We thank Ian Bolding for assistance with surgical preparation of subjects and Elizabeth Sumner for her meticulous editing. These studies were completed as part of a team funded by The Moody Project for Translational Traumatic Brain Injury Research.

<ol>
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L., Capra, B., Eidson, K., Garcia, J., Kennedy, D., Uchida, T., 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=Dose-dependent+neuronal+injury+after+traumatic+brain+injury.">Dose-dependent neuronal injury after traumatic brain injury.</a> Brain Research. 1044, 144-154 (2005).</li><li>Bramlett, H. M., Detrich, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-Term+consequences+of+Traumatic+Brain+Injury:+Current+Status+of+Potential+Mechanisms+of+Injury+and+Neurological+Outcomes.">Long-Term consequences of Traumatic Brain Injury: Current Status of Potential Mechanisms of Injury and Neurological Outcomes.</a> J. Neurotrauma. 32, 1-15 (2015).</li><li>Pierce, J. E. S., Smith, D. H., Trojanowski, J. Q., McIntosh, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enduring+cognitive+neurobehavioral+and+histopathological+changes+persist+for+up+to+one+year+following+severe+experimental+brain+injury+in+rats.">Enduring cognitive neurobehavioral and histopathological changes persist for up to one year following severe experimental brain injury in rats.</a> Neuroscience. 87 (2), 359-369 (1998).</li><li>Smith, D. H., Chen, X. H., Pierce, J. E. S., Wolf, J. A., Trojanowski, J. Q., Graham, D. I., McIntosh, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progressive+atrophy+and+neuron+death+for+one+year+following+brain+trauma+in+the+rat.">Progressive atrophy and neuron death for one year following brain trauma in the rat.</a> J Neurotrauma. 14 (10), 715-727 (1997).</li><li>Schallert, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+tests+for+preclinical+intervention+assessment.">Behavioral tests for preclinical intervention assessment.</a> NeuroRx. 3 (4), 497-504 (2006).</li><li>Sell, S. L., Avila, M. A., Yu, G., Vergara, L., Prough, D. S., Grady, J. J., DeWitt, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypertonic+resuscitation+improves+neuronal+and+behavioral+outcomes+after+traumatic+brain+injury+plus+hemorrhage.">Hypertonic resuscitation improves neuronal and behavioral outcomes after traumatic brain injury plus hemorrhage.</a> Anesthesiology. 108 (5), 873-881 (2008).</li><li>Hamm, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurobehavioral+assessment+of+outcome+following+traumatic+brain+injury+in+rats:+an+evaluation+of+selected+measures.">Neurobehavioral assessment of outcome following traumatic brain injury in rats: an evaluation of selected measures.</a> J Neurotrauma. 18 (11), 1207-1216 (2001).</li><li>Feeney, D. M., Gonzalez, A., Law, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amphetamine,+haloperidol,+and+experience+interact+to+affect+rate+of+recovery+after+motor+cortex+injury.">Amphetamine, haloperidol, and experience interact to affect rate of recovery after motor cortex injury.</a> Science. 217, 855-857 (1982).</li><li>Hamm, R. J., Temple, M. D., Pike, B. R., O'Dell, D. M., Buck, D. L., Lyeth, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Working+memory+deficits+following+traumatic+brain+injury+in+the+rat.">Working memory deficits following traumatic brain injury in the rat.</a> J Neurotrauma. 13, 317-323 (1996).