Funding for this project was provided by ARRA funds from NINDS grant NS045647.

1. Materials Required for Training and Testing
<ol>
	<li>Testing chamber. Any container large enough to accommodate one animal and two scent cups. Our laboratory utilized modified operant chambers (Med Associates Inc.) with floor bars removed to make space for scent cups (Figure 1).</li>
	<li>Scent cups and cup holder. Cups sufficiently wide and deep to allow for digging and some mechanism/holder to stabilize them (Figure 2).</li>
	<li>Scented sands. Our testing protocol used scents previously established to not show response bias in rats (cocoa, basil, cumin, coffee)4. Each scented sand was mixed at a ratio of 1 g odorant to 110 g unscented, clean playground "sand box" sand and can be stored in airtight containers for up to 6 months.</li>
	<li>Reinforcer. We utilized fruit loop cereal pieces as a reinforcer. We recommend exposing animals to the reinforcer (via placement in their home cages) 3 to 5 days prior to training to overcome neophobic responses.</li>
	<li>Stop watch or timer.</li>
</ol>
2. Animal Handling Prior to Training
<ol>
	<li>Handling. As animals will be placed in and removed from the chambers repeatedly throughout training/testing, we recommend handling animals three to five days prior to training in order to familiarize them with human contact.</li>
	<li>Motivating. In order to motivate animals to perform on this task, some level of food deprivation is necessary. Food restriction should be initiated at least three days prior to both training and testing and animals may be maintained at 90% of free-feeding weight. Typically amounts of 15-20 g of chow per day are sufficient, but animal weights should be checked every other day while food restricting.</li>
</ol>
3. Dig Training
Training an animal to dig in sand can be accomplished in an average of eight sessions over a four day period of time. During dig training, the chamber should be set up by placing two scent cups into the mechanism/holder and placing the scent cup setup into the chamber. The general procedure is detailed below with the goal of shaping the animal from consuming the reinforcer to completely digging in sand to retrieve it.
<ol>
	<li>Session 1 - chamber habituation and magazine training. Completely fill the scent cups with the reinforcer (no sand). Place the animal into the apparatus for 30 min allowing the animal to freely consume the reinforcer.</li>
	<li>Session 2 - sand habituation. Completely fill the scent cups with the reinforcer and pour a small amount of unscented sand into each cup, filling no more than half of the cup. Place the animal into the apparatus for 30 min allowing the animal to freely consume the reinforcer.</li>
	<li>Session 3 - surface digging. Fill the scent cups with an even mixture of reinforcer and unscented sand, leaving multiple reinforcers exposed. Place the animal into the apparatus for 30 min allowing the animal to freely consume the reinforcer. In the process of consuming the exposed reinforcers, the animal should uncover additional reinforcers below the surface of the sand. The animal should shift from using its mouth to using its forepaws to remove each reinforcer from the sand.</li>
	<li>Session 4 - digging. Fill the scent cups with a mixture of 1 part reinforcer to 3 parts unscented sand, leaving 1-2 reinforcers exposed. Place the animal into the chamber for 30 min allowing the animal to freely consume the reinforcer. The animal should begin to dig with its forepaws to uncover additional reinforcers and remove them from the sand. Animals that do not begin to uncover the reinforcer after a 10 min period of time should be assisted (the researcher should uncover one reinforcer at a time until the animal learns).</li>
	<li>Session 5 - full digging. Fill the scent cups with a mixture of 1 part reinforcer to 3 parts unscented sand, leaving no reinforcers exposed. Place the animal into the apparatus for 30 min allowing the animal to freely consume the reinforcer. Animals should be assisted as needed. Some animals may need multiple instances of this session in order to demonstrate digging behavior.</li>
	<li>Session 6 - trial structure habituation. Place three reinforcers in deep, middle, and shallow positions in the sand in each scent cup. Place the animal into the chamber until all three are retrieved from each cup, assisting as needed. Once all have been retrieved, place the animal into a holding apparatus for 15-30 sec and reset the scent cups as described in this step. Then, place the animal back into the chamber and repeat the process until the animal has completed four total trials. The animal should become habituated to being placed in the holding apparatus and start digging immediately when placed into the chamber. The animal should also demonstrate full digging behavior and retrieve the reinforcer at the bottom of the scent cup.</li>
	<li>Session 7 - single reinforcers. Place a single reinforcer at half of the depth of the sand in each cup. Place the animal into the chamber and allow it to retrieve both reinforcers, and then place it into the holding apparatus for 30 sec. Repeat this process for four total trials. The animal should begin to dig faster and require minimal assistance from the researcher.</li>
	<li>Session 8 - mock discrimination testing. Fill cups as described in session 7. Give the animal 30 sec to retrieve both reinforcers. Place the animal into the holding apparatus when both have been retrieved or 30 sec has elapsed. Repeat this for the number of trials that will be used in the testing phase. Animals should learn to dig rapidly when placed back into the chamber.</li>
	<li>Additional sessions. Depending on initial discrimination testing, the experimenter may wish to perform additional sessions to familiarize the animal with the given scents. Rats will tend to initially exhibit neophobia and prefer the familiar scents previously associated with reinforcement10.</li>
</ol>
4. Discrimination Testing
<ol>
	<li>The researcher should choose a scent pairing, including which scent will be baited with the reinforcer (correct discrimination) and which will not. Additional decisions, such as number of discriminations and reversals, maximum number of days and trials, and whether to pretest prior to injury will be largely based on experimental question (see additional notes below).</li>
	<li>Rats can be tested and data recorded by hand by the researcher. The researcher should record for each trial: correct or incorrect choice, latency to initiate digging, and trials in which a rat times out on.
	<ol class="lalpha">
		<li>The number of trials administered should be at the discretion of the researcher (see additional notes below).</li>
		<li>The length of the trial should be at the discretion of the researcher (see additional notes below).</li>
		<li>The researcher should randomize the trial structure (left-right location of the scent cups), with the caveat that no more than three trials in a row are on a given side. This will help reduce side bias.</li>
	</ol>
	</li>
	<li>In order to initiate each trial, place the animal into the chamber facing the rear, equidistant from each cup. Placing an animal closer to one cup may increase the chance of developing a bias.</li>
	<li>If the animal correctly discriminates, allow it to retrieve and consume the reinforcer, and then remove it to the holding apparatus for 30 sec. If it times out or chooses incorrectly, remove it immediately, before it "corrects" itself, and place it into the holding apparatus for 30 sec.</li>
	<li>Repeat this process for the chosen number of trials.</li>
	<li>Once an animal achieves a pre-determined criterion (i.e. &gt; 80% accuracy per day for three consecutive days), the animal may move on to the next discrimination/reversal and repeat the above process.</li>
</ol>
5. Additional Notes
<ol>
	<li>Testing in our laboratory found that training and testing work best using one-half of a fruit loop cereal piece to avoid satiation.</li>
	<li>During training, some animals may need more help than others. We recommend doing what is necessary to achieve digging behavior. However, once testing starts, we never attempt to intervene and improve a rat's digging behavior.</li>
	<li>Varying the total number of trials may be sufficient for different labs. Testing in our laboratory have used 6, 8, and 12 trials per session, 1 session per day, and have found a preference for the 8 trial setup.</li>
	<li>Varying the length of the trial may be necessary in some experimental setups (e.g., animals under a stress paradigm may need increased trial times or fewer total trials).</li>
	<li>A preset criterion should be used in order to determine when an animal has mastered a discrimination. Testing in our laboratory have required an accuracy of greater than 80% (5/6 trials, 7/8 trials, or 10/12 trials) per day for 3 consecutive days. However, a higher criterion (85%) could also be used.</li>
	<li>Preset thresholds/maximums should be used for the maximum number of days spent on a given scent. This will be largely dictated by research question. For example, our laboratory studies TBI and, as of result of the progressive nature of TBI, rats that have not hit criterion are moved on to a novel discrimination/reversal after 12 days of testing.</li>
	<li>Deciding whether to pretest or just posttest is a question that is largely determined by experimental direction. Pretesting allows one to perform a memory assessment on the animal post injury, which may be relevant depending on the injury type. However, post testing allows one to examine differences in acquisition rate on the discriminations between groups. Our laboratory has and will continue to use both.</li>
</ol>

