We thank G. Pruzzo for his excellent technical assistance and the IIT mechanical workshop for help in building our new apparatus. This research was supported by the Istituto Italiano di Tecnologia, the Marie Curie FP7-Reintegration-Grant N&#176;268247, and by the Italian Ministry of Health Grants for Young Researchers (GR-2010-2315883).

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M., Braver, T. S., Carter, C. S., Poldrack, R. A., Robbins, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CNTRICS+final+task+selection:+executive+control.">CNTRICS final task selection: executive control.</a> Schizophrenia bulletin. 35 (1), 115-135 (2009).</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=Dissociation+in+prefrontal+cortex+of+affective+and+attentional+shifts.">Dissociation in prefrontal cortex of affective and attentional shifts.</a> Nature. 380 (6569), 69-72 (1996).</li><li>Birrell, J. M., Brown, V. 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The authors declare that they have no financial conflicts of interest.

In this study we present a novel automated two-chamber ID/ED &#34;Operon&#34; task for mice that is able to reliably measure cognitive flexibility through reversal learning, attentional set formation and shifting. This paradigm is analogous to the WCST and ID/ED tasks commonly used in human and nonhuman primates and overcomes the major limitations of the previous versions for rodents. This Operon paradigm can be used as a new effective tool for large drug and/or genetic screenings relevant to cognitive (dys)functions in mice with high relevance to translational medicine.
This automated task have the following advantages over previously used ID/ED task for rodents: (1) it has less labor-intensive procedures than the manual versions (e.g., the software regulates all the phases of the task, greatly decreasing interventions by the experimenter); (2) it eliminates any source of subjectivity in the measured parameters (e.g.,&#160;the experimenter is not required to judge whether or not the animal has actually made a choice response); (3) it eliminates any possibility that mice might follow reinforcement-related cues in order to make a response (i.e., the reward is always automatically delivered in the center magazine); (4) it avoids arbitrary environmental conditions (e.g.,&#160;use of mazes and apparatus of different sizes/materials, use of manually prepared cue stimuli); (5) it allows manipulation of three distinct dimensions with a large range of different stimuli, in accordance to the equivalent human tasks used in the clinical setting. Here we present data only from mice, but similar advantages might be expected also for rats.
There are critical steps in the task, which are internal construct-validity parameters that can be used in order to identify areliable attentional set-shifting performance: i) poorer performance in the EDS compared to previous stages ii) a general improvement from IDS to IDS2; iii) a specific improvement in consecutive reversal stages, as the more an animal is trained in within-set reversals, the better it should perform on subsequent within-set reversals20; iv) better performance in the EDSRe compared to the EDS. This novel ID/ED operon task presents all these features, consistent with previous studies in primates using the CANTAB ID/ED task and in rodents using the manual &#34;digging&#34; version12,13. Moreover, each stage of this automated task was learnt in an equivalent number of trials irrespective of the relevant/irrelevant dimension used (i.e.,&#160;odor, texture and light)19. This demonstrated that all the stimuli used have similar salience and are suitable for attentional set-formation and/or shifting. Our experiments demonstrate that the difficulty in solving the EDS is also highlighted by the increase in response latencies. Processing time increases to maintain optimal accuracy during harder discriminations21. Thus, considering the latency to respond as an index of speed of information processing, decision-making and problem solving18, the slower latencies during the EDS might reflect a strategy adopted to face the difficulty in solving the set-shifting.
We have shown that our novel apparatus can be effectively used with the more &#39;classical&#39; attentional set-shifting paradigm employing only two dimensions throughout the test (i.e.,&#160;the two-dimension) or with a stuck-in-set paradigm, implying the use of three dimensions. Depending on the cognitive domain of interest, it is possible to choose the most appropriate protocol. Both of these paradigms have been previously used in humans, primates and rodents7,12-17. However, the stuck-in-set procedure is able to distinguish between different components of set-shifting and is a more selective measure of frontal lobe functioning in human patients7,22. Furthermore, one problem that might occur in the two-dimension paradigm is the learned irrelevance that might bias the results. Learned irrelevance perseveration refers to the inability to attend to and learn about information that was previously irrelevant7. This situation can occur in the EDS phase of the two-dimension paradigm, as the subject is required to shift the attention to the previously irrelevant dimension. In this case, it is basically impossible to discern whether an EDS deficit is due to the inability to shift attention away from a previously relevant dimension and/or the inability to now shift the attention to a previously irrelevant dimension. Impairment may then just reflect the active inhibition of responding to a dimension previously made irrelevant by its random association with reinforcing feedback. In contrast, in the stuck-in-set condition, failure to shift to the new relevant dimension cannot be attributed to any prior learning about this dimension since it had not been experienced previously. Failure, therefore, reflects perseveration to the previously relevant dimension. In conclusion, even if in normal wild-type mice the &#39;stuck-in-set&#39; and the &#39;two-dimension&#39; attentional set-shifting paradigms might produce similar results, we expressly prefer the stuck-in-set perseveration for the reasons discussed above.

