Research described here was supported by a grant from the Natural Sciences and Engineering Research Council of Canada to S.B.F.

<ol>
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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effects+of+inactivation+of+the+orbitofrontal+cortex+on+strategy+set-shifting+and+reversal+learning.">Differential effects of inactivation of the orbitofrontal cortex on strategy set-shifting and reversal learning.</a> Neurobiol Learn Mem. 89, 567-573 (2008).</li><li>Boulougouris, V., Dalley, J. W., 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=Effects+of+orbitofrontal,+infralimbic+and+prelimbic+cortical+lesions+on+serial+spatial+reversal+learning+in+the+rat.">Effects of orbitofrontal, infralimbic and prelimbic cortical lesions on serial spatial reversal learning in the rat.</a> Behav Brain Res. 179, 219-228 (2007).</li><li>Tseng, K. Y., Chambers, R. A., Lipska, B. 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Publication of and free access to this manuscript was supported by Med Associates, Inc.

The development of behavioral tasks to measure higher-order cognitive constructs in rodents is essential to advance knowledge of the neurobiology of cognition. With well-constructed and validated tasks, rodents can be assessed on tasks of complexity rivaling those of primates or even humans. Here we have shown how two aspects of executive function, strategy shifting and reversal learning, can be investigated in rodents using automated operant techniques. Using these automated tasks, we have replicated previous findings in cross-maze and digging tasks regarding the neural substrates of set-shifting and reversal learning11,13,18-21,27,29, suggesting that the operant tasks are valid assessments of these constructs.
These automated tasks have a number of benefits and advantages over existing non-automated cross-maze and digging tasks. Most compelling is the superior rate of data collection in the automated operant version. Each day&#8217;s training or testing takes only 30-60 minutes, and is fully computer-controlled requiring minimal supervision by the experimenter. Moreover, several animals can be tested simultaneously with a multi-chamber operant setup. Each task series, from shaping to final testing, can be completed in approximately 2-3 weeks. Another important advantage of the automated tasks is the precise control of stimulus presentation, thus minimizing the possibility of experimenter error. For example, the order of presentation of cue location on each trial is randomized and controlled by the computer, rather than by an experimenter manually consulting a trial-by-trial list. The timing between trials is precisely measured and consistent, and is not confounded by the time it takes an experimenter to, e.g., remove a rat from the cross-maze or rearrange the digging containers. Reinforcement delivery is automatic and is not subject to experimenter error (e.g., forgetting to bait the correct arm of a cross-maze). Data collection is similarly improved, with automatic recording of response patterns including the measurement of exact response latencies. In the absence of other motor abnormalities, changes in response latencies can be used to infer evidence of altered processing speed and/or to judge the level of cognitive complexity of a task21,22.
The automated tasks also retain one important advantage of the cross-maze tasks: the ability to conduct a detailed analysis of the types of errors made on the shift or reversal day. Distinguishing between set-shifting errors that replicate the previous day&#8217;s strategy (perseverative or regressive errors) and errors that represent previously untried strategies (never-reinforced errors) can assist in characterizing specific deficits in behavioral flexibility. In particular, perseverative errors occurring early in testing reflect an animal&#8217;s inability to abandon the previous strategy, while later-occurring regressive errors reflect an animal&#8217;s inability to maintain the new strategy once perseveration has ceased20. Never-reinforced errors may indicate a failure to acquire the new strategy, or an inability to respond systematically according to a rule20. Previous findings16,17,20 demonstrating dissociable neuroanatomical bases of these types of errors are also valuable in interpreting the results of these tasks.
Our procedures have been developed and optimized for use with rats. This being said, other groups have used similar procedures for testing set-shifting abilities in mice31. However, certain modifications need to be employed with mice to accommodate for species differences. These include longer presentation of the visual cue light prior to lever extension, training over multiple days using 30 trials/day and incorporation of a time-out punishment after incorrect choices. Although these modifications make this assay less amenable for use with pharmacological challenges, it could prove useful for assessing cognitive flexibility in genetically altered mice (although it is unclear whether these modifications would preserve the frontal cortex sensitivity of the task).
