Production of this manuscript and video was supported by National Institute on Drug Abuse/National Institutes of Health grant R15 DA016285-02, the Western Washington University Biomedical Research Activities in Neuroscience Initiative, and Western Washington University.

1. Animal Subjects
<ol>
 <li>Male Long-Evans rats are 3 months old at the start of a study. Rats are Simonsen-derived (Gilroy, California, USA) bred in the Western Washington University vivarium and are housed individually on a 12-h reverse day/night cycle (lights off at 0700) with Purina Mills Inc. Mazuri Rodent Pellets (Gray Summit, MO, USA) and water available ad libitum.</li>
 <li>All training and testing takes place between 0900-1300 with cohorts of rats always trained and tested at the same time daily.</li>
 <li>Rats are weighed each Monday, Wednesday, and Friday for the duration of the study.</li>
 <li>Immediately prior to the training phase, rats are deprived of water for approximately 20 h to encourage sucrose self-administration on the first day of training.</li>
</ol>
2. Behavioral Procedures
All operant behavioral procedures use standard operant conditioning chambers (Med Associates Inc., St. Albans, VT, USA) with an infusion pump (Razel Scientific Instruments, St. Albans, VT, USA) located on top of each operant conditioning chamber. Operant conditioning chambers are enclosed in sound-attenuating cabinets with ventilation fans (Western Washington University, USA). All experimental conditions and data collection are controlled by Med PC IV software (Med Associates Inc., St. Albans, VT, USA).
Operant conditioning chambers (30 x 20 x 24 cm) contain two levers (one stationary and one retractable), a tone generator, a white stimulus light above the retractable lever, and a red house light on the opposite wall. The infusion pump delivers sucrose into a reward receptacle to the right of the active lever. Levers are 10 cm from the grid floor. Four infrared photobeams crisscross each chamber. The front beams are each 10.5 cm from the side walls and the side beams each 6 cm from the side walls. Each beam is 4.5 cm above the stainless steel bar floor. The beams are set to count the number of complete breaks. The total number of beam breaks are recorded during Training and craving Testing.
With the exception of water restriction prior to the first Training session, rats are provided food and water ad libitum both in home cages and in the operant conditioning chambers. Chambers are in a dedicated test room that, as with the vivarium, is on a reverse light cycle. Rats are transported in home cages between the rooms on a cart that is covered with light blocking fabric. At the end of each session, rats are returned to home cages.
There are three phases to the typical experiment: Training, Forced Abstinence, and Testing. Testing is the session wherein craving is assessed. We assess craving in two general testing procedures, depending on the study. These two procedures are described in sections 2.3.1 and 2.3.2.
<ol>
 <li>Training
 <ol>
 <li>In the typical experiment, rats spend 2 h/day for 10 consecutive days in operant conditioning chambers where they are allowed to press the retractable (active) lever for a 0.2 ml delivery of 10 % sucrose solution into the receptacle to the right of the lever.</li>
 <li>This response also activates a compound stimulus consisting of a tone (2 kHz, 15 dB over ambient noise) and the white light. The compound stimulus lasts for 5 s and is followed by a 40-s time out, during which presses on the active lever are recorded but have no programmed consequence.</li>
 <li>A response on the inactive (stationary) lever has no programmed consequence, but presses are recorded. The numbers of beam breaks are recorded during Training and Testing. All response and locomotor measures are recorded as time courses (2 minute bins), but are usually reported as totals per session.</li>
 </ol>
 </li>
 <li>Forced Abstinence. Rats remain in home cages for the duration of forced abstinence. The typical forced abstinence periods we use are to have rats tested the day immediately following the final day of Training (Day 1 of forced abstinence) or 30 days following the final day of Training (Day 30 of forced abstinence).</li>
 <li>Testing
 <ol>
 <li>Extinction without cues. In this Testing procedure, rats are first allowed to press the active lever in conditions identical to Training with the exception that the response has no consequences and the session duration is 1 h. Rats experience at least six of these extinction sessions on the Test day. Each extinction session is separated by a 5 minute period where the active lever is retracted and the house light is turned off. "Extinction" is indicated if active lever responding has been reduced to less than 20 presses/h. Some rats require a seventh session. The next session (after another 5 minute delay) is the Testing session where rats can then press for the tone+light cue that was initially paired with each delivery of sucrose during Training.</li>
