This study was supported by Human Frontier Science Program and the Sasakawa Scientific Research Grant from the Japan Science Society.

All procedures for the use of animals were approved by the Animal Care and Use Committee at the Okinawa Institute of Science and Technology.
1. Animals
<ol>
	<li>Obtain male Long-Evans rats (250-300 g on arrival).</li>
	<li>Upon arrival, house a group of two or three rats together for a week and later separate them into individual cages. Note that this experimental design involves food-restriction and needs to hold an animal in each cage to control the amount food consumed.</li>
	<li>Provide all animals with food and water ad libitum, and house them under standard conditions (12 hr/12 hr light/dark cycle, at 23 &#176;C).</li>
	<li>5 days before the beginning of behavioral experiments, food-restrict animals at approximately 85% of their average weight with free access to water throughout experiments.</li>
	<li>Handle animals for 5 min a day for a minimum of 5 days before the beginning of experiments in order to familiarize them with an experimenter.</li>
</ol>
2. Hardware and Software for Behavioral Tests and Analyses
<ol>
	<li>Hardware
	<ol>
		<li>Use an operant chamber equipped with a sound attenuating box.</li>
		<li>The chamber is incorporated with several additional attachments: two receptacle levers on the front panel, two light cues just above the levers, a magazine spot with a sensor for detection of head entry between two levers, a food dispenser, a pure tone generator and a house light on the rear panel.</li>
	</ol>
	</li>
	<li>Software
	<ol>
		<li>Control all the behavioral events under a programmed code written by Trans IV software. Use Med-PC IV software to signal and detect all the behavioral events during training and a test.</li>
		<li>Use Trans IV for writing code for behavioral testing. Open a new file for writing.</li>
		<li>Once the program is written, translate and compile it within the software. Modify the code if errors are detected and retry.</li>
		<li>Upon successful debugging of a program, check if user&#39;s code works properly by conducting a trial-run before actual initiation of experiments.</li>
		<li>Using MED-PC IV, run behavioral experiments. Open the software, click &#34;open session&#34; and assign a single program to each box.</li>
		<li>After all the programs have been properly assigned to each box, send signals necessary to start experiments.</li>
		<li>After the completion of the task, save data as clicking &#34;Save Data&#34; or write code to automatically save data (for details, see programmer&#39;s manual for MED-PC IV).</li>
		<li>Once data is exported to a designated folder, rename the data as acquisition date and animal ID numbers for later use. The collected data is imported into Matlab for behavioral analyses described below (Protocol, 3.7).</li>
	</ol>
	</li>
</ol>
3. Behavioral Training and Testing
<ol>
	<li>Habituation and Magazine training.
	<ol>
		<li>Present no levers to animals during those phases.</li>
		<li>During the habituation phase, place animals in an operant chamber for 20 minutes a day. On the same day, give 10 sucrose pellets (45 mg) to animals in their home cage, which familiarize them to the sucrose reward.</li>
		<li>Next, begin magazine training. Place animals in the chamber and give 20 sucrose pellets (1 pellet per a minute) to the animals, providing an opportunity to learn the location of the food tray and acquisition of the pellets. 
		NOTE: Those phases can be skipped due to potential confusion of animals on obtaining a pellet without an operant response, even though we adopted them to make animals familiarized with a chamber and eating from a food tray.</li>
	</ol>
	</li>
	<li>Continuous reinforcement schedule
	<ol>
		<li>Train animals on a continuous reinforcement schedule to obtain a reward by pressing a lever. A session of this training lasts until 60 pellets have been received (60 lever-presses) or 40 min has elapsed.</li>
		<li>Present either the left or right lever throughout the first half of a session (until 30 pellets are obtained), followed by the presentation of the opposite lever during the second half. The order is alternated on a daily basis.</li>
		<li>Continue this reinforcement schedule until animals have successfully obtained 60 rewards for at least 2 consecutive days. Note that there may be high variability among animals of how quickly they make the first lever-press and this may affect the progress of reinforcement. In this case, put a few sucrose pellets on a presented lever to motivate them to approach toward the lever when no response has been made over the first session. 
		NOTE: A possible alternative is to put sucrose pellets when the lever is first presented to animals in order to make the completion of this phase faster.</li>
	</ol>
	</li>
	<li>Time schedule for a trial in lever-press training and testing.
	<ol>
		<li>Start a single trial with a 3 sec (s) tone.</li>
		<li>2 sec after the termination of tone, present two levers and allow animals to press either lever within 10 sec. In case that no response is made within 10 s, retract both levers and count this trial as an omission trial.</li>
		<li>In the case of the lever press training, present only a single lever on either side.</li>