Of course, there are also limitations to these tasks. Some of these limitations arise from the automated nature of the task, while others are related to the parameters of the task itself. With regard to the latter, the set-shifting task described here (as well as the cross-maze set-shifting task26) utilize a restricted set of stimuli and responses. Unlike the digging task, on which novel exemplars (e.g., unfamiliar scents or digging media) can be used to construct new attentional sets at each stage11,19, the operant set-shifting task necessarily requires choosing between two stimuli that are familiar to the animal &#8212; either the left vs. right cue light, or the left vs. right position. This means that the operant and cross-maze set-shifting tasks involve response conflict as well as strategy shifting, although the concept of shifting one&#8217;s strategy to a new, previously irrelevant stimulus dimension is preserved20,23. On a related note, the set-shifting and reversal operant tasks as described here do not allow for a third stimulus dimension, as in the digging task which may include digging media, odor, and texture11,19. However, we do not consider this a fatal flaw, as the operant set-shifting task still requires the animal to suppress the previously relevant discrimination strategy and attend to a previously ignored stimulus dimension. Additionally, it seems conceivable that modifications to the equipment and task parameters could support the addition of a third stimulus dimension, such as auditory cues or odor, although these additions would likely make learning more difficult and less amenable to single-day pharmacological tests.
Finally, a potential limitation of any operant-based task is the loss of direct information regarding rat behavior &#8212; i.e., the experimenter is no longer watching the rat. We feel that the advantages in objectivity and data collection speed conferred by automation more than make up for this loss, and cameras mounted in the operant chambers are a relatively easy way to restore individual visual access if desired.
There are a number of steps that can be taken to maximize success using these operant tasks. First, the importance of handling the animals before training begins cannot be overstated; as with any behavioral task, well-handled animals are easier to work with, are less stressed, and tend to produce less variable data. Second, some pilot testing may be necessary to determine the best time of day to conduct testing; we test during the light cycle, and find that performance is optimal when animals are tested near the end of this cycle (e.g., approximately 4:00 pm for a light cycle ending at 7:00 pm). Third, care should be taken to confirm that stable performance is established at each pretraining stage before an animal is advanced to the next step. For example, consistent and robust performance at the retractable lever training stage is an excellent predictor of proficient performance on the &#8220;set&#8221; discrimination task. Regarding the equipment, although all steps are automated, experimenter intervention remains necessary to confirm that all components are in working order. For example, an equipment check should be run daily (or more than once a day, if large number of animals are being tested) to ensure that all lights, levers, and reward delivery systems are operational. In particular, malfunctions in reward delivery systems (particularly pellet dispensers) can drastically affect performance. An unusually high number of omissions on a given day may indicate an issue with reward delivery equipment, and thus data output should be checked every day by an experimenter familiar with the task and expected performance levels. In the absence of an equipment malfunction, a high number of omissions may indicate other problems with motivation or animal health. If an animal is otherwise healthy, food restriction may be increased to take the animal to 80-85% of the free-feeding weight for a short time until performance recovers.
These set-shifting and reversal tasks can be used in a variety of experimental paradigms. For example, the effects of manipulations such as lesions, developmental treatments, dietary manipulations, long-term pharmacological treatment, or genetic modifications could be investigated. While the effect of a treatment on the set-shifting or reversal stage may be of primary interest, note that since such chronic or permanent treatments must necessarily be administered before training begins, effects on multiple stages of performance (particularly on the initial discrimination or &#8220;set&#8221;) must also be examined21. The use of acute manipulations, such as pharmacological treatments or temporary neuroanatomical inactivations, are particularly well-suited to these tasks. In such cases, the addition of a third group (as illustrated in Figure 2) is useful; thus, the primary experimental group receives the manipulation of interest on the day of shift or reversal, while one control group receives the manipulation on the day of initial discrimination or &#8220;set&#8221; to test for broad effects on learning, and a second control group receives no manipulations (or sham treatments) on both days20,22. Note that for such acute manipulation studies, it is advisable to match rats for performance during the learning of the initial set and allocate them to the experimental group and (second) control group accordingly. This minimizes the possibility that treatment-induced differences in performance may be confounded by individual variations in how readily rats learn to discriminate between stimuli. Furthermore, if an experiment requires testing of multiple cohorts over weeks or months, each cohort should include animals from all experimental groups. For example, a study testing the effects of acute pharmacological manipulations during a shift may require 48 rats in total and 3 experimental groups, tested in three cohorts of 16 animals each. In this case, each cohort should contain 5-6 rats in each experimental group. Ideally, the statistical analyses should include a factor that confirms there were no differences in performance across each cohort of rats. Finally, these operant tasks may be particularly useful for applying in vivo recording techniques, including microdialysis, voltammetry, and electrophysiology, due to components such as the controlled environment, precise timing of stimulus presentation and responses, and restricted movements of the animals which are not available or practical in the cross-maze or digging tasks.

