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 border="1">
<tbody>
<tr>
<td>Item</td>
<td>Company</td>
<td>Catalog number</td>
</tr>
<tr>
<td>Enrichment Cage 		</td>
<td>Quality Cage Company</td>
<td>FH-36&frac12;T-N</td>
</tr>
<tr>
<td>Operant Chamber 		</td>
<td>Med Associates</td>
<td>MED-008-CT-B3</td>
</tr>
<tr>
<td>SCH 23390		</td>
<td>Sigma</td>
<td>D054</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 Simple Composite Phenotype Scoring System for Evaluating Mouse Models of Cerebellar Ataxia

We describe a protocol for the rapid and sensitive quantification of disease severity in mouse models of cerebella ataxia. It is derived from previously published phenotype assessments in several disease models, including spinocerebellar ataxias, Huntington s disease and spinobulbar muscular atrophy. Measures include hind limb clasping, ledge test, gait and kyphosis. Each measure is recorded on a scale of 0-3, with a combined total of 0-12 for all four measures. The results effectively discriminate between affected and non-affected individuals, while also quantifying the temporal progression of neurodegenerative disease phenotypes. Measures may be analyzed individually or combined into a composite phenotype score for greater statistical power. The ideal combination of the four described measures will depend upon the disorder in question. We present an example of the protocol used to assess disease severity in a transgenic mouse model of spinocerebellar ataxia type 7 (SCA7).
Albert R. La Spada and Gwenn A. Garden contributed to this manuscript equally.

We describe a protocol for the rapid and sensitive quantification of disease severity in mouse models of cerebellar ataxia. Measures include hind limb clasping, ledge test, gait and kyphosis. This protocol effectively discriminates between affected and non-affected individuals, and detects the progression of affected individuals over time.

We describe a protocol for the rapid and sensitive quantification of disease severity in mouse models of cerebella ataxia. It is derived from previously ...

The Vermicelli and Capellini Handling Tests: Simple quantitative measures of dexterous forepaw function in rats and mice

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended upon this work to develop a simple quantitative measure of forepaw dexterity that is sensitive to lateralized impairments and age-dependent changes. Rodents learn skillful forepaw and digit movements to manage thin pasta pieces, which they eagerly consume. We have previously described methods for quantifying vermicelli handling in rats and showed that the measures are very sensitive to forelimb impairments resulting from unilateral ischemic lesions, middle cerebral artery occlusions and unilateral striatal dopamine depletion [Allred, R.P., Adkins, D.L., Woodlee, M.T., Husbands, L.C., Maldonado M.A., Kane, J.R., Schallert, T. &amp; Jones, T.A. The Vermicelli Handling Test: a simple quantitative measure of dexterous forepaw function in rats. J. Neurosci. Methods 170, 229-244 (2008)]. Here we present a more detailed protocol for this test in rats and compare it with a newly developed version for mice, the Capellini Handling Test. Rats and mice are videotaped while handling short lengths of uncooked vermicelli or capellini pasta, respectively, with a camera positioned to optimize the view of paw movements. Slow motion video playback allows for the identification of forepaw adjustments, defined as any distinct removal and replacement of the paw, or of any number of digits, on the pasta piece after eating commences. Forepaw adjustments per piece are averaged over trials per each testing session. Repeated testing permits sensitive quantitative analysis of changes in forepaw dexterity over time. Protocols for pre-testing habituation and handling practice, as well as procedures for characterizing atypical handling patterns, are described. Because rats and mice perform the pasta handling tests slightly differently, species-specific differences in administration and scoring of these tests are highlighted. All animal use was in accordance with protocols approved by the University of Texas at Austin Animal Care and Use Committee.

The Vermicelli and Capellini Handling Tests of forepaw dexterity take advantage of the natural inclination of rodents to manipulate food items using skillful forepaw and digit movements. Animals are videotaped while handling short strands of uncooked dry pasta. Slow motion video playback allows for the quantification of forepaw adjustments.

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended ...