		<li>In most of experimental conditions, there was a light stimulus above one of levers. Turn the light cue on immediately after the tone ceases and turn off when animals have either made a response or in 10 sec after lever insertion when no response has been made.</li>
		<li>Set inter-trial intervals at 20~30 sec.</li>
	</ol>
	</li>
	<li>Lever-press training
	<ol>
		<li>In this training phase, present no light to animals.</li>
		<li>Train animals under lever-press training for 5-8 sessions with the same time schedule for a trial as testing sessions described below.</li>
		<li>In this training, present either left or right lever randomly, and the lever has to be pressed within 10 sec after the lever is presented, or the trial is counted as an omission without a response. A session for the lever press training comprised of 80 trials.</li>
		<li>Once the animals have scored fewer than 10% of omissions out of 80 trials, move them to the following side-bias test.</li>
	</ol>
	</li>
	<li>Side-bias test
	<ol>
		<li>Perform a side-bias test to determine the animal&#39;s preference to either left or right lever1. A trial entails two lever-presses on both sides.</li>
		<li>Place animals in an operant chamber and allow them to choose either lever. In the next attempt, animals have to select the opposite lever in order to obtain a reward. If animal&#39;s second attempt is on the same side of the first response, give no reward and continue the trial until a response is made on the opposite side.</li>
		<li>Conduct a total of 7 trials to determine animal&#39;s side preference.</li>
	</ol>
	</li>
	<li>Testing.
	<ol>
		<li>A daily session consists of 80 trials.</li>
		<li>Prepare three different conditions for set-shifting procedures as shown in Figure 1.</li>
		<li>All the three conditions require animals to similarly change behavioral strategies from choosing one lever that is consistently on the same side (Phase 1, response strategy) to following a light cue that indicates a correct side (Phase 2, visual cue strategy).</li>
		<li>Begin with initial learning of a response strategy (Phase 1) for 4 sessions, in which animals have to press a lever based on the location of levers. In this phase, set the correct side to the lever opposite to their preference based on a preliminary side-bias test as mentioned above.</li>
		<li>Next, commence visual cue learning (Phase 2) for 10 sessions. A light cue illuminated above either lever indicates a correct lever. In this shifting phase, three different patterns of attentional shifts can be compared among three experimental conditions as described below (3.3.6-3.3.8).</li>
		<li>In set-shift condition 1 (Figure 1A), give no light in phase 1, but a light cue indicates the correct side in phase 2. In condition 1, therefore, animals need to attend to a novel stimulus.</li>
		<li>In set-shift condition 2 (Figure 1B), present a light cue above the correct lever in phase 1, and again in phase 2. In this condition, the light cue had been relevant, but not necessarily required for making a choice in phase 1. Thus animals have to attend to a previously relevant cue.</li>
		<li>In set-shift condition 3 (Figure 1C), turn on a light cue randomly above either left or right lever in phase 1. Thus it has to be ignored. In phase 2 animals are required to pay attention to the light stimulus that has been previously irrelevant.</li>
	</ol>
	</li>
	<li>Behavioral analysis.
	<ol>
		<li>Throughout sessions, measure the percentage of correct responses on a daily basis, excluding omission trials.</li>
		<li>Count errors accumulated during 10 sessions of visual cue learning and classify them into perseverative, regressive or never-reinforced errors as described in a previous study1. There, a detailed analysis of error types suggests separate functions in set-shifting.</li>
		<li>Define perseverative errors as incorrect responses to the previously correct lever while animal&#39;s performance was still below chance level4,6,13,14,15. Similar criteria have been used in previous studies1,3,7,11.</li>
		<li>Based on a principled way, determine the criterion to separate between perseverative and regressive errors as the point at which the animal first scored fewer than 8 out of 10 incorrect responses (probability of making 8/10 errors or more = 0.054, based on cumulative binomial distribution) in a moving window of 10 trials.</li>
		<li>In order to find this point, start calculating a moving average of the 10 trial window from 1st trial and then advance it by one trial at a time until &#60;8/10 errors are measured. Conduct this analysis across all trials in which a light cue is illuminated above the previously incorrect lever during visual cue strategy.</li>
		<li>Define all the subsequent errors committed after this point as regressive errors.</li>
		<li>During visual cue learning, count never-reinforced errors when animals responded to a previously incorrect lever on which the light cue was not illuminated. Divide them into an early or late portion based on the learning phase; errors made in the first half of 10 sessions (session 1-5) are considered early and those in the second half (session 6-10) are regarded as late ones.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