Attentional set-shifting is a measure of cognitive flexibility and executive functions1. It refers to the ability to switch between arbitrary internal rules (&#34;cognitive-attentional sets&#34;). The most widely used neuropsychological tasks for measuring attentional set-shifting and cognitive flexibility in humans are the Wisconsin Card Sorting Test (WCST)2 and a more recent and more refined version: the intra- and extra-dimensional attentional set-shifting (ID/ED) of the Cambridge Neuropsychological Test Automated Battery (CANTAB)3,4. These tasks have been used to identify specific cognitive abnormalities in a wide range of mental disorders including autism5, schizophrenia6, Parkinson&#39;s disease7, obsessive compulsive disorders8 and attention-deficit/hyperactive disorders9. The clinical relevance and solid methodological approach of the WCST and the ID/ED tests have attracted interest in the implementation of similar tests in preclinical research10,11. These tasks allow for the selective measurement within the same subject of different cognitive abilities such as discriminative learning, reversal learning, formation of an attentional set, shifting of attention within the same dimension (i.e., intra-dimensional shift: IDS) and between different perceptual dimensions (i.e., extra-dimensional shift: EDS). This is crucial because distinct brain circuits, as well as neuropathology might alter these distinct cognitive functions in different ways. For example, a double dissociation or functional specialization effect has been demonstrated between the lateral (in monkeys and humans) / medial (in rodents) and orbital regions of the PFC in the attentional set-shifting tasks. While the orbitofrontal cortex is more involved in the reversal phases of these tasks, the lateral/medial PFC region governs the extra-dimensional shift stages12-14.
Rodent versions of these attentional set-shifting tests for primates have been successfully generated13-16. However, some aspects of these rodent versions have been limiting their applications and use. For instance, these tasks are manually run and therefore highly labor-intensive and difficult to standardize. Moreover, the presence of food reinforcers inside the stimuli might result in an ambiguous interpretation of animal responses and potential bias in choice-making10. These characteristics have limited the throughput of testing and, more importantly, their application in large-scale genetic and/or drug-screening studies.
To overcome these limitations and to enhance the potential applications of ID/ED shift paradigms in rodents, we present here a novel two-chamber operant-based task to test cognitive flexibility in mice. This novel task closely mimics the ID/ED task used in human and non-human primates and circumvents the problems of earlier rodent versions.

<table border="0" cellpadding="0" cellspacing="0" width="1386">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Plexiglas walls and aluminum floor</td>
			<td style="width:264px;">Custom made</td>
			<td style="width:141px;"></td>
			<td style="width:744px;">(16 x 16 x 16 cm for each chamber)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plexiglas door&#160;</td>
			<td>Custom made</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PC</td>
			<td>Dell Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MED-PC IV software&#160;</td>
			<td>(Med Associates, St. Albans, VT, USA)</td>
			<td>custom made</td>
			<td>Use an operant code in order to automatically control the presentation of the stimuli and the sequence of the stages</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nose-poke hole&#160;</td>
			<td>Med Associates, St. Albans, VT, USA)</td>
			<td>ENV-314M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Food magazine&#160;</td>
			<td>Med Associates, St. Albans, VT, USA)</td>
			<td>ENV-303M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pellet dispenser for food reinforcement&#160;</td>
			<td>Med Associates, St. Albans, VT, USA)</td>
			<td>ENV-203-14P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Houselight&#160;</td>
			<td>Med Associates, St. Albans, VT, USA)</td>
			<td>ENV-315M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dilution olfactometer&#160;</td>
			<td>(Med Associates, St. Albans, VT, USA)</td>
			<td>PHM-275</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Liquid odorants</td>
			<td>(Sigma Aldrich, Dorset, UK)</td>
			<td></td>
			<td>(see Table 1)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mineral oil</td>
			<td>(Sigma Aldrich, Dorset, UK)</td>
			<td>M5904</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Air pump and vacuum</td>
			<td>(Med Associates, St. Albans, VT, USA)</td>
			<td>PHM-280</td>
			<td>Vacuum is recommended for scents removal at the end of each trial</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Light-emitting diodes (LED)</td>
			<td></td>
			<td></td>
			<td>High-intensity, transparent gem, 3 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Floor textures</td>
			<td></td>
			<td></td>
			<td>3x3 cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reinforcement pellets</td>
			<td>TestDiet</td>
			<td>5TUL 14mg</td>
			<td>For mice</td>
		</tr>
	</tbody>
</table>