High-level cognitive processes including rule generation, behavioral selection, and strategy evaluation are collectively referred to as &#8220;executive function&#8221; or &#8220;behavioral flexibility1.&#8221; Such processes are crucial to normal cognitive function, and may be impaired in such diverse disorders as schizophrenia, alcoholism, depression, stroke, and Alzheimer&#8217;s disease2-7. The regulation of executive function processes is primarily mediated by areas in the frontal cortex, including the dorsolateral prefrontal cortex and the orbitofrontal cortex in humans8-10.
The development of tasks to assess executive function and/or behavioral flexibility in nonhuman animals, particularly rodents, has greatly advanced the understanding of the neurobiology of cognition11-14. Such tasks have made it possible to separately measure distinct components of behavioral flexibility, including strategy shifting and reversal learning. Strategy shifting refers to the ability to actively suppress a previously learned response strategy while acquiring a new, competing strategy, particularly across stimulus dimensions (extradimensional shift) &#8212; e.g., switching from performing a visually-based discrimination (red vs. green, where red is &#8220;correct&#8221; and tactile stimuli are irrelevant) to performing a tactile discrimination (smooth vs. rough, where smooth is &#8220;correct&#8221; and visual stimuli are now irrelevant). On the other hand, reversal learning also involves a change in response strategy, but within the same stimulus dimension &#8212; e.g., in the &#8220;red vs. green&#8221; example, if red was previously correct, a reversal would dictate that green is now correct, while tactile stimuli would remain irrelevant.
Several tasks have been developed to investigate behavioral flexibility in rodents. The cross-maze task requires an animal to first learn either a direction-based rule (e.g., &#8220;always turn right&#8221;) or a visual-based rule (e.g., &#8220;always turn toward the visual cue&#8221;) to a certain criterion of performance. Then, the animal is required to unexpectedly shift either across modality to the opposite rule (strategy shifting, originally referred to as a &#8220;nonreversal shift&#8221;15) or shift within modality to the opposite contingency (reversal learning)13,14,16. Such tasks are sensitive to disruptions in cortical and subcortical networks, involving the prefrontal cortex, thalamus, and striatum1,13,14,16-18. Another type of attentional set-shifting task (sometimes referred to as the digging task) requires training animals to discriminate between two containers that differ along two or three stimulus dimensions (digging media, odor, and/or external texture). Similar to the cross-maze task, animals are then required to shift either across dimensions (strategy shifting) or within the same dimension (reversal learning), and these tasks are similarly sensitive to frontal cortex manipulations11,19. An advantage of this task is that during the extra-dimensional strategy shift, rats are presented with novel sets of stimuli (exemplars), which ensures that performance impairments during this stage are likely attributable to disruptions in the ability to shift attentional set to different aspects of compound stimuli, rather than an impaired ability to stop approaching a specific stimulus previously associated with reward. However, this feature also makes it more difficult to ascertain the specific nature of a deficit during a set shift.
Although the tasks described above have been well documented in the literature, they both suffer from a number of procedural disadvantages, primarily the length of time it takes to test animals. In both the cross-maze task and the digging task, only one animal may be tested at a time; furthermore, testing must be administered in real time by a dedicated experimenter, and may take up to several hr per day per animal. In addition, the presentation of stimuli and the recording of behavioral responses in both types of tasks are manually controlled by an experimenter, and are thus vulnerable to human error and subjective interpretation.
Here, we describe an automated method for assessing strategy shifting and reversal learning in the rat, using operant procedures that streamline stimulus control and data presentation, and dramatically improve the rate of data collection and throughput20,21. The methods used to shape and train rats are described, as well as the components of the task itself and the analysis of the resulting data. We have found that like the cross-maze and digging tasks, these automated tasks are sensitive to disruptions in prefrontal and subcortical circuitry, as well as to a neurodevelopmental manipulation that models schizophrenia20-23.