</li><li>Morris, R. G., Hagan, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allocentric+spatial+learning+by+hippocampectomised+rats:+A+further+test+of+the+"Spatial+Mapping"+and+"Working+Memory"+Theories+of+hippocampal+function.">Allocentric spatial learning by hippocampectomised rats: A further test of the "Spatial Mapping" and "Working Memory" Theories of hippocampal function.</a> The Quart J of Exp Psych. 38 (4), 365-395 (1986).</li><li>Dixon, C. E., Lyeth, B. G., Povlishock, J. T., Findling, R. L., Hamm, R. J., Marmarou, A., Young, H. F., Hayes, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fluid-percussion+model+of+experimental+brain+injury+in+the+rat.">A fluid-percussion model of experimental brain injury in the rat.</a> J. Neurosurg. 67, 110-119 (1987).</li><li>DeWitt, D. S., Smith, T. G., Deyo, D. J., Miller, K. R., Uchida, T., Prough, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=L-arginine+and+superoxide+dismutase+prevent+or+reverse+cerebral+hypoperfusion+after+fluid-percussion+traumatic+brain+injury.">L-arginine and superoxide dismutase prevent or reverse cerebral hypoperfusion after fluid-percussion traumatic brain injury.</a> J. Neurotrauma. 14, 223-233 (1997).</li><li>R Core Team. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> R: A Language and Environment for Statistical Computing. , (2017).</li><li>Pohlert, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Pairwise Multiple comparison of Mean Ranks Package (PMCMR) R package. , (2014).</li><li>Verma, P., Hellemans, K. G., Choi, F. Y., Yu, W., Weinberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+phase+and+sex+effects+on+depressive/anxiety-like+behaviors+and+HPA+axis+responses+to+acute+stress.">Circadian phase and sex effects on depressive/anxiety-like behaviors and HPA axis responses to acute stress.</a> Physiol Behav. 99, 276-285 (2010).</li><li>Schallert, T., Woodlee, M. T., Fleming, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+Focal+Ischemic+Injury:+Behavior-Brain+Interactions+and+Issues+of+Animal+Handling+and+Housing.">Experimental Focal Ischemic Injury: Behavior-Brain Interactions and Issues of Animal Handling and Housing.</a> ILAR J. 44 (2), 130-143 (2003).</li><li>Ruis, J. F., Rietveld, W. J., Buys, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+parametric+photic+entrainment+of+circadian+rhythms+in+the+rat.">Properties of parametric photic entrainment of circadian rhythms in the rat.</a> Physiol Behav. 50, 1233-1239 (1991).</li><li>Ferguson, S. A., Maier, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+seasonal/circannual+effects+of+laboratory+rodent+behavior.">A review of seasonal/circannual effects of laboratory rodent behavior.</a> Physiol Behav. 119, 130-136 (2003).</li><li>Fujimoto, S. T., Longhi, L., Saatman, K. E., McIntosh, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+and+cognitive+function+evaluation+following+experimental+traumatic+brain+injury.">Motor and cognitive function evaluation following experimental traumatic brain injury.</a> Neurosci &amp; Biobehav Reviews. , (2004).</li><li>Gold, E. M., Su, D., Lopez-Velazquez, L., Haus, D. L., Perez, H., Lacuesta, G. A., Anderson, A. J., Cummings, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+assessment+of+long-term+deficits+in+rodent+models+of+traumatic+brain+injury.">Functional assessment of long-term deficits in rodent models of traumatic brain injury.</a> Regen. Med. 8 (4), 483-516 (2013).</li></ol>