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.

Figure 2 shows an example of the ID/ED task. Pairs of stimuli (either &#39;Discrimination 1&#39; or &#39;Discrimination 2&#39;) are randomly presented in each stage, and the mouse must choose the correct stimulus in each pair. In this example table, the correct exemplar is reported in bold. In the first stage (SD or simple discrimination), the stimuli presented in the two nose-poke holes differed in one of three dimensions (e.g., O1: Vanilla vs. O2: Lavender) and the mouse is rewarded for choosing the correct exemplar (e.g., O1). Once the subject reaches the criterion in this stage, the next stage (CD or compound discrimination) begins, where the same exemplars of the relevant dimension are presented overlaid at random by exemplars of a second, but irrelevant dimension, introduced as a confounding factor (e.g., L1: blue light vs.L2: yellow light). Two different discriminations are possible in this stage (either &#39;Discrimination 1&#39; or &#39;Discrimination 2&#39;). In the next stage (CDRe or compound discrimination reversal), the reward contingencies are reversed but the exemplars and the relevant dimension are unchanged: the mouse has to learn that the previously correct stimulus is now incorrect (e.g., lavender odor is now rewarded). In the next stage (IDS or intra-dimensional shift), new exemplars (both odors and lights) are introduced but the relevant dimension (odor in this example) remains the same (e.g., strawberry is the correct choice). In the next stage (IDR or intra-dimensional reversal), the reward contingencies are reversed. After a second intra-dimensional shift (IDS2 and its reversal), new exemplars are introduced to test the extra-dimensional shift (EDS) in which the relevant dimension is changed. In the &#39;stuck-in-set&#39; EDS, the mouse has to focus on the new dimension (e.g.,&#160;the Texture, T1: coarse sandpaper vs. T2: fine sandpaper), while the previously relevant dimension (in this case, odor) is now the irrelevant dimension. In the &#39;two-dimension&#39; EDS, the previously irrelevant dimension (in this case, light) is now the relevant dimension. In the final stage (EDSRe or extra-dimensional reversal), the reward contingencies are reversed.
In order to obtain reliable results, the stimulus dimensions used in the task should be equally well-learned. As shown in Figure 3, visual, tactile and odor discriminations in this novel apparatus required similar time (F(2,64)=0.36; p=0.69) and similar number of trials (F(2,64)=0.059; p=0.94) to reach the criterion, suggesting that animals are able to perform simple discriminations regardless the dimension presented.
If a reliable attentional set has been developed over the testing phases of the task, the performance of a normal wild-type mouse should be poorer in the EDS stage compared to previous and following stages, as reported in previous studies in rodents and primates12,13. In particular, a robust increase of time and trials needed to reach criteria should be found in the EDS compared to the IDS stages. As illustrated in Figure 4, in our experiment with the &#39;stuck-in-set&#39; protocol, analysis of performance of the mice revealed a discrimination effect for the number of trials (F(8,168)=9.23; p&#60;0.0001) and time (F(8,168)=8.62; p&#60;0.0001) required to reach criteria. Indeed, mice needed more trials and more time to solve the EDS stage compared to the CD, IDS, IDS2 and EDSRe stages (p&#60;0.05; Figure 4A-B). Similarly, analysis of performance tested with the &#39;two-dimension&#39; protocol (Figure 5) showed a significant discrimination effect for the number of trials (F(8,72)=3.66; p&#60;0.005) and time (F(8,72)=4.65; p&#60;0.0005) needed to reach criteria. Indeed, we show that mice required more trials (p&#60;0.05) and more time (p&#60;0.05) to solve the EDS compared to CD, IDS, IDS2 and EDSRe (Figure 5A-B). No differences in shifting abilities should be observed between mice tested with different dimensions.