<ol>
	<li>Birrell, J. M., Brown, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medial+frontal+cortex+mediates+perceptual+attentional+set+shifting+in+the+rat.">Medial frontal cortex mediates perceptual attentional set shifting in the rat.</a> J. Neurosci. 20, 4320-4324 (2000).</li><li>Bunsey, M., Eichenbaum, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conservation+of+hippocampal+memory+function+in+rats+and+humans.">Conservation of hippocampal memory function in rats and humans.</a> Nature. 379, 255-257 (1996).</li><li>Eichenbaum, H., Fagan, A., Cohen, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+olfactory+discrimination+learning+set+and+facilitation+of+reversal+learning+after+medial-temporal+damage+in+rats:+Implications+for+an+account+of+preserved+learning+abilities+in+amnesia.">Normal olfactory discrimination learning set and facilitation of reversal learning after medial-temporal damage in rats: Implications for an account of preserved learning abilities in amnesia.</a> J. Neurosci. 6, 1876-1884 (1986).</li><li>Kaiser, D. H., Means, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Value+transfer+across+odor+stimuli+using+probability+of+reinforcement+in+the+rat.">Value transfer across odor stimuli using probability of reinforcement in the rat.</a> Behav. Processes. 73, 164-169 (2006).</li><li>Martens, K. M., Vonder Haar, C., Hutsell, B. A., Hoane, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Additional+options+for+behavioral+testing+in+rodent+models+of+traumatic+brain+injury:+A+simple+discrimination+task+used+as+a+novel+method+of+testing+decision-making+behavior.">Additional options for behavioral testing in rodent models of traumatic brain injury: A simple discrimination task used as a novel method of testing decision-making behavior.</a> J. Neurotrauma. , (2012).</li><li>Writing Group Members. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Executive+summary:+Heart+disease+and+stroke+statistics+-+2012+update.">Executive summary: Heart disease and stroke statistics - 2012 update.</a> Circulation. 125, 188-197 (2012).</li><li>Narayan, R. K., 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=Clinical+trials+in+head+injury.">Clinical trials in head injury.</a> J. Neurotrauma. 19, 503-557 (2002).</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. Biobehav. Rev. 28, 365-378 (2004).</li><li>Mitchell, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experiments+on+neophobia+in+wild+and+laboratory+rats:+A+reevaluation.">Experiments on neophobia in wild and laboratory rats: A reevaluation.</a> J. Comp. Physiol. Psychol. 90, 190-197 (1976).</li><li>Baum, 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=On+two+types+of+deviation+from+the+matching+law:+Bias+and+undermatching.">On two types of deviation from the matching law: Bias and undermatching.</a> J. Exp. Anal. Behav. 22, 231-242 (1974).</li><li>Swets, J. A., Dawes, R. M., Monahan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Psychological+science+can+improve+diagnostic+decisions.">Psychological science can improve diagnostic decisions.</a> Psychological Science in the Public Interest. 1, 1-26 (2000).</li><li>Stuss, D. T., Murphy, K. J., Binns, M. A., Alexander, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staying+on+the+job:+The+frontal+lobes+control+individual+performance+variability.">Staying on the job: The frontal lobes control individual performance variability.</a> Brain. 126, 2363-2380 (2003).</li><li>Burkett, E. E., Bunnell, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Septal+lesions+and+the+retention+of+DRL+performance+in+the+rat.">Septal lesions and the retention of DRL performance in the rat.</a> J. Comp. Physiol. Psychol. 62, 468-471 (1966).</li><li>Porter, M. C., Burk, J. A., Mair, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+the+effects+of+hippocampal+or+prefrontal+cortical+lesions+on+three+versions+of+delayed+non-matching-to-sample+based+on+positional+or+spatial+cues.">A comparison of the effects of hippocampal or prefrontal cortical lesions on three versions of delayed non-matching-to-sample based on positional or spatial cues.</a> Behav. Brain Res. 109, 69-81 (2000).</li><li>Walton, M. E., Bannerman, D. M., Alterescu, K., Rushworth, M. F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+specialization+within+medial+frontal+cortex+of+the+anterior+cingulate+for+evaluating+effort-related+decisions.">Functional specialization within medial frontal cortex of the anterior cingulate for evaluating effort-related decisions.</a> J. Neurosci. 23, 6475-6479 (2003).</li></ol>