The authors have nothing to disclose.

When conducting any type of behavioral testing, it is critical to be consistent. This detail includes many considerations that seem insignificant but have a major impact on the response of the animal. An important step that cannot be overlooked is acclimation of animals to their home-cage/housing situation prior to any experiment. This preparation reduces the effects of the animals&#39; physiological stress response, which can alter behavioral outcomes18. Similarly, it is absolutely essential that every effort is made to handle all animals in the same manner. This consistency includes, as previously mentioned, acclimation to housing and also acclimation to handling and transportation between rooms prior to training or testing. This concept cannot be overstated. Sloppy animal handling is disastrous to any behavioral testing19. Likewise, every effort needs to be made to test animals at the same time of day, whether during their dark or light cycle. For the tests discussed here, testing during light or dark stage is acceptable, as long as the tests are performed consistently. Testing done at different times during the circadian cycle has been shown to alter behavioral outcomes18,20. Additionally, the handler as well as the animal must be in a stress free, calm state in order to maximize the accuracy of results.
Particularly in the case of the Neuroscore, false positives and negatives are common. False positives typically occur when an animal is not fully habituated to handling and testing. The animal must be completely relaxed so the observed response is reflexive and not due to muscles tightening from reacting out of stress or fear. A tense handler can influence the results by transmitting stress to the animal. Therefore, holding the rat too tight or too loose can both be problematic. Additionally, if the handler is nervous, this can confound the reaction of the rats. There is also the risk that an inexperienced observer will misinterpret the rat&#39;s response. Good training and a lot of practice are essential to the success and consistency of the Neuroscore.
In general, the chief concern with these tests is the lack of a large difference, and sometimes no difference, between treatment groups. Since animals can react differently to different handlers, noises, times of day, and potentially, seasons21, every effort must be made to reduce all possible confounding factors.
The results of the Beam-Balance and Beam-Walk tasks shown here demonstrate that these tests are useful early after injury to detect deficits in vestibulomotor function. These deficits typically resolve over time1,14. In this model, by 6 months after injury, the injury-induced deficits have resolved. The results of the 6 month time point indicate that there are no differences between NAIVE, SHAM, or injured rats; however, all the rats have been relaxing in their home cages for 6 months, aging and gaining weight. Thus, by the time they are re-tested at 6 months post-surgery (or equivalent in the case of NA&#207;VE), they are essentially becoming old and fat, and therefore all the groups do not perform as well as they did compared to their baseline Day 0 results.
Another important consideration is that the behavior test used is the correct test. For example, the tests employed here are thought to represent the function of specific brain areas. One example is the vestibular system, which is important for balance. Brain areas involved in sensorimotor function, such as the cortex including sensorimotor cortex, the thalamus, corticospinal neurons, basal ganglia, nigro-striatum, to name a few, are all involved in vestibulomotor coordination. Thus, deficits in the Beam-Balance or Beam-Walk indicate potential deficits in these areas. Furthermore, the hippocampus and prefrontal cortex are involved in the learning and memory functions tested by the working memory water maze. Even when the correct test is chosen, the limitations of the tests that are employed must be kept in mind. For example, none of the tests presented here are sensitive to deficits in mood, such as depression, anxiety, or social interactions such as aggression, decision making, or impulsivity. To reiterate, it is imperative to choose the appropriate test for the behavior and brain area to be evaluated.
Interpretation and analysis of behavioral data must be approached with caution. It is highly recommended to include power analyses of each type of test separately, because, using a behavioral outcome as a measure of a neural deficit, is by its nature, a crude measure of a subtle effect. Furthermore, different tests require different types of statistical analyses. For example, the Neuroscore and Beam-Balance tests described are dependent on the interpretation of a trained observer to score the behavior using an ordinal scale. These types of data are not continuous and not normally distributed, so nonparametric statistics should be used, such as the Kruskal-Wallis test, as demonstrated in Sections 6.1 and 6.2. Alternatively, the Beam-Walk and working memory water maze tests produce data that are continuous and normally distributed, so parametric statistics can be used, such as one-way ANOVA or repeated-measures two-way ANOVA, as demonstrated in Sections 6.3 and 6.4.
The behavioral tasks presented here have stood the test of time and give reproducible results, particularly when paired with the FPI model in rats, although many other methods of behavioral testing for brain injury do exist. The neuroscore is a short assessment performed with a minimum of equipment. Other tests of reflexes and strength are available and could be incorporated into a neurological assessment, such as the lateral pulsion task, the akinesia test, the inclined plane test, and grip strength (see Fujimoto et al.22 and Gold et al.23). The Beam-Balance and Beam-Walk tasks described are measures of vestibulomotor deficits after injury. Vestibulomotor coordination can be considered one measure of gross locomotor behavior, while other measures of gross locomotor deficits include the Rotarod, the rotating pole, and open field activity. The ability to swim, measured as swim speed during the water maze, is also an indication of gross motor coordination22,23. The working memory water maze task completes this set of tests by detecting both reference memory deficits (indicated by Trial 1) and working memory deficits (indicated by Trial 2 or the difference between Trial 1 and Trial 2). Other measures of cognitive function include the eight arm radial maze, the Barnes maze, the novel object recognition test, and different variations of the water maze. These variations include the original Morris water maze and the Lashley III maze (again see Fujimoto et al.22 and Gold et al.23). This battery of tests has proven to be useful early after injury and, in varying degrees, out to 12 months after injury1.
Additionally, the tasks demonstrated here can be used with different strains, sex, and ages of rats; however, accommodations may need to be made for differing sizes and in cases of greater frailty. For example, older, heavier rats need a wider beam for the Beam-Balance task and aged, frail rats, may need shorter duration of swim times in the water maze. Thus, there is room for flexibility in these tests and potential for development of new tests to accommodate different situations and hypotheses.