 <li>Extinction with cues. In this abbreviated Testing procedure, rats are allowed to press the active lever in conditions identical to Training except that sucrose is not delivered.</li>
 </ol>
 </li>
 <li>Inactive lever pressing is used as a measure of lever discrimination. It is also, along with photobeam breaks, used as a measure of non-specific motor activation. Motoric vs. motivational changes complicate the interpretation of craving behavior. The inactive lever responding and photobeam break data are useful in clarifying motor vs. motivation contributions to craving, as are comparison groups in pharmacological studies where rats responding at a high rate for sucrose or another food are challenged with a potential craving affecting compound (e.g. 7,8).</li>
</ol>
3. Representative Manipulations to Affect Sucrose Craving after Variable Periods of Forced Abstinence
The general method described here provides a way to probe the behavioral and neurobiological determinants of craving after variable periods of forced abstinence. Described here are methods related to Representative Results provided in section 4.
<ol>
 <li>Satiation. Some rats received bottles of 10 % sucrose in their home cages for the 17 h immediately prior to Testing 8. Rats in this study were tested using the Extinction without cues procedure.</li>
 <li>Environmental Enrichment. During the forced-abstinence period, some rats remained in individual housing (Controls) while others were moved into a large (36 " width x 20 " depth x 40 " height; Quality Cage Products, Portland, OR, USA) cage with three cohorts (Enriched). The Enriched animals had several toys in their environment including PVC tubing and were also given novel toys every M,W,F 10. Rats in this study were tested using the Extinction without cues procedure.</li>
 <li>Pharmacological challenge. On the Testing day, rats were pretreated with a dose of the dopamine D1 antagonist SCH 23390. Rats in this study were tested using the Extinction with cues procedure.</li>
</ol>
4. Representative Results:
<img alt="Figure 1" src="/files/ftp_upload/3335/3335fig1.jpg" /> 
Figure 1 Representative Training and Testing data for an incubation of sucrose craving experiment.
Data points indicate means &plusmn; SEMs. Group size for Figure 1a is 77 rats and for Figure 1b group sizes ranged from 11-12 per group. * significant difference from Day 1 group, p &lt; 0.05. This figure is adapted from data in 8 with permission of Springer.
<img alt="Figure 2" src="/files/ftp_upload/3335/3335fig2.jpg" /> 
Figure 2 Effect of sucrose satiation on sucrose craving after 1 or 30 days of forced abstinence. Group sizes are 8-9 per group. * significant difference from Day 1 group, &dagger; significant difference from Control group, p &lt; 0.05. This figure is adapted from 9 with permission of Elsevier.
<img alt="Figure 3" src="/files/ftp_upload/3335/3335fig3.jpg" /> 
Figure 3 One month of environmental enrichment reduced sucrose craving. Group sizes are each 8 rats. * significant difference from Day 1, &dagger; significant difference from Control group, p &lt; 0.05. This figure is adapted from 10 with permission of Wolters Kluwer Health.
<img alt="Figure 4" src="/files/ftp_upload/3335/3335fig4.jpg" /> 
Figure 4 Systemic SCH 23390 decreased sucrose craving more effectively after one day of forced abstinence. Group sizes are 8-12 per group. * significant difference from Day 1 group, &dagger; significant difference from 0 dose group, p &lt; 0.05. This figure is adapted from 8 with permission of Springer.
<ol>
 <li>Figure 1 indicates representative Training and Testing data for rats self-administering sucrose (Figure 1 TOP) and then responding in Extinction with the sucrose-paired cue available (Figure 1 BOTTOM). As shown, rats respond selectively on the active lever during Training and during Testing. Interestingly, inactive responding and locomotor activity show some incubation, although the effect is most robust on active lever responding.</li>
 <li>Figure 2 shows the effect of satiation with sucrose on sucrose craving after 1 or 30 days of forced abstinence. Satiation reduced Extinction without cues responding after either period of forced abstinence, but only reduced responding for the sucrose cue alone in rats that had only one day of forced abstinence.</li>
 <li>As shown in Figure 3, environmental enrichment reduced incubated Extinction without cue responding and responding for the sucrose cue alone behaviors to levels found after only one day of forced abstinence.</li>
 <li>Figure 4 depicts an abstinence-dependent effect of SCH 23390 on Extinction with cues responding. SCH 23390 was most effective at reducing sucrose craving after 1, vs. 30, days of forced abstinence.</li>
</ol>

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All procedures followed the guidelines outlined in the "Principles of Laboratory Animal Care" (NIH publication no. 86-23) and were approved by the Western Washington University Institutional Animal Care and Use Committee.