We developed new variations on the established set-shifting paradigm for use in rats. Using those paradigms, cholinergic lesions of the striatum were found to impair set-shifting, suggesting a specific role of striatal cholinergic interneurons in set-shifting: suppression of an old rule and facilitation of exploration for a new rule. The effects differed between dorsomedial and ventral striatum, in accordance with the different role of these structures in learning.
A set-shifting task has been widely used to test behavioral flexibility in species ranging from humans to rodents1,4,5,7,8,9,12. The term, &#34;set&#34; is defined as the property of the stimulus relevant to behavior in a given trial20,21. The present study introduced new variations in which a subject was required to change behavioral strategy based on a change in which set is relevant. The new versions should be compared carefully with other studies using set-shifting. In a typical set-shifting paradigm, a subject initially forms a relevant set to guide behavior and ignores the irrelevant set. After the set-shift, the subject has to attend to the previously irrelevant set. Among the three conditions we proposed here, only condition 3 involves a set-shift. Condition 1 and 2 differ from such set-shift tasks in that either a novel stimulus or a subset of a compound stimulus becomes relevant. Learning curves and the number of perseverative errors of intact rats revealed differences in the initial acquisition and reacquisition between three conditions. Thus, each condition measures different functions: acquisition of a response to a novel cue, attention to a relevant but not crucial cue, and attention to the irrelevant cue. These new variations are useful for investigating neural mechanisms for different forms of behavioral flexibility.
Rats have many advantages for studying the neural mechanisms underlying behavioral flexibility, including their large size making them suitable for stereotaxic surgery, the availability of transgenic strains, and cognitive ability. Previous studies have established T-maze based or automated version of the set-shifting task in rats1,7,8,9,11. In case that an automated version is not available, three different manipulations introduced in this article are applicable to a T-maze based set-shift task3,7. Also, other stimulus dimensions with different sensory modality such as an odor cue can be combined22, which further extends variations.
It has previously been shown that inactivation of DMS or VS impairs set-shifting when it requires attending to a previously irrelevant stimulus7,9. This is also the case in condition 3 of the present study. However, an important question which remains to be answered is whether impaired set-shifting is derived from being unable to alter action strategies (such as from a response strategy to a visual cue strategy) during a shift, or the inability to pay attention to a stimulus that had been irrelevant in the initial discrimination. It is impossible to decide between these two possibilities by examining a single experimental context only. To dissociate the specific attentional deficit from more general impairment of shifting strategies, we have sought to create new variations of the set-shifting task, using two different conditions requiring the same shift but different types of attention.
Using these additional conditions, we could separate neural substrates underlying a shift of strategies in different contexts. For example, perseveration of VS lesioned rats in condition 1 where a new stimulus was introduced allowed us to reveal a potential mechanism of ventral cholinergic system in attentional processes and approach to the novelty important for the new rule. On the other hand, we did not observe general effects of DMS lesions on a strategic shift. Rather, it was specific to the situation in which a stimulus contingency changed and animals needed to pay attention to a previously irrelevant cue. Two additional conditions successfully control for a general impairment of shifting strategies. This enabled us to conclude that DMS and VS cholinergic systems have a common role in suppression of an old strategy and facilitation of exploratory behavior, even though they work in different environmental contexts, and neither one has a general role in shifting strategies itself.
In conclusion, new set-shifting variations make it possible to analyze rat&#39;s cognitive flexibility in more detail and help further understanding of neural mechanisms for behavioral flexibility under different environmental contexts. Future studies testing the involvement of other key brain regions such as prefrontal cortex and hippocampus would be encouraged using a variety of contexts as introduced in this article.

