This research was supported by grants R01-MH090127, P30-MH089868 from the National Institute of Mental Health and award number UL1TR001120 from the National Center for Advancing Translational Sciences.&#160; The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIMH or NCATS. Additionally, we would like to gratefully acknowledge Dr. David Morilak for his valuable expertise in guiding the early method development.

NOTE: Animals used in this study are male C57Bl/6J mice between 4 and 6 months of age for optimal results. Mice are maintained on a reverse light cycle in order to conveniently test them during the active (dark) phase. If behavior is performed in a room separate from housing, mice are allowed at least 1 hr to acclimate following transportation to the testing room. Behavioral testing is performed under red light conditions minimize disruption to the normal activity of the mice as little as possible while still allowing the experimenter adequate illumination to visually monitor the testing session.


NOTE: All aspects of the experimental procedure described herein were carried out in accord with the Guide for the Care and Use of Laboratory Animals, 8th edition (NRC) and were approved by the Institutional Animal Care and Use Committee at the University of Texas Health Science Center at San Antonio.
1. Experimental Setup
<ol>
	<li>Fill ceramic ramekins (non-porous; 1.5&#8221; depth x 2.5&#39; diameter), to inner rim with digging medium (approximately 1&#8221; from bottom); filter paper rounds cut in half and taped to inside back of ramekin (Figure 1A)</li>
	<li>Break lightly sweetened dry breakfast cereal (food reward) &#8211; Honey Nut or sugar coated varieties, into approximately 20 mg pieces for acclimation, training and testing stages.</li>
	<li>Make an attentional set shifting task (AST) chamber (Figure 1B): Acclimate train and test each mouse in individual chambers; recommend production of at least 8 chambers.
	<ol>
		<li>Build a 12&#8221; x 8&#8221; x 7&#8221; AST chamber; walls and start gate made of opaque white acrylic that is easy to clean with 70% ethanol. The guillotine-style, removable starting gate separates chamber into a waiting area (5&#8221; x 8&#8221;) and testing area (7&#8221; x 8&#8221;).</li>
		<li>Include a Clear 5&#8221;x7&#8221; removable partition that separates two sides of testing area evenly; cut three &#188;&#8221; diameter holes into bottom portion of partition to allow sniffing access, spaced 1&#8221; apart and 1&#8221; from the front of the partition (Figure 1C).</li>
	</ol>
	</li>
</ol>
2. Procedure Overview (Figure 2)
<ol>
	<li>Prior to AST, house mice individually and allow them to adjust to a reverse light cycle for at least one week.</li>
	<li>Acclimate, train and test each animal in individual testing chambers. Assign each chamber to one mouse and clean with 70% ethanol after finishing the final day of testing. This will minimize stress during testing by making the chamber smell like &#8220;home-cage&#8221; or &#8220;self&#8221;.</li>
	<li>Scent the testing pots 2 days prior to testing to allow the scent to dissipate; mice will not dig in pots that have a very strong odor. Using a syringe with a 25 G&#160;needle, add approximately 0.1 ml of essential oil to the top of the filter paper.</li>
</ol>
3. Handling (Days 1-8)
<ol>
	<li>Handle mice starting 8 days prior to the first day of food restriction. This will reduce stress associated with handling during testing.</li>
	<li>Weigh and record body weights each day of handling. Handle each mouse 2-3 min&#160;per day.</li>
	<li>If multiple experimenters are to be involved in testing a set of mice, ensure that each experimenter participates in the handling process to increase familiarity with experimenters and reduce stress and anxiety experienced on training or testing days.</li>
	<li>Continue handling and recording weights throughout the duration of testing.</li>
</ol>
4. Food restriction (Days 9-12)
<ol>
	<li>Beginning 4 days prior to the start of acclimation, place mice on a restricted diet to maintain mice at 80-85% of their free feeding body weight.&#160;Give 1 gram of food per mouse per day. If body weight falls below the specified range give 2 grams per day until they return to the appropriate range.</li>
	<li>Place 2 ramekins at the front of each home cage in order to allow mice to acclimate to the testing pots. These are the same spots used to train the mouse to dig for a food reward.</li>
	<li>Add 2 grams of food and 1 piece of food reward (half a piece of cereal in each) in the ramekins each day for 4 days.</li>
	<li>If behavior is to be performed in a separate room than the one in which the mice are housed, beginning on day 1 of food restriction, transport mice to the testing room for at least 1 hr&#160;per day prior to returning to the housing room to allow them to acclimate to the testing room.</li>
	<li>On the last day of food restriction, before the start of acclimation, change the home cage bedding. The cage bedding will not be changed again until after testing is complete to reduce stress or anxiety experienced during training and testing (unless absolutely necessary).</li>