The procedures we describe here could be identified as reinstatement procedures, although this is technically inaccurate as lever pressing for the cue is not extinguished prior to the craving test session. In a typical reinstatement procedure, responding with reward-paired cues is extinguished and then "reinstated" with administration of a non-contingent presentation of the reward (priming-induced reinstatement) or a stressor such as footshock (stress-induced reinstatement) 11. The extinction of lever pressi...

<table><tbody><tr><td>Enrichment Cage </td><td>Quality Cage Company</td><td>FH-36&amp;#189;T-N</td><td></td></tr><tr><td>Operant Chamber </td><td>Med Associates, Inc.</td><td>MED-008-CT-B3</td><td></td></tr><tr><td>SCH 23390</td><td>Sigma-Aldrich</td><td>D054</td><td></td></tr></tbody></table>

a general method for evaluating incubation of sucrose craving in rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and relapse to food taking 1. Food craving conceptualized in this way is akin to drug craving in response to drug-paired cues. A rich literature has been developed around understanding the behavioral and neurobiological determinants of drug craving; we and others have been focusing recently on translating techniques from basic addiction research to better understand addiction-like behaviors related to food 2-4.
As done in previous studies of drug craving, we examine sucrose craving behavior by utilizing a rat model of relapse. In this model, rats self-administer either drug or food in sessions over several days. In a session, lever responding delivers the reward along with a tone+light stimulus. Craving behavior is then operationally defined as responding in a subsequent session where the reward is not available. Rats will reliably respond for the tone+light stimulus, likely due to its acquired conditioned reinforcing properties 5. This behavior is sometimes referred to as sucrose seeking or cue reactivity. In the present discussion we will use the term "sucrose craving" to subsume both of these constructs.
In the past decade, we have focused on how the length of time following reward self-administration influences reward craving. Interestingly, rats increase responding for the reward-paired cue over the course of several weeks of a period of forced-abstinence. This "incubation of craving" is observed in rats that have self-administered either food or drugs of abuse 4,6. This time-dependent increase in craving we have identified in the animal model may have great potential relevance to human drug and food addiction behaviors.
Here we present a protocol for assessing incubation of sucrose craving in rats. Variants of the procedure will be indicated where craving is assessed as responding for a discrete sucrose-paired cue following extinction of lever pressing within the sucrose self-administration context (Extinction without cues) or as responding for sucrose-paired cues in a general extinction context (Extinction with cues).

Watch this Scientific Journal Video about A General Method for Evaluating Incubation of Sucrose Craving in Rats at JoVE.com

A General Method for Evaluating Incubation of Sucrose Craving in Rats

Responding for food or drug-paired cues increases over the course of a period of abstinence and this may relate to an increased susceptibility to relapse behaviors. Here we detail a procedure for evaluating this "incubation of craving" in rats that have self-administered sucrose.

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

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13.2K Views.  Western Washington University. Responding for food or drug-paired cues increases over the course of a period of abstinence and this may relate to an increased susceptibility to relapse behaviors. Here we detail a procedure for evaluating this "incubation of craving" in rats that have self-administered sucrose.

Video: A General Method for Evaluating Incubation of Sucrose Craving 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 ...

Behavior

A Method for Evaluating the Reinforcing Properties of Ethanol in Rats without Water Deprivation, Saccharin Fading or Extended Access Training

Operant oral self-administration methods are commonly used to study the reinforcing properties of ethanol in animals. However, the standard methods require saccharin/sucrose fading, water deprivation and/or extended training to initiate operant responding in rats. This paper describes a novel and efficient method to quickly initiate operant responding for ethanol that is convenient for experimenters and does not require water deprivation or saccharin/sucrose fading, thus eliminating the potential confound of using sweeteners in ethanol operant self-administration studies. With this method, Wistar rats typically acquire and maintain self-administration of a 20% ethanol solution in less than two weeks of training. Furthermore, blood ethanol concentrations and rewards are positively correlated for a 30 min self-administration session. Moreover, naltrexone, an FDA-approved medication for alcohol dependence that has been shown to suppress ethanol self-administration in rodents, dose-dependently decreases alcohol intake and motivation to consume alcohol for rats self-administering 20% ethanol, thus validating the use of this new method to study the reinforcing properties of alcohol in rats.