In a normal wild-type mouse, the first reversal learning (i.e., CDRe) is more difficult than the initial discrimination (i.e., CD). In agreement, as evident from Figures 4 and 5, mice needed more trials (p&#60;0.05; Figure 4-5A) and more time (p&#60;0.05; Figure 4-5B) to complete this stage. Moreover, reversal performance should improve from CDRe to IDSRe to IDS2Re, as we show in our experiment with both the &#39;stuck-in-set&#39; and &#39;two-dimension&#39; protocols. These results further strengthen the evidence of the formation of an attentional set through the task.
Throughout the task, the mice should improve their speed to respond over consecutive stages. Accordingly, the analysis of the latency to respond showed a significant discrimination effect (F(8,200)=42.59; p&#60;0.0001). In particular, as demonstrated by representative results in Figure 4C the latency to poke in the IDS2 stage is decreased compared to that in the IDS, CD and SD stages (p&#60;0.0005). Moreover, the latency to respond increased during the EDS stage compared to the previous IDS2 and IDS2Re and successive EDSRe stages (p&#60;0.05). In line with these results, the analysis of the latency to respond during the &#39;two-dimension&#39; task also showed a significant effect of discrimination (F(7,63)=9.98; p&#60;0.0005). As shown in Figure 5C, the latency to make a choice was increased during the EDS compared to previous IDS2 and IDS2Re and successive EDSRe (p&#60;0.05). Since the latency to respond has been considered an index of decisional processing18, these results further suggest that the mice encountered some problems processing the new discriminative rule during the EDS. Based on the behavioral performance of wild-type mice (Figure 4-5), we determined that the minimal number of sample size (by R power analysis) for each experimental group should be 8.
<img alt="Figure 3" src="/files/ftp_upload/53503/53503fig3.jpg" /> 
Figure 3. Simple discriminations of light, odor and texture are equivalent. (A) Number of trials and (B) time required to reach the criterion on simple discriminations with only light, odor or texture stimuli. Values represent mean &#177; SEM throughout Figures 2-4. Data originally published in &#39;The ultimate Intra- and Extradimensional Attentional Set-Shifting Task for Mice&#39;19.
<img alt="Figure 4" src="/files/ftp_upload/53503/53503fig4.jpg" /> 
Figure 4. Wild-type C57BL6J male mice performance in the &#39;stuck-in-set&#39; ID/ED Operon task. (A) Trials, (B) time (in minutes) and days needed to reach the criterion in the different stages of the ID/ED Operon task using a &#39;stuck-in-set&#39; ID/ED paradigm. (C) Time (in seconds) elapsed between the opening of the divider door and a nose-poke response (latency to respond) during the different stages of the task. A total of 26 mice were tested; 4 mice were excluded because they were not reliably poking to retrieve the food reinforcement during the training or were not able to finish the entire procedure. A: *p &#60;0.05 versus CD, IDS, IDS2, IDS2Re and EDSRe; B: *p &#60;0.05 versus CD, IDS2, IDS2Re and EDSRe. A and B: #p&#60;0.05, ##p&#60;0.005 versus CD, IDSRe and IDS2Re. C: *p &#60;0.05 versus IDS2, IDS2Re and EDSRe. Note that the mice were able to complete the entire task in 5-9 days in all experiments reported in Figures 3-4. Data originally published in &#39;The ultimate Intra- and Extradimensional Attentional Set-Shifting Task for Mice&#39;19. <a href="https://www.jove.com/files/ftp_upload/53503/53503fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/53503/53503fig5.jpg" />
Figure 5. Wild-type C57BL6J male mice performance in the &#39;two-dimension&#39; ID/ED Operon task. (A) Trials, (B) time (in minutes) and days needed to reach the criterion in the different stages of the ID/ED Operon task using a two-dimension paradigm. (C)Time (in seconds) elapsed between the opening of the divider door and a nose-poke response (latency to respond) during the different stages of the task. A total of 13 mice were tested; 3 mice were excluded because they were not reliably poking to retrieve the food reinforcement during the training or were not able to finish the entire procedure. A and B:#p&#60;0.05 versus CD and IDS2Re, *p &#60;0.05 versus CD, IDS, IDS2, EDSRe. C:*p &#60;0.05 versus IDS2, IDS2Re and EDSRe. Data originally published in &#39;The ultimate Intra- and Extradimensional Attentional Set-Shifting Task for Mice&#39;19. <a href="https://www.jove.com/files/ftp_upload/53503/53503fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/53503/53503fig6.jpg" /> 