Behavioral flexibility is a key requirement for survival in a changing world. One of the established behavioral paradigms for testing this ability is set-shifting, in which a shift of attention from one stimulus dimension to another is necessary for changing action strategies after a change in rule. Several brain regions such as the prefrontal cortex and striatum are implicated in set-shifting2,3,4,5. Neural mechanisms for this function have been investigated across several species including humans5, monkeys6 and rats1,7,8,9. However, the rat versions of set-shifting tasks have not been as extensively developed. The cost-effectiveness of rats, their appropriate size for stereotaxic surgery, and the availability of recently developed genetic methods10, motivate further development of set-shifting paradigms for use in rats.
A typical set-shifting paradigm for rats requires a change between two behavioral strategies: for example, a response strategy and a visual-cue strategy. Rats initially have to choose one of two available options (such as left or right levers in an operant automated version1 or left or right arms in a T-maze version7,8,9,11). After a set shift, they have to switch to using a visual-cue strategy, such as a light cue indicating the correct side. In those conventional set-shifting tasks, it is necessary to shift attention from one stimulus dimension to another dimension that had been previously irrelevant.
In addition to changing to a dimension that had been previously irrelevant, there is also the logical possibility that a stimulus was previously relevant, or previously absent and now novel. Real life situations in nature may entail attention to a novel, or historically relevant but not crucial cue. Therefore, we considered these subtypes of set-shift, in a new variation of rodent set-shifting based on a previously established automated set-shifting task1.
We have recently demonstrated the use of the new version of set-shifting paradigms in an experiment to determine the effect of neurochemically specific lesions of the striatum12. In our previous study, we targeted cholinergic interneurons releasing acetylcholine (ACh) of the dorsomedial or ventral striatum since ACh and those subregions have been implicated in behavioral flexibility. All the experimental conditions demanded the same strategic shift but each involved different types of attentional shift: to a novel, previously relevant or previously irrelevant cue. We here describe detailed procedures of the paradigms, and highlight representative results suggesting that striatal cholinergic systems play a fundamental role in set-shifting, which is dissociable between different striatal subregions depending on behavioral contexts12.

<table border="0" cellpadding="0" cellspacing="0" width="687">
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		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Standard Modular Test Chamber</td>
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			<td style="width:119px;">ENV-008</td>
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			<td height="21" style="height:21px;">Low Profile Retractable Response Lever</td>
			<td>Med Associates</td>
			<td>ENV-112CM</td>
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			<td height="21" style="height:21px;">Stimulus Light for Rat</td>
			<td>Med Associates</td>
			<td>ENV-221M</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Switchable Dual Pellet/Dipper Receptacle for Rat</td>
			<td>Med Associates</td>
			<td>ENV-202RM-S</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Head Entry Detector for Rat Receptacles</td>
			<td>Med Associates</td>
			<td>ENV-254-CB</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Modular Pellet Dispenser; 45 mg for Rat</td>
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			<td>ENV-203M-45</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Sonalert Module for Rat</td>
			<td>Med Associates</td>
			<td>ENV-223AM</td>
			<td>4.5 kHz available (ENV-223HAM)</td>
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		<tr height="21">
			<td height="21" style="height:21px;">House Light for Rat Chambers</td>
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			<td height="21" style="height:21px;">SmartCtrl Interface Module, 8 input/16 output</td>
			<td>Med Associates</td>
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			<td height="21" style="height:21px;">SmartCtrl Connection Panel, 8 input/16 output</td>
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			<td height="21" style="height:21px;">45 mg Tablet-Fruit Punch</td>
			<td>TestDiet</td>
			<td>1811255</td>
			<td>Several flavors available</td>
		</tr>
	</tbody>
</table>

