1 ml blood samples from the tail vein of rats 15 min after 3 voluntary swims or dives. 
		NOTE: Preliminary experiments indicated that 15 min is sufficient time to allow for the production and release of corticosterone into the circulation and cause a peak in plasma corticosterone levels. Since corticosterone levels have a circadian rhythm, schedule all blood draws at the same time of day and corresponded with the timing of training sessions.</li>
	</ol>
	</li>
</ol>

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M., et al. <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+brainstem+neurons+by+underwater+diving+in+the+rat.">Activation of brainstem neurons by underwater diving in the rat.</a> Front. Physiol. 3, 1-13 (2012).</li><li>McCulloch, P. F., Panneton, W. M. <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+brainstem+catecholaminergic+neurons+during+voluntary+diving+in+rats.">Activation of brainstem catecholaminergic neurons during voluntary diving in rats.</a> Brain Res. 984, 42-53 (2003).</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=Globosa+neurons:+a+distinct+subgroup+of+noradrenergic+neurons+in+the+caudal+pons+of+rats.">Globosa neurons: a distinct subgroup of noradrenergic neurons in the caudal pons of rats.</a> Brain Res. 964, 164-167 (2003).</li><li>Panneton, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mammalian+diving+response:+an+enigmatic+reflex+to+preserve+life.">The mammalian diving response: an enigmatic reflex to preserve life.</a> Physiology. 28, 284-297 (2013).</li></ol>

The author is a consultant for Stoelting Company, and provided the overall design and specifications of the McCulloch Dive Tank Maze to them for commercial purposes.

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.
After being placed in the start area most rats will begin their underwater swim within 20 sec. However, occasionally a rat will take up to 5 min or more before initiating its voluntary dive. While it may be tempting at this stage to force the rats into the underwater tunnel, this generally should be avoided to prevent the rats from associating the water with a negative experience. Rodents can be stubborn and may initially refuse to dive during the training sessions, but once they realize the only way to exit the water is by completing the dive through the maze, they usually initiate their dives soon after being placed in the start area.
A critical aspect of the repetitious and methodical training procedures is that they are conducted in such a way to reduce the stress experienced by the rats. Letting rats explore their environment, especially while in the finishing area between trials, seems to further reduce stress. Rats will often re-enter the water to swim, and/or submerge their head underwater while still sitting on the finish area platform. This suggests that rats are not inherently water aversive. Additionally, while it is not uncommon for rats to produce fecal pellets during swimming or diving, or when waiting in the finish area, daily training results in fewer fecal pellets 17. In general the less stressed the rats are during the training, the fewer pellets they will produce. Any fecal pellets that are produced are removed from the water or finish area as soon as possible to keep the water relatively clean.
Rats may occasionally get bloody noses while swimming and diving, which may be a result of insufflation of water into the nasal passages. The appearance of blood may be due to osmotic stresses within the nasal mucosa. While in the finish area between trials the rats will groom themselves. As a consequence blood from the nose can get redistributed over the rats head and snout, giving the rats a slight reddish tinge, especially around the eyes, during the course of a training session.&#160;Also, an occasional rat may find diving stressful and/or have negative diving experiences (i.e., by turning around and getting lost while diving underwater (see note after 5.4.3. on how to prevent this from occurring)). In these rats porphyrin may appear in the corners of their eyes, signaling a stress response.
The size of the diving tank will to some degree determine the room requirements. The described tank is designed to have rats swim 2-3 cm under the surface of the water through a 5 m long Plexiglas maze to give an underwater swimming duration of 10-15 sec 17,19,24,26,27. Should an experiment be designed to measure responses from a longer duration dive, or from a deeper dive underwater, the tank may need to be re-designed. The room requirements could then also change to fit the dimensions of a re-designed diving tank. If there is no floor drain available in the procedure room, the water from the tank can be collected in a large container, such as a 60 gal garbage can, which can then be emptied elsewhere in a convenient manner.
The Fos technique can be utilized with other neuronal detection methods to further identify and characterize neurons that are part of the brainstem circuitry of the diving response. For instance, Fos detection in conjunction with tyrosine hydroxylase staining has identified catecholaminergic neurons in the A1, C1, A2, A5 and sub-coeruleus areas (Figure 6; 26), and globosa neurons within the lateral A7 area 26,27, that are activated during voluntary diving. Also, Fos detection in conjunction with the retrograde tracer Cholera toxin has identified the cell bodies of cardiac vagal motorneurons within the external formation of the nucleus ambiguus that are activated during voluntary diving 20.
Investigating the central nervous integration of the cardiorespiratory responses to diving is important for a number of reasons 6,8,28. The diving response enables animals, including humans, to remain submerged underwater without breathing for extended periods of time. The diving response represents a functional reorganization of brainstem homeostatic control, and demonstrates one of the most powerful patterns of autonomic reflexes observed in animals. The diving response may also be important clinically in humans as part of the trigemino-cardiac reflex, nasopharyngeal reflex, and/or sudden infant death syndrome. Finally, an understanding of the neuronal circuitry that exists within the brainstem of rats will help determine how cortical afferent signals can modify basic brainstem autonomic reflexes. All of these considerations make study of the central aspects of the mammalian diving response inherently worthwhile and interesting. Using the described procedures to train rats to voluntarily dive underwater will allow better investigation of the central aspects of the mammalian diving response than will the use of forced dived animals. This is because the training procedures as described 1) reduce activation of CNS stress circuitry, and 2) do not activate CNS reward circuitry because external rewards are not used.

