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

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

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

<ol>
	<li>Butler, P. J., Jones, D. R., Lowenstein, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Advances in Comparative Physiology and Biochemistry. 8, 179-364 (1982).</li><li>Butler, P. J., Jones, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiology+of+diving+birds+and+mammals).">Physiology of diving birds and mammals).</a> Physiol. Rev. 77, 837-899 (1997).</li><li>Butler, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+regulation+in+diving+birds+and+mammals.">Metabolic regulation in diving birds and mammals.</a> Resp. Physiol. Neurobiol. 141, 297-315 (2004).</li><li>Foster, G. E., Sheel, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+diving+response,+its+function,+and+its+control.">The human diving response, its function, and its control.</a> Scand. J. Med. Sci. Sports. 15, 3-12 (2005).</li><li>Lindholm, P., Lundgren, C. E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+physiology+and+pathophysiology+of+human+breath-hold+diving.">The physiology and pathophysiology of human breath-hold diving.</a> J. Appl. Physiol. 106, 284-292 (2009).</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=Animal+models+for+investigating+the+central+control+of+the+mammalian+diving+response.">Animal models for investigating the central control of the mammalian diving response.</a> Front. Physiol. 3, 1-16 (2012).</li><li>Krogh, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+progress+of+physiology.">The progress of physiology.</a> Am. J. Physiol. 90, 243-251 (1929).</li><li>Panneton, W. M., Gan, Q., Juric, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+rat:+a+laboratory+model+for+studies+of+the+diving+response.">The rat: a laboratory model for studies of the diving response.</a> J. Appl. Physiol. 108, 811-820 (2010).</li><li>Lin, Y. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autonomic+nervous+control+of+cardiovascular+responses+during+diving+in+the+rat.">Autonomic nervous control of cardiovascular responses during diving in the rat.</a> Am. J. Physiol. 227, 601-605 (1974).</li><li>Lin, Y. C., Baker, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+output+and+its+distribution+during+diving+in+the+rat.">Cardiac output and its distribution during diving in the rat.</a> Am. J. Physiol. 228, 733-737 (1975).</li><li>Huang, T. F., Peng, Y. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+chemoreceptors+in+diving+bradycardia+in+the+rat.">Role of the chemoreceptors in diving bradycardia in the rat.</a> Jap. J. Physiol. 26, 395-401 (1976).</li><li>Fahlman, A., Bostrom, B. L., Dillon, K. H., Jones, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetic+component+of+the+forced+diving+bradycardia+response+in+mammals.">The genetic component of the forced diving bradycardia response in mammals.</a> Front. Physiol. 2, 1-7 (2011).</li><li>Blix, A. S., Folkow, B., Shepher, J. T., Abboud, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of Physiology. , 917-944 (1984).</li><li>Kooyman, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diverse+Divers.">Diverse Divers.</a> 200, (1989).</li><li>MacArthur, R. A., Karpan, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+rates+of+muskrats+diving+under+simulated+field+conditions:+persistence+of+the+bradycardia+response+and+factors+modifying+its+expression.">Heart rates of muskrats diving under simulated field conditions: persistence of the bradycardia response and factors modifying its expression.</a> Can. J. Zool. 67, 1783-1792 (1989).</li><li>McCulloch, P. F., Jones, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical+influences+on+diving+bradycardia+in+muskrats+(Ondatra+zibethicus).">Cortical influences on diving bradycardia in muskrats (Ondatra zibethicus).</a> Physiol. Zool. 63, 1098-1117 (1990).</li><li>McCulloch, P. F., Dinovo, K. M., Connolly, T. 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+cardiovascular+and+endocrine+responses+to+voluntary+and+forced+diving+in+trained+and+untrained+rats.">The cardiovascular and endocrine responses to voluntary and forced diving in trained and untrained rats.</a> Am. J. Physiol. Regul. Integr. Comp. Physiol. 298, 224-234 (2010).</li><li>Panneton, W. M., Gan, Q., Dahms, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiorespiratory+and+neural+consequences+of+rats+brought+past+their+aerobic+dive+limit.">Cardiorespiratory and neural consequences of rats brought past their aerobic dive limit.</a> J. Appl. Physiol. 109, 1256-1269 (2010).</li><li>Chotiyanonta, J. S., DiNovo, K. M., 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=Bilateral+sectioning+of+the+anterior+ethmoidal+nerves+does+not+eliminate+the+diving+response+in+voluntarily+diving+rats.">Bilateral sectioning of the anterior ethmoidal nerves does not eliminate the diving response in voluntarily diving rats.</a> Physiol. Reports. 1, (2013).</li><li>Panneton, W. M., Anch, A. M., Panneton, W. M., Gan, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parasympathetic+preganglionic+cardiac+motoneurons+labeled+after+voluntary+diving.">Parasympathetic preganglionic cardiac motoneurons labeled after voluntary diving.</a> Front. Physiol. 5, 1-10 (2014).</li><li>McCulloch, P. F., Ollenberger, G. P., Bekar, L. K., West, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trigeminal+and+chemoreceptor+contributions+to+bradycardia+during+voluntary+dives+in+rats.">Trigeminal and chemoreceptor contributions to bradycardia during voluntary dives in rats.</a> Am. J. Physiol. Regul. Integr. Comp. Physiol. 273, 814-822 (1997).</li><li>Ollenberger, G. P., Matte, G., Wilkinson, A. A., West, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relative+distribution+of+blood+flow+in+rats+during+surface+and+submerged+swimming.">Relative distribution of blood flow in rats during surface and submerged swimming.</a> Comp. Biochem. Physiol. A. 119, 271-277 (1998).</li><li>Ollenberger, G. P., West, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+regional+cerebral+blood+flow+in+voluntarily+diving+rats.">Distribution of regional cerebral blood flow in voluntarily diving rats.</a> J. Exp. Biol. 201, 549-558 (1998).</li><li>McCulloch, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+the+trigeminal+medullary+dorsal+horn+during+voluntary+diving+in+rats.">Activation of the trigeminal medullary dorsal horn during voluntary diving in rats.</a> Brain Res. 1051, 194-198 (2005).</li><li>Panneton, W. 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>

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

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 ...

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 ...

Assessing Dominant-Submissive Behavior in Adult Rats Following Traumatic Brain Injury

Competition over resources such as food, territory, and mates significantly influences relationships within animal species and is mediated through social hierarchies that are often based on dominant-submissive relationships. The dominant-submissive relationship is a normal behavioral pattern among the individuals of a species. Traumatic brain injury is a frequent cause of social interaction impairment and the reorganization of dominant-submissive relationships in animal pairs. This protocol describes submissive behavior in adult male Sprague-Dawley rats after the induction of traumatic brain injury using a fluid-percussion model compared to naive rats through a series of dominant-submissive tests performed between 29 days and 33 days after induction. The dominant-submissive behavior test shows how brain injury can induce submissive behavior in animals competing for food. After traumatic brain injury, the rodents were more submissive, as indicated by them spending less time at the feeder and being less likely to arrive first at the trough compared to the control animals. According to this protocol, submissive behavior develops after traumatic brain injury in adult male rats.

The present protocol describes a rat model of fluid percussion-induced traumatic brain injury followed by a series of behavioral tests to understand the development of dominant and submissive behavior. Using this model of traumatic brain injury in conjunction with specific behavioral tests enables the study of social impairments following brain injury.

Competition over resources such as food, territory, and mates significantly influences relationships within animal species and is mediated through social ...

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 ...