new variations for strategy set-shifting in the rat

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

We used the strategy set-shifting task described above to investigate the role of cholinergic interneurons in behavioral flexibility. We compared the effect on the task of an immunotoxin-induced selective lesion of cholinergic interneurons in dorsomedial (DMS), ventral striatum (VS) and saline-injected control. All animals had to switch from choosing a lever based on the side (left or right), to choosing based on a cue light above the correct lever. We used three experimental conditions of set-shift in which the cue light was either: (1) novel, (2) previously relevant (indicating the correct lever), or (3) previously irrelevant (randomly assigned).
In these three experimental conditions, the initial acquisition of response strategy was intact in all the treatment groups, suggesting that cholinergic loss in the striatum had no effects on initial learning (Figure 2A-2C). These results are consistent with previous studies showing that inactivation of DMS or VS did not affect initial discrimination7,9 and that application of cholinergic antagonists systemically16 or locally to the striatum17,18,19 left initial learning intact.
In set-shift condition 1 (Figure 2A, novel cue), the percentage of correct responses was not significantly different. However, the number of perseverative errors was significantly increased in the VS lesion group than controls. During set-shift condition 2 (Figure 2B, previously relevant cue) neither learning performance nor types of errors were altered by the lesions. In contrast, in set-shift condition 3 (Figure 2C, previously irrelevant cue), the number of perseverative errors was significantly different between groups. In particular, there was a significant increase in perseverative errors after DMS lesions. Compared to the control group, the number of never-reinforced errors was significantly decreased in both DMS and VS lesion groups, which was evident in the early but not in the late phase of visual cue learning.
In summary, VS cholinergic lesions disrupted a strategic shift when a novel stimulus was given as a new important cue, causing more perseverative errors. On the other hand, DMS lesions affected set-shifting only when attention to a previously irrelevant stimulus was required, resulting in a different distribution of error types.
<img alt="Figure 1" src="/files/ftp_upload/55005/55005fig1.jpg" /> 
Figure 1: Three different conditions for a set-shift. A flow chart of three variations (A, B and C) of the set-shifting paradigm. A yellow circle shows a visual cue. Reprinted with permission from Aoki et al.12. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55005/55005fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55005/55005fig2.jpg" /> 
Figure 2: Behavioral results in the set-shifting task. Percentage of correct responses in both response and visual cue strategy (left), types of errors committed over 10 sessions of visual cue strategy (middle), and early and late components of never-reinforced errors (right) are shown for each experimental condition (A, a shift of behavioral strategy required attention to a novel stimulus, B, to a previously relevant stimulus, C, to a previously irrelevant stimulus). Final group size is follows: condition 1, n = 16 (control), n = 19 (DMS), n = 14 (VS); condition 2, n = 13 (control), n = 18 (DMS), n = 14 (VS); condition 3, n = 21 (control), n = 17 (DMS), n = 16 (VS). Data are shown as Means &#177; SEM. Asterisks *, **, and *** are p &#60;0.05, &#60;0.01 and &#60;0.001, respectively. Reprinted with permission from Aoki et al.12. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55005/55005fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about New Variations for Strategy Set-shifting in the Rat at JoVE.com

New Variations for Strategy Set-shifting in the Rat

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

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

new-variations-for-strategy-set-shifting-in-the-rat

Nanyang Technological University

Research

JoVE Journal

Behavior

8.1K Views.  Okinawa Institute of Science and Technology. Set-shifting, a form of behavioral flexibility, requires an attentional shift from one stimulus dimension to another. We extended an established rodent set-shifting task1 by requiring attention to different stimuli according to context. The task was combined with specific lesions to identify neuron subtypes underlying a successful shift.

Video: New Variations for Strategy Set-shifting in the Rat

A Procedure to Study the Effect of Prolonged Food Restriction on Heroin Seeking in Abstinent Rats

In human drug addicts, exposure to drug-associated cues or environments that were previously associated with drug taking can trigger relapse during abstinence. Moreover, various environmental challenges can exacerbate this effect, as well as increase ongoing drug intake.
The procedure we describe here highlights the impact of a common environmental challenge, food restriction, on drug craving that is expressed as an augmentation of drug seeking in abstinent rats.
Rats are implanted with chronic intravenous i.v. catheters, and then trained to press a lever for i.v. heroin over a period of 10-12 days. Following the heroin self-administration phase the rats are removed from the operant conditioning chambers and housed in the animal care facility for a period of at least 14 days. While one group is maintained under unrestricted access to food (sated group), a second group (FDR group) is exposed to a mild food restriction regimen that results in their body weights maintained at 90% of their nonrestricted body weight. On day 14 of food restriction the rats are transferred back to the drug-training environment, and a drug-seeking test is run under extinction conditions (i.e.&#160;lever presses do not result in heroin delivery).
The procedure presented here results in a highly robust augmentation of heroin seeking on test day in the food restricted rats. In addition, compared to the acute food deprivation manipulations we have used before, the current procedure is a more clinically relevant model for the impact of caloric restriction on drug seeking. Moreover, it might be closer to the human condition as the rats are not required to go through an extinction-training phase before the drug-seeking test, which is an integral component of the popular reinstatement procedure.