The methods presented here are designed to detect functional deficits in specific brain areas induced by an experimental model of TBI in the rat. Four different behavior tests will be described. First, the short neurological assessment, referred to as the neuroscore, can be performed without requiring any specialized equipment but does require practice; this test detects deficits in reflexes. Second, the Beam-Balance test detects deficits in the ability to balance. This task requires the handler to score the rat based on an ordinal scale and requires some training of the handler. The Beam-Balance test requires a narrow beam and is sensitive to deficits in the vestibular system. The third test assesses vestibulomotor coordination. This test is known as the Beam-Walk task, and although some pre-training of the rat is required, this procedure is more objective than the previous two as the latency to traverse the beam is an objective measure not dependent on subjective scoring. This difference is because the time to traverse a narrow beam to reach a safe box is measured. The Beam-Walk test requires a longer beam than the Beam-Balance as well as an escape box. This test measures deficits in both motor coordination and balance and thus is sensitive to damage to the cerebellum and motor related brain areas. The working memory version of the Morris water maze (MWM-WM) primarily tests hippocampal function and integration with the prefrontal cortex or executive function. The version of the Morris water maze shown can also be used to detect deficits in reference memory1.
These methods were chosen based on their well-established track record in the literature. Each one has been effective in many hands from different laboratories with multiple strains of rats over numerous years of research. However, in the past, post-injury measures up to two weeks after injury were considered &#34;chronic&#34; time points. Thus, to establish behavioral techniques for the study of chronic effects of TBI in rodents, these well-known methods needed to be evaluated for sensitivity to detect TBI induced deficits at longer time points after injury. While there are now several rodent models of TBI, the FPI model is one of the most widely used, and is applied in this study. This model was first published in the 1950&#39;s2, and since then, more than 1,000 papers have employed FPI in rats3. The neuropathology of this type of injury has been well-described by us4 and others5,6,7. Briefly, neuronal injury in the hippocampus has been shown to be dose-dependent using Fluoro-Jade staining at short times after injury, i.e., 24-48 h; while gross atrophy and cavitation including thinning of the internal capsule and cortex has been reported at one year after injury6,7.
The most meaningful representation of brain function is assessed by using behavioral outcome measures after an experimental brain injury. However, the vast majority of FPI experiments that use behavioral outcomes make measures relatively early, typically from 1 to 14 days after injury. Using the methods demonstrated here, some behavioral deficits can be detected out to 12 months after injury1.Neurological function, gross vestibulomotor function, and fine motor coordination were assessed on post-injury days (PIDs) 1-3 and at 3, 6, and 12 months after surgery, using a short neurological assessment (Neuroscore; modified from Schallert8), the Beam-Balance task, and the Beam-Walk task9,10,11. Reference and working memory were assessed using a working memory version of the Morris water maze1,12,13.

<table>
	<tbody>
		<tr>
			<td>Sprague-Dawley rats</td>
			<td>Charles Rivers Laboratories 
			251 Ballardvale St 
			Wilmington, MA 01887-1096 
			Phone: 800-522-7287</td>
			<td>CD-IGS rats, strain code 001</td>
			<td>male, albino, 300-350g at arrival</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Beam-Balance</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Beam</td>
			<td>home built</td>
			<td></td>
			<td>wood, 25&#34; l x 1&#34; h x 3/4&#34; w sealed with polyurethane varnish</td>
		</tr>
		<tr>
			<td>C-clamp</td>
			<td>Home Depot</td>
			<td>1422-C</td>
			<td>2 1/2&#34;</td>
		</tr>
		<tr>
			<td>barrier</td>
			<td>Home Depot</td>
			<td></td>
			<td>styrofoam, 18&#34; x 17 1/2&#34;</td>
		</tr>
		<tr>
			<td>table (for both BB &#38; BW)</td>
			<td>generic office supply</td>
			<td></td>
			<td>37&#34; h x 30&#34; w x 60&#34; l</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Beam-Walk</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Beam</td>
			<td>home built</td>
			<td></td>
			<td>wood 38-1/2&#34; l x 1-3/4&#34; h x 1&#34; w sealed with polyurethane varnish (~ 37&#34; off floor)</td>
		</tr>
		<tr>
			<td>escape box</td>
			<td>home built</td>
			<td></td>
			<td>woodpainted black 12 1/2 &#34; l x 9&#34; h x 7-1/4&#34; w</td>
		</tr>
		<tr>
			<td>nails (pegs)</td>
			<td></td>
			<td></td>
			<td>2&#34;</td>
		</tr>
		<tr>
			<td>hinges</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>clamps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>white noise machine</td>
			<td>San Diego Instruments 
			9155 Brown Deer Rd, Suite 8 San Diego, CA 92121 
			Phone: (858)530-2600</td>
			<td></td>
			<td>http://www.sandiegoinstruments.com/libraries/misc/datasheets/whitenoise.pdf</td>
		</tr>
		<tr>
			<td>light</td>
			<td>Home Depot</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Morris Water Maze</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>fiberglass pool</td>
			<td>manufacturer unknown</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>(similar to one made by SDI)</td>
			<td>San Diego Instruments</td>
			<td>7000-0723</td>
			<td>72&#34; diameter x 30&#34; deep (~ 500 gal)</td>
		</tr>
		<tr>
			<td>plexiglass platform</td>
			<td>hand-made by Maggie Parsley</td>
			<td></td>
			<td>10 cm diameter, 26&#34; tall with silicone applied to the surface of the platform to provide a gripping surface</td>
		</tr>
		<tr>
			<td>(similar to one made by SDI)</td>
			<td>SDI</td>
			<td>7500-0272</td>
			<td></td>
		</tr>
		<tr>
			<td>plexiglass animal boxes w/ lids</td>
			<td>UTMB Machine Shop</td>
			<td></td>
			<td>2 boxes, 10&#34; w x 16&#34; L x 9&#34; h</td>
		</tr>
		<tr>
			<td>spot lights/ heat lamps</td>
			<td>Home Depot</td>
			<td></td>
			<td>3 around pool, 2 over boxes to dry animals</td>
		</tr>
		<tr>
			<td>AnyMaze</td>
			<td>San Diego Instruments 
			9155 Brown Deer Rd, Suite 8 San Diego, CA 92121 
			Phone: (858)530-2600</td>
			<td>/9001</td>
			<td>http://www.sandiegoinstruments.com/any-maze-video-tracking/</td>
		</tr>
	</tbody>
</table>