No conflicts of interest declared.

In this video, we have shown how the Dig task can be conducted using relatively low-cost materials and, with some experience, testing can be conducted fairly rapidly (~10 min/animal). This allows for the task to be easily incorporated into existing test batteries for TBI, stroke, and other brain injuries. The Dig task is also robust in that it can assess several different aspects of cognition, including evaluations of previous learning, reversals, and novel acquisitions (e.g., set shifting). Most importantly, the Dig task has been shown to be effective in assessing frontal function following brain damage. In addition, a similar dig paradigm has been used to study value transfer in rats following hippocampal damage4, and thus could easily be studied following TBI.
The primary benefits of tasks such as this one are the ability to assess other forms of cognition than what is typically tested in most brain injury studies. There currently exists a lack of simple, rapidly acquired tasks for assessing frontally-mediated cognition following brain damage. In the field of experimental TBI, there is a strong reliance on mazes as the sole assessment of cognition9. Discrimination and choice behavior are important aspects of frontally-mediated cognition which need to be evaluated following brain damage. These have been studied under many different paradigms (delayed matching-to-sample, differential reinforcement of low rate of responding, effort-based decision making, etc) in the field of the experimental analysis of behavior, but are lacking in studies of brain injury14-16. The development of novel therapeutics for the treatment of brain damage requires validation across multiple modalities of sensory, motor, and cognitive function. Tasks such as this could become vital to fully assessing cognitive function under therapeutic treatment.
While the Dig task represents a step in the right direction for assessing brain function after injury, there are still limitations to it. It is intended to be simple to construct, administer, and analyze, but this means that there are upper limits to what it can measure. Currently, two choices are all that can be assessed under the given paradigm. Additionally, the Dig task is not sensitive enough to handle other aspects of frontal dysfunction, such as impulsivity, that may interfere with an animal's decision-making ability.
While the Dig task has been shown to be successful for us in evaluating brain damage in young animals, there are still several things that can be done with the task to further refine and improve it. These include assessing special populations (age, sex, strain), comparing it directly to the operant paradigms it is adapted from, performing a direct comparison of this task with other tasks that assess frontal dysfunction following injury, and exploring additional ways to analyze and compare the data generated from the task. This task is designed to address some of the issues in our field by testing frontal function in multiple ways following brain damage. However, one task cannot measure all the cognitive processes that are impacted by a brain injury. Thus, we advocate that people continue to develop and improve upon tasks such as the one described in this protocol to more fully assess functional impairments following brain injuries. In doing so, we can increase the effectiveness of drug and therapy development for brain injury by assessing across multiple measures.

