Name: three laboratory procedures for assessing different manifestations of impulsivity in rats
Uploaded: 2019-03-17T13:00:00
Description: Watch this Scientific Journal Video about Three Laboratory Procedures for Assessing Different Manifestations of Impulsivity in Rats at JoVE.com

We would like to thank Florencia Mata, Mar&#237;a Elena Ch&#225;vez, Miguel Burgos, and Alejandro Tapia for providing technical assistance. We also wish to thank Sarah Gordon Frances for her useful comments on a previous draft of this article and Vladimir Ordu&#241;a for kindly providing raw data from a published paper. Thanks to Claudio Nallen for creating the diagram in Figure 1. We are grateful to the Direcci&#243;n de Investigaci&#243;n of the Universidad Iberoamericana Ciudad de M&#233;xico for funding proofreading/editing services and the video producing expenses.

The three protocols described in this section require the use of rats as subjects. Most laboratory rat strains are suitable; for example, Wistar, Long-Evans, Sprague-Dawley, etc. The Ethics Committee of the Universidad Iberoamericana, following the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council, 1996), approved the laboratory protocols to be described.
1. Animal Housing and Preparation
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The present article provided a description of a miscellaneous variety of protocols for screening impulsivity in rats. It is argued that these particular protocols are favored for their ease of programming and data analysis and require fewer operating and stimulus devices than other available alternatives. There are several crucial steps for the effective implementation of these protocols, such as (1) yielding a research question, (2) selecting an appropriate study design, (3) programming the selected protocol, (4) conduc...