Figure 6. Timeline of the entire procedure of the protocol to test ID/ED task.
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Stage</td>
			<td>Description</td>
			<td>Additional notes</td>
			<td>References</td>
		</tr>
		<tr>
			<td>Simple Discrimination (SD)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Compound Discrimination (CD)</td>
			<td>Stimuli vary in two perceptual dimensions, such as color and shape for visual stimuli in the human task, or between texture and odor stimuli for rodents</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reversal learning (CDRe &#8211; IDSRe &#8211; IDS2Re &#8211; EDSRe)</td>
			<td>Two exemplars within a perceptual dimension have their reinforcement contingencies reversed so that what was previously correct is then incorrect and vice versa)</td>
			<td>In serial reversal learning, performance improves with consecutive within-set reversals. Thus, reversal stages (i.e., CDRe, IDSRe, IDS2Re) not only assess function of different cortical areas, but also serve 1) to form the cognitive- attentional set challenged by the EDS stage, and 2) to prevent superstitious conditioning to unintended aspects of the stimulus</td>
			<td>- Lesions of the orbitofrontal cortex in mice (Bissonette et al., 2008) and monkyes (Dias et al., 1996) impaired Reversal learning 
			- fMRI during performance on the Reversal learning showed activation of the orbitofrontal cortex (Hampshire and Owen, 2006)</td>
		</tr>
		<tr>
			<td>Intradimensional shift (IDS &#8211; IDS2)</td>
			<td>Novel exemplars are introduced, but the same dimension is reinforced</td>
			<td>IDS stages serve as a crucial internal control (i.e., EDS should be more difficult than IDS), and also contribute to form the cognitive-attentional set</td>
			<td>Dopamine depletion in the PFC impaired attentional set formation (Robbins and Roberts, 2007). 6-OHDA lesioned monkeys did not show the classical reduction the reduction in errors from the first (IDS1) to the last (IDS5) discrimination, which should&#160; reflect acquisition of an attentional set&#160;</td>
		</tr>
		<tr>
			<td rowspan="2">Extradimensional shift (EDS)</td>
			<td>&#8216;stuck-in-set&#8217; protocol: previously irrelevant stimulus dimension is replaced by a new stimulus dimension which immediately becomes relevant</td>
			<td>failure to shift to the new relevant dimension cannot be attributed to any prior learning about this dimension since it had not been previously experienced. Failure, therefore, reflects perseveration to the previously relevant dimension</td>
			<td>- Lesions of the mPFC have been show to impair EDS in mice (Bissonette et al., 2008), and mokeys (Dias et al., 1996) 
			- Frontal lobe patients are impaired in the stuck-in-set &#39;perseveration&#39; condition but not in the &#8216;two dimension&#39; condition (Owen et al., 1993) 
			- Dopamine in the mPFC modulated EDS performance in mice (Papaleo et al., 2008; Scheggia et al., 2014) and rats (Tunbridge et al., 2004)</td>
		</tr>
		<tr>
			<td>&#8216;two dimensions&#8217; protocol: the previously irrelevant dimension is reinforced</td>
			<td>an apparent failure to shift attentional set may arise when a subject is able to shift attention away from a previously relevant dimension (when it becomes irrelevant) but is, nevertheless, unable to refocus attention on the newly relevant dimension</td>
			<td>- Lesion of the mPFC in rats (Birrell and Brown, 2000) impaired EDS shift 
			- fMRI during performance on the EDS has been shown activation of the ventro-lateral PFC (Hampshire and Owen, 2006)&#160;</td>
		</tr>
	</tbody>
</table>
Table 2: Stages of the ID/ED Operon task. Description of the stages of the task, including references to lesion and pharmacological studies addressing the role of brain areas involved in the different constructs tested during the task.