This protocol describes a novel and efficient method to quickly initiate operant responding for ethanol in rats that, contrary to standard methods, does not require water deprivation or saccharin/sucrose fading to initiate responding.

Operant oral self-administration methods are commonly used to study the reinforcing properties of ethanol in animals. However, the standard methods ...

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 ...

A Conditioned Place Preference Protocol for Measuring Incubation of Craving in Rats

A major cause of repeated relapses is a craving for the drug. Drug craving increases progressively during the abstinence period, a phenomenon termed incubation of drug craving. Here, we describe a morphine conditioned place preference (CPP) protocol for measuring the incubation of craving in rats. In this protocol, a CPP paradigm mainly employing somatosensory cues is used to establish a long-term reward memory of morphine. A three-chamber CPP box that differs in the texture of the chamber floor is constructed. First, the animals are tested for their baseline preference to the two side chambers for three consecutive days. Then, they are injected intraperitoneally with morphine/saline and put into their non-preferred/preferred chamber for 45 min. After 6 days of conditioning, their preference to the side chambers is tested for 15 min at different time points after the last conditioning session. With this paradigm, the reward memory of morphine could last for at least 18 days. To test whether the above-mentioned protocol can model increased craving, the number of entrances into the two side chambers are counted during the abstinence period. The results show that the entrances increased, suggesting that the CPP paradigm could mimic the incubation of craving. Future studies can employ this model to study neural mechanisms underlying long-term memory and incubation of craving.

Here, a morphine conditioned place preference (CPP) protocol is described to measure incubation of craving in rats.

A major cause of repeated relapses is a craving for the drug. Drug craving increases progressively during the abstinence period, a phenomenon termed ...

A Proboscis Extension Response Protocol for Investigating Behavioral Plasticity in Insects: Application to Basic, Biomedical, and Agricultural Research

Insects modify their responses to stimuli through experience of associating those stimuli with events important for survival (e.g., food, mates, threats). There are several behavioral mechanisms through which an insect learns salient associations and relates them to these events. It is important to understand this behavioral plasticity for programs aimed toward assisting insects that are beneficial for agriculture. This understanding can also be used for discovering solutions to biomedical and agricultural problems created by insects that act as disease vectors and pests. The Proboscis Extension Response (PER) conditioning protocol was developed for honey bees (Apis mellifera) over 50 years ago to study how they perceive and learn about floral odors, which signal the nectar and pollen resources a colony needs for survival. The PER procedure provides a robust and easy-to-employ framework for studying several different ecologically relevant mechanisms of behavioral plasticity. It is easily adaptable for use with several other insect species and other behavioral reflexes. These protocols can be readily employed in conjunction with various means for monitoring neural activity in the CNS via electrophysiology or bioimaging, or for manipulating targeted neuromodulatory pathways. It is a robust assay for rapidly detecting sub-lethal effects on behavior caused by environmental stressors, toxins or pesticides.
We show how the PER protocol is straightforward to implement using two procedures. One is suitable as a laboratory exercise for students or for quick assays of the effect of an experimental treatment. The other provides more thorough control of variables, which is important for studies of behavioral conditioning. We show how several measures for the behavioral response ranging from binary yes/no to more continuous variable like latency and duration of proboscis extension can be used to test hypotheses. And, we discuss some pitfalls that researchers commonly encounter when they use the procedure for the first time.

The Proboscis Extension Response (PER) conditioning protocol, developed for the honey bee (Apis mellifera), provides an ecologically-relevant and easily quantifiable means for studying several different mechanisms of learning in many insect species.

Insects modify their responses to stimuli through experience of associating those stimuli with events important for survival (e.g., food, mates, threats). ...

A Complex Diving-For-Food Task to Investigate Social Organization and Interactions in Rats

For many species, where status is a vital motivator that can affect health, social hierarchies influence behavior. Social hierarchies that include dominant-submissive relationships are common in both animal and human societies. These relationships can be affected by interactions with others and with their environment, making them difficult to analyze in a controlled study. Rather than a simple dominance hierarchy, this formation has a complicated presentation that allows rats to avoid aggression. Status can be stagnant or mutable, and results in complex societal stratifications. Here we describe a complex diving-for-food task to investigate rodent social hierarchy and behavioral interactions. This animal model may allow us to assess the relationship between a wide range of mental illnesses and social organization, as well as to study the effectiveness of therapy on social dysfunction.