	<li>Maintain mice on food restriction until the end of testing. On days 13-17, give mice food after acclimation, training, or testing based on an estimate of the amount of food reward consumed (generally &#60;1g of food pellet).</li>
</ol>
5. Acclimation (Days 13-14)
<ol>
	<li>Beginning 2 days prior to the start of training, perform acclimation during the dark cycle under red light.</li>
	<li>Spread a small amount of dirty bedding from the home cage in the chamber, this will reduce the stress of being in a new environment by introducing a familiar &#8220;home-cage&#8221; or &#8220;self&#8221; smell to the testing chamber (the same chamber will be used for a single mouse for the duration of training and testing).</li>
	<li>Place a clean ramekin with water in the waiting area of the chamber; mice tend not to eat if they do not have access to water.</li>
	<li>Place the two ramekins that have been used for food restriction in the testing area with a food reward (~20 mg cereal piece) in each.</li>
	<li>Place the mouse in the waiting area; remove the start gate and allow the mouse to explore the chamber for 1 hr.</li>
	<li>Continuously add food rewards (~20 mg cereal piece) to the empty ramekins in order to encourage the mice to explore the testing area and the pots frequently.</li>
	<li>Ensure that the mouse can see the experimenter throughout the acclimation period so that the presence of the experimenter is not an added stressor during testing.</li>
</ol>
6. Training (Day 15)
<ol>
	<li>Place the mouse in the waiting area.</li>
	<li>Place the empty ramekins, each containing a food reward (~20 mg cereal piece), into the testing area and lift the start gate. Allow mouse 3 min&#160;to retrieve both food rewards. Repeat this step several times.</li>
	<li>Gradually add sawdust/mouse cage bedding to the pots in subsequent trials. Allow the mouse 3 min&#160;to retrieve the food reward from each pot before ending the trial. Once the mouse has retrieved the food reward from each pot, proceed to the next trial. If the mouse does not make attempts at digging once the food reward is partially covered, hints may be provided (i.e., sprinkle cereal dust over area of the food reward, make an indention in the sawdust over area of food reward, uncover part of the food reward).</li>
	<li>Continue adding sawdust/bedding after each trial until the food reward is fully covered and the mouse reliably demonstrates the ability to dig in a full pot to find the food reward.</li>
	<li>If a mouse has not reliably demonstrated the ability to find a food reward in a full pot within 2 hr&#160;of the start of training, continue to a second day of training. Maintain the mouse on food restriction and repeat this procedure the next day.</li>
	<li>If the mouse fails to dig for a food reward after two successive days, exclude it from the remainder of the experiment, and annotate as a &#8220;failed to dig&#8221; in the experimental record.</li>
</ol>
7. Testing (Days 16-17):
<ol>
	<li>On day one, test simple discrimination (SD), compound discrimination (CD), reversal (R1), and intra-dimensional shift (IDS).</li>
	<li>On day two, test the intra-dimensional shift 2 (IDS2), intra-dimensional shift 3 (IDS3), reversal 2 (R2; optional), extra-dimensional shift (EDS).</li>
	<li>Pair media and scents as shown in Table 1.</li>
	<li>At the time of handling on testing day, give mice one food reward to avoid a lack of focus during testing due to hunger. From this point until the end of the testing day, do not provide any food except rewards for correct choices.</li>
	<li>At the start of testing, using forceps (limits the spread of scents between pots), place a piece of the food reward in the pot that indicates a correct choice. The food reward should be fully covered by media (see Figure 3). To avoid a scent cue from the food reward, sprinkle cereal dust over all pots at the start of the stage.</li>
	<li>Place the mouse in the waiting area with the start gate closed. Place the pots on either side of the testing chamber. The trial begins when the start gate is removed and the timer is started. Allow each mouse 3 min&#160;per trial to make a choice. Record the choice (correct or incorrect) and time until the mouse made a choice for each trial. 
	NOTE: A &#8220;choice&#8221; is indicated by an attempt to dig in either pot. A dig is only recorded as a choice if the mouse has displaced the media and poked its nose far enough in the space it creates to find the food reward or displaced enough of the media to uncover the food reward. If a mouse has not made a choice within 3 min, this is recorded as no choice (incorrect).</li>
	<li>Once a choice has been made for either pot, remove the pot that was not chosen from the testing chamber. If a correct choice was made, allow the mouse to finish the food reward before placing the mouse back in the waiting area. If an incorrect choice is made, allow the mouse to explore the pot to show that there is no food reward for that choice before returning the mouse to the waiting area.</li>
	<li>At the end of each trial, place the mouse back in the waiting area and replace the start gate.</li>