Brain damage resulting from traumatic brain injury (TBI) and stroke is one of the leading causes of death and disability in the United States. Together, these disorders result in a total of nearly 2.5 million incidences, with nearly 500,000 resulting in death or impairment6-7. Despite this heavy toll on society, very few treatments have been developed to treat either TBI or stroke. To some extent, the lack of available treatment options can be attributed to inadequate behavioral assessments of novel drugs and therapies8. In particular, there is a lack of tasks to accurately and sensitively evaluate the effects of treatments on the various cognitive deficits following brain injury in the preclinical literature. Currently the majority of cognitive assessments that are performed evaluate spatial memory using a maze task such as the Morris water maze9. There are multiple reasons for this lack of testing including the time it takes to fully characterize and evaluate cognitive deficits, the heavy reliance upon expensive equipment and expertise to utilize operant tasks, and the speed at which these evaluations can be performed. To this end, we have adapted a basic scent discrimination task, the Dig task, to assess cognition following brain injury. The Dig task is designed to be a cheap, rapid, and effective tool for evaluating cognitive dysfunction following brain injury from a TBI or stroke.
The Dig task relies on the basic operant principles of learning and decision making. As it is only a two-choice decision paradigm, this task represents one of the simplest discriminations that can be made. In the protocol below the details for setting up the task as well as the training methodology will be described. We have found that this task is strongest when assessing frontally-mediated cognitive deficits compared to unilateral parietal deficits; however, it has yet to be tested under a model of hippocampal damage5. Ideally, this task would be suited for evaluating the effectiveness of a treatment or therapy on recovery of frontal deficits following an injury. However, it is possible that with modifications this task could also be used to explore more complex decision-making behavior such as impulsivity and generalized matching.

<table border="1">
 <tr>
 <td>Material Name</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>EQUIPMENT</td>
 </tr>
 <tr>
 <td>Operant Chamber</td>
 <td>Med Associates</td>
 <td>ENV-008CT</td>
 <td>Amount: 1 Standard operant chamber, no external devices.</td>
 </tr>
 <tr>
 <td>Plexiglas</td>
 <td>Local Hardware Store</td>
 <td>N/A</td>
 <td>Amount: 1 sheet Cut into proper size to fit into chamber.</td>
 </tr>
 <tr>
 <td>PVC Pipe End Caps</td>
 <td>Local Hardware Store</td>
 <td>N/A</td>
 <td>Amount: 2 / scent cup setup.</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>REAGENTS </td>
 </tr>
 <tr>
 <td>Sand</td>
 <td>Local Hardware Store</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Various Spices</td>
 <td>Local Grocery Store</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Reinforcer</td>
 <td>Local Grocery Store</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 	<td></td>
 <td></td>
 <td></td>
 <td>*Equipment is listed for construction per chamber.</td>
 </tr>
 </table>

the dig task: a simple scent discrimination reveals deficits following frontal brain damage

Cognitive impairment is the most frequent cause of disability in humans following brain damage, yet the behavioral tasks used to assess cognition in rodent models of brain injury is lacking. Borrowing from the operant literature our laboratory utilized a basic scent discrimination paradigm1-4 in order to assess deficits in frontally-injured rats. Previously we have briefly described the Dig task and demonstrated that rats with frontal brain damage show severe deficits across multiple tests within the task5. Here we present a more detailed protocol for this task. Rats are placed into a chamber and allowed to discriminate between two scented sands, one of which contains a reinforcer. The trial ends after the rat either correctly discriminates (defined as digging in the correct scented sand), incorrectly discriminates, or 30 sec elapses. Rats that correctly discriminate are allowed to recover and consume the reinforcer. Rats that discriminate incorrectly are immediately removed from the chamber. This can continue through a variety of reversals and novel scents. The primary analysis is the accuracy for each scent pairing (cumulative proportion correct for each scent). The general findings from the Dig task suggest that it is a simple experimental preparation that can assess deficits in rats with bilateral frontal cortical damage compared to rats with unilateral parietal damage. The Dig task can also be easily incorporated into an existing cognitive test battery. The use of more tasks such as this one can lead to more accurate testing of frontal function following injury, which may lead to therapeutic options for treatment. All animal use was conducted in accordance with protocols approved by the Institutional Animal Care and Use Committee.

