The Operant Conditioning Chamber is a behavioral task used in neuroscience to study executive function by assessing behavioral flexibility. Executive function refers to high-level cognitive processes including rule generation, behavioral selection, and strategy evaluation, and is primarily regulated by areas in the frontal cortex (Lie et al, 2006). Such processes are crucial to normal cognitive function, and may be impaired in multiple neurological and psychiatric disorders such as schizophrenia, depression, stroke, and neurodegenerative diseases (e.g. Snyder, 2013).

The Operant Conditioning Chamber allows the study of behavioral flexibility – the adaptive changes in an animal’s behavior in response to environmental changes – by training animals to perform specific actions (pressing a lever) in response to specific stimuli (light cue), and then assessing their ability to actively suppress the initially learned response strategy to acquire a new, competing response.

This type of behavioral tasks is commonly used in the study of the neurobiology of cognition (e.g. Bissonette et al, 2008), allowing to separately assess different aspects of behavioral flexibility, namely strategy shifting and reversal learning. Such tasks are sensitive to disruptions in prefrontal and subcortical circuitry, to neurodevelopmental and neurological manipulations modeling psychiatric or neurological diseases, and to the effect of therapeutic interventions on those models (Brady and Floresco, 2015, Enomoto et al, 2011).

The Operant Conditioning Chamber was created by BF Skinner in the early 1930s (Krebs, 1983). Since then, numerous modifications have been introduced, including its automation. Using an automated Operant Conditioning Chamber allows the assessment of strategy shifting and reversal learning in rodent using a simplified procedure with improved rate of data collection and throughput.

The apparatus is made up of a chamber equipped with (at least) two retractable levers, two stimulus lights, and a reinforcement dispenser. The levers are placed on either side of the reinforcement dispenser with one stimulus light located above each lever.

Stimulus presentation, lever operation, and data collection can be automatically controlled through the Noldus EthoVision® XT software. The position of the cue light, the lever selected by the animal, whether the animal made a correct, incorrect or no response (omission), and the latency to make a choice can all be automatically recorded.

The purpose of the Operant Conditioning Chamber is to assess executive function/behavioral flexibility in rodents in a control vs. disease model/intervention group, by assessing strategy shifting and/or reversal learning.

There are several versions of protocols to be used with the Operant Conditioning Chamber, depending in the specific behavioral assessment to be made.

Below are examples of protocols that allow the assessment of different aspects of behavioral flexibility. A more detailed version of these protocols can be found in Brady & Floresco, 2015.

Pre-training for the Operant Conditioning Chamber

Animals can be housed individually or in groups, depending on the experimental needs. Single housing should be used for experiments requiring food-restriction, such as to allow a better control of food intake.

After a few days of habituation to the testing facilities, without handling or food restriction, animals should be handled daily for around 5 min, and for at least 3 days before beginning behavioral testing.

The animals’ weight should be recorded on the first day of handling, when food restriction begins. Over the 3 days of handling, animals’ daily food intake should be gradually reduced to bring them to a target weight of 85-90% of their initial weight. At the end of each handling session, 10-20 reward pellets should be placed in the animal’s cage to habituate them to the reinforcement that will be used in the tasks.

Each animal should always be tested in the same operant chamber and at approximately the same time of day.

Evaluation of executive function/behavioral flexibility using the Operant Conditioning Chamber

Training – may take about 10-20 days.

1. a) Training to press extended lever

Place the animal in the chamber and deliver one reinforcement for each lever press. Perform daily 30-min training sessions until animals meet a minimum response criterion (e.g. 50-60 presses per session for two consecutive days for both levers extended).

1. b) Training to press retractable lever

Proceed as in a) such as to familiarize the animals with the extension and retraction of the levers. Alternate lever extensions such that there are 45 left-lever trials and 45 right-lever trials, but no more than two consecutive trials extending the same lever.

Extend the selected lever. If the animal presses the lever within 10 sec, deliver one reinforcement. If it does not respond within 10 sec, retract the lever and record an omission.

Begin trials every 20 sec throughout the session. Continue retractable lever training sessions until animals meet a minimum criterion of five or fewer omissions for two consecutive days. For studies using acute manipulations (e.g., drug testing), use a fixed number of days to ensure that all rats receive similar exposure to the levers.

1. c) Assessment of side preference.

Conduct side preference testing immediately after the last session of retractable lever training. Do not illuminate the stimulus lights during this phase of training.

Perform 7 trials, each composed of 2-8 sub-trials separated by a 20-sec interval. On each sub-trial, extend both levers into the chamber for 10 sec or until a lever press response is made.

Reinforce the response on the first sub-trial of each trial, and record it as the “initial response”; do not reinforce responses on the same lever on subsequent sub-trials within the same trial. Following the initial response on each trial, reinforce the first response on the opposite lever, and then terminate that trial.