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><tbody><tr><td>McCulloch Diving Tank Maze</td><td>Stoelting Company</td><td>60139</td><td></td></tr><tr><td>1 inch internal diameter tubing&amp;nbsp;</td><td>Fisher</td><td>14-169-63</td><td>Used to fill or drain tank</td></tr><tr><td>Plexiglas rodent restraint device (Economy flat bottomed restrainer)</td><td>Braintree</td><td>FB-M/L&amp;nbsp;</td><td>For forced dives</td></tr><tr><td>Telemetric transmitters&amp;nbsp;</td><td>DSI</td><td>Model PA-C40 (270-0040-008)</td><td>Used to transmit pulsatile arterial blood pressure</td></tr><tr><td>Hand-held antenna wand</td><td>DSI</td><td>Model RLA 3000 (272-5007)</td><td>Used to ensure radio antenna is near to transmitter while rat is negotiating underwater maze</td></tr><tr><td>Intramedic PE50, 0.023&quot; ID</td><td>Fisher</td><td>14-170-12B</td><td>Used as trailing arterial cannula</td></tr><tr><td>N95 mask - Moldex #2300N Series</td><td>Fisher</td><td>19-003-246D</td><td>Used to limit inhalation of rat allergens</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). Additionally, both trained and untrained rats find forced diving to be the most stressful (Figure 2; 17).
The cardiovascular responses from swimming, voluntary diving and forced diving rats have been recorded using implanted telemetry devices (Figure 3; 8,17-20) and trailing cannulae (Figure 4; 21-23). Immediately upon voluntary submersion, and within a single beat, heart rate decreases by 78% and mean arterial blood pressure decreases by 25% 17. These results show that voluntarily diving rats exhibit the same cardiorespiratory changes typically seen in other diving animals. Trailing arterial cannulae have been used to inject the muscarinic antagonist atropine, which eliminates the bradycardia associated with voluntary diving (Figure 4; 21), and to determine the distribution of cardiac output 22, including cerebral blood flow 23, during voluntary diving. Trailing cannulae have also been used to show that rats ignore increasing arterial hypoxemia and hypercapnia while they are submerged 18, and that pre-existing chemoreceptor drive does not have any effect on the cardiovascular responses to voluntary diving 21.
Neurons within laminae I and II of the ventral medullary dorsal horn (MDH) express Fos during voluntary diving, and these neurons may constitute the initial brainstem afferent relay of the diving response (Figure 5; 24). Important brainstem cardiorespiratory control areas, such as the caudal pressor area (CPA), nucleus tractus solitaries (NTS), rostral ventrolateral medulla (RVLM), and peribrachial regions, all show increased Fos labeling during voluntary diving compared with swimming 25. Neurons in chemosensitive regions of the brainstem express Fos after long duration forced dives 18.
<img alt="Figure 1" fo:content-width="4in" src="/files/ftp_upload/52093/52093fig1highres.jpg" width="400" /> 
Figure 1: Schematic of Diving Tank. A Plexiglas tank (100 x 60 x 15 cm) was used to create a simple maze consisting of five 1 m long channels. The tank was filled with 30 &#176;C tap water, and rats were initially trained to negotiate the maze by swimming on the surface of the water, from the Start Area (top left) to the Finish Area (bottom right). The rats were then trained to dive through the maze, kept underwater by horizontal Plexiglas pieces placed 2-3 cm below the water surface. [This figure has been modified from 26]
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/52093/52093fig2highres.jpg" width="500" /> 
Figure 2: Corticosterone measurements. Blood draws from rat tail veins were used to measure corticosterone concentrations (mean &#177; SE) from rats left in their cages (Na&#239;ve), rats handled for 10 min/day (Handled), rats trained to swim and dive (Trained), and rats that received no swim or dive training (Untrained). Corticosterone was measured after trained rats had completed their swim training (Left set of bars), after trained rats had completed their voluntary dive training (Center set of bars), and after trained rats had completed their forced dive training (Right set of bars). 1 indicates value is significantly greater than Na&#239;ve; 2 indicates value is significantly greater than Handled; 3 indicates value is significantly greater than Trained; * indicates that in Trained rats value during forced dive is significantly greater than during voluntary dive. [This figure has been modified from 17]