There are two main variables that can be recorded in the Dig task: Correct/Incorrect and Latency. However, non-responses can also be recorded and analyzed as they may be important when considering motivation. These can be used to generate measures for analysis such as accuracy (# correct/total trials), response bias11, and latency. These measures can be used to assess several different types of learning, depending upon the design of the experiment (see additional notes above). Pretesting of animals allows a researcher to evaluate memory for a learned discrimination. Reversals of learned discriminations allow a researcher to see how well an animal can adapt to a changing contingency. Learning of novel discriminations allows researchers to compare learning across different scent pairings as well as evaluate capacity for novel learning.
Depending upon the design of the study (see additional notes above), these measures can be analyzed in a repeated measures design or can be collapsed into a single variable. Previously, we collapsed accuracy into a measure of cumulative proportion correct in order to analyze each scent discrimination separately5. This occurred by summing the number of correct discriminations and dividing by the total number of trials for each discrimination. A series of one-way between-subjects analysis of variances (ANOVAs) were then used to examine group differences for each post-injury scent discrimination. However, more power may be obtained for analysis by utilizing advanced statistical techniques such as mixed effects modeling. One other alternative is to utilize signal-detection theory for data analysis. This is a popular approach in the human decision-making literature and allows the researcher to examine most of the data from a given subject in a single graph and quickly assess the degree of accuracy, bias, and deficits12. Choice of analytic approach will be largely driven by experimental question, whether one wants to compare across types of learning, evaluate one single type of learning, or apply a decision-making approach to the data.
Following bilateral frontal brain injury, our laboratory has found that rats suffer large deficits under virtually all types of learning in this task. Frontally-injured animals showed a deficit in memory for a previously learned discrimination as well as difficulty learning the reversal of that scent pairing (Figure 3). Frontal animals also had difficulty learning novel discriminations (Figure 3). In accordance with previous research13, there was also a large increase in variability in the frontally injured animals (Figure 3).
In contrast, unilateral parietal injuries in our laboratory have shown no deficits across all the discriminations in this task. Unilateral rats showed no deficits in memory for a previously learned discrimination and also acquired the reversal discrimination very rapidly (Figure 4). They learned novel scent pairings very rapidly as well (Figure 4). In addition, the variability within the unilaterally-injured group was very low, with most rats performing optimally on the task.
<img alt="Figure 1" src="/files/ftp_upload/50033/50033fig1.jpg" /> 
Figure 1. Testing in our laboratory was conducted in a bank of standard operant chambers (Med Associates Inc.) that were adapted for use in this task by removing four consecutive floor-bars to open up space for two scent cups.
<img alt="Figure 2" src="/files/ftp_upload/50033/50033fig2.jpg" /> 
Figure 2. Testing in our laboratory utilized PVC pipe end-caps (6 cm tall and 5.5 cm in diameter) as the scent cups and a piece of Plexiglas (10 x 22 cm) with two drilled circular holes (5.5 cm in diameter) as the scent cup holder.
<img alt="Figure 3" fo:content-width="5in" fo:src="/files/ftp_upload/50033/50033fig3highres.jpg" src="/files/ftp_upload/50033/50033fig3.jpg" /> 
Figure 3. This graph shows typical performance of sham (blue) vs. frontally-injured (red) rats across several different phases of testing in two different formats. In panel A, the data is represented as a bar graph with white dots representing data points for individual rats in each group. In panel B, the same data is graphed in a line graph. There was no difference on the pre-injury discrimination acquisition. However, following injury and on the same discrimination, sham rats perform at 93% cumulative percent correct while injured rats perform at 65% cumulative percent correct. When a reversal of the discrimination was administered, injured rats drop to below chance performance. Similarly, on a novel scent discrimination frontally-injured rats had difficulty learning the novel pairing and stayed at below chance performance. <a href="https://www.jove.com/files/ftp_upload/50033/50033fig3large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 4" fo:content-width="5in" fo:src="/files/ftp_upload/50033/50033fig4highres.jpg" src="/files/ftp_upload/50033/50033fig4.jpg" /> 
Figure 4. This graph shows typical performance of sham (blue) vs. unilateral parietally-injured rats (green) across several different phases of testing in two different formats. In panel A, the data is represented as a bar graph with white dots representing data points for individual rats in each group. In panel B, the same data is graphed in a line graph. There is no difference between the groups on any of the discriminations. Unilaterally-injured rats performed at or, in some cases, slightly higher than sham levels following injury. <a href="https://www.jove.com/files/ftp_upload/50033/50033fig4large.jpg" target="_blank"> Click here to view larger figure</a>.

Watch this Scientific Journal Video about The Dig Task: A Simple Scent Discrimination Reveals Deficits Following Frontal Brain Damage at JoVE.com

The Dig Task: A Simple Scent Discrimination Reveals Deficits Following Frontal Brain Damage

In this protocol, a novel application of scent discrimination is described. The Dig task is a reasonably inexpensive task that can be used to assess frontally-mediated cognition following brain damage.

Cognitive impairment is the most frequent cause of disability in humans following brain damage, yet the behavioral tasks used to assess cognition in ...

the-dig-task-simple-scent-discrimination-reveals-deficits-following

Research

JoVE Journal

Neuroscience

13.5K Views.  Southern Illinois University at Carbondale. In this protocol, a novel application of scent discrimination is described. The Dig task is a reasonably inexpensive task that can be used to assess frontally-mediated cognition following brain damage.