detecting behavioral deficits in rats after traumatic brain injury

With the increasing incidence of traumatic brain injury (TBI) in both civilian and military populations, TBI is now considered a chronic disease; however, few studies have investigated the long-term effects of injury in rodent models of TBI. Shown here are behavioral measures that are well-established in TBI research for times early after injury, such as two weeks, until two months. Some of these methods have previously been used at later times after injury, up to one year, but by very few laboratories. The methods demonstrated here are a short neurological assessment to test reflexes, a Beam-Balance to test balance, a Beam-Walk to test balance and motor coordination, and a working memory version of the Morris water maze that can be sensitive to deficits in reference memory. Male rats were handled and pre-trained to neurological, balance, and motor coordination tests prior to receiving parasagittal fluid percussion injury (FPI) or sham injury. Rats can be tested on the short neurological assessment (neuroscore), the beam-balance, and the Beam-Walk multiple times, while testing on the water maze can only be done once. This difference is because rats can remember the task, thus confounding the results if repeated testing is attempted in the same animal. When testing from one to three days after injury, significant differences are detected in all three non-cognitive tasks. However, differences in the Beam-Walk task were not detectable at later time points (after 3 months). Deficits were detected at 3 months in the Beam-Balance and at 6 months in the neuroscore. Deficits in working memory were detected out to 12 months after injury, and a deficit in a reference memory first appeared at 12 months. Thus, standard behavioral tests can be useful measures of persistent behavioral deficits after FPI.

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Detecting Behavioral Deficits in Rats After Traumatic Brain Injury

The goal of the behavioral tests presented here is to detect functional deficits in rats after traumatic brain injury. Four specific tests are presented that detect deficits in behaviors to reflect the damage to specific brain areas at times extending to one year after injury.

With the increasing incidence of traumatic brain injury (TBI) in both civilian and military populations, TBI is now considered a chronic disease; however, ...

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17.8K Views.  University of Texas Medical Branch. The goal of the behavioral tests presented here is to detect functional deficits in rats after traumatic brain injury. Four specific tests are presented that detect deficits in behaviors to reflect the damage to specific brain areas at times extending to one year after injury.

Video: Detecting Behavioral Deficits in Rats After Traumatic Brain Injury

Uncovering Beat Deafness: Detecting Rhythm Disorders with Synchronized Finger Tapping and Perceptual Timing Tasks

A set of behavioral tasks for assessing perceptual and sensorimotor timing abilities in the general population (i.e., non-musicians) is presented here with the goal of uncovering rhythm disorders, such as beat deafness. Beat deafness is characterized by poor performance in perceiving durations in auditory rhythmic patterns or poor synchronization of movement with auditory rhythms (e.g., with musical beats). These tasks include the synchronization of finger tapping to the beat of simple and complex auditory stimuli and the detection of rhythmic irregularities (anisochrony detection task) embedded in the same stimuli. These tests, which are easy to administer, include an assessment of both perceptual and sensorimotor timing abilities under different conditions (e.g., beat rates and types of auditory material) and are based on the same auditory stimuli, ranging from a simple metronome to a complex musical excerpt. The analysis of synchronized tapping data is performed with circular statistics, which provide reliable measures of synchronization accuracy (e.g., the difference between the timing of the taps and the timing of the pacing stimuli) and consistency. Circular statistics on tapping data are particularly well-suited for detecting individual differences in the general population. Synchronized tapping and anisochrony detection are sensitive measures for identifying profiles of rhythm disorders and have been used with success to uncover cases of poor synchronization with spared perceptual timing. This systematic assessment of perceptual and sensorimotor timing can be extended to populations of patients with brain damage, neurodegenerative diseases (e.g., Parkinson&#8217;s disease), and developmental disorders (e.g., Attention Deficit Hyperactivity Disorder).

Behavioral tasks that allow for the assessment of perceptual and sensorimotor timing abilities in the general population (i.e., non-musicians) are presented. Synchronization of finger tapping to the beat of an auditory stimuli and detecting rhythmic irregularities provides a means of uncovering rhythm disorders.

A set of behavioral tasks for assessing perceptual and sensorimotor timing abilities in the general population (i.e., non-musicians) is presented here ...