<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/52093/52093fig3highres.jpg" width="500" /> 
Figure 3: Arterial blood pressure traces from telemetric transmitters. Raw data traces showing pulsatile arterial blood pressure during swimming (left column), voluntary diving (middle column), and forced diving (right column) from rats trained to swim and dive through the maze (bottom row) and from rats that had not had the training procedure (top row). Diving underwater (both voluntary and forced submergence) produced an immediate bradycardia and slower onset increase in arterial pressure, whereas swimming on the surface of the water caused no such cardiovascular changes. Bars under traces indicate periods of submergence. Breaks in trace indicate periods when the telemetric signal was lost. [This figure has been modified from 17]
<img alt="Figure 4" fo:content-width="5in" src="/files/ftp_upload/52093/52093fig4highres.jpg" width="500" /> 
Figure 4: Atropine eliminates diving bradycardia. Original recordings of pulsatile arterial blood pressure of voluntarily diving rats (A) before and (B) after atropine pre-treatment. Traces were obtained using a trailing arterial cannula. Before atropine pre-treatment, arterial pressure decreased slightly upon submersion, but then increased to greater than pre-dive for the remainder of the dive. Heart rate was determined from adjacent pulse pressure intervals. Upon submersion there was an immediate and substantial bradycardia that was sustained for the duration of the dive. After parasympathetic blockade by atropine pre-treatment the bradycardia was eliminated. There was also an increase in arterial pressure during the dive. The bar under the trace indicates the period of submergence. [McCulloch, unpublished]
<img alt="Figure 5" fo:content-width="6in" src="/files/ftp_upload/52093/52093fig5highres.jpg" width="600" /> 
Figure 5: Fos labeling within the MDH. Photomicrographs of the trigeminal medullary dorsal horn (MDH) and the spinal trigeminal tract (sp5) in rats trained to dive underwater. (A) In a control rat that did not repetitively dive there is no Fos labeling. (B) In a swimming rat there is very little Fos label in the MDH (large arrowhead) or paratrigeminal nucleus (small arrow) within sp5. (C) In a diving rat there is more Fos labeling ventrally in both the MDH (large arrowhead) and paratrigeminal nucleus (small arrows) compared to the swimming and control rat. Insert in panel (A) indicates the rostral-caudal location of panels A-C. Scale bar in panel C is 100 &#956;m. [This figure has been modified from 24]
<img alt="Figure 6" fo:content-width="5in" src="/files/ftp_upload/52093/52093fig6highres.jpg" width="500" /> 
Figure 6: Activated catecholaminergic neurons from diving rats. Photomicrographs show the medullas of a non-diving control rat (A, C, and E) and a voluntarily diving rat (B, D, and F). The brain tissue was immunohistologically processed for both Fos and tyrosine hydroxylase (TH), producing brown TH somas and black Fos nuclei. Open arrowheads identify single-labeled TH-positive neurons, while solid arrows identify Fos+TH double-labeled neurons. A1 neurons are identified in A and B. C1 neurons are identified in C and D. A5 neurons are identified in E and F. More Fos and TH double-labeled are seen in the A1, C1, and A5 regions of the diving rat than in the non-diving control rat. Calibration bar in E is for panels A-F, and is 250 &#956;m. Calibration bar in inset in F is for all insets, and is 50 &#956;m. [This figure has been modified from 26]