Video: The Dig Task: A Simple Scent Discrimination Reveals Deficits Following Frontal Brain Damage

The Vermicelli and Capellini Handling Tests: Simple quantitative measures of dexterous forepaw function in rats and mice

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended upon this work to develop a simple quantitative measure of forepaw dexterity that is sensitive to lateralized impairments and age-dependent changes. Rodents learn skillful forepaw and digit movements to manage thin pasta pieces, which they eagerly consume. We have previously described methods for quantifying vermicelli handling in rats and showed that the measures are very sensitive to forelimb impairments resulting from unilateral ischemic lesions, middle cerebral artery occlusions and unilateral striatal dopamine depletion [Allred, R.P., Adkins, D.L., Woodlee, M.T., Husbands, L.C., Maldonado M.A., Kane, J.R., Schallert, T. &amp; Jones, T.A. The Vermicelli Handling Test: a simple quantitative measure of dexterous forepaw function in rats. J. Neurosci. Methods 170, 229-244 (2008)]. Here we present a more detailed protocol for this test in rats and compare it with a newly developed version for mice, the Capellini Handling Test. Rats and mice are videotaped while handling short lengths of uncooked vermicelli or capellini pasta, respectively, with a camera positioned to optimize the view of paw movements. Slow motion video playback allows for the identification of forepaw adjustments, defined as any distinct removal and replacement of the paw, or of any number of digits, on the pasta piece after eating commences. Forepaw adjustments per piece are averaged over trials per each testing session. Repeated testing permits sensitive quantitative analysis of changes in forepaw dexterity over time. Protocols for pre-testing habituation and handling practice, as well as procedures for characterizing atypical handling patterns, are described. Because rats and mice perform the pasta handling tests slightly differently, species-specific differences in administration and scoring of these tests are highlighted. All animal use was in accordance with protocols approved by the University of Texas at Austin Animal Care and Use Committee.

The Vermicelli and Capellini Handling Tests of forepaw dexterity take advantage of the natural inclination of rodents to manipulate food items using skillful forepaw and digit movements. Animals are videotaped while handling short strands of uncooked dry pasta. Slow motion video playback allows for the quantification of forepaw adjustments.

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended ...

Chromatin Immunoprecipitation from Dorsal Root Ganglia Tissue following Axonal Injury

Axons in the central nervous system (CNS) do not regenerate while those in the peripheral nervous system (PNS) do regenerate 
to a limited extent after injury (Teng et al., 2006). It is recognized that transcriptional programs essential for neurite and axonal outgrowth are 
reactivated upon injury in the PNS (Makwana et al., 2005). However the tools available to analyze neuronal gene regulation in vivo are limited and 
often challenging.
The dorsal root ganglia (DRG) offer an excellent injury model system because both the CNS and PNS are innervated by a 
bifurcated axon originating from the same soma. The ganglia represent a discrete collection of cell bodies where all transcriptional events occur, 
and thus provide a clearly defined region of transcriptional activity that can be easily and reproducibly removed from the animal. Injury of nerve 
fibers in the PNS (e.g. sciatic nerve), where axonal regeneration does occur, should reveal a set of transcriptional programs that are distinct from 
those responding to a similar injury in the CNS, where regeneration does not take place (e.g. spinal cord). Sites for transcription factor binding, 
histone and DNA modification resulting from injury to either PNS or CNS can be characterized using chromatin immunoprecipitation (ChIP). 
Here, we describe a ChIP protocol using fixed mouse DRG tissue following axonal injury. This powerful combination provides a means for characterizing the pro-regeneration chromatin environment necessary for promoting axonal regeneration.

We present a method for chromatin immunoprecipitation from dorsal root ganglia tissue following axonal injury. The approach can be used to identify specific transcription factor binding sites and epigenetic modification of histone and DNA important for the regeneration of injured axons in both the peripheral and central nervous system.

Axons in the central nervous system (CNS) do not regenerate while those in the peripheral nervous system (PNS) do regenerate 
to a limited extent after ...

Morris Water Maze Test for Learning and Memory Deficits in Alzheimer&#39;s Disease Model Mice

The Morris Water Maze (MWM) was first established by neuroscientist Richard G. Morris in 1981 in order to test hippocampal-dependent learning, including acquisition of spatial memoryand long-term spatial memory 1. The MWM is a relatively simple procedure typically consisting of six day trials, the main advantage being the differentiation between the spatial (hidden-platform) and non-spatial (visible platform) conditions 2-4. In addition, the MWM testing environment reduces odor trail interference 5. This has led the task to be used extensively in the study of the neurobiology and neuropharmacology of spatial learning and memory. The MWM plays an important role in the validation of rodent models for neurocognitive disorders such as Alzheimer&#x2019;s Disease 6, 7. In this protocol we discussed the typical procedure of MWM for testing learning and memory and data analysis commonly used in Alzheimer&#x2019;s disease transgenic model mice.