	<li>Ensure that the mouse meets the criterion of 8 correct trials consecutively to advance to the next stage of AST. If a mouse does not obtain 8 correct choices consecutively within 50 trials, it fails that stage and cannot move on to complete the test. 6 consecutive no choices (3 min&#160;without a choice) is a failure to participate and prohibits the mouse from moving on to the next stage.</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 (11), 4320-4324 (2000).</li><li>Garner, J. P., Thogerson, C. M., Wurbel, H., Murray, J. D., Mench, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+neuropsychology:+validation+of+the+Intra-Dimensional+Extra-Dimensional+set+shifting+task+for+mice.">Animal neuropsychology: validation of the Intra-Dimensional Extra-Dimensional set shifting task for mice.</a> Behav Brain Res. 173 (1), 53-61 (2006).</li><li>Nagahara, A. H., Bernot, T., Tuszynski, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+cognitive+deficits+in+rhesus+monkeys+mirror+human+deficits+on+an+automated+test+battery.">Age-related cognitive deficits in rhesus monkeys mirror human deficits on an automated test battery.</a> Neurobiol Aging. 31 (6), 1020-1031 (2010).</li><li>Rock, P. L., Roiser, J. P., Riedel, W. J., Blackwell, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cognitive+impairment+in+depression:+a+systematic+review+and+meta-analysis.">Cognitive impairment in depression: a systematic review and meta-analysis.</a> Psychol Med. , 1-12 (2013).</li><li>Stuss, D. 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=Wisconsin+Card+Sorting+Test+performance+in+patients+with+focal+frontal+and+posterior+brain+damage:+effects+of+lesion+location+and+test+structure+on+separable+cognitive+processes.">Wisconsin Card Sorting Test performance in patients with focal frontal and posterior brain damage: effects of lesion location and test structure on separable cognitive processes.</a> Neuropsychologia. 38 (4), 388-402 (2000).</li><li>Bissonette, G. B., 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=Double+dissociation+of+the+effects+of+medial+and+orbital+prefrontal+cortical+lesions+on+attentional+and+affective+shifts+in+mice.">Double dissociation of the effects of medial and orbital prefrontal cortical lesions on attentional and affective shifts in mice.</a> J Neurosci. 28 (44), 11124-11130 (2008).</li><li>Chase, E. A., Tait, D. S., 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=Lesions+of+the+orbital+prefrontal+cortex+impair+the+formation+of+attentional+set+in+rats.">Lesions of the orbital prefrontal cortex impair the formation of attentional set in rats.</a> Eur J Neurosci. 36 (3), 2368-2375 (2012).</li><li>McAlonan, K., 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=Orbital+prefrontal+cortex+mediates+reversal+learning+and+not+attentional+set+shifting+in+the+rat.">Orbital prefrontal cortex mediates reversal learning and not attentional set shifting in the rat.</a> Behav Brain Res. 146 (1-2), 97-103 (2003).</li><li>Dias, R., Robbins, T. W., Roberts, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primate+analogue+of+the+Wisconsin+Card+Sorting+Test:+effects+of+excitotoxic+lesions+of+the+prefrontal+cortex+in+the+marmoset.">Primate analogue of the Wisconsin Card Sorting Test: effects of excitotoxic lesions of the prefrontal cortex in the marmoset.</a> Behav Neurosci. 110 (5), 872-886 (1996).</li><li>Bissonette, G. B., Powell, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversal+learning+and+attentional+set-shifting+in+mice.">Reversal learning and attentional set-shifting in mice.</a> Neuropharmacology. 62 (3), 1168-1174 (2012).</li><li>Colacicco, G., Welzl, H., Lipp, H. P., Wurbel, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attentional+set-shifting+in+mice:+modification+of+a+rat+paradigm,+and+evidence+for+strain-dependent+variation.">Attentional set-shifting in mice: modification of a rat paradigm, and evidence for strain-dependent variation.</a> Behav Brain Res. 132 (1), 95-102 (2002).</li><li>Kos, T., Nikiforuk, A., Rafa, D., Popik, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+NMDA+receptor+antagonists+on+attentional+set-shifting+task+performance+in+mice.">The effects of NMDA receptor antagonists on attentional set-shifting task performance in mice.</a> Psychopharmacology (Berl). 214 (4), 911-921 (2011).</li><li>Tait, D. S., Chase, E. A., 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=Attentional+Set-Shifting+in+Rodents:+a+Review+of+Behavioural+Methods+and+Pharmacological+Results.">Attentional Set-Shifting in Rodents: a Review of Behavioural Methods and Pharmacological Results.</a> Curr Pharm Des. , (2013).</li><li>Lapiz-Bluhm, M. D., 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=Behavioural+assays+to+model+cognitive+and+affective+dimensions+of+depression+and+anxiety+in+rats.">Behavioural assays to model cognitive and affective dimensions of depression and anxiety in rats.</a> J Neuroendocrinol. 20 (10), 1115-1137 (2008).</li><li>Brigman, J. L., Bussey, T. J., Saksida, L. M., Rothblat, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discrimination+of+multidimensional+visual+stimuli+by+mice:+intra-+and+extradimensional+shifts.">Discrimination of multidimensional visual stimuli by mice: intra- and extradimensional shifts.</a> Behav Neurosci. 119 (3), 839-842 (2005).</li></ol>