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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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12.2K Views.  Midwestern University. 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.

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

Shallow Water (Paddling) Variants of Water Maze Tests in Mice

When Richard Morris devised his water maze in 19817, most behavioral work was done in rats. However, the greater understanding of mouse genetics led to the mouse becoming increasingly important. But researchers found that some strains of mutant mice were prone to problems like passively floating or diving when they were tested in the Morris water maze11. This was unsurprising considering their natural habitat; rats swim naturally (classically, the "sewer rat"), whereas mice evolved in the dry areas of central Asia.
To overcome these problems, it was considered whether shallow water would be a sufficient stimulus to provide escape motivation for mice. This would also avoid the problems of drying the small creatures with a towel and then putting them in a heated recovery chamber to avoid hypothermia, which is a much more serious problem than with rats; the large ratio of surface area to volume of a mouse makes it particularly vulnerable to rapid heat loss.
Another consideration was whether a more natural escape strategy could be used, to facilitate learning. Since animals that fall into water and swim away from the safety of the shore are unlikely to pass on their genes, animals have evolved a natural tendency to swim to the edge of a body of water. The Morris water maze, however, requires them to swim to a hidden platform towards the center of the maze - exactly opposite to their evolved behavior. Therefore the paddling maze should incorporate escape to the edge of the apparatus. This feature, coupled with the use of relatively non-aversive shallow water, embodies the "Refinement" aspect of the "3 Rs" of Russell and Burch8.
Various types of maze design were tried; the common feature was that the water was always shallow (2 cm deep) and escape was via a tube piercing the transparent wall of the apparatus. Other tubes ("false exits") were also placed around the walls but these were blocked off. From the inside of the maze all false exits and the single true exit looked the same. Currently a dodecagonal (12-sided) maze is in use in Oxford, with 12 true/false exits set in the corners. In a recent development a transparent paddling Y-maze has been tested successfully.

Shallow Water Paddling Variants of Water Maze Tests in Mice

Mice can swim, but many strains appear to find this activity stressful. To overcome this problem mazes have been devised where escape from shallow water is used to motivate behaviour. These have been demonstrated to support learning at least as good as the traditional and widely used Morris water maze.

When Richard Morris devised his water maze in 19817, most behavioral work was done in rats. However, the greater understanding of mouse genetics led to ...

The Forced Swim Test as a Model of Depressive-like Behavior

The goal of the present protocol is to describe the forced swim test (FST), which is one of the most commonly used assays for the study of depressive-like behavior in rodents. The FST is based on the assumption that when placing an animal in a container filled with water, it will first make efforts to escape but eventually will exhibit immobility that may be considered to reflect a measure of behavioral despair. This test has been extensively used because it involves the exposure of the animals to stress, which was shown to have a role in the tendency for major depression. Additionally, the FST has been shown to share some of the factors that are influenced or altered by depression in humans, including changes in food consumption, sleep abnormalities and drug-withdrawal-induced anhedonia. The main advantages of this procedure are that it is relatively easy to perform and that its results are easily and quickly analyzed. Moreover, its sensitivity to a broad range of antidepressant drugs that makes it a suitable screening test is one of the most important features leading to its high predictive validity. Despite its appeal, this model has a number of disadvantages. First, the issue of chronic augmentation is problematic in this test because in real life patients need to be treated for at least several weeks before they experience any relief from their symptoms. Last, due to the aversiveness of the FST, it is important to take into account possible influences it might have on brain structure/function if brain analyses are to be carried out following this procedure.