Morris Water Maze Test for Learning and Memory Deficits in Alzheimer&#039;s Disease Model Mice

The Morris Water Maze is a behavioral task to test hippocampal-dependent learning and memory. It has been widely used in the study of neurobiology, neuropharmacology and neurocognitive disorders in rodent models.

The Morris Water Maze (MWM) was first established by neuroscientist Richard G. Morris in 1981 in order to test hippocampal-dependent learning, including ...

Investigations on Alterations of Hippocampal Circuit Function Following Mild Traumatic Brain Injury

Traumatic Brain Injury (TBI) afflicts more than 1.7 million people in the United States each year and even mild TBI can lead to persistent neurological impairments 1. Two pervasive and disabling symptoms experienced by TBI survivors, memory deficits and a reduction in seizure threshold, are thought to be mediated by TBI-induced hippocampal dysfunction 2,3. In order to demonstrate how altered hippocampal circuit function adversely affects behavior after TBI in mice, we employ lateral fluid percussion injury, a commonly used animal model of TBI that recreates many features of human TBI including neuronal cell loss, gliosis, and ionic perturbation 4-6.
	Here we demonstrate a combinatorial method for investigating TBI-induced hippocampal dysfunction. Our approach incorporates multiple ex vivo physiological techniques together with animal behavior and biochemical analysis, in order to analyze post-TBI changes in the hippocampus. We begin with the experimental injury paradigm along with behavioral analysis to assess cognitive disability following TBI. Next, we feature three distinct ex vivo recording techniques: extracellular field potential recording, visualized whole-cell patch-clamping, and voltage sensitive dye recording. Finally, we demonstrate a method for regionally dissecting subregions of the hippocampus that can be useful for detailed analysis of neurochemical and metabolic alterations post-TBI.
	These methods have been used to examine the alterations in hippocampal circuitry following TBI and to probe the opposing changes in network circuit function that occur in the dentate gyrus and CA1 subregions of the hippocampus (see Figure 1). The ability to analyze the post-TBI changes in each subregion is essential to understanding the underlying mechanisms contributing to TBI-induced behavioral and cognitive deficits. 
	The multi-faceted system outlined here allows investigators to push past characterization of phenomenology induced by a disease state (in this case TBI) and determine the mechanisms responsible for the observed pathology associated with TBI.

A multi-faceted approach to investigating functional changes to hippocampal circuitry is explained. Electrophysiological techniques are described along with the injury protocol, behavioral testing and regional dissection method. The combination of these techniques can be applied in similar fashion for other brain regions and scientific questions.

Traumatic Brain Injury (TBI) afflicts more than 1.7 million people in the United States each year and even mild TBI can lead to persistent neurological ...

A Simple Alternative to Stereotactic Injection for Brain Specific Knockdown of miRNA

MicroRNAs (miRNAs) are key regulators of gene expression. In the brain, vital processes like neurodevelopment and neuronal functions depend on the correct expression of microRNAs. Perturbation of microRNAs in the brain can be used to model neurodegenerative diseases by modulating neuronal cell death. Currently, stereotactic injection is used to deliver miRNA knockdown agents to specific location in the brain. Here, we discuss strategies to design antagomirs against miRNA with locked nucleotide modifications (LNA). Subsequently describe a method for brain specific delivery of antagomirs, uniformly across different regions of the brain. This method is simple and widely applicable since it overcomes the surgery, associated injury and limitation of local delivery in stereotactic injections. We prepared a complex of neurotropic, cell-penetrating peptide Rabies Virus Glycoprotein (RVG) with antagomir against miRNA-29 and injected through tail vein, to specifically deliver in the brain. The antagomir design incorporated features that allow specific targeting of the miRNA and formation of non-covalent complexes with the peptide. The knock-down of the miRNA in neuronal cells, resulted in apoptotic cell death and associated behavioural defects. Thus, the method can be used for acute models of neuro-degeneration through the perturbation of miRNAs.

MicroRNAs play crucial roles in the brain and are potential targets for modeling neuro-degeneration. However, perturbing miRNA levels is challenging due to the short length of miRNA and inaccessibility of the brain tissue. This video presents a method for antagomir design and brain specific delivery using a neuropeptide in mice.

MicroRNAs (miRNAs) are key regulators of gene expression.  In the brain, vital processes like neurodevelopment and neuronal functions depend on the ...

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We demonstrate a simple way to tap into the fly reward system, modify experiences related to natural reward, and use voluntary ethanol consumption as a measure for changes in reward states. The approach serves as a relevant tool to study the neurons and genes that play a role in experience-mediated changes of internal state. The protocol is composed of two discrete parts: exposing the flies to rewarding and nonrewarding experiences, and assaying voluntary ethanol consumption as a measure of the motivation to obtain a drug reward. The two parts can be used independently to induce the modulation of experience as an initial step for further downstream assays or as an independent two-choice feeding assay, respectively. The protocol does not require a complicated setup and can therefore be applied in any laboratory with basic fly culture tools.

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for inducing rewarding and nonrewarding experiences in fruit flies (Drosophila melanogaster) using voluntary ethanol consumption as a measure for changes in reward states.