No conflicts of interest declared.

Deficits in cognitive flexibility mediated by the PFC represent a significant disability associated with a variety of neurological and neuropsychiatric disorders, and can have a major impact on outcomes and quality of life of individuals suffering from these disorders. Optimization of the AST for use in mice is vital to investigating the genetic underpinnings in diseases coupled to prefrontal cortical mediated cognitive dysfunction. We have found that the method described above is a reliable measure of a specific cogniti...

The attentional set shifting task (AST) was developed as a measure of attention and cognitive flexibility in rats over a decade ago1,2. AST is modeled after the intradimensional /extradimensional component of the Cambridge Neuropsychological Test Automated Battery (CANTAB) which is used to identify cognitive dysfunction in humans and non-human primates3,4. While the ability to learn simple rules remains intact, deficits in learning to modify a response when the rules have changed are found in patients suffering from a variety of neuropsychiatric disorders (i.e., schizophrenia, obsessive compulsive disorder, depression), neurodegenerative disorders (i.e., Parkinson&#8217;s disease) and in patients with lesions to the pre-frontal cortex5. More generally, these patients are described as having deficits in cognitive flexibility. Analogous deficits have been shown in both non-human primates and rodents when lesions to the prefrontal cortex have been induced1,6-8. These deficits indicate a state of cognitive inflexibility or an impaired ability to shift attentional set.
An attentional set is formed when a subject learns that a set of rules can be applied to complex stimuli in order to differentiate relevant from irrelevant cues. For example, in the AST, animals will learn to pay attention and respond to the relevant cue (i.e., digging medium) and ignore an irrelevant cue (i.e., odor), by pairing a food reward with the medium. This association is then reinforced in subsequent tasks where the type of digging medium and odor changes, but the paired association between medium and reward remains. This reinforced rule forms a cognitive set. Two stages within the AST protocol measure aspects of cognitive flexibility: reversal and the extra-dimensional shift. At the reversal stage, the previously negative stimuli within one dimension (medium in this example) is now positive, which challenges the animal to ignore the positive stimuli from the previous stage. For example, if felt digging medium was the positive stimuli in the previous stage and paper was the negative stimuli, now the reverse is true. This challenges the animal&#8217;s flexibility in that it must maintain the attentional set (i.e., medium is the relevant dimension) while altering the rule learned for stimulus and reward pair within a dimension. The formation of an attentional set is challenged at the extra-dimensional shift stage, when the irrelevant dimension (odor in this example) becomes the relevant dimension. A perseverative response, as indicated by a continued choice using the previously learned rule, at either stage reflects a deficit in cognitive flexibility.
Lesion studies in both non-human primates and rodents have shown specific regions of the prefrontal cortex can be attributed to the ability to perform particular stages of the AST1,9. Lesions to the orbitofrontal cortex (OFC), a subregion of the prefrontal cortex (PFC), have been shown to induce deficits in reversal learning on the AST8. Additionally, lesions to the medial prefrontal cortex (MPFC) lead to specific difficulties in performing the extra-dimensional shift.
While numerous studies using the AST have been performed in rats and non-human primates, relatively few have utilized AST as a measure of cognitive function in mice. Given the ease of genetic manipulation in mice, and the critical need to measure prefrontal cortex function in studying a variety of disorders, adapting and validating this behavioral measure in mice is an important addition to research on diseases associated with PFC dysfunction. The protocol detailed here is a modification of the procedure by Birrell and Brown1 that is optimized for application in mice and reflects many of the mouse-specific adaptations that have been previously reported2,6,10-13.