This protocol describes a method of examining social hierarchy in a rat model. Rats perform a complex diving-for-food task in which they form a distinct hierarchy according to their willingness to dive underwater and swim to obtain a food pellet. This method is used to understand decision making and social relationships among highly social animals in small groups.

For many species, where status is a vital motivator that can affect health, social hierarchies influence behavior. Social hierarchies that include ...

A Procedure to Study Stress-Induced Relapse of Heroin Seeking after Punishment-Imposed Abstinence

The punishment-imposed abstinence procedure models the self-imposed abstinence that humans initiate due to the adverse consequences associated with drug-taking. This model has been implemented in experiments using different types of substances of abuse such as methamphetamine, cocaine, and alcohol. However, punishment-induced abstinence in heroin-trained animals has not been demonstrated. Furthermore, acute stress is a key trigger for relapse in humans and animal models. It was previously demonstrated that acute food deprivation robustly induced reinstatement of extinguished cocaine and heroin seeking. The procedure described here can be used to assess the effects of acute stress exposure on heroin seeking after punishment-imposed abstinence. A total of 8 rats were implanted with chronic intravenous (i.v.) catheters and trained to self-administer heroin (0.1 mg/kg/infusion) for 18 days under a seek-take chained schedule. Completing the seek link gave access to the take lever, which was paired with a heroin infusion. The seek lever was programmed with a variable interval 60 schedule of reinforcement (VI60), and the take lever was programmed with a fixed-ratio 1 reinforcement schedule (FR1). Following self-administration training, a mild foot shock was delivered on 30% of the completed seek links instead of the extension of the take lever. Footshock intensity was increased by 0.1 mA per daily session from 0.2 mA to 1.0 mA. Heroin-seeking tests were performed after 24 h of food deprivation (FD) or sated conditions. Rats under acute food deprivation condition robustly increased heroin seeking after punishment-imposed abstinence.

A procedure that demonstrates a robust acute food deprivation-induced relapse to heroin seeking after punishment-imposed abstinence is described. A punishment-imposed abstinence model was successfully implemented using the seek and take chain schedule for heroin self-administration. Heroin-seeking tests are then performed following 24 h of food-deprivation stress.

The punishment-imposed abstinence procedure models the self-imposed abstinence that humans initiate due to the adverse consequences associated with ...

Simultaneous Detection of c-Fos Activation from Mesolimbic and Mesocortical Dopamine Reward Sites Following Naive Sugar and Fat Ingestion in Rats

This study uses cellular c-fos activation to assess effects of novel ingestion of fat and sugar on brain dopamine (DA) pathways in rats. Intakes of sugars and fats are mediated by their innate attractions as well as learned preferences. Brain dopamine, especially meso-limbic and meso-cortical projections from the ventral tegmental area (VTA), has been implicated in both of these unlearned and learned responses. The concept of distributed brain networks, wherein several sites and transmitter/peptide systems interact, has been proposed to mediate palatable food intake, but there is limited evidence empirically demonstrating such actions. Thus, sugar intake elicits DA release and increases c-fos-like immunoreactivity (FLI) from individual VTA DA projection zones including the nucleus accumbens (NAC), amygdala (AMY) and medial prefrontal cortex (mPFC) as well as the dorsal striatum. Further, central administration of selective DA receptor antagonists into these sites differentially reduce acquisition and expression of conditioned flavor preferences elicited by sugars or fats. One approach by which to determine whether these sites interacted as a distributed brain network in response to sugar or fat intake would be to simultaneous evaluate whether the VTA and its major mesotelencephalic DA projection zones (prelimbic and infralimbic mPFC, core and shell of the NAc, basolateral and central-cortico-medial AMY) as well as the dorsal striatum would display coordinated and simultaneous FLI activation after oral, unconditioned intake of corn oil (3.5%), glucose (8%), fructose (8%) and saccharin (0.2%) solutions. This approach is a successful first step in identifying the feasibility of using cellular c-fos activation simultaneously across relevant brain sites to study reward-related learning in ingestion of palatable food in rodents.