Allow up to six subsequent responses on the same lever within a trial, after which give a forced sub-trial – extend only the opposite lever until a response is made. Thus, within each trial (containing up to 8 sub-trials), an animal is required to respond at least once on each lever.

Define each animal’s side preference as the side on which the majority of initial responses took place (at least 4/7 trials). If an animal disproportionately responds to one lever throughout the session (defined as greater than a 2:1 ratio), record that side as the animal’s preference.

Begin testing on the next consecutive day after the side preference test.

Testing

General procedures

Animals may be tested in one of three sequences, each involving two different tasks. Conduct each sequence on consecutive days; test animals in one task (“Set” – the initial discrimination learning) on one day, followed by a second task (“Shift” or “Reversal”) on the following day.

Strategy shifting

Set-shifting: Cue to Response

1. Cue task (Set task in this sequence) – reinforcement for responding on the lever below the illuminated stimulus light (cue).
   1. Begin each trial with both levers retracted.
   2. Illuminate either the left or right stimulus light for 3 sec; then extend both levers into the chamber for 10 sec or until a response occurs.
   3. Reinforce only a correct response on the signaled lever. Upon a response on either lever, retract the levers.
   4. Begin trials every 20 sec throughout the session. No more than two consecutive trials should occur with the same stimulus light illuminated.
   5. Continue trials until an animal has reached criterion (e.g. 10 consecutive correct responses) and has completed a minimum of 30 trials, or until 150-200 trials are completed without reaching criterion.
   6. If criterion is not reached on the first day, test the animal on the Cue task again on the second day, but remove the requirement to complete a minimum of 30 trials. If criterion is not reached on the second day, test the animal on the third day following the same procedure. Remove animals that do not reach criterion within 3 days.

2. Response task (Shift task in this sequence) – reinforcement for responding on the lever opposite their side preference, regardless of stimulus light (cue) illumination.
   1. Begin each trial with both levers retracted.
   2. Illuminate either the left or right stimulus light for 3 sec; then extend both levers into the chamber for 10 sec or until a response occurs.
   3. Reinforce only a response on the correct position lever – opposite of the animal’s side preference. Upon a response on either lever, retract the levers.
   4. Begin trials every 20 sec throughout the session. No more than two consecutive trials should occur with the same stimulus light illuminated.
   5. Continue trials until an animal has reached criterion (e.g. 10 consecutive correct responses) or until 150 trials are completed without reaching criterion.
   6. If criterion is not reached on the first day, test the animal on the Response task again on the second day. If criterion is not reached on the second day, test the animal on the third day following the same procedure. For animals that do not reach criterion within 3 days, assign a maximal score for trials to criterion that represents the number of trials experienced (i.e., 450 trials for 3 days of 150 trials each).

Set-shifting: Response to Cue.

This sequence benefits from the addition of the visual-cue light pre-exposure condition to pre-training (see modifications).

1. Response task (Set task in this sequence) – reinforcement for responding on the lever opposite their side preference, regardless of stimulus light (cue) illumination.
   1. Proceed with testing as detailed in the Response task (2) above.
   2. Have animals complete a minimum of 30 trials on this task.
2. Cue task (Shift task in this sequence) – reinforcement for responding on the lever below the illuminated stimulus light (cue).
   1. Proceed with testing as detailed in the Cue task (1) above. The minimum of 30 trials completed is not needed.

Reversal learning: reversal of response.

1. Response task (Set task in this sequence) – reinforcement for responding on the lever opposite their side preference, regardless of stimulus light (cue) illumination.
   1. Proceed with testing detailed in the Response task above.
   2. Have animals complete a minimum of 30 trials on this task.
2. Reversal of the response task (Reversal task) – reinforcement for responding on the opposite lever as on the first task, i.e., the lever corresponding to their original side preference.
   1. Proceed with testing as detailed in the Response task above, with the exception that the reinforced lever position is now equal to the animal’s original side preference.