<table><tbody><tr><td>Testing Chamber</td><td></td><td></td><td>Fabricated in-house, specifications can be found in protocol</td></tr><tr><td>3 oz. Ramekins</td><td>BIA Cordon Bleu</td><td>900002S12</td><td>Purchased on amazon.com</td></tr><tr><td>Essential Oils:</td><td>Fronteir Natural Products Co-Op</td><td></td><td>Purchased on http://www.frontiercoop.com/prodlist.php?ct=anpceoeo</td></tr><tr><td>Clove Bud</td><td></td><td>191114</td><td></td></tr><tr><td>Red Thyme</td><td></td><td>191140</td><td></td></tr><tr><td>Lemon</td><td></td><td>190810</td><td></td></tr><tr><td>Cinnamon Leaf</td><td></td><td>191111</td><td></td></tr><tr><td>Rosemary</td><td></td><td>191133</td><td></td></tr><tr><td>Citronella Java</td><td></td><td>191112</td><td></td></tr><tr><td>Vanilla in jojoba oil</td><td></td><td>191231</td><td></td></tr><tr><td>Anise Seed</td><td></td><td>191102</td><td></td></tr><tr><td>Ginger</td><td></td><td>191121</td><td></td></tr><tr><td>Nutmeg</td><td></td><td>191161</td><td></td></tr><tr><td>Filter Paper, 9.0 cm diameter</td><td>VWR International</td><td>28310-048</td><td>Purchased on https://us.vwr.com/</td></tr><tr><td>Digging Media</td><td></td><td></td><td></td></tr><tr><td>Raffia</td><td>Ashland 8 oz Raffia</td><td></td><td>Purchased on http://www.michaels.com/&lt;br/&gt; Cut into 1/2&quot; to 1&quot; strips</td></tr><tr><td>Green Felt</td><td>Creatology Basic Felt Forest/Dark Green 36 in x 36 in&amp;nbsp;</td><td></td><td>Purchased on http://www.michaels.com/&lt;br/&gt; cut into 1 cm x 1 cm squares</td></tr><tr><td>Brown foam</td><td>Creatology Fun Foam 18 x 12 in sheets (light brown/tan)</td><td></td><td>Purchased on http://www.michaels.com/&lt;br/&gt; cut into 1/2 cm x 2 cm rectangles</td></tr><tr><td>Crepe paper (yellow)</td><td>Celebrate It Paper Crinkle 4 oz yellow jumbo</td><td></td><td>Purchased on http://www.michaels.com/&lt;br/&gt; Cut into 1/2&quot; to 1&quot; strips</td></tr><tr><td>Ribbon (turqoise)</td><td>Celebrate It Wide Ribbon 4 in x 10 yd 100% polyester&amp;nbsp;</td><td></td><td>Purchased on http://www.michaels.com/&lt;br/&gt; cut into 1 cm x 1 cm squares</td></tr><tr><td>Metallic&amp;nbsp;</td><td>Celebrate It Metallic Crinkle Gold 2 oz</td><td></td><td>Purchased on http://www.michaels.com/&lt;br/&gt; Cut into 1/2&quot; to 1&quot; strips</td></tr><tr><td>Googly Eyes, 4 mm</td><td></td><td>ME4144</td><td>Purchased on http://factorydirectcraft.com/index.php</td></tr><tr><td>Black Sequins, 3 mm</td><td></td><td>8780BK</td><td></td></tr><tr><td>Pink Pom Poms, 1/4 inch</td><td></td><td>1018221</td><td></td></tr><tr><td>Pipe Cleaners, red</td><td></td><td>108430</td><td></td></tr><tr><td>Bedding material/sawdust</td><td></td><td></td><td>Obtained from in-house animal resources</td></tr><tr><td>Timer</td><td>Control Company</td><td>5000</td><td>Purchased on http://www.control3.com/5000p.htm</td></tr><tr><td>Lightly sweetened dry breakfast cereal loops</td><td>HEB&amp;nbsp;</td><td></td><td>Honey nut toasted oats used in the protocol described, any lightly sweet coated breakfast cereal is recommended</td></tr></tbody></table>

the attentional set shifting task: a measure of cognitive flexibility in mice

Cognitive impairment, particularly involving dysfunction of circuitry within the prefrontal cortex (PFC), represents a core feature of many neuropsychiatric and neurodevelopmental disorders, including depression, post-traumatic stress disorder, schizophrenia and autism spectrum disorder. Deficits in cognitive function also represent the most difficult symptom domain&#160;to successfully treat, as serotonin reuptake inhibitors and tricyclic antidepressants have only modest effects. Functional neuroimaging studies and postmortem analysis of human brain tissue implicate the PFC as being a primary region of dysregulation in patients with these disorders. However, preclinical behavioral assays used to assess these deficits in mouse models which can be readily manipulated genetically and could provide the basis for studies of new treatment avenues have been underutilized. Here we describe the adaptation of a behavioral assay, the attentional set shifting task (AST), to be performed in mice to assess prefrontal cortex mediated cognitive deficits. The neural circuits underlying behavior during the AST are highly conserved across humans, nonhuman primates and rodents, providing excellent face, construct and predictive validity.

The typical dependent measure in this test is the number of trials per stage to meet criterion (or 8 correct consecutive choices). Figure 4A shows the average number of trials to meet criterion at each stage in untreated C57BL/6J using the AST. As mice form an attentional set on the relevant cue dimension (odor or media), performance will improve, as indicated by a reduction in trials to meet criterion in successive intradimensional shifts, and the reversal stages and the extradimensional shift will requ...

Watch this Scientific Journal Video about The Attentional Set Shifting Task: A Measure of Cognitive Flexibility in Mice at JoVE.com

The Attentional Set Shifting Task: A Measure of Cognitive Flexibility in Mice

The goal of this protocol is to perform a behavioral assay such as the attentional set shifting task (AST) to assess prefrontal cortex-mediated cognitive flexibility in mice.