This protocol describes the forced swim test, which is used for the study of depressive-like behavior in rodents. This procedure involves placing an animal in a container filled with water that eventually will lead to the exhibition of immobility behavior, which is considered to reflect behavioral despair.

The goal of the present protocol is to describe the forced swim test (FST), which is one of the most commonly used assays for the study of depressive-like ...

The Double-H Maze: A Robust Behavioral Test for Learning and Memory in Rodents

Spatial cognition research in rodents typically employs the use of maze tasks, whose attributes vary from one maze to the next. These tasks vary by their behavioral flexibility and required memory duration, the number of goals and pathways, and also the overall task complexity. A confounding feature in many of these tasks is the lack of control over the strategy employed by the rodents to reach the goal, e.g., allocentric (declarative-like) or egocentric (procedural) based strategies. The double-H maze is a novel water-escape memory task that addresses this issue, by allowing the experimenter to direct the type of strategy learned during the training period. The double-H maze is a transparent device, which consists of a central alleyway with three arms protruding on both sides, along with an escape platform submerged at the extremity of one of these arms.
Rats can be trained using an allocentric strategy by alternating the start position in the maze in an unpredictable manner (see protocol 1; &#167;4.7), thus requiring them to learn the location of the platform based on the available allothetic cues. Alternatively, an egocentric learning strategy (protocol 2; &#167;4.8) can be employed by releasing the rats from the same position during each trial, until they learn the procedural pattern required to reach the goal. This task has been proven to allow for the formation of stable memory traces.
Memory can be probed following the training period in a misleading probe trial, in which the starting position for the rats alternates. Following an egocentric learning paradigm, rats typically resort to an allocentric-based strategy, but only when their initial view on the extra-maze cues differs markedly from their original position. This task is ideally suited to explore the effects of drugs/perturbations on allocentric/egocentric memory performance, as well as the interactions between these two memory systems.

The goal of this protocol is to investigate spatial cognition in rodents. The double-H water maze is a novel test, which is particularly useful to elucidate the different components of learning, consolidation and memory, as well as the interplay of memory systems.

Spatial cognition research in rodents typically employs the use of maze tasks, whose attributes vary from one maze to the next. These tasks vary by their ...

A New Method for Inducing a Depression-Like Behavior in Rats

Contagious depression is a phenomenon that is yet to be fully recognized and this stems from insufficient material on the subject. At the moment, there is no existing format for studying the mechanism of action, prevention, containment, and treatment of contagious depression. The purpose of this study, therefore, was to establish the first animal model of contagious depression.
Healthy rats can contract depressive behaviors if exposed to depressed rats. Depression is induced in rats by subjecting them to several manipulations of chronic unpredictable stress (CUS) over 5 weeks, as described in the protocol. A successful sucrose preference test confirmed the development of depression in the rats. The CUS-exposed rats were then caged with na&#239;ve rats from the contagion group (1 na&#239;ve rat/2 depressed rats in a cage) for an additional 5 weeks. 30 social groups were created from the combination of CUS-exposed rats and na&#239;ve rats.
This proposed depression-contagion protocol in animals consists mainly of cohabiting CUS-exposed and healthy rats for 5 weeks. To ensure that this method works, a series of tests are carried out - first, the sucrose preference test upon inducing depression to rats, then, the sucrose preference test, alongside the open field and forced-swim tests at the end of the cohabitation period. Throughout the experiment, rats are given tags and are always returned to their cages after each test.
A few limitations to this method are the weak differences recorded between the experimental and control groups in the sucrose preference test and the irreversible traumatic outcome of the forced swim test. These may be worth considering for suitability before any future application of the protocol. Nonetheless, following the experiment, na&#239;ve rats developed contagion depression after 5 weeks of sharing the same cage with the CUS-exposed rats.