A Procedure to Observe Context-induced Renewal of Pavlovian-conditioned Alcohol-seeking Behavior in Rats

Environmental contexts in which drugs of abuse are consumed can trigger craving, a subjective Pavlovian-conditioned response that can facilitate drug-seeking behavior and prompt relapse in abstinent drug users. We have developed a procedure to study the behavioral and neural processes that mediate the impact of context on alcohol-seeking behavior in rats. Following acclimation to the taste and pharmacological effects of 15% ethanol in the home cage, male Long-Evans rats receive Pavlovian discrimination training (PDT) in conditioning chambers. In each daily (Mon-Fri) PDT session, 16 trials each of two different 10 sec auditory conditioned stimuli occur. During one stimulus, the CS+, 0.2 ml of 15% ethanol is delivered into a fluid port for oral consumption. The second stimulus, the CS-, is not paired with ethanol. Across sessions, entries into the fluid port during the CS+ increase, whereas entries during the CS- stabilize at a lower level, indicating that a predictive association between the CS+ and ethanol is acquired. During PDT each chamber is equipped with a specific configuration of visual, olfactory and tactile contextual stimuli. Following PDT, extinction training is conducted in the same chamber that is now equipped with a different configuration of contextual stimuli. The CS+ and CS- are presented as before, but ethanol is withheld, which causes a gradual decline in port entries during the CS+. At test, rats are placed back into the PDT context and presented with the CS+ and CS- as before, but without ethanol. This manipulation triggers a robust and selective increase in the number of port entries made during the alcohol predictive CS+, with no change in responding during the CS-. This effect, referred to as context-induced renewal, illustrates the powerful capacity of contexts associated with alcohol consumption to stimulate alcohol-seeking behavior in response to Pavlovian alcohol cues.

A procedure to study the capacity of an alcohol associated environmental context to trigger the renewal of alcohol-seeking behavior in rats is described.

Environmental contexts in which drugs of abuse are consumed can trigger craving, a subjective Pavlovian-conditioned response that can facilitate ...

Operant Procedures for Assessing Behavioral Flexibility in Rats

Executive functions consist of multiple high-level cognitive processes that drive rule generation and behavioral selection. An emergent property of these processes is the ability to adjust behavior in response to changes in one&#8217;s environment (i.e., behavioral flexibility). These processes are essential to normal human behavior, and may be disrupted in diverse neuropsychiatric conditions, including schizophrenia, alcoholism, depression, stroke, and Alzheimer&#8217;s disease. Understanding of the neurobiology of executive functions has been greatly advanced by the availability of animal tasks for assessing discrete components of behavioral flexibility, particularly strategy shifting and reversal learning. While several types of tasks have been developed, most are non-automated, labor intensive, and allow testing of only one animal at a time. The recent development of automated, operant-based tasks for assessing behavioral flexibility streamlines testing, standardizes stimulus presentation and data recording, and dramatically improves throughput. Here, we describe automated strategy shifting and reversal tasks, using operant chambers controlled by custom written software programs. Using these tasks, we have shown that the medial prefrontal cortex governs strategy shifting but not reversal learning in the rat, similar to the dissociation observed in humans. Moreover, animals with a neonatal hippocampal lesion, a neurodevelopmental model of schizophrenia, are selectively impaired on the strategy shifting task but not the reversal task. The strategy shifting task also allows the identification of separate types of performance errors, each of which is attributable to distinct neural substrates. The availability of these automated tasks, and the evidence supporting the dissociable contributions of separate prefrontal areas, makes them particularly well-suited assays for the investigation of basic neurobiological processes as well as drug discovery and screening in disease models.

The ability to assess executive functions such as behavioral flexibility in rats is useful for investigating the neurobiology of cognition in both intact animals and disease models. Here we describe automated tasks for assessing strategy shifting and reversal learning, which are particularly sensitive to disruptions in prefrontal cortical networks.

Executive functions consist of multiple high-level cognitive processes that drive rule generation and behavioral selection. An emergent property of these ...

The Modified Hole Board - Measuring Behavior, Cognition and Social Interaction in Mice and Rats

This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure multiple dimensions of unconditioned behavior in small laboratory mammals (e.g., mice, rats, tree shrews and small primates). This paradigm is a valuable alternative for the use of a behavioral test battery, since a broad behavioral spectrum of an animal&#8217;s behavioral profile can be investigated in one single test.
The apparatus consists of a box, representing the &#8216;protected&#8217; area, separated from a group compartment. A board, on which small cylinders are staggered in three lines, is placed in the center of the box, representing the &#8216;unprotected&#8217; area of the set-up. The cognitive abilities of the animals can be measured by baiting some cylinders on the board and measuring the working and reference memory. Other unconditioned behavior, such as activity-related-, anxiety-related- and social behavior, can be observed using this paradigm. Behavioral flexibility and the ability to habituate to a novel environment can additionally be observed by subjecting the animals to multiple trials in the mHB, revealing insight into the animals&#8217; adaptive capacities.
Due to testing order effects in a behavioral test battery, na&#239;ve animals should be used for each individual experiment. By testing multiple behavioral dimensions in a single paradigm and thereby circumventing this issue, the number of experimental animals used is reduced. Furthermore, by avoiding social isolation during testing and without the need to food deprive the animals, the mHB represents a behavioral test system, inducing if any, very low amount of stress.