Impulsivity can be conceptualized as a behavioral dimension associated with maladaptive outcomes1. Despite the widespread use of this term, there is no universal consensus on its precise definition. In fact, several authors have defined impulsivity by giving examples of impulsive behaviors or their consequences, rather than delineating which distinctive aspects govern the phenomenon. For instance, impulsivity is assumed to involve an inability to wait, plan, inhibit prepotent behaviors, or an insensitivity to delayed outcomes2, and it has been considered a core vulnerability to addictive behavior3. Bari and Robbins4 have characterized impulsivity as the co-occurrence of strong impulses, being triggered by dispositional and situational variables, and dysfunctional inhibitory processes. A different definition was provided by Dalley and Robbins, who stated that impulsivity could be regarded as a predisposition to rapid, often premature, actions without appropriate insight5. Yet, another definition of impulsivity, proposed by Sosa and dos Santos6, is a behavior tendency that deviates an organism from maximizing available rewards due to the acquired control exerted over the organism&#39;s responding by stimuli incidentally related to those rewards.
Due to the behavioral processes related to impulsivity, its neurophysiological substrate involves structures in common with those of motivated behavior, decision making and reward valuing. This is supported by studies that show that structures of the cortico-striatal pathway (e.g., nucleus accumbens [NAc], prefrontal cortex [PFC], amygdala, and caudate putamen [CPU]), as well as the ascending monoaminergic neurotransmitter system, participate in the expression of impulsive behavior7. However, the neural substrate of impulsivity is more complex than that. Although NAc and PFC are involved in impulsive behavior, these structures are part of a more complex system, and also are composed by substructures that have different functions (for more detailed documentation, see Dalley and Robbins5).
Regardless of the controversies about its nature and biological substrate, this behavioral dimension is known to vary across individuals, in which case it can be considered as a trait, and within individuals, in which case it can be considered as a state8. Impulsivity has long been recognized as a feature of some psychiatric conditions, such as attention-deficit/hyperactivity disorder (ADHD), substance abuse, and manic episodes9. There seems to be a high consensus that impulsivity is composed by multiple dissociable factors, including unwillingness to wait (i.e., delay discounting), incapacity to refrain prepotent responses (i.e., inhibitory deficit), difficulty to focus on relevant information (i.e., inattention), and a tendency to engage in risky situations (i.e., sensation seeking)5,10,11. Each of these factors can be assessed through special behavioral tasks, which are usually assigned to two broad categories: choice and response inhibition (these may have different labels between each authors&#39; taxonomies). Some important features of such behavioral tasks are that they could be applied across several animal species2&#160;and that they allow&#160;studying impulsivity in controlled laboratory conditions.
Modeling a behavioral dimension with laboratory non-human animals has a number of advantages including the possibility of measuring specific, operationalized behavioral tendencies, allowing the researchers to largely reduce confounding variables (e.g., contamination by past life events4) and to implement experimental manipulations such as chronic pharmacological administration, performing neurotoxic lesions, or genetic manipulations. Most of these protocols have analogue versions for humans, which make comparisons easy5. Importantly, using analogues of these laboratory protocols in humans is effective to aid diagnosis of psychiatric conditions, such as ADHD (especially when more than one protocol is applied12).
Like any other psychological measurement, laboratory protocols for assessing impulsivity must comply with particular criteria in order to achieving the goal of providing insight into the phenomenon under study. To be considered as an appropriate model of impulsive behavior a laboratory protocol should be reliable, and possess (at least, in some degree) face, construct, and/or predictive validity13. Reliability could imply either that an effect upon the measurement would replicate if a manipulation is conducted two or more times, or that the measurement is consistent over time or across different situations14,15. The former feature would be especially useful for experimental studies, while the latter would be so for correlational studies14. Face validity refers to the degree in which what is measured resembles the phenomenon that is supposed to be modeled, as to being, for example, affected by the same variables. Predictive validity refers to the ability of a measure to forecast future performance in protocols, which aim to measure the same or a related construct. Finally, construct validity refers to whether the protocol reproduces behavior that is theoretically sound regarding the process or processes assumed to be involved in the phenomenon under study. However, although these are highly desirable features, one should be cautious when stating that a protocol is valid purely based on these criteria16.
There are several protocols to measure impulsivity in laboratory settings. However, the present article presents only three such methods: intertemporal choice, differential reinforcement of low rates, and feature-negative discrimination. Intertemporal procedures aim to assess the delay discounting (i.e., the difficulty of delayed outcomes to control behavior) component of impulsivity. The basic rationale of this protocol is confronting subjects with two rewards that differ in both magnitude and delay17. One alternative provides a small immediate reward (termed smaller sooner, SS) and the other provides a larger but delayed reward (termed larger later, LL). The proportion of responses to the SS alternative can be used as an index of impulsivity18. In differential reinforcement of low rates procedures, the factor of impulsivity to be assessed is response inhibition (i.e., incapacity to withhold prepotent responses) when there is a negative punishment contingency upon inappropriate responding. The rationale of this protocol is introducing subjects to a situation in which the only way of obtaining rewards is to pause their responding19. Finally, feature-negative discrimination procedure evaluates response inhibition when there is no explicit punishment upon inappropriate responding. The rationale of this protocol (also known as Pavlovian conditioned inhibition or the A+/AX- procedure) is to evaluate subjects&#39; ability to withhold unnecessary responses20.
These procedures stand out in comparison to others as having some convenient features. For example, the procedures presented here are suitable for being conducted in minimally equipped conditioning chambers (also known as &#39;the Skinner box&#39;). Figure 1 shows a diagram of a typical conditioning chamber. Conditioning chambers are useful research instruments due to a number of advantages. They allow automated collection of a relatively large volume of data, maximizing the number of subjects assessed for unity of time and space21. Moreover, behavioral studies conducted in conditioning chambers require minimal researcher intervention, which reduces the time and effort invested by laboratory staff, unlike other available methods (e.g., non-automated T-mazes, set-shifting boxes)21. Minimizing researchers&#39; intervention also help in reducing researchers&#39; bias, decreasing effects of researchers&#39; learning curve, and a reduction of handling-induced stress22. Typical conditioning chambers are fairly standardized to be used with medium sized rodents, such as rats (R. norvegicus), but can be employed to study other taxa, like similar-sized marsupials (e.g., D. albiventris, and L. crassicaudata23). There are also commercial conditioning chambers adapted for smaller (e.g., mice [M. musculus]) and larger (e.g., non-human primates) species. Setting up and conducting the protocols presented in this article require minimal programming skills and demand a quite low number of attainable input and output devices, unlike more sophisticated alternative methods (e.g., 5-choice serial reaction time task [5-CSRTT]24 and sign-tracking25).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59070/59070fig1.jpg" /> 
Figure 1: Diagram of a conditioning chamber prototype. The main components of the conditioning chamber include: (1) left lever, (2) food receptacle (equipped with lateral infrared diodes to detect head entries), (3) focalized light, (4) speaker for tone emission (rear view), (5) house light (rear view), (6) food dispenser. <a href="https://www.jove.com/files/ftp_upload/59070/59070fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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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.

The three protocols described in this article may be each conducted alone or in conjunction with other procedures; this will depend on the research question, which in turn will determine the study design. Some examples of study designs that are compatible with these protocols are: (1) time series studies, which aim to describe longitudinal changes in performance; (2) quantification of individual variability, which aims to determine the reliability of the measures; (3) cross-sectional corr...

Watch this Scientific Journal Video about Three Laboratory Procedures for Assessing Different Manifestations of Impulsivity in Rats at JoVE.com

Three Laboratory Procedures for Assessing Different Manifestations of Impulsivity in Rats

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 ...