Cognitive impairment, particularly involving dysfunction of circuitry within the prefrontal cortex (PFC), represents a core feature of many ...

the-attentional-set-shifting-task-measure-cognitive-flexibility

Universidad San Sebastian

Research

JoVE Journal

Behavior

27.1K Views.  University of Texas Health Science Center at San Antonio. The goal of this protocol is to perform a behavioral assay such as the attentional set shifting task (AST) to assess prefrontal cortex-mediated cognitive flexibility in mice.

Video: The Attentional Set Shifting Task: A Measure of Cognitive Flexibility in Mice

The Crossmodal Congruency Task as a Means to Obtain an Objective Behavioral Measure in the Rubber Hand Illusion Paradigm

The rubber hand illusion (RHI) is a popular experimental paradigm. Participants view touch on an artificial rubber hand while the participants' own hidden hand is touched. If the viewed and felt touches are given at the same time then this is sufficient to induce the compelling experience that the rubber hand is one's own hand. The RHI can be used to investigate exactly how the brain constructs distinct body representations for one's own body. Such representations are crucial for successful interactions with the external world. To obtain a subjective measure of the RHI, researchers typically ask participants to rate statements such as "I felt as if the rubber hand were my hand". Here we demonstrate how the crossmodal congruency task can be used to obtain an objective behavioral measure within this paradigm.
The variant of the crossmodal congruency task we employ involves the presentation of tactile targets and visual distractors. Targets and distractors are spatially congruent (i.e. same finger) on some trials and incongruent (i.e. different finger) on others. The difference in performance between incongruent and congruent trials - the crossmodal congruency effect (CCE) - indexes multisensory interactions. Importantly, the CCE is modulated both by viewing a hand as well as the synchrony of viewed and felt touch which are both crucial factors for the RHI.
The use of the crossmodal congruency task within the RHI paradigm has several advantages. It is a simple behavioral measure which can be repeated many times and which can be obtained during the illusion while participants view the artificial hand. Furthermore, this measure is not susceptible to observer and experimenter biases. The combination of the RHI paradigm with the crossmodal congruency task allows in particular for the investigation of multisensory processes which are critical for modulations of body representations as in the RHI.

We demonstrate how an objective measure can be employed in the widely employed rubber hand illusion paradigm. This measure is obtained by modifying the well-established crossmodal congruency task. This task allows the investigation of multisensory processes which are critical for modulations of body representations as in the rubber hand illusion.

The rubber hand illusion (RHI) is a popular experimental paradigm. Participants view touch on an artificial rubber hand while the participants' own hidden ...

The 5-Choice Serial Reaction Time Task: A Task of Attention and Impulse Control for Rodents

This protocol describes the 5-choice serial reaction time task, which is an operant based task used to study attention and impulse control in rodents. Test day challenges, modifications to the standard task, can be used to systematically tax the neural systems controlling either attention or impulse control. Importantly, these challenges have consistent effects on behavior across laboratories in intact animals and can reveal either enhancements or deficits in cognitive function that are not apparent when rats are only tested on the standard task. The variety of behavioral measures that are collected can be used to determine if other factors (i.e., sedation, motivation deficits, locomotor impairments) are contributing to changes in performance. The versatility of the 5CSRTT is further enhanced because it is amenable to combination with pharmacological, molecular, and genetic techniques.

This protocol describes the 5-choice serial reaction time task, which is an operant based task used to study attention and impulse control in rodents. Test day challenges, which are modifications of the standard task, increase flexibility of the task and can be combined with other manipulations to more fully characterize behavior.

This protocol describes the 5-choice serial reaction time task, which is an operant based task used to study attention and impulse control in rodents. ...

A Dual Task Procedure Combined with Rapid Serial Visual Presentation to Test Attentional Blink for Nontargets

When viewers search for targets in a rapid serial visual presentation (RSVP) stream, if two targets are presented within about 500 msec of each other, the first target may be easy to spot but the second is likely to be missed. This phenomenon of attentional blink (AB) has been widely studied to probe the temporal capacity of attention for detecting visual targets. However, with the typical procedure of AB experiments, it is not possible to examine how the processing of non-target items in RSVP may be affected by attention. This paper describes a novel dual task procedure combined with RSVP to test effects of AB for nontargets at varied stimulus onset asynchronies (SOAs). In an exemplar experiment, a target category was first displayed, followed by a sequence of 8 nouns. If one of the nouns belonged to the target category, participants would respond &#8216;yes&#8217; at the end of the sequence, otherwise participants would respond &#8216;no&#8217;. Two 2-alternative forced choice memory tasks followed the response to determine if participants remembered the words immediately before or after the target, as well as a random word from another part of the sequence. In a second exemplar experiment, the same design was used, except that 1) the memory task was counterbalanced into two groups with SOAs of either 120 or 240 msec and 2) three memory tasks followed the sequence and tested remembrance for nontarget nouns in the sequence that could be anywhere from 3 items prior the target noun position to 3 items following the target noun position. Representative results from a previously published study demonstrate that our procedure can be used to examine divergent effects of attention that not only enhance targets but also suppress nontargets. Here we show results from a representative participant that replicated the previous finding.&#160;

This paper describes a novel dual task procedure combined with Rapid Serial Visual Presentation to look at attentional blink at varied stimulus onset asynchronies. This experimental procedure differed from others in that it tested attentional blink for nontargets.