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

High Resolution Quantitative Synaptic Proteome Profiling of Mouse Brain Regions After Auditory Discrimination Learning

The molecular synaptic mechanisms underlying auditory learning and memory remain largely unknown. Here, the workflow of a proteomic study on auditory discrimination learning in mice is described. In this learning paradigm, mice are trained in a shuttle box Go/NoGo-task to discriminate between rising and falling frequency-modulated tones in order to avoid a mild electric foot-shock. The protocol involves the enrichment of synaptosomes from four brain areas, namely the auditory cortex, frontal cortex, hippocampus, and striatum, at different stages of training. Synaptic protein expression patterns obtained from trained mice are compared to na&#239;ve controls using a proteomic approach. To achieve sufficient analytical depth, samples are fractionated in three different ways prior to mass spectrometry, namely 1D SDS-PAGE/in-gel digestion, in-solution digestion and phospho-peptide enrichment. 
High-resolution proteomic analysis on a mass spectrometer and label-free quantification are used to examine synaptic protein profiles in phospho-peptide-depleted and phospho-peptide-enriched fractions of synaptosomal protein samples. A commercial software package is utilized to reveal proteins and phospho-peptides with significantly regulated relative synaptic abundance levels (trained/na&#239;ve controls). Common and differential regulation modes for the synaptic proteome in the investigated brain regions of mice after training were observed. Subsequently, meta-analyses utilizing several databases are employed to identify underlying cellular functions and biological pathways.

The identification of molecules and pathways controlling synaptic plasticity and memory is still a major challenge in neuroscience. Here, a workflow is described addressing the relative quantification of synaptic proteins supposedly involved in the molecular reorganization of synapses during learning and memory consolidation in an auditory learning paradigm.

The molecular synaptic mechanisms underlying auditory learning and memory remain largely unknown. Here, the workflow of a proteomic study on auditory ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

Measurement &amp; Analysis of the Temporal Discrimination Threshold Applied to Cervical Dystonia

The temporal discrimination threshold (TDT) is the shortest time interval at which an observer can discriminate two sequential stimuli as being asynchronous (typically 30-50 ms). It has been shown to be abnormal (prolonged) in neurological disorders, including cervical dystonia, a phenotype of adult onset idiopathic isolated focal dystonia. The TDT is a quantitative measure of the ability to perceive rapid changes in the environment and is considered indicative of the behavior of the visual neurons in the superior colliculus, a key node in covert attentional orienting. This article sets out methods for measuring the TDT (including two hardware options and two modes of stimuli presentation). We also explore two approaches of data analysis and TDT calculation. The application of the assessment of temporal discrimination to the understanding of the pathogenesis of cervical dystonia and adult onset idiopathic isolated focal dystonia is also discussed.

Methods for the measurement and analysis of the temporal discrimination threshold are presented, and its application to the study of the pathogenesis of cervical dystonia are discussed.

The temporal discrimination threshold (TDT) is the shortest time interval at which an observer can discriminate two sequential stimuli as being ...

A Simple and Reproducible Method to Prepare Membrane Samples from Freshly Isolated Rat Brain Microvessels

The blood-brain barrier (BBB) is a dynamic barrier tissue that responds to various pathophysiological and pharmacological stimuli. Such changes resulting from these stimuli can greatly modulate drug delivery to the brain and, by extension, cause considerable challenges in the treatment of central nervous system (CNS) diseases. Many BBB changes that affect pharmacotherapy, involve proteins that are localized and expressed at the level of endothelial cells. Indeed, such knowledge on BBB physiology in health and disease has sparked considerable interest in the study of these membrane proteins. From a basic science research standpoint, this implies a requirement for a simple but robust and reproducible method for isolation of microvessels from brain tissue harvested from experimental animals. In order to prepare membrane samples from freshly isolated microvessels, it is essential that sample preparations be enriched in endothelial cells but limited in the presence of other cell types of the neurovascular unit (i.e., astrocytes, microglia, neurons, pericytes). An added benefit is the ability to prepare samples from individual animals in order to capture the true variability of protein expression in an experimental population. In this manuscript, details regarding a method that is utilized for isolation of rat brain microvessels and preparation of membrane samples are provided. Microvessel enrichment, from samples derived, is achieved by using four centrifugation steps where dextran is included in the sample buffer. This protocol can easily be adapted by other laboratories for their own specific applications. Samples generated from this protocol have been shown to yield robust experimental data from protein analysis experiments that can greatly aid the understanding of BBB responses to physiological, pathophysiological, and pharmacological stimuli.

Here, a method for isolation of rat brain microvessels and for the preparation of membrane samples is described. This protocol has the clear advantage of producing enriched microvessel samples with acceptable protein yield from individual animals. Samples can then be used for robust protein analyses at the brain microvascular endothelium.

The blood-brain barrier (BBB) is a dynamic barrier tissue that responds to various pathophysiological and pharmacological stimuli. Such changes resulting ...