<table border="0" cellpadding="0" cellspacing="0" width="823">
	<tbody>
		<tr height="34">
			<td height="34" style="height:34px;width:231px;">Name of Material/ Equipment</td>
			<td style="width:153px;">Company</td>
			<td style="width:139px;">Catalog Number</td>
			<td style="width:301px;">Comments/Description</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:231px;">Behavioral Chamber Package with Retractable Levers</td>
			<td style="width:153px;">Med Associates, Inc.</td>
			<td style="width:139px;">MED-008-B2</td>
			<td style="width:301px;">Required components include two retractable levers, two stimulus lights, houselight, and reinforcement delivery system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">MED-PC software</td>
			<td style="width:153px;">Med Associates, Inc.</td>
			<td style="width:139px;">SOF-735</td>
			<td style="width:301px;"></td>
		</tr>
		<tr height="42">
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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.

Acute, reversible inactivation of the prefrontal cortex can be accomplished by infusion of the local anesthetic bupivacaine hydrochloride (0.75%, 0.5 &#181;l) into the prelimbic region via a surgically implanted infusion cannula20 approximately 10 min prior to testing. Furthermore, the effects of inactivation during either the first task (&#8220;Set&#8221;) or the second task (&#8220;Shift&#8221; or &#8220;Reversal&#8221;) can be assessed to investigate possible general effects on learning. Figure 2 illustrates the results of such inactivations on animals performing the Cue-to-Response strategy-shifting sequence. Prefrontal inactivation on the first day, the Cue/&#8220;Set&#8221; task, did not impair performance (Figure 2A), suggesting that the medial prefrontal cortex is not necessary for initial discrimination learning. However, prefrontal inactivation on the second day, the Response/&#8220;Shift&#8221; task, significantly impaired performance in that animals required a substantially greater number of trials to reach criterion performance (Figure 2B). When the prefrontal cortex was inactivated, animals made more perseverative-like errors than never-reinforced errors on the Shift task (Figure 2C). These findings replicate previous data regarding the importance of the medial prefrontal cortex for strategy shifting and, in particular, in suppressing a previously learned strategy13,20.
Conversely, animals trained in the Reversal of Response sequence did not show this prefrontal dependency. Animals receiving inactivation of the prefrontal cortex on the &#8220;Reversal&#8221; day did not differ from saline-infused animals on either the initial response discrimination (Figure 3A) or the subsequent reversal (Figure 3B)20.These findings are consistent with previous research showing that the orbitofrontal cortex, not the medial prefrontal cortex, regulates reversal learning on a variety of tasks12,19,26, including an operant task similar to the one described here27.
<img alt="Figure 2" src="/files/ftp_upload/52387/52387fig2highres.jpg" /> 
Figure 2. Inactivation of the Prefrontal Cortex Impairs Strategy Shifting. A, Trials to criterion on the initial Cue discrimination task (&#8220;Set&#8221;) by rats receiving infusions of saline or bupivacaine (Bupi) into the medial prefrontal cortex on the set day. Prefrontal inactivation had no effect on initial acquisition. B, Trials to criterion on the shift to the Response task (&#8220;Shift&#8221;) following medial prefrontal infusions of either saline or bupivacaine on the shift day. Inactivation of the prefrontal cortex impaired the strategy shift to the response task. C, Types of errors committed by animals on the shift day. Prefrontal inactivation prior to the shift task (&#8220;sal-bupi&#8221; group) led to an increase in perseverative-like errors. *, p &#60; .05 vs. saline-saline. This figure has been modified from Floresco et al.20 <a href="https://www.jove.com/files/ftp_upload/52387/52387fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52387/52387fig3highres.jpg" /> 
Figure 3. Inactivation of the Prefrontal Cortex Leaves Reversal Learning Intact. A, Trials to criterion during the initial response discrimination training by rats that would subsequently receive infusions of saline or bupivacaine (Bupi) into the medial prefrontal cortex prior to reversal training. No differences were seen. B, Trials to criterion during the reversal of the response discrimination, following medial prefrontal infusions of either saline or bupivacaine. Prefrontal inactivation did not impair reversal learning. This figure has been modified from Floresco et al.20