This protocol describes the modified hole board, which is a behavioral test set-up that comprises the characteristics of an open field and a traditional hole board. This set-up enables the differential analysis of unconditioned behavior of small laboratory mammals as well as the analysis of cognitive abilities.

This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure ...

The Rodent Psychomotor Vigilance Test (rPVT): A Method for Assessing Neurobehavioral Performance in Rats and Mice

The human Psychomotor Vigilance Test (PVT) is a widely used procedure for measuring changes in fatigue and sustained attention. The present article describes a rodent version of the PVT&#8212;termed the &#34;rPVT&#34;&#8212;that measures similar aspects of attention (i.e., performance accuracy, motor speed, premature responding, and lapses in attention). Data are presented that demonstrate both the short- and long-term usefulness of the rPVT when employed with laboratory rats. Rats easily learn the rPVT, and learning to perform the basic procedure takes less than two weeks of training. Once acquired, rat performances in the rPVT show a high degree of similarity to these same performance measures in the human PVT, including similarities in, lapses in attention, reaction times, vigilance decrements across session time (i.e., the human &#34;time-on-task&#34; effects), and the response-stimulus interval (RSI) effect described for humans. Thus the rPVT can be an extremely valuable tool for assessing the effects of a wide range of variables on sustained attention quite similar to human PVT performances, and thus can be useful for developing novel treatments for neurobehavioral dysfunctions.

The Rodent Psychomotor Vigilance Test rPVT: A Method for Assessing Neurobehavioral Performance in Rats and Mice

A rat version of the human Psychomotor Vigilance Test (PVT) is described that measures aspects of attention similar to those measured with the human PVT, including aspects of human vigilance such as performance accuracy, motor speed, premature responding, and lapses in attention.

The human Psychomotor Vigilance Test (PVT) is a widely used procedure for measuring changes in fatigue and sustained attention. The present article ...

Assessing Spatial Memory Impairment in a Mouse Model of Traumatic Brain Injury Using a Radial Water Tread Maze

Despite the recent increase in use of mouse models in scientific research, researchers continue to use cognitive tasks that were originally designed and validated for rat use. The Radial Water Tread (RWT) maze test of spatial memory (designed specifically for mice and requiring no swimming) has been shown previously to successfully distinguish between controlled cortical impact-induced TBI mice and sham controls. Here, a detailed protocol for this task is presented. The RWT maze capitalizes on the natural tendency of mice to avoid open areas in favor of hugging the sides of an apparatus (thigmotaxis). The walls of the maze are lined with nine escape holes placed above the floor of the apparatus, and mice are trained to use visual cues to locate the escape hole that leads out of the maze. The maze is filled with an inch of cold water, sufficient to motivate escape but not deep enough to require that the mouse swim. The acquisition period takes only four training days, with a test of memory retention on day five and a long-term memory test on day 12. The results reported here suggest that the RWT maze is a feasible alternative to rat-validated, swimming-based cognitive tests in the assessment of spatial memory deficits in mouse models of TBI.

Here we present a protocol for a mouse-specific test of cognition that does not require swimming. This test can be used to successfully distinguish controlled cortical impact-induced traumatic brain injury mice from sham controls.

Despite the recent increase in use of mouse models in scientific research, researchers continue to use cognitive tasks that were originally designed and ...

Using Laser Doppler Imaging and Monitoring to Analyze Spinal Cord Microcirculation in Rat

Laser Doppler flowmetry (LDF) is a noninvasive method for blood flow (BF) measurement, which makes it preferable for measuring microcirculatory alterations of the spinal cord. In this article, our goal was to use both Laser Doppler imaging and monitoring to analyze the change of BF after spinal cord injury. Both the laser Doppler image scanner and the probe/monitor were being employed to obtain each readout. The data of LDPI provided a local distribution of BF, which gave an overview of perfusion around the injury site and made it accessible for comparative analysis of BF among different locations. By intensely measuring the probing area over a period of time, a combined probe was used to simultaneously measure the BF and oxygen saturation of the spinal cord, showing overall spinal cord perfusion and oxygen supply. LDF itself has a few limitations, such as relative flux, sensitivity to movement, and biological zero signal. However, the technology has been applied in clinical and experimental study due to its simple setup and rapid measurement of BF.

