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....

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.

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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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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

A Method for Evaluating the Reinforcing Properties of Ethanol in Rats without Water Deprivation, Saccharin Fading or Extended Access Training

Operant oral self-administration methods are commonly used to study the reinforcing properties of ethanol in animals. However, the standard methods require saccharin/sucrose fading, water deprivation and/or extended training to initiate operant responding in rats. This paper describes a novel and efficient method to quickly initiate operant responding for ethanol that is convenient for experimenters and does not require water deprivation or saccharin/sucrose fading, thus eliminating the potential confound of using sweeteners in ethanol operant self-administration studies. With this method, Wistar rats typically acquire and maintain self-administration of a 20% ethanol solution in less than two weeks of training. Furthermore, blood ethanol concentrations and rewards are positively correlated for a 30 min self-administration session. Moreover, naltrexone, an FDA-approved medication for alcohol dependence that has been shown to suppress ethanol self-administration in rodents, dose-dependently decreases alcohol intake and motivation to consume alcohol for rats self-administering 20% ethanol, thus validating the use of this new method to study the reinforcing properties of alcohol in rats.

This protocol describes a novel and efficient method to quickly initiate operant responding for ethanol in rats that, contrary to standard methods, does not require water deprivation or saccharin/sucrose fading to initiate responding.

Operant oral self-administration methods are commonly used to study the reinforcing properties of ethanol in animals. However, the standard methods ...

Pavlovian Conditioned Approach Training in Rats

Cues that are contingently paired with unconditioned, rewarding stimuli can acquire rewarding properties themselves through a process known as the attribution of incentive salience, or the transformation of neutral stimuli into attractive, &#34;wanted&#39; stimuli capable of motivating behavior. Pavlovian conditioned approach (PCA) develops after the response-independent presentation of a conditioned stimulus (CS; e.g., a lever) that predicts the delivery of an unconditioned stimulus (US; e.g., a food pellet) and can be used to measure incentive salience. During training, three patterns of conditioned responses (CRs) can develop: sign-tracking behavior (CS-directed CR), goal-tracking behavior (US-directed CR), and an intermediate response (both CRs). Sign-trackers attribute incentive salience to reward-related cues and are more vulnerable to cue-induced reinstatement of drug-seeking as well as other addiction-related behaviors, making PCA a potentially valuable procedure for studying addiction vulnerability. Here, we describe materials and methods used to elicit PCA behavior from rats as well as analyze and interpret PCA behavior in individual experiments.

Here, we present a protocol to elicit Pavlovian conditioned approach behavior in rats. This procedure can be used to measure individual differences in the tendency to approach and attribute incentive salience to reward-related cues and investigate addiction vulnerability.

Cues that are contingently paired with unconditioned, rewarding stimuli can acquire rewarding properties themselves through a process known as the ...

Paradigms of Lower Extremity Electrical Stimulation Training After Spinal Cord Injury

Skeletal muscle atrophy, increased adiposity and reduced physical activity are key changes observed after spinal cord injury (SCI) and are associated with numerous cardiometabolic health consequences. These changes are likely to increase the risk of developing chronic secondary conditions and impact the quality of life in persons with SCI. Surface neuromuscular electrical stimulation evoked resistance training (NMES-RT) was developed as a strategy to attenuate the process of skeletal muscle atrophy, decrease ectopic adiposity, improve insulin sensitivity and enhance mitochondrial capacity. However, NMES-RT is limited to only a single muscle group. Involving multiple muscle groups of the lower extremities may maximize the health benefits of training. Functional electrical stimulation-lower extremity cycling (FES-LEC) allows for the activation of 6 muscle groups, which is likely to evoke greater metabolic and cardiovascular adaptation. Appropriate knowledge of the stimulation parameters is key to maximizing the outcomes of electrical stimulation training in persons with SCI. Adopting strategies for long-term use of NMES-RT and FES-LEC during rehabilitation may maintain the integrity of the musculoskeletal system, a pre-requisite for clinical trials aiming to restore walking after injury. The current manuscript presents a combined protocol using NMES-RT prior to FES-LEC. We hypothesize that muscles conditioned for 12 weeks prior to cycling will be capable of generating greater power, cycle against higher resistance and result in greater adaptation in persons with SCI.