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

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 ...

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12.1K Views.  Istituto Italiano di Tecnologia. 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.

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

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. ...

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 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 ...

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 ...

A Protocol for Housing Mice in an Enriched Environment

Environmental enrichment (EE) is a housing environment for mice that boosts mental and physical health compared to standard laboratory housing. Our recent studies demonstrate that environmental enrichment decreases adiposity, increases energy expenditure, resists diet induced obesity, and causes cancer remission and inhibition in mice. EE typically consists of larger living space, a variety of &#8216;toys&#8217; to interact with, running wheels, and can include a number of other novel environmental changes. All of this fosters a more complex social engagement, cognitive and physical stimulations. Importantly, the toy location and type of toy is changed regularly, which encourages the mice to adapt to a frequently changing and novel environment. Many variables can be manipulated in EE to promote health effects in mice. Thus these approaches are difficult to control and must be properly managed to successfully replicate the associated phenotypes. Therefore, the goal of this video is to demonstrate how EE is properly set up and maintained to assure a complex, challenging, and controlled environment so that other researchers can easily reproduce the protective effects of EE against obesity and cancer.

Environmental enrichment for mice requires a complex and challenging setup, as well as comprehensive husbandry and handling techniques to assure robust metabolic and anti-cancer effects in the mice. This protocol provides detailed procedures to reproduce the above mentioned effects in mice.

Environmental enrichment (EE) is a housing environment for mice that boosts mental and physical health compared to standard laboratory housing. Our recent ...

Quantifying Social Motivation in Mice Using Operant Conditioning

In this protocol, social motivation is measured in mice through a pair of operant conditioning paradigms. To conduct the experiments, two-chambered shuttle boxes were equipped with two operant levers (left and right) and a food receptacle in one chamber, which was then divided from the second chamber by an automated guillotine door covered by a wire grid. Different stimulus mice, rotated across testing days, served as a social stimulus behind the wire grid, and were only visible following the opening of the guillotine door. Test mice were trained to lever press in order to open the door and gain access to the stimulus partner for 15 sec. The number of lever presses required to obtain the social reward progressively increased on a fixed schedule of 3. Testing sessions ended after test mice stopped lever pressing for 5 consecutive minutes. The last reinforced ratio or breakpoint can be used as a quantitative measure of social motivation. For the second paradigm, test mice were trained to discriminate between left and right lever presses in order to obtain either a food reward or the social reward. Mice were rewarded for every 3 presses of each respective lever. The number of food and social rewards can be compared as a measurement of the value placed upon each reward. The ratio of each reward type can also be compared between mouse strains and the change in this ratio can be monitored within testing sessions to measure satiation with a given reward type. Both of these operant conditioning paradigms are highly useful for the quantification of social motivation in mouse models of autism and other disorders of social behavior.

This article describes a new protocol for the quantitative measurement of social motivation in mice using operant conditioning for a social reward. The protocol is useful for the measurement of social motivation in mouse models of autism and other disorders of social behavior.

In this protocol, social motivation is measured in mice through a pair of operant conditioning paradigms. To conduct the experiments, two-chambered ...

Measuring Delay Discounting in Humans Using an Adjusting Amount Task

Delay discounting refers to a decline in the value of a reward when it is delayed relative to when it is immediately available. Delay discounting tasks are used to identify indifference points, which reflect equal preference for two dichotomous reward alternatives differing in both delay and magnitude. Indifference points are key to assessing the shape of a delay-discounting gradient because they allow us to isolate the effect of delay on value. For example, if at a 1 week delay and a maximum of $1,000, the indifference point is at $700 we know that, for that participant, a 1-week delay corresponds to a 30% reduction in value. This video outlines an adjusting amount delay-discounting task that identifies indifference points relatively quickly and is inexpensive and easy to administer. Once data have been collected, non-linear regression techniques are typically used to generate discounting curves. The steepness of the discounting curve reflects the degree of impulsive choice of a group or individual. These techniques have been used with a wide range of commodities and have identified populations that are relatively impulsive. For example, people with substance abuse problems discount delayed rewards more steeply than control participants. Although degree of discounting varies as a function of the commodity examined, discounting of one commodity correlates with discounting of other commodities, which suggests that discounting may be a persistent pattern of behavior1.

Delay discounting refers to a decline in the value of a reward when it is delayed relative to when it is immediately available. We outline a computer-based delay discounting task that is easy to implement and allows for the quantification of the degree of delay discounting in human participants.

Delay discounting refers to a decline in the value of a reward when it is delayed relative to when it is immediately available. Delay discounting tasks ...