Data presented in Figure 4 provides an example of how requiring rats to perform &#8220;reminder&#8221; trials using the old rule prior to a strategy shift can aid in data interpretation. In this study (Enomotor and Floresco, unpublished observations), rats were matched for performance on acquiring a visual cue rule on Day 1 (Figure 4A). On Day 2, rats received vehicle or 0.2 mg/kg haloperidol. At the start of the Day 2 test session, they were given 20 trials where they were required to respond using the visual cue rule acquired on Day 1, after which the rule switched mid-session to a response discrimination. As displayed in Figure 4B, this treatment impaired retrieval of the visual cue rule during the first 20 reminder trials of the session. Subsequently, haloperidol-treated rats required fewer trials to achieve criterion (Figure 4C) and made fewer perseverative errors (Figure 4D) on the strategy shift. Note that had we not used the reminder trials prior to the shift, these data may have been interpreted as an improvement in set shifting by haloperidol. However, the impairment during the rule retrieval phase suggests that these effects are better understood as impaired memory for the previously acquired rule, which may have led to less response conflict when rats were required to learn a novel rule and thus faster shifting. 
<img alt="Figure 4" src="/files/ftp_upload/52387/52387fig4highres.jpg" /> 
Figure 4. Impaired Rule Retrieval and Facilitated Set-Shifting Induced by Haloperidol Treatment. A, Trials to criterion on visual cue discrimination from rats that were to receive vehicle (saline) or the dopamine D2 antagonist haloperidol (0.2 mg/kg) prior to the strategy shifting sequence on the following day. Animals in both groups showed comparable pre-drug performance. B, At the beginning of testing on Day 2, rats received 20 reminder trials where they were required to continue to respond using the visual cue rule from Day 1. Treatment with haloperidol significantly decreased accuracy during these reminder trials. C, After the 20 reminder trials, the rule shifted mid-session to a response discrimination. Haloperidol treated rats required fewer trials to achieve criterion during the shift. D. Haloperidol treatment also reduced perseverative errors. Although these data could suggest improved performance, the impairment in rule retrieval display in B indicates that the apparent &#8220;enhanced strategy shifting&#8221; is more likely attributable to reduced interference from the previously acquired rule. Enomoto and Floresco, unpublished observations. *, p &#60; .05 vs. vehicle.
The neonatal ventral hippocampal lesion (NVHL) manipulation has been used to model some aspects of schizophrenia in rats28, particularly cognitive impairments29,30. Briefly, an excitotoxic lesion is administered to the hippocampus of 7-day old rats, and testing is carried out in adults (60+ days postnatal). This models the hypothesized developmental trajectory of schizophrenia28. Figure 5 illustrates performance of NVHL and control rats on the pre-exposed version of the Set-Shifting: Response to Cue sequence. NVHL rats are unimpaired at learning the first rule (Response/&#8221;Set&#8221;, Figure 5A), but are dramatically impaired at shifting to the new rule (Cue/&#8221;Shift&#8221;) as shown by the increase in the number of trials required to reach criterion (Figure 5B). Moreover, this deficit was due primarily to an increase in perseverative errors, as shown in Figure 5C, suggesting prefrontal abnormalities20,21. These results confirm previous findings of impaired strategy shifting in NVHL animals using the cross-maze task29.
Similar to the data from prefrontally-inactivated animals shown above, NVHL animals were not impaired at reversal learning (Figure 6A,B), although they were slower to respond (Figure 6C,D). This negative finding implies that the observed strategy shifting deficits are not attributable to a simple inability to switch between stimuli21.
<img alt="Figure 5" src="/files/ftp_upload/52387/52387fig5highres.jpg" /> 
Figure 5. Impaired Set-Shifting in the NVHL Model of Schizophrenia. Performance on the pre-exposed version of the set-shifting sequence (Response-to-Cue) in NVHL and sham control animals. A, NVHL animals were unimpaired on the Response (&#8220;Set&#8221;) task. B, However, NVHL animals required significantly more trials than shams to reach criterion on the Visual Cue (&#8220;Shift&#8221;) task. C, Errors on the &#8220;Shift&#8221; day. NVHL animals made more perseverative errors than sham animals, but did not differ on regressive or never-reinforced errors. *, p &#60; .05 vs. sham. This figure has been modified from Placek et al.21
<img alt="Figure 6" src="/files/ftp_upload/52387/52387fig6highres.jpg" /> 
Figure 6. Lack of NVHL Impairment on Reversal Learning. A,B, NVHL and sham animals did not differ in their ability to acquire either the initial Response Learning task (&#8220;Set&#8221;), or the Response Reversal. C,D, NVHL animals were slower than shams to respond on both the &#8220;Set&#8221; and the &#8220;Reversal&#8221; tasks. This figure has been modified from Placek et al.21
Finally, pilot testing has indicated that animals are virtually unable to learn a reversal of the Cue task, i.e., to press the lever opposite the illuminated stimulus light. Five of six animals tested completed 450 reversal trials (3 days) without reaching criterion, and the sixth animal required 418 trials (Brady, unpublished observations; data not shown). This is likely because the stimulus lights are very salient and attractive cues that make it very difficult for rats to direct responding away from them. Thus, this testing sequence is not recommended.