This protocol describes a new model by which healthy rats could contract depression over a given time periodthrough contagion by exposure to chronic unpredictable stressed (CUS) rats.

Contagious depression is a phenomenon that is yet to be fully recognized and this stems from insufficient material on the subject. At the moment, there is ...

Using Clicker Training and Social Observation to Teach Rats to Voluntarily Change Cages

Cage cleaning is a routinely performed husbandry procedure and is known to induce stress in laboratory rats. As stress can have a negative impact on well-being and can affect the comparability and reproducibility of research results, the amount of stress experienced by laboratory animals should be minimized and avoided when possible. Further, the direct contact between the rat and animal caretaker during the cage change bears hygiene risks and therefore possibly negatively impacts the well-being of the rats and the quality of the research. 
Our protocol aims to improve the routinely performed cage changing procedure. For this reason, we present a feasible protocol that enables rats to learn via clicker training and observation to voluntarily change to a clean cage. This training helps to reduce stress caused by the physical disturbance and handling associated with the cage changes and concurrently enables a reduction in direct contact between animal and animal caretaker after the training phase is completed.
The implementation of clicker training to rats is fast and easy. Rats are generally interested in the training and efficiently learn the desired behavior, which entails changing cages through a pipe. Even without training, the rats learn to perform the desired behavior by observation, as 80% of the observational learning group successfully changed cages when tested. The training further helps to establish a relationship of trust between trainer and animal. As hygiene and well-being are both very important in animal experiments, this protocol might also help to improve high-quality research.

This protocol introduces a method of cage change for rats via clicker training. Rats learn the desired behavior not only by direct training but also by observational learning. The implementation of this fast and easy protocol might help to improve well-being and hygiene in rodent facilities.

Cage cleaning is a routinely performed husbandry procedure and is known to induce stress in laboratory rats. As stress can have a negative impact on ...

A Complex Diving-For-Food Task to Investigate Social Organization and Interactions in Rats

For many species, where status is a vital motivator that can affect health, social hierarchies influence behavior. Social hierarchies that include dominant-submissive relationships are common in both animal and human societies. These relationships can be affected by interactions with others and with their environment, making them difficult to analyze in a controlled study. Rather than a simple dominance hierarchy, this formation has a complicated presentation that allows rats to avoid aggression. Status can be stagnant or mutable, and results in complex societal stratifications. Here we describe a complex diving-for-food task to investigate rodent social hierarchy and behavioral interactions. This animal model may allow us to assess the relationship between a wide range of mental illnesses and social organization, as well as to study the effectiveness of therapy on social dysfunction.

This protocol describes a method of examining social hierarchy in a rat model. Rats perform a complex diving-for-food task in which they form a distinct hierarchy according to their willingness to dive underwater and swim to obtain a food pellet. This method is used to understand decision making and social relationships among highly social animals in small groups.

For many species, where status is a vital motivator that can affect health, social hierarchies influence behavior. Social hierarchies that include ...

Behavioral Training Procedures for Head-fixed Virtual Reality in Mice

Virtual reality (VR) combined with head-fixation is increasingly being utilized in behavioral neuroscience studies as it allows complex behavioral assays to be performed in head-fixed mice. This enables precise behavioral recordings while incorporating various neurophysiological techniques that require head-fixation to minimize movement-related signal noise during neural recordings. However, despite the growing use of VR, there is little published data on the detailed methodology of how to implement it. In this study, a training protocol is developed whereby male and female C57B16/J mice are trained to run down a virtual linear corridor, the length of which is increased from 1-3 m over multiple training sessions. Building upon this foundation, this study investigated the feasibility of mice performing complex behaviors within VR using a Y-maze paradigm. The task required navigating to the arm with black walls from the choice point in the Y-maze. After reaching a criterion of two consecutive days equal to or greater than 70% correct, the mice progressed to increasingly difficult sensory discrimination. The findings provide important details on the methodologies useful for the successful training of mice in VR and demonstrate that mice exhibit learning capabilities in navigating the Y-maze. The methodology presented not only offers insights into training duration in VR-based assays but also underscores the potential for probing intricate behaviors in mice, opening avenues for more comprehensive neuroscience investigations.