A procedure that allows a demonstration of robust augmentation of drug seeking in food-restricted rats is described. Following heroin self-administration training, rats go through an abstinence period, in a drug-free environment, during which they are mildly food restricted. Drug seeking is then tested in the drug-associated environment.

In human drug addicts, exposure to drug-associated cues or environments that were previously associated with drug taking can trigger relapse during ...

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

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

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

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

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 ...

Infant Auditory Processing and Event-related Brain Oscillations

Rapid auditory processing and acoustic change detection abilities play a critical role in allowing human infants to efficiently process the fine spectral and temporal changes that are characteristic of human language. These abilities lay the foundation for effective language acquisition; allowing infants to hone in on the sounds of their native language. Invasive procedures in animals and scalp-recorded potentials from human adults suggest that simultaneous, rhythmic activity (oscillations) between and within brain regions are fundamental to sensory development; determining the resolution with which incoming stimuli are parsed. At this time, little is known about oscillatory dynamics in human infant development. However, animal neurophysiology and adult EEG data provide the basis for a strong hypothesis that rapid auditory processing in infants is mediated by oscillatory synchrony in discrete frequency bands. In order to investigate this, 128-channel, high-density EEG responses of 4-month old infants to frequency change in tone pairs, presented in two rate conditions (Rapid: 70 msec ISI and Control: 300 msec ISI) were examined. To determine the frequency band and magnitude of activity, auditory evoked response averages were first co-registered with age-appropriate brain templates. Next, the principal components of the response were identified and localized using a two-dipole model of brain activity. Single-trial analysis of oscillatory power showed a robust index of frequency change processing in bursts of Theta band (3 - 8 Hz) activity in both right and left auditory cortices, with left activation more prominent in the Rapid condition. These methods have produced data that are not only some of the first reported evoked oscillations analyses in infants, but are also, importantly, the product of a well-established method of recording and analyzing clean, meticulously collected, infant EEG and ERPs. In this article, we describe our method for infant EEG net application, recording, dynamic brain response analysis, and representative results.

High-density electroencephalography (dEEG) is being used increasingly to study brain development and plasticity in the early years of life. Here we present an application of sophisticated analysis techniques that builds on traditional EEG recording to understand the oscillatory dynamics of rapid auditory processing in the infant brain.

Rapid auditory processing and acoustic change detection abilities play a critical role in allowing human infants to efficiently process the fine spectral ...

Using the Activity-based Anorexia Rodent Model to Study the Neurobiological Basis of Anorexia Nervosa

Anorexia nervosa (AN) is a psychiatric illness characterized by excessively restricted caloric intake and abnormally high levels of physical activity. A challenging illness to treat, due to the lack of understanding of the underlying neurobiology, AN has the highest mortality rate among psychiatric illnesses. To address this need, neuroscientists are using an animal model to study how neural circuits may contribute toward vulnerability to AN and may be affected by AN. Activity-based anorexia (ABA) is a bio-behavioral phenomenon described in rodents that models the key symptoms of anorexia nervosa. When rodents with free access to voluntary exercise on a running wheel experience food restriction, they become hyperactive &#8211; running more than animals with free access to food. Here, we describe the procedures by which ABA is induced in adolescent female C57BL/6 mice. On postnatal day 36 (P36), the animal is housed with access to voluntary exercise on a running wheel. After 4 days of acclimation to the running wheel, on P40, all food is removed from the cage. For the next 3 days, food is returned to the cage (allowing animals free food access) for 2 hr daily. After the fourth day of food restriction, free access to food is returned and the running wheel is removed from the cage to allow the animals to recover. Continuous multi-day analysis of running wheel activity shows that mice become hyperactive within 24 hr following the onset of food restriction. The mice run even during the limited time during which they have access to food. Additionally, the circadian pattern of wheel running becomes disrupted by the experience of food restriction. We have been able to correlate neurobiological changes with various aspects of the animals&#8217; wheel running behavior to implicate particular brain regions and neurochemical changes with resilience and vulnerability to food-restriction induced hyperactivity.

Here we present a protocol to induce activity-based anorexia (ABA) in female adolescent mice. ABA is a condition of hyperactivity evoked by imposing food restriction on rodents with access to a running wheel. This phenomenon is being used as a model to study the underlying neurobiology of anorexia nervosa.