Watch this Scientific Journal Video about Operant Procedures for Assessing Behavioral Flexibility in Rats at JoVE.com

Operant Procedures for Assessing Behavioral Flexibility in Rats

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 ...

operant-procedures-for-assessing-behavioral-flexibility-in-rats

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20.7K 조회수.  St. Mary's College of Maryland. 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. 

비디오: Operant Procedures for Assessing Behavioral Flexibility in Rats

A Novel Procedure for Evaluating the Reinforcing Properties of Tastants in Laboratory Rats: Operant Intraoral Self-administration

This paper describes a novel method for studying the bio-behavioral basis of addiction to food. This method combines the surgical component of taste reactivity with the behavioral aspects of operant self-administration of drugs. Under very brief general anaesthesia, rats are implanted with an intraoral (IO) cannula that allows delivery of test solutions directly in the oral cavity. Animals are then tested in operant self-administration chambers whereby they can press a lever to receive IO infusions of test solutions. IO self-administration has several advantages over experimental procedures that involve drinking a solution from a spout&#160;or operant responding for solid pellets or solutions delivered in a receptacle. Here, we show that IO self-administration can be employed to study self-administration of high fructose corn syrup (HFCS). Rats were first tested for self-administration on a progressive ratio (PR) schedule, which assesses the maximum amount of operant behavior that will be emitted for different concentrations of HFCS (i.e.&#160;8%, 25%, and 50%). Following this test, rats self-administered these concentrations on a continuous schedule of reinforcement (i.e.&#160;one infusion for each lever press) for 10 consecutive days (1 session/day; each lasting 3 hr), and then they were retested on the PR schedule. On the continuous reinforcement schedule, rats took fewer infusions of higher concentrations, although the lowest concentration of HFCS (8%) maintained more variable self-administration. Furthermore, the PR tests revealed that 8% had lower reinforcing value than 25% and 50%. These results indicate that IO self-administration can be employed to study acquisition and maintenance of responding for sweet solutions. The sensitivity of the operant response to differences in concentration and schedule of reinforcement makes IO self-administration an ideal procedure to investigate the neurobiology of voluntary intake of sweets.

The current study evaluates a novel procedure for assessing the reinforcing effects of palatable solutions in laboratory rats: intraoral self-administration. To this end, operant responding (i.e.&#160;lever pressing) for intraoral infusions of sweet solutions at different concentrations was measured on continuous and progressive ratios schedules of reinforcement.

This paper describes a novel method for studying the bio-behavioral basis of addiction to food. This method combines the surgical component of taste ...

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

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

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

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

Moderate Prenatal Alcohol Exposure and Quantification of Social Behavior in Adult Rats

Alterations in social behavior are among the major negative consequences observed in children with Fetal Alcohol Spectrum Disorders (FASDs). Several independent laboratories have demonstrated robust alterations in the social behavior of rodents exposed to alcohol during brain development across a wide range of exposure durations, timing, doses, and ages at the time of behavioral quantification. Prior work from this laboratory has identified reliable alterations in specific forms of social interaction following moderate prenatal alcohol exposure (PAE) in the rat that persist well into adulthood, including increased wrestling and decreased investigation. These behavioral alterations have been useful in identifying neural circuits altered by moderate PAE1, and may hold importance for progressing toward a more complete understanding of the neural bases of PAE-related alterations in social behavior. This paper describes procedures for performing moderate PAE in which rat dams voluntarily consume ethanol or saccharin (control) throughout gestation, and measurement of social behaviors in adult offspring.

The goal of the protocol presented here is to describe procedures to expose rats to moderate levels of alcohol during prenatal brain development and to quantify resulting alterations in social behavior during adulthood.

Alterations in social behavior are among the major negative consequences observed in children with Fetal Alcohol Spectrum Disorders (FASDs). Several ...