The article describes the experimental procedures for the commonly used linear track virtual reality (VR) paradigm in mice as well as determining the feasibility of running complex VR tasks by testing a Y-shaped signal discrimination task.

Virtual reality (VR) combined with head-fixation is increasingly being utilized in behavioral neuroscience studies as it allows complex behavioral assays ...

Exploring Maternal Exercise for the Well-Being of Rat Offspring

Swimming Exercise Protocol and Care Methods for Pregnant Rats

The developmental origins of health and disease concept highlights the impact of early environments on chronic non-communicable diseases like diabetes, cardiovascular disease, and cancer. Studies using animal models have investigated how maternal factors such as undernutrition, overnutrition, obesity, and exposure to chemicals or hypoxia affect fetal development and offspring health, leading to issues like low birth weight, high blood pressure, dyslipidemia, and insulin resistance. Given the increasing prevalence of overweight and obesity among reproductive-age women, effective interventions are critical. Maternal exercise during pregnancy has emerged as a key intervention, benefiting both mother and offspring and reducing the risk of disease. This study compares the differences of three exercise models on pregnant rats: voluntary wheel running, motorized treadmills, and swimming. Swimming is the most beneficial option due to its safe and controlled intensity levels. This protocol details the rat breeding methods, swimming training during pregnancy, and post-breeding nursing protocols. This model, suitable for various rat and mouse species, is useful for studying the benefits of maternal exercise on offspring health and intergenerational wellness.

Author Spotlight: Enhancing the Offspring Health in Rats with Maternal Exercise During Pregnancy

Here, we present a protocol to delineate rat breeding methods, swimming training procedures, and post-breeding nursing protocols for pregnant rats after training. This protocol provides an animal model for studying the effects of maternal exercise during pregnancy on offspring and its underlying mechanisms.

The developmental origins of health and disease concept highlights the impact of early environments on chronic non-communicable diseases like diabetes, ...

Developmental Biology

The Morris Water Maze

The Morris water maze is one of the most widely used behavioral tests for studying spatial learning and memory. In the initial phases of this task, rodents must swim to a platform to escape from a pool of water. The platform is then hidden under the water&#8217;s surface, so that the animal is required to remember it&#8217;s location in order to escape.This simple yet powerful maze design can be used to assay cognitive function, study animal models of neurodegenerative disease, and test potential drug therapies.
This video provides an introduction to the Morris water maze and the principles surrounding its use, including a discussion of the different types of memory tested in the maze, important points to consider when designing and conducting this experiment, and the procedures for setup and running of the test. Several applications of the maze are examined, such as investigating how radiation treatment may lead to memory impairment. Finally, other types of water mazes, such as the 8-arm radial maze, are introduced to show how this paradigm can be adapted to engage different types of memory.

The Morris water maze is one of the most widely used behavioral tests for studying spatial learning and memory. In the initial phases of this task, ...

Education

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Advanced Biology

Neuroscience

Novel Tank Diving Assay: A Method to Study Innate Stress Responses in Zebrafish

Source: Chin J.S.R., et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/59092/behavioral-approaches-to-studying-innate-stress-in-zebrafish" target="_blank">Behavioral Approaches to Studying Innate Stress in Zebrafish</a>. J. Vis. Exp. (2019)
The article describes a behavioral method to assess stress in zebrafish. In response to stress, zebrafish tends to swim at bottom of a tank until stress is removed. The sample protocol describes stepwise method for performing the assay.

Source: Chin J.S.R., et al. Behavioral Approaches to Studying Innate Stress in Zebrafish. J. Vis. Exp. (2019)
The article describes a behavioral method to ...

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

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