When viewers search for targets in a rapid serial visual presentation (RSVP) stream, if two targets are presented within about 500 msec of each other, the ...

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 ...

An Operant Intra-/Extra-dimensional Set-shift Task for Mice

Alterations in executive control and cognitive flexibility, such as attentional set-shifting abilities, are core features of several neuropsychiatric diseases. The most widely used neuropsychological tests for the evaluation of attentional set-shifting in human subjects are the Wisconsin Card Sorting Test (WCST) and the CANTAB Intra-/Extra-dimensional set shift task (ID/ED). These tasks have proven clinical relevance and have been modified and successfully adapted for research in animal models. However, currently available tasks for rodents present several limitations, mainly due to their manual-based testing procedures, which are hampering translational advances in psychiatric medicine. To overcome these limitations and to better mimic the original version in primates, we present the development of a novel operant-based two-chamber ID/ED "Operon" task for rodents. We demonstrated the effectiveness of this novel task to measure different facets of cognitive flexibility in mice including attentional set formation and shifting, and reversal learning. Moreover, we show the high flexibility of this task in which three different perceptual dimensions can be manipulated with a high number of stimuli cues for each dimension. This novel ID/ED Operon task can be an effective preclinical tool for drug testing and/or large genetic screening relevant to the study of executive dysfunction and cognitive symptoms found in psychiatric disorders.

Attentional set-shifting cognitive abnormalities are a core feature of many psychiatric diseases. Here, we present the development of a novel automatic system for the effective study of attentional set-shifting abilities, executive functions and other cognitive abilities in mice with high translational validity to human studies.

Alterations in executive control and cognitive flexibility, such as attentional set-shifting abilities, are core features of several neuropsychiatric ...

The Emotional Stroop Task: Assessing Cognitive Performance under Exposure to Emotional Content

The emotional Stroop effect (ESE) is the result of longer naming latencies to ink colors of emotion words than to ink colors of neutral words. The difference shows that people are affected by the emotional content conveyed by the carrier words even though they are irrelevant to the color-naming task at hand. The ESE has been widely deployed with patient populations, as well as with non-selected populations, because the emotion words can be selected to match the tested pathology. The ESE is a powerful tool, yet it is vulnerable to various threats to its validity. This report refers to potential sources of confounding and includes a modal experiment that provides the means to control for them. The most prevalent threat to the validity of existing ESE studies is sustained effects and habituation wrought about by repeated exposure to emotion stimuli. Consequently, the order of exposure to emotion and neutral stimuli is of utmost importance. We show that in the standard design, only one specific order produces the ESE.

The emotional Stroop effect (ESE) is the result of longer naming latencies to ink colors of emotion words than those of neutral words. This report refers to potential sources of confounding and includes a modal experiment that provides the means to control for them.

The emotional Stroop effect (ESE) is the result of longer naming latencies to ink colors of emotion words than to ink colors of neutral words. The ...

New Variations for Strategy Set-shifting in the Rat

Behavioral flexibility is crucial for survival in changing environments. Broadly defined, behavioral flexibility requires a shift of behavioral strategy based on a change in governing rules. We describe a strategy set-shifting task that requires an attentional shift from one stimulus dimension to another. The paradigm is often used for testing cognitive flexibility in primates. However, the rodent version has not been as extensively developed. We have recently extended an established set-shifting task in the rat1 by requiring attention to different stimuli according to context. All the experimental conditions required animals to choose either a left or right lever. Initially, all animals had to choose on the basis of the location of the lever. Subsequently, a change in the rule occurred, which required a shift in set from location-based rule to a rule in which the correct lever was indicated by a light cue. We compared performance on three different versions of the task, in which the light stimulus was either novel, previously relevant, or previously irrelevant. We found that specific neurochemical lesions selectively impaired the ability to make particular types of set shift as measured by the performance on the different versions of the task.

Set-shifting, a form of behavioral flexibility, requires an attentional shift from one stimulus dimension to another. We extended an established rodent set-shifting task1 by requiring attention to different stimuli according to context. The task was combined with specific lesions to identify neuron subtypes underlying a successful shift.

Behavioral flexibility is crucial for survival in changing environments. Broadly defined, behavioral flexibility requires a shift of behavioral strategy ...

