Name: an operant intra-/extra-dimensional set-shift task for mice
Uploaded: 2016-01-22T14:00:00
Duration: 8 min 35 s
Description: Watch this Scientific Journal Video about An Operant Intra-/Extra-dimensional Set-shift Task for Mice at JoVE.com

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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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.

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			<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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			<td height="21" style="height:21px;">Light-emitting diodes (LED)</td>
			<td></td>
			<td></td>
			<td>High-intensity, transparent gem, 3 mm</td>
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			<td height="21" style="height:21px;">Floor textures</td>
			<td></td>
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			<td>3x3 cm</td>
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			<td height="21" style="height:21px;">Reinforcement pellets</td>
			<td>TestDiet</td>
			<td>5TUL 14mg</td>
			<td>For mice</td>
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</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

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