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

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

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

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

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

Battery of Behavioral Tests Assessing General Locomotion, Muscular Strength, and Coordination in Mice

Behavioral testing is used in pre-clinical trials to assess the phenotypic effects and outcomes that a particular disease or treatment has on the animal&#39;s wellbeing and health. There are numerous behavioral tests that may be applied. We selected a test for general locomotion, the open field test (OFT); a test for muscular strength, the mesh test (MT); and a test for coordination, the rotarod test (RR). Testing can be accomplished on a weekly or monthly basis. As a test for general locomotion, the OFT works by objectively monitoring movement parameters while the mouse is in an open field apparatus. The field is generally a 2&#39; x 2&#39; box, and the movements are recorded through laser sensing or through video capture. The mouse is placed in the center of the open field and allowed to move freely for the test. The MT uses the latency for a mouse to fall off an inverted screen as a measure of muscular strength. A mouse is placed on a screen, which is inverted over a clear box, and is timed for their latency to fall. Three trials are performed, with the best of the three trials scored for that day. A score of 60 s is the maximum time a mouse is left inverted. Mice are given a 5-min rest period between mesh test trials. Lastly, an accelerated protocol on the RR assesses motor coordination and endurance. During a trial, a mouse walks on a rotating rod as it increases in speed from 4 rpm to 40 rpm over 5 min. The trial ends when the mouse touches the magnetized pressure sensor upon falling. Each mouse undergoes three trials, and the best trial is scored for that day. This combined behavioral data allows for the global assessment of mobility, coordination, strength, and movement of the test animals. At least two out of the three behavioral testing measures must show improvement for an animal to qualify as having overall improved motor function.

Behavioral testing is used in pre-clinical trials to assess the phenotypic effects of a disease or treatment on an animal's wellbeing. In order to globally assess motor functioning, we selected tests for general locomotion, muscular strength, and coordination: the open field test, the mesh test, and the rotarod test, respectively.

Behavioral testing is used in pre-clinical trials to assess the phenotypic effects and outcomes that a particular disease or treatment has on the ...

Detecting Behavioral Deficits in Rats After Traumatic Brain Injury

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

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

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

A Wireless, Bidirectional Interface for In Vivo Recording and Stimulation of Neural Activity in Freely Behaving Rats

In vivo electrophysiology is a powerful technique to investigate the relationship between brain activity and behavior at a millisecond and micrometer scale. However, current methods mostly rely on tethered cable recordings or only use unidirectional systems, allowing either recording or stimulation of neural activity, but not at the same time or same target. Here, a new wireless, bidirectional device for simultaneous multichannel recording and stimulation of neural activity in freely behaving rats is described. The system operates through a single portable head stage that both transmits recorded activity and can be targeted in real-time for brain stimulation using a telemetry-based multichannel software. The head stage is equipped with a preamplifier and a rechargeable battery, allowing stable long-term recordings or stimulation for up to 1 h. Importantly, the head stage is compact, weighs 12 g (including battery) and thus has minimal impact on the animal&#180;s behavioral repertoire, making the method applicable to a broad set of behavioral tasks. Moreover, the method has the major advantage that the effect of brain stimulation on neural activity and behavior can be measured simultaneously, providing a tool to assess the causal relationships between specific brain activation patterns and behavior. This feature makes the method particularly valuable for the field of deep brain stimulation, allowing precise assessment, monitoring, and adjustment of stimulation parameters during long-term behavioral experiments. The applicability of the system has been validated using the inferior colliculus as a model structure.

A Wireless, Bidirectional Interface for In Vivo Recording and Stimulation of Neural Activity in Freely Behaving Rats

A wireless, bidirectional system for multi-channel neural recordings and stimulation in freely behaving rats is introduced. The system is light and compact, thus having minimal impact on the animal&#180;s behavioral repertoire. Moreover, this bidirectional system provides a sophisticated tool to assess causal relationships between brain activation patterns and behavior.

In vivo electrophysiology is a powerful technique to investigate the relationship between brain activity and behavior at a millisecond and micrometer ...