A Procedure for Studying the Footshock-Induced Reinstatement of Cocaine Seeking in Laboratory Rats

The most insidious aspect of drug addiction is the high propensity for relapse. Animal models of relapse, known as reinstatement procedures, have been used extensively to study the neurobiology and phenomenology of relapse to drug use. Although procedural variations have emerged over the past several decades, the most conventional reinstatement procedures are based on the drug self-administration (SA) model. In this model, an animal is trained to perform an operant response to obtain drug. Subsequently, the behavior is extinguished by withholding response-contingent reinforcement. Reinstatement of drug seeking is then triggered by a discrete event, such as an injection of the training drug, re-exposure to drug-associated cues, or exposure to a stressor 1.
Reinstatement procedures were originally developed to study the ability of acute non-contingent exposure to the training drug to reinstate drug seeking in rats and monkeys 1, 2. Reinstatement procedures have since been modified to study the role of environmental stimuli, including drug-associated cues and exposure to various forms of stress, in relapse to drug seeking 1, 3, 4.
Over the past 15 years, a major focus of the reinstatement literature has been on the role of stress in drug relapse. One of the most commonly used forms of stress for studying this relationship is acute exposures to mild, intermittent, electric footshocks. The ability of footshock stress to induce reinstatement of drug seeking was originally demonstrated by Shaham and colleagues (1995) in rats with a history of intravenous heroin SA5. Subsequently, the effect was generalized to rats with histories of intravenous cocaine, methamphetamine, and nicotine SA, as well as oral ethanol SA 3, 6.
Although footshock-induced reinstatement of drug seeking can be achieved reliably and robustly, it is an effect that tends to be sensitive to certain parametrical variables. These include the arrangement of extinction and reinstatement test sessions, the intensity and duration of footshock stress, and the presence of drug-associated cues during extinction and testing for reinstatement. Here we present a protocol for footshock-induced reinstatement of cocaine seeking that we have used with consistent success to study the relationship between stress and cocaine seeking.

Animal models of relapse, known as reinstatement procedures, have been used extensively to study the role of stress in relapse to drug seeking. Here, we report on a method for inducing the reinstatement of cocaine seeking in laboratory rats via acute exposures to mild, intermittent electric footshock.

The most insidious aspect of drug addiction is the high propensity for relapse.  Animal models of relapse, known as reinstatement procedures, have been ...

Preparation of Synaptoneurosomes from Mouse Cortex using a Discontinuous Percoll-Sucrose Density Gradient

Synaptoneurosomes (SNs) are obtained after homogenization and fractionation of mouse brain cortex. They are resealed vesicles or isolated terminals that break away from axon terminals when the cortical tissue is homogenized. The SNs retain pre- and postsynaptic characteristics, which makes them useful in the study of synaptic transmission. They retain the molecular machinery used in neuronal signaling and are capable of uptake, storage, and release of neurotransmitters.
The production and isolation of active SNs can be problematic using medias like Ficoll, which can be cytotoxic and require extended centrifugation due to high density, and filtration and centrifugation methods, which can result in low activity due to mechanical damage of the SNs. However, the use of discontinuous Percoll-sucrose density gradients to isolate SNs provides a rapid method to produce good yields of translationally active SNs. The Percoll-sucrose gradient method is quick and gentle as it employs isotonic conditions, has fewer and shorter centrifugation spins and avoids centrifugation steps that pellet SNs and cause mechanical damage.

A method to prepare translationally active, intact synaptoneurosomes (SNs) from mouse brain cortex is described. The method uses a discontinuous Percoll-sucrose density gradient allowing for the quick preparation of active SNs.

Synaptoneurosomes (SNs) are obtained after homogenization and fractionation of mouse brain cortex. They are resealed vesicles or isolated terminals that ...

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the use of IUE for investigating behavior or neuropathology in the adult brain has been limited by insufficient methods for monitoring of IUE transfection success by non-invasive techniques in postnatal animals. 
 For the present study, E16 rats were used for IUE. After intraventricular injection of the nucleic acids into the embryos, positioning of the tweezer electrodes was critical for targeting either the developing cortex or the hippocampus. 
 Ventricular co-injection and electroporation of a luciferase gene allowed monitoring of the transfected cells postnatally after intraperitoneal