Conducting behavioral procedures to assess impulsivity in rats provides insight into the environmental, neurophysiological, and neurochemical basis of impulsivity, that is, about the factors that trigger and underlie those behaviors. This protocol allow the study of impulsivity with a good balance of efficiency and accessibility, and instruction of data suitable for quantitative analysis. The performance in these protocols may be considered as an analog of impulsiveness in humans.Hence, human versions of them serve for the diagnosis and treatment of related psychiatric disorders such as ADHD or pathological gambling. Demonstrating the procedure with Patsy Moreno will be Gustavo Lago, a technician, and Pablo Saavedra, an undergraduate student from our laboratory. After beginning the food restriction regime, add 60 food pellets to the food receptacle to habituate food neophobia before introducing the rats to the conditioning chambers without the initiation of any protocol for 30 minutes to habituate the exploring responses.After the habituation stage, introduce the rats into the conditioning chambers for two additional daily 30-minute sessions of delivering a food pellet every 45 seconds to help the rats identify the source of the pellets. For intertemporal choice and differential reinforcement of low rates of responding, or DRL protocols, place one or two levers into the chamber and deliver food for every level press in conjunction with a free food pellet delivery every 45 seconds. For an intertemporal choice protocol, select the values for the delay and the magnitude of the reward.For example, choices for the larger-later, or LL alternative, deliver five food pellets after a 20-second fixed delay and choices for the small-sooner, or SS alternative, deliver one food pellet immediately. Select a finishing criterion, and end the sessions automatically after the completion of the specified criterion, for example, after 40 choice trials or after 50 minutes. Combine each alternative with a lever within the conditioning chamber, counterbalancing the laterality of the alternatives among the subjects, and make SS and LL alternatives available upon the accomplishment of a variable interval schedule.In free choice trials, when one of the levers is pressed after a certain interval has elapsed, the associated alternative will be activated. Both levers will be retracted, and the consequence associated with the SS or LL alternatives after the accomplishment of a variable interval schedule of reinforcement will be activated. After reward delivery, perform a time-out condition, adjusting the duration of this condition to equate the average duration of the intertrial intervals for both alternatives.The next choice trial begins after the completion of the time-out. If a subject selects one alternative for two consecutive trials, the program will determine that the next trial will be a forced trial of the remaining alternative, that is, in the next trial, both levers will be available, but only one will operate to ensure that the subject experiences the outcomes associated with both alternatives. For DRL programming, select the value of the minimum time after which responding will produce a reward.After the beginning of a session or after any lever-press response, start a countdown timer from the selected time value to zero. If the subject displays a response before the timer reaches the value of zero, the timer will reset so that the subject must wait for a new opportunity to obtain a reward. If subject emit a response after the timer reaches the value of zero, deliver a food pellet.The timer will be reset after two seconds to allow the animal to consume the pellet. For feature-negative discrimination, first select stimuli durations, intertrial interval durations, and finishing criteria for the sessions. After presenting a stimulus A in A-plus trials, deliver a food pellet.After presenting the same stimulus A accompanied by an additional stimulus X in AX-minus trials, do not deliver any food. After setting up the protocols in the computer software, clean the inner walls, ceiling, and grill floor of the operant chambers with an appropriate disinfectant solution to remove odors from the previous sessions or previous studies. Use the computer to manually activate and monitor the crucial inputs and outputs to make sure they work and check that the food dispenser holds enough food to be delivered during the session.Move the housing cages with the rats inside close to the conditioning chambers, and open the housing cage. Gently place eat rat in its corresponding conditioning chamber before closing the conditioning chambers in the isolating shells. Initiate the program and wait until the program is finished.If the data is not saved automatically, save the output files of the session in the computer drive or elsewhere. When the program is finished, gently return the rats to their corresponding housing cages and give complementary food to the animals according to the selected food restriction regime. In this intertemporal choice procedure, the log-ratio of the lever response rate associated with the SS alternative was higher in spontaneously hypertensive rats compared to wild-type rats, suggesting the ADHD model rats prefer an immediate reward at the expense of a richer but delayed alternative.In this representative DRL protocol experiment, the longitudinal data of a single rat with a 10-second temporal restraint on responding demonstrates that over time, the animal acquired experience in the task, eventually learning to respond around 10 seconds. This pharmacological experiment tested whether impulsive performance in a DRL procedure with a temporal restriction of 10 seconds was decreased with a treatment consisting of low levels of an antipsychotic drug. Blue density plots represent the distribution of responses when saline is administered, and salmon plots represent the distribution of such responses when haloperidol was administered.The upper embedded plots show response rates, and lower ones show reward rates for saline and haloperidol with the same color code as the former plots. It can be noted that the response rate was reduced for three to five subjects, specifically for those subjects with a high proportion of burst responses in the saline condition. Typically, in the feature-negative discrimination protocol, responding during the single stimulus plus food trials and during the compound stimulus without food trials do not differ substantially in early sessions.However, after a few sessions, the rats respond differentially in both types of trials, revealing that the addition of a second stimulus counteracts the response tendency controlled by the first. Importantly, the subjects exhibit quite robust individual differences in both types of trials. Take care that the appropriate protocols are loaded in the corresponding conditioning chambers, that the rats are correctly assigned to each chamber, and that all of the chambers are closed.After a baseline procedure has been completed, behavioral, pharmacological, or surgical treatments could be administered in order to test their effect on performance. The results are not always conclusive, so researchers might want to consider whether the observed data are dependent upon the selected parameters or are in fact induced by confounding factors. Be sure to handle the rats appropriately to avoid harming the rats and to avoid the rats harming you.

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