The Knob Supination Task: A Semi-automated Method for Assessing Forelimb Function in Rats

Tasks that accurately measure dexterity in animal models are critical to understand hand function. Current rat behavioral tasks that measure dexterity largely use video analysis of reaching or food manipulation. While these tasks are easy to implement and are robust across disease models, they are subjective and laborious for the experimenter. Automating traditional tasks or creating new automated tasks can make the tasks more efficient, objective, and quantitative. Since rats are less dexterous than primates, central nervous system (CNS) injury produces more subtle deficits in dexterity, however, supination is highly affected in rodents and crucial to hand function in primates. Therefore, we designed a semi-automated task that measures forelimb supination in rats. Rats are trained to reach and grasp a knob-shaped manipulandum and turn the manipulandum in supination to receive a reward. Rats can acquire the skill within 20 &plusmn; 5 days. While the early part of training is highly supervised, much of the training is done without direct supervision. The task reliably and reproducibly captures subtle deficits after injury and shows functional recovery that accurately reflects clinical recovery curves. Analysis of data is performed by specialized software through a graphical user interface that is designed to be intuitive. We also give solutions to common problems encountered during training, and show that minor corrections to behavior early in training produce reliable acquisition of supination. Thus, the knob supination task provides efficient and quantitative evaluation of a critical movement for dexterity in rats.

This manuscript describes a semi-automated task that quantifies supination in rats. Rats reach, grasp, and supinate a spherical manipulandum. The rat is rewarded with a pellet if the turn angle exceeds a criterion set by the user. This task increases throughput, sensitivity to injury, and objectivity compared to traditional tasks.

Tasks that accurately measure dexterity in animal models are critical to understand hand function. Current rat behavioral tasks that measure dexterity ...

Eye Tracking During Visually Situated Language Comprehension: Flexibility and Limitations in Uncovering Visual Context Effects

The present work is a description and an assessment of a methodology designed to quantify different aspects of the interaction between language processing and the perception of the visual world. The recording of eye-gaze patterns has provided good evidence for the contribution of both the visual context and linguistic/world knowledge to language comprehension. Initial research assessed object-context effects to test&#160;theories of modularity in language processing. In the introduction, we describe how subsequent investigations have taken the role of the wider visual context in language processing as a research topic in its own right, asking questions such as how our visual perception of events and of speakers contributes to&#160;comprehension informed by&#160;comprehenders&#39; experience. Among the examined aspects of the visual context are actions, events, a speaker&#39;s gaze, and emotional facial expressions, as well as spatial object configurations. Following an overview of the eye-tracking method and its different applications, we list the key steps of the methodology in the protocol, illustrating how to successfully use it to study visually-situated language comprehension. A final section presents three sets of representative results and illustrates the benefits and limitations of eye tracking for investigating the interplay between the perception of the visual world and language comprehension.

The present article reviews an eye-tracking methodology for studies on language comprehension. To obtain reliable data, key steps of the protocol must be followed. Among these are the correct set-up of the eye tracker (e.g., ensuring good quality of the eye and head images) and accurate calibration.

The present work is a description and an assessment of a methodology designed to quantify different aspects of the interaction between language processing ...

Three Laboratory Procedures for Assessing Different Manifestations of Impulsivity in Rats

The present article provides a guide for the conduction and analysis of three conditioning-based protocols to evaluate impulsivity in rats. Impulsivity is a meaningful concept because it is associated with psychiatric conditions in humans and with maladaptive behavior in non-human animals. It is believed that impulsivity is composed of separate factors. There are laboratory protocols devised to assess each of these factors using standardized automated equipment. Delay discounting is associated with the incapacity to be motivated by delayed outcomes. This factor is evaluated through intertemporal choice protocols, which consist&#160;of presenting the individual with a choice situation involving an immediate reward and a larger but delayed reward. Response inhibition deficit is associated with the incapacity to withhold prepotent responses. Differential reinforcement of low rates (DLR) and feature-negative discrimination protocols assess the response inhibition deficit factor of impulsivity. The former imposes a condition to a motivated individual in which most wait a minimum period of time for a response to be rewarded. The latter evaluates the capacity of individuals to refrain from food seeking responses when a signal of the absence of food is presented. The purpose of these protocols is to construct an objective quantitative measure of impulsivity, which serves to make cross-species comparisons, allowing the possibility of translational research. The advantages of these particular protocols include their easy set-up and application, which stems from the relatively small amount of equipment needed and the automated nature of these protocols.

We present three protocols that assess different forms of impulsivity in rats and other small mammals. Intertemporal choice procedures evaluate the tendency to discount the value of delayed outcomes. Differential reinforcement of low rates and feature-negative discrimination evaluate response inhibition capacity with and without punishment for inappropriate responses, respectively.

The present article provides a guide for the conduction and analysis of three conditioning-based protocols to evaluate impulsivity in rats. Impulsivity is ...