The goal of this study is to identify reward-related distributed brain networks by delineating a reliable immunohistological technique using cellular c-fos activation to measure simultaneous changes in dopamine pathways and terminal sites after novel ingestion of fat and sugar in rats.

This study uses cellular c-fos activation to assess effects of novel ingestion of fat and sugar on brain dopamine (DA) pathways in rats. Intakes of sugars ...

A Novel Behavioral Assay to Investigate Gustatory Responses of Individual, Freely-moving Bumble Bees (Bombus terrestris)

Generalist pollinators like the buff-tailed bumble bee, Bombus terrestris, encounter both nutrients and toxins in the floral nectar they collect from flowering plants. Only a few studies have described the gustatory responses of bees toward toxins in food, and these experiments have mainly used the proboscis extension response on restrained honey bees. Here, a new behavioral assay is presented for measuring the feeding responses of freely-moving, individual worker bumble bees to nutrients and toxins. This assay measures the amount of solution ingested by each bumble bee and identifies how tastants in food influence the microstructure of the feeding behavior.
The solutions are presented in a microcapillary tube to individual bumble bees that have been previously starved for 2-4 hr. The behavior is captured on digital video. The fine structure of the feeding behavior is analyzed by continuously scoring the position of the proboscis (mouthparts) from video recordings using event logging software. The position of the proboscis is defined by three different behavioral categories: (1) proboscis is extended and in contact with the solution, (2) proboscis is extended but not in contact with the solution and (3) proboscis is stowed under the head. Furthermore the speed of the proboscis retracting away from the solution is also estimated.
In the present assay the volume of solution consumed, the number of feeding bouts, the duration of the feeding bouts and the speed of the proboscis retraction after the first contact is used to evaluate the phagostimulatory or the deterrent activity of the compounds tested.
This new taste assay will allow researchers to measure how compounds found in nectar influence the feeding behavior of bees and will also be useful to pollination biologists, toxicologists and neuroethologists studying the bumble bee&#39;s taste system.

A Novel Behavioral Assay to Investigate Gustatory Responses of Individual, Freely-moving Bumble Bees Bombus terrestris

A novel behavioral assay is described for investigating the short term gustatory responses of the mouthparts of freely-moving bumble bees (Bombus terrestris) toward nutrients and toxins in solution.

Generalist pollinators like the buff-tailed bumble bee, Bombus terrestris, encounter both nutrients and toxins in the floral nectar they collect from ...

A Protocol for Measuring Cue Reactivity in a Rat Model of Cocaine Use Disorder

Cocaine use disorder (CUD) follows a trajectory of repetitive self-administration during which previously neutral stimuli gain incentive value. Cue reactivity, the sensitivity to cues previously linked with the drug-taking experience, plays a prominent role in human craving during abstinence. Cue reactivity can be assessed as the attentional orientation toward drug-associated cues that is measurable as appetitive approach behavior in both preclinical and human studies. Herein describes an assessment of cue reactivity in rats trained to self-administer cocaine. Cocaine self-administration is paired with the presentation of discrete cues that act as conditioned reinforcers (i.e., house light, stimulus light, infusion pump sounds). Following a period of abstinence, lever presses in the cocaine self-administration context accompanied by the discrete cues previously paired with cocaine infusion are measured as cue reactivity. This model is useful to explore neurobiological mechanisms underlying cue reactivity processes as well as to assess pharmacotherapies to suppress cue reactivity and therefore, modify relapse vulnerability. Advantages of the model include its translational relevance, and its face and predictive validities.&#160;The primary limitation of the model is that the cue reactivity task can only be performed infrequently and must only be used in short duration (e.g., 1 hour), otherwise rats will begin to extinguish the pairing of the discrete cues with the cocaine stimulus. The model is extendable to any positively reinforcing stimulus paired with discrete cues; though particularly applicable to drugs of abuse, this model may hold future applications in fields such as obesity, where palatable food rewards can act as positively reinforcing stimuli.

Cue reactivity is conceptualized as sensitivity to cues linked with drug-taking experiences that contribute to craving and relapse in abstinent humans. Cue reactivity is modeled in rats by measuring attentional orientation toward drug-associated cues that results in appetitive approach behavior in a cue reactivity test following self-administration and forced abstinence.

Cocaine use disorder (CUD) follows a trajectory of repetitive self-administration during which previously neutral stimuli gain incentive value. Cue ...