Anorexia nervosa (AN) is a psychiatric illness characterized by excessively restricted caloric intake and abnormally high levels of physical activity. A ...

The Social Dimension of Stress: Experimental Manipulations of Social Support and Social Identity in the Trier Social Stress Test

In many situations humans are influenced by the behavior of other people and their relationships with them. For example, in stressful situations supportive behavior of other people as well as positive social relationships can act as powerful resources to cope with stress. In order to study the interplay between these variables, this protocol describes two effective experimental manipulations of social relationships and supportive behavior in the laboratory. In the present article, these two manipulations are implemented in the Trier Social Stress Test (TSST)&#8212;a standard stress induction paradigm in which participants are subjected to a simulated job interview. More precisely, we propose (a) a manipulation of the relationship between different protagonists in the TSST by making a shared social identity salient and (b) a manipulation of the behavior of the TSST-selection committee, which acts either supportively or unsupportively. These two experimental manipulations are designed in a modular fashion and can be applied independently of each other but can also be combined. Moreover, these two manipulations can also be integrated into other stress protocols and into other standardized social interactions such as trust games, negotiation tasks, or other group tasks.

Previous research on the social dimension of stress has focused on two important variables: social identity and social support. This protocol introduces an effective experimental manipulation of these two social variables and describes their implementation in a standard stress induction paradigm (Trier Social Stress Test).

In many situations humans are influenced by the behavior of other people and their relationships with them. For example, in stressful situations ...

Assessing Working Memory in Children: The Comprehensive Assessment Battery for Children &#8211; Working Memory (CABC-WM)

The Comprehensive Assessment Battery for Children - Working Memory (CABC-WM) is a computer-based battery designed to assess different components of working memory in young school-age children. Working memory deficits have been identified in children with language-based learning disabilities, including dyslexia1,2 and language impairment3,4, but it is not clear whether these children exhibit deficits in subcomponents of working memory, such as visuospatial or phonological working memory. The CABC-WM is administered on a desktop computer with a touchscreen interface and was specifically developed to be engaging and motivating for children. Although the long-term goal of the CABC-WM is to provide individualized working memory profiles in children, the present study focuses on the initial success and utility of the CABC-WM for measuring central executive, visuospatial, phonological loop, and binding constructs in children with typical development. Immediate next steps are to administer the CABC-WM to children with specific language impairment, dyslexia, and comorbid specific language impairment and dyslexia.

Working memory predicts a significant amount of variance for a variety of cognitive tasks, including speaking, reading, and writing. However, few tools are available to assess working memory in children. We present an innovative, computer-based battery that comprehensively assesses different components of working memory in school-age children.

The Comprehensive Assessment Battery for Children - Working Memory (CABC-WM) is a computer-based battery designed to assess different components of ...

Ultrasound Images of the Tongue: A Tutorial for Assessment and Remediation of Speech Sound Errors

Diagnostic ultrasound imaging has been a common tool in medical practice for several decades. It provides a safe and effective method for imaging structures internal to the body. There has been a recent increase in the use of ultrasound technology to visualize the shape and movements of the tongue during speech, both in typical speakers and in clinical populations. Ultrasound imaging of speech has greatly expanded our understanding of how sounds articulated with the tongue (lingual sounds) are produced. Such information can be particularly valuable for speech-language pathologists. Among other advantages, ultrasound images can be used during speech therapy to provide (1) illustrative models of typical (i.e.&#160;&#34;correct&#34;) tongue configurations for speech sounds, and (2) a source of insight into the articulatory nature of deviant productions. The images can also be used as an additional source of feedback for clinical populations learning to distinguish their better productions from their incorrect productions, en route to establishing more effective articulatory habits.
Ultrasound feedback is increasingly used by scientists and clinicians as both the expertise of the users increases and as the expense of the equipment declines. In this tutorial, procedures are presented for collecting ultrasound images of the tongue in a clinical context. We illustrate these procedures in an extended example featuring one common error sound, American English /r/. Images of correct and distorted /r/ are used to demonstrate (1) how to interpret ultrasound images, (2) how to assess tongue shape during production of speech sounds, (3), how to categorize tongue shape errors, and (4), how to provide visual feedback to elicit a more appropriate and functional tongue shape. We present a sample protocol for using real-time ultrasound images of the tongue for visual feedback to remediate speech sound errors. Additionally, example data are shown to illustrate outcomes with the procedure.