Spinal cord injury is a traumatic medical condition that may result in elevated risks of chronic secondary metabolic disorders. Here, we presented a protocol using surface neuromuscular electrical stimulation-resistance training in conjunction with functional electrical stimulation-lower extremities cycling as a strategy to ameliorate several of these medical problems.

Skeletal muscle atrophy, increased adiposity and reduced physical activity are key changes observed after spinal cord injury (SCI) and are associated with ...

An Automated Method to Determine the Performance of Drosophila in Response to Temperature Changes in Space and Time

Temperature is a ubiquitous environmental factor that affects how species distribute and behave. Different species of Drosophila fruit flies have specific responses to changing temperatures according to their physiological tolerance and adaptability. Drosophila flies also possess a temperature sensing system that has become fundamental to understanding the neural basis of temperature processing in ectotherms. We present here a temperature-controlled arena that permits fast and precise temperature changes with temporal and spatial control to explore the response of individual flies to changing temperatures. Individual flies are placed in the arena and exposed to pre-programmed temperature challenges, such as uniform gradual increases in temperature to determine reaction norms or spatially distributed temperatures at the same time to determine preferences. Individuals are automatically tracked, allowing the quantification of speed or location preference. This method can be used to rapidly quantify the response over a large range of temperatures to determine temperature performance curves in Drosophila or other insects of similar size. In addition, it can be used for genetic studies to quantify temperature preferences and reactions of mutants or wild-type flies. This method can help uncover the basis of thermal speciation and adaptation, as well as the neural mechanisms behind temperature processing.

An Automated Method to Determine the Performance of Drosophila in Response to Temperature Changes in Space and Time

Here we present a protocol to automatically determine the locomotor performance of Drosophila at changing temperatures using a programmable temperature-controlled arena that produces fast and accurate temperature changes in time and space.

Temperature is a ubiquitous environmental factor that affects how species distribute and behave. Different species of Drosophila fruit flies have specific ...

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 ...

Simultaneous Recordings of Cortical Local Field Potentials and Electrocorticograms in Response to Nociceptive Laser Stimuli from Freely Moving Rats

Electrocortical responses, elicited by laser heat pulses that selectively activate nociceptive free nerve endings, are widely used in many animal and human studies to investigate the cortical processing of nociceptive information. These laser-evoked brain potentials (LEPs) consist of several transient responses that are time-locked to the onset of laser stimuli. However, the functional properties of the LEP responses are still largely unknown, due to the lack of a sampling technique that can simultaneously record neural activities at the surface of the cortex (i.e., electrocorticogram [ECoG] and scalp electroencephalogram [scalp EEG]) and inside the brain (i.e., local field potential [LFP]). To address this issue, we present here an animal protocol using freely moving rats. This protocol is composed of three main procedures: (1) animal preparation and surgical procedures, (2) a simultaneous recording of ECoG and LFP in response to nociceptive laser stimuli, and (3) data analysis and feature extraction. Specifically, with the help of a 3D-printed protective shell, both ECoG and LFP electrodes implanted on the rat&#39;s skull were securely held together. During data collection, laser pulses were delivered on the rat&#39;s forepaws through gaps in the bottom of the chamber when the animal was in spontaneous stillness. Ongoing white noise was played to avoid the activation of the auditory system by the laser-generated ultrasounds. As a consequence, only nociceptive responses were selectively recorded. Using the standard analytical procedures (e.g., band-pass filtering, epoch extraction, and baseline correction) to extract stimulus-related brain responses, we obtained results showing that LEPs with a high signal-to-noise ratio were simultaneously recorded from ECoG and LFP electrodes. This methodology makes the simultaneous recording of ECoG and LFP activities possible, which provides a bridge of electrocortical signals at the mesoscopic and macroscopic levels, thereby facilitating the investigation of nociceptive information processing in the brain.

We developed a technique that simultaneously records both electrocorticography and local field potentials in response to nociceptive laser stimuli from freely moving rats. This technique helps establish a direct relationship of electrocortical signals at the mesoscopic and macroscopic levels, which facilitates the investigation of nociceptive information processing in the brain.

Electrocortical responses, elicited by laser heat pulses that selectively activate nociceptive free nerve endings, are widely used in many animal and ...