Behavioral measures

1. Trials to criterion on both the Set task and the Shift task (measure of accuracy) – the number of trials required to complete 10 consecutive trials, including those 10 trials. The number of omissions should be factored out – e.g., if a rat requires 100 trials to achieve criterion and makes 10 omissions, the actual trials to criterion is 90.
2. Errors to criterion on both the Set task and the Shift task (measure of accuracy) – number of errors before criterion; can be more sensitive than Trials to Criterion and is not affected by increased omission rates.
3. Perseverative or regressive shift errors in a set-shifting sequence – count an error when an animal responds incorrectly on the Shift task by following the rule that was correct on the previous day’s Set task. To divide into perseverative or regressive:
   1. Divide the Shift session into blocks of 16 consecutive completed trials (do not include omitted trials). Within each block, identify shift errors. There will be a maximum of 8 possible errors in each 16-trial block.
   2. Score identified errors as perseverative until less than six of them are made within a block.
   3. Beginning with the next block and continuing through the end of the task, score errors as regressive.
   4. If the animal was tested in the Shift task on more than one day, continue scoring errors as if the blocks were contiguous.
4. Never-reinforced shift errors in a set-shifting sequence – count an error when an animal responds incorrectly on the Shift task with a response that was not correct on either the Set or the Shift task.
5. Perseverative and regressive reversal errors. To divide into perseverative or regressive:
   1. Divide the Reversal session into blocks of 16 consecutive completed trials. Count the errors in each block (max 16 errors).
   2. Score errors as perseverative until fewer than 10 of them are made within a block.
   3. Beginning with the next block and continuing through the end of the task, score errors as regressive.
   4. If the animal was tested in the Reversal task on more than one day, continue scoring errors as if the blocks were contiguous.
6. Toward-distractor and away-from-distractor reversal errors.
   1. Toward-distractor: stimulus light was illuminated above the incorrect, pressed lever.
   2. Away-from-distractor: stimulus light was illuminated above the correct, unpressed lever.
7. Number of omitted trials –provides a broad measure of the animal’s motivation level.
8. Response latencies – the time elapsed between lever extension and a response; provide a rough measure of motor function and/or speed of processing.

Modifications

During retractable lever training, pre-exposure to stimulus lights may be employed to decrease the novelty of the lights. Pre-exposing rats to the lights makes the response-to cue shift more difficult and dependent on the medial prefrontal cortex. Prefrontal inactivation will not impair this type of shift if this pre-training procedures are not employed (Floresco et al 2008; Placek et al 2013).

Difficulty can also be added to these tasks by adding additional levers for example.

Sample Data

The data obtained with the Operant Conditioning Chamber can be visualized by graphing any of the behavioral measures described above: trials to criterion or errors to criterion on both the Set task and the Shift task; perseverative or regressive shift errors; never-reinforced shift errors; perseverative and regressive reversal errors; toward-distractor and away-from-distractor reversal errors; omitted trials; or response latencies.

Below are presented example graphs showing the effect of brain injury on trials to criterion and shift errors. Further examples can be found in Brady & Floresco, 2015.

Using graphs similar to these to compare the behavioral flexibility between control and disease or treatment groups allows for easy visualization of the effects on executive function. Animals with impaired behavioral flexibility (due to prefrontal inactivation, for example) may take longer to reach criterion and commit a larger number of errors.

The main strength of the Operant Conditioning Chamber is the possibility of performing a multiple of behavioral assessments associated with cognitive function. This automated version of the operant conditioning chamber dramatically improves the rate of data collection and throughput.

A possible disadvantage is the lengthy training.

Keep in mind that experiments entailing food restriction may require specific approval of an institutional regulatory body before any procedures may begin.

Summary and Key Points

• The Operant Conditioning Chamber is used to test behavioral flexibility as an index of executive function.
• This test assesses the animals’ capacity to actively suppress an initially learned response strategy to acquire a new, competing response.
• Different groups have adapted this test in order to collect data regarding different aspects of behavioral flexibility.
• Animals with neurological of psychiatric disease models may show impaired behavioral flexibility.
• The maze has been used to study cognitive mechanisms and the effect of diseases and treatments on executive function.

References

Bissonette GB, et al (2008). Double dissociation of the effects of medial and orbital prefrontal cortical lesions on attentional and affective shifts in mice. J Neurosci, 28:11124-11130.

Brady AM, Floresco SB (2015). Operant procedures for assessing behavioral flexibility in rats. J Vis Exp, (96):e52387.

Enomoto T, et al (2011). Reducing prefrontal gamma-aminobutyric acid activity induces cognitive, behavioral, and dopaminergic abnormalities that resemble schizophrenia. Biol Psychiatry. 69, 432-441.

Floresco SB, et al (2008). Inactivation of the medial prefrontal cortex of the rat impairs strategy set-shifting, but not reversal learning, using a novel, automated procedure. Behav Brain Res. 190, 85-96.

Krebs JR (1983). Animal behaviour. From Skinner box to the field. Nature, 304(5922):117.

Lie, CH et al (2006). Using fMRI to decompose the neural processes underlying the Wisconsin Card Sorting Test. NeuroImage, 30:1038-1049.

Placek K, et al (2013). Impairments in set-shifting but not reversal learning in the neonatal ventral hippocampal lesion model of schizophrenia: Further evidence for medial prefrontal deficits. Behav Brain Res, 256C, 405-413.

Snyder HR (2013). Major depressive disorder is associated with broad impairments on neuropsychological measures of executive function: a meta-analysis and review. Psychol Bull, 139, 81-132.