Here we present a combination of laser Doppler perfusion imaging (LDPI) and laser Doppler perfusion monitoring (LDPM) to measure spinal cord local blood flows and oxygen saturation (SO2), as well as a standardized procedure for introducing spinal cord trauma on rat.

Laser Doppler flowmetry (LDF) is a noninvasive method for blood flow (BF) measurement, which makes it preferable for measuring microcirculatory ...

Stress-Enhanced Fear Learning, a Robust Rodent Model of Post-Traumatic Stress Disorder

Fear behaviors are important for survival, but disproportionately high levels of fear can increase the vulnerability for developing psychiatric disorders such as post-traumatic stress disorder (PTSD). To understand the biological mechanisms of fear dysregulation in PTSD, it is important to start with a valid animal model of the disorder. This protocol describes the methodology required to conduct stress-enhanced fear learning (SEFL) experiments, a preclinical model of PTSD, in both rats and mice. SEFL was developed to recapitulate critical aspects of PTSD, including long-term sensitization of fear learning caused by an acute stressor. SEFL uses aspects of Pavlovian fear conditioning but produces a distinct and robust sensitized fear response far greater than normal conditional fear responses. The trauma procedure involves placing a rodent in a conditioning chamber and administering 15 unsignaled shocks randomly distributed over 90 minutes (for rat experiments; for mouse experiments, 10 unsignaled shocks randomly distributed over 60 minutes are used). On day 2, rodents are placed in a novel conditioning context where they receive a single shock; then, on day 3 they are placed back in the same context as on day 2 and tested for changes in freezing levels. Rodents that previously received the trauma display enhanced levels of freezing on the test day compared to those that received no shocks on the first day. Thus, with this model, a single highly stressful experience (the trauma) produces extreme fear of the stimuli associated with the traumatic event.

Here we describe the detailed methodology required to conduct stress-enhanced fear learning (SEFL) experiments, a preclinical model of post-traumatic stress disorder, in both rats and mice. The model utilizes aspects of Pavlovian fear conditioning and freezing as an index of enhanced fear in rodents.

Fear behaviors are important for survival, but disproportionately high levels of fear can increase the vulnerability for developing psychiatric disorders ...

Novel Object Recognition and Object Location Behavioral Testing in Mice on a Budget

Ethologically relevant behavioral testing is a critical component of any study that uses mouse models to study the cognitive effects of various physiological or pathological changes. The object location task (OLT) and the novel object recognition task (NORT) are two effective behavioral tasks commonly used to reveal the function and relative health of specific brain regions involved in memory. While both of these tests exploit the inherent preference of mice for the novelty to reveal memory for previously encountered objects, the OLT primarily evaluates spatial learning, which relies heavily on hippocampal activity. The NORT, in contrast, evaluates non-spatial learning of object identity, which relies on multiple brain regions. Both tasks require an open-field-testing arena, objects with equivalent intrinsic value to mice, appropriate environmental cues, and video recording equipment and the software. Commercially available systems, while convenient, can be costly. This manuscript details a simple, cost-effective method for building the arenas and setting up the equipment necessary to perform the OLT and NORT. Furthermore, the manuscript describes an efficient testing protocol that incorporates both OLT and NORT and provides typical methods for data acquisition and analysis, as well as representative results. Successful completion of these tests can provide valuable insight into the memory function of various mouse model systems and appraise the underlying neural regions that support these functions.

Here we provide a protocol which includes comprehensive instructions for the economical establishment of murine object location and novel object recognition behavioral testing, including the design, cost, and construction of required equipment as well as execution of behavioral testing, data collection, and analysis.

Ethologically relevant behavioral testing is a critical component of any study that uses mouse models to study the cognitive effects of various ...

Assessing the Autonomic and Behavioral Effects of Passive Motion in Rats using Elevator Vertical Motion and Ferris-Wheel Rotation

The overall goal of this study is to assess the autonomic and behavioral effects of passive motion in rodents using the elevator vertical motion and Ferris-wheel rotation devices. These assays can help confirm the integrity and normal functioning of the autonomic nervous system. They are coupled to quantitative measures based on defecation counting, open-field examination, and balance beam crossing. The advantages of these assays are their simplicity, reproducibility, and quantitative behavioral measures. The limitations of these assays are that the autonomic reactions could be epiphenomena of non-vestibular disorders and that a functioning vestibular system is required. Examination of disorders such as motion sickness will be greatly aided by the detailed procedures of these assays.

Protocols are presented to assess the autonomic and behavioral effects of passive motion in rodents using elevator vertical motion and Ferris-wheel rotation.

The overall goal of this study is to assess the autonomic and behavioral effects of passive motion in rodents using the elevator vertical motion and ...