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 Assessment of Visual Function via Optomotor Response and Cognitive Function via Y-Maze in Diabetic Rats

The optomotor response and the Y-maze are behavioral tests useful for assessing visual and cognitive function, respectively. The optomotor response is a valuable tool to track changes in spatial frequency (SF) and contrast sensitivity (CS) thresholds over time in a number of retinal disease models, including diabetic retinopathy. Similarly, the Y-maze can be used to monitor spatial cognition (as measured by spontaneous alternation) and exploratory behavior (as measured by a number of entries) in a number of disease models that affect the central nervous system. Advantages of the optomotor response and the Y-maze include sensitivity, speed of testing, the use of innate responses (training is not needed), and the ability to be performed on awake (non-anesthetized) animals. Here, protocols are described for both the optomotor response and the Y-maze and examples of their use shown in models of Type I and Type II diabetes. Methods include preparation of rodents and equipment, performance of the optomotor response and the Y-maze, and post-test data analysis.

Neural degeneration in both eyes and brain as a result of diabetes can be observed through behavioral tests carried out on rodents. The Y-maze, a measure of spatial cognition, and the optomotor response, a measure of visual function, both provide insight into potential diagnoses and treatments.

The optomotor response and the Y-maze are behavioral tests useful for assessing visual and cognitive function, respectively. The optomotor response is a ...

Physical Activity Measurement in Children Accepting Table Tennis Training

An increasing body of evidence now shows that the majority of children in China experience lower levels of physical activity (PA) than the recommended guideline. Table tennis is a compound and technically difficult game that is popular in China; undertaking table tennis training in clubs can help children to elevate their levels of PA. Given that children cannot complete self-evaluated questionnaires themselves and caregiver-based observations are not suitable for children, we hypothesized that an actigraphy-based method can be an objective method to measure PA. In the present study, we describe a procedure that can be used to evaluate PA levels using an actigraphic device and software. Furthermore, since hip-worn devices are known to reduce compliance, we attempted to assess the agreement between hip-worn and wrist-worn device data. Collectively, our results indicate that these devices are suitable for measuring PA and leisure time physical activity (LTPA) levels. Together with subjective questionnaires, both hip-worn and wrist-worn devices are highly suitable for evaluating PA in Chinese children undergoing table tennis training in clubs.

This study proposes an accelerometer-based method to objectively measure physical activity (PA) and leisure time physical activity (LTPA) in Chinese children accepting table tennis training in clubs.

An increasing body of evidence now shows that the majority of children in China experience lower levels of physical activity (PA) than the recommended ...

Positive Reinforcement-Based Training for Routine Rabbit Husbandry Procedures

Training Laboratory Rabbits to Refine Routine Husbandry Procedures

Non-aversive handling and training techniques for laboratory animals are required to facilitate experimental and routine husbandry procedures, improving both animal welfare and scientific quality. Clicker training was utilized to develop training protocols for rabbits to refine stressful routine husbandry procedures usually associated with lifting (i.e., being picked up from the floor)/restraining (i.e., being held in the arms of a human) them. Thirteen female New Zealand White rabbits were trained over three weeks. All rabbits learned the predefined goal behaviors: they followed the target stick, jumped onto the weighing scale, entered a transport box, and reared while placing their front paws onto the trainer&#39;s hand. In addition, ten animals jumped from the floor onto the sitting trainer&#39;s lap and allowed the trainer to lift their paws off the surface while sitting on the trainer&#39;s lap. For some individuals, the protocols had to be adapted by additional interim steps. At the end of the training, the rabbits reliably showed the expected goal behaviors, even after short and long training breaks. With few exceptions, a familiar person other than the trainer could elicit the goal behaviors from the rabbits (generalization), though further sessions were required for generalization. In the voluntary approach test, the rabbits preferred interacting with the trainer in the 1st trial but spent as much time with an unfamiliar person as with the trainer in the 2nd trial. The behavioral observations suggested that picking the rabbits up with the transport box, as described in the protocol, instead of restraining them with the scruff of their neck and lifting them on the arm, was less aversive. All in all, the training protocols were feasible and can serve as a refinement strategy in laboratory animal facilities. In the interest of animal welfare, the training protocols should be applied wherever possible.

Author Spotlight: Training of Laboratory Animals for Gentle and Stress-Free Handling

Training protocols involving positive reinforcement for routine husbandry procedures in rabbits were developed and proved to be successful, as the rabbits reliably showed the trained behaviors at the weekly general examination. Picking the rabbits up with the transport box was less aversive than the conventional handling technique.

Non-aversive handling and training techniques for laboratory animals are required to facilitate experimental and routine husbandry procedures, improving ...