Ultrasound imaging can be used to display the shape and movements of the tongue in real time during speech. The images can be used to determine the nature of speech sound errors. Visual feedback of the tongue can be used to facilitate improvements in speech sound production in clinical populations.

Diagnostic ultrasound imaging has been a common tool in medical practice for several decades. It provides a safe and effective method for imaging ...

A New Method for Inducing a Depression-Like Behavior in Rats

Contagious depression is a phenomenon that is yet to be fully recognized and this stems from insufficient material on the subject. At the moment, there is no existing format for studying the mechanism of action, prevention, containment, and treatment of contagious depression. The purpose of this study, therefore, was to establish the first animal model of contagious depression.
Healthy rats can contract depressive behaviors if exposed to depressed rats. Depression is induced in rats by subjecting them to several manipulations of chronic unpredictable stress (CUS) over 5 weeks, as described in the protocol. A successful sucrose preference test confirmed the development of depression in the rats. The CUS-exposed rats were then caged with na&#239;ve rats from the contagion group (1 na&#239;ve rat/2 depressed rats in a cage) for an additional 5 weeks. 30 social groups were created from the combination of CUS-exposed rats and na&#239;ve rats.
This proposed depression-contagion protocol in animals consists mainly of cohabiting CUS-exposed and healthy rats for 5 weeks. To ensure that this method works, a series of tests are carried out - first, the sucrose preference test upon inducing depression to rats, then, the sucrose preference test, alongside the open field and forced-swim tests at the end of the cohabitation period. Throughout the experiment, rats are given tags and are always returned to their cages after each test.
A few limitations to this method are the weak differences recorded between the experimental and control groups in the sucrose preference test and the irreversible traumatic outcome of the forced swim test. These may be worth considering for suitability before any future application of the protocol. Nonetheless, following the experiment, na&#239;ve rats developed contagion depression after 5 weeks of sharing the same cage with the CUS-exposed rats.

This protocol describes a new model by which healthy rats could contract depression over a given time periodthrough contagion by exposure to chronic unpredictable stressed (CUS) rats.

Contagious depression is a phenomenon that is yet to be fully recognized and this stems from insufficient material on the subject. At the moment, there is ...

Efficiently Recording the Eye-Hand Coordination to Incoordination Spectrum

The objective analysis of eye movements has a significant history and has been long proven to be an important research tool in the setting of brain injury. Quantitative recordings have a strong capacity to screen diagnostically. Concurrent examinations of the eye and upper limb movements directed toward shared functional goals (e.g., eye-hand coordination) serve as an additional robust biomarker-laden path to capture and interrogate neural injury, including acquired brain injury (ABI). While quantitative dual-effector recordings in 3-D afford ample opportunities within ocular-manual motor investigations in the setting of ABI, the feasibility of such dual recordings for both eye and hand is challenging in pathological settings, particularly when approached with research-grade rigor. Here we describe the integration of an eye tracking system with a motion tracking system intended primarily for limb control research to study a natural behavior. The protocol enables the investigation of unrestricted, three-dimensional (3D) eye-hand coordination tasks. More specifically, we review a method to assess eye-hand coordination in visually guided saccade-to-reach tasks in subjects with chronic middle cerebral artery (MCA) stroke and compare them to healthy controls. Special attention is paid to the specific eye- and limb-tracking system properties in order to obtain high fidelity data from participants post-injury. Sampling rate, accuracy, permissible head movement range given anticipated tolerance and the feasibility of use were several of the critical properties considered when selecting an eye tracker and an approach. The limb tracker was selected based on a similar rubric but included the need for 3-D recording, dynamic interaction and a miniaturized physical footprint. The quantitative data provided by this method and the overall approach when executed correctly has tremendous potential to further refine our mechanistic understanding of eye-hand control and help inform feasible diagnostic and pragmatic interventions within the neurological and rehabilitative practice.&#160;

Cerebral injury can damage both ocular and somatic motor systems. Characterization of motor control post-injury affords biomarkers that assist in disease detection, monitoring, and prognosis. We review a method to measure eye-hand movement control in health and in pathologic incoordination, with look-and-reach paradigms to assess coordination between eye and hand.

The objective analysis of eye movements has a significant history and has been long proven to be an important research tool in the setting of brain ...