Determining Pain Detection and Tolerance Thresholds Using an Integrated, Multi-Modal Pain Task Battery

Human pain models are useful in the assessing the analgesic effect of drugs, providing information about a drug&#39;s pharmacology and identify potentially suitable therapeutic populations. The need to use a comprehensive battery of pain models is highlighted by studies whereby only a single pain model, thought to relate to the clinical situation, demonstrates lack of efficacy. No single experimental model can mimic the complex nature of clinical pain. The integrated, multi-modal pain task&#160;battery presented here encompasses the electrical stimulation task, pressure stimulation task, cold pressor task, the UVB inflammatory model which includes a thermal task and a paradigm for inhibitory conditioned pain modulation. These human pain models have been tested for predicative validity and reliability both in their own right and in combination, and can be used repeatedly, quickly, in short succession, with minimum burden for the subject and with a modest quantity of equipment. This allows a drug to be fully characterized and profiled for analgesic effect which is especially useful for drugs with a novel or untested mechanism of action.

Human pain models are valuable tools used to assess the analgesic potential of novel compounds and predict their clinical efficacy, especially when used in an integrated manner. Although implementation of these models is complex, with proper execution, the pain models described in this protocol can provide predictive and reliable results.

Human pain models are useful in the assessing the analgesic effect of drugs, providing information about a drug&#39;s pharmacology and identify ...

Task Interruption and Resumption Paradigm for Testing the Activation and Pursuit of an Abstract Thinking Goal

This protocol is based on the task interruption and resumption paradigm, the premise of which is that active goals lead to persistent behavior and thus a higher resumption rate after a period of delay or interruption. The task interruption and resumption protocol described in this research is tailored to test the activation of cognitive goals (e.g., a goal to think more abstractly). Cognitive goals may be pursued even during the interruption period; thus, to prevent this, the protocol involves cognitive distraction. The protocol consists of several stages. Specifically, the initial stage includes the goal activation process, where the treatment (versus control) condition receives a manipulation to activate the cognitive goal being tested by the researcher. In the next stage, participants are presented with the introduction of a task that is perceived to either satisfy or not satisfy the cognitive goal of interest. Importantly, this task is interrupted a few seconds after it begins. The task interruption forces a delay period and introduces a cognitive distraction to prevent the automatic pursuit and fulfillment of the cognitive goal. After the interruption period, participants are given a choice between resuming the interrupted task and abandoning the interrupted task to complete an alternative task instead. Among participants whose cognitive goals had been activated at the earlier stage, the task resumption rate should be higher if the task was perceived as an opportunity to satisfy (versus not satisfy) the goal. Such a finding would provide empirical evidence that the cognitive goal has been activated and pursued. In previous research, this protocol has been used to test whether causal uncertainty activates an abstract thinking goal. Adapting the protocol to test the activation of other cognitive goals is also discussed.

This protocol was designed to test the activation and pursuit of cognitive goals (e.g., an abstract thinking goal) using the task interruption and resumption paradigm. The protocol is suitable for cognitive goals that are automatically pursued once activated, as the distraction procedure prevents goal pursuit during the interruption period.

This protocol is based on the task interruption and resumption paradigm, the premise of which is that active goals lead to persistent behavior and thus a ...

Operant Conditioning Task to Measure Song Preference in Zebra Finches

An operant conditioning paradigm is used to test the song preference of female zebra finches. Finches are placed in a two-chambered cage with a connecting opening and indicate their preference for a song by landing on a perch within each chamber. By interrupting the infrared beam from a photoelectric sensor above each perch, the bird activates the playback of a song through a speaker located on each side of the cage. Freely available software is used to trigger the song playback from each perch. To determine the song preference of each animal, her chamber preference is first identified by triggering no song playback when she lands on each perch. This chamber preference is then compared to her song preference. A minimum activity threshold is set to ensure the preference is real. Using this method, we show that paired females prefer the song of their partner. This method was used to understand the contribution of dopamine to the formation and maintenance of song preference.

We describe a technique to evaluate song preference in zebra finches. Females are placed in a two-chambered cage and song preference is measured by the number of times she triggers the playback of one song by landing on a perch within one chamber, compared with triggering a different song in the second chamber. Perch landings are counted using infrared sensors.

An operant conditioning paradigm is used to test the song preference of female zebra finches. Finches are placed in a two-chambered cage with a connecting ...

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 ...