T-maze Forced Alternation and Left-right Discrimination Tasks for Assessing Working and Reference Memory in Mice

Forced alternation and left-right discrimination tasks using the T-maze have been widely used to assess working and reference memory, respectively, in rodents. In our laboratory, we evaluated the two types of memory in more than 30 strains of genetically engineered mice using the automated version of this apparatus. Here, we present the modified T-maze apparatus operated by a computer with a video-tracking system and our protocols in a movie format. The T-maze apparatus consists of runways partitioned off by sliding doors that can automatically open downward, each with a start box, a T-shaped alley, two boxes with automatic pellet dispensers at one side of the box, and two L-shaped alleys. Each L-shaped alley is connected to the start box so that mice can return to the start box, which excludes the effects of experimenter handling on mouse behavior. This apparatus also has an advantage that in vivo microdialysis, in vivo electrophysiology, and optogenetics techniques can be performed during T-maze performance because the doors are designed to go down into the floor. In this movie article, we describe T-maze tasks using the automated apparatus and the T-maze performance of &alpha;-CaMKII+/- mice, which are reported to show working memory deficits in the eight-arm radial maze task. Our data indicated that &alpha;-CaMKII+/- mice showed a working memory deficit, but no impairment of reference memory, and are consistent with previous findings using the eight-arm radial maze task, which supports the validity of our protocol. In addition, our data indicate that mutants tended to exhibit reversal learning deficits, suggesting that &alpha;-CaMKII deficiency causes reduced behavioral flexibility. Thus, the T-maze test using the modified automatic apparatus is useful for assessing working and reference memory and behavioral flexibility in mice.

This article presents the protocol of T-maze tests using a modified automated apparatus for assessing the learning and memory functions in mice.

Forced alternation and left-right discrimination tasks using the T-maze have been widely used to assess working and reference memory, respectively, in ...

Neuroscience

Assessment of Stress Effects on Cognitive Flexibility using an Operant Strategy Shifting Paradigm

Stress affects cognitive function. Whether stress enhances or impairs cognitive function depends on several factors, including the 1) type, intensity, and duration of the stressor; 2) type of cognitive function under study; and 3) timing of the stressor in relation to learning or executing the cognitive task. Furthermore, sex differences among the effects of stress on cognitive function have been widely documented. Described here is an adaptation of an automated operant strategy shifting paradigm to assess how variations in stress affect cognitive flexibility in male and female Sprague Dawley rats. Specifically, restraint stress is used before or after training in this operant-based task to examine how stress affects cognitive performance in both sexes. Particular brain areas associated with each task in this automated paradigm have been well-established (i.e., the medial prefrontal cortex and orbitofrontal cortex). This allows for targeted manipulations during the experiment or the assessment of particular genes and proteins in these regions upon completion of the paradigm. This paradigm also allows for the detection of different types of performance errors that occur after stress, each of which has defined neural substrates. Also identified are distinct sex differences in perseverative errors after a repeated restraint stress paradigm. The use of these techniques in a preclinical model may reveal how stress affects the brain and impairs cognition in psychiatric disorders, such as post-traumatic stress disorder (PTSD) and major depressive disorder (MDD), which display marked sex differences in prevalence.

Stressful life events impair cognitive function, increasing the risk of psychiatric disorders. This protocol illustrates how stress affects cognitive flexibility using an automated operant strategy shifting paradigm in male and female Sprague Dawley rats. Specific brain areas underlying particular behaviors are discussed, and translational relevance of results are explored.

Stress affects cognitive function. Whether stress enhances or impairs cognitive function depends on several factors, including the 1) type, intensity, and ...

Chronic Stress Shifts Effort-Related Choice Behavior in a Y-Maze Barrier Task in Mice

Mood disorders, including major depressive disorder, can be precipitated by chronic stress. The Y-maze barrier task is an effort-related choice test that measures motivation to expend effort and obtain reward. In mice, chronic stress exposure significantly impacts motivation to work for a higher value reward when a lesser value reward is freely available compared to unstressed mice. Here we describe the chronic corticosterone administration paradigm, which produces a shift in effortful responding in the Y-maze barrier task. In the Y-maze task, one arm contains 4 food pellets, while the other arm contains only 2 pellets. After mice learn to select the high reward arm, barriers with progressively increasing height are then introduced into the high reward arm over multiple test sessions. Unfortunately, most chronic stress paradigms (including corticosterone and social defeat) were developed in male mice and are less effective in female mice. Therefore, we also discuss chronic non-discriminatory social defeat stress (CNSDS), a stress paradigm we developed that is effective in both male and female mice. Repeating results with multiple distinct chronic stressors in male and female mice combined with increased usage of translationally relevant behavior tasks will help to advance the understanding of how chronic stress can precipitate mood disorders.

The Y-maze barrier task is a behavior test that examines motivation to expend effort for reward. Here, we discuss testing multiple well-validated chronic stressors including chronic corticosterone and social defeat stress with this behavior, as well as the novel chronic non-discriminatory social defeat stress (CNSDS), which is effective in females.

Mood disorders, including major depressive disorder, can be precipitated by chronic stress. The Y-maze barrier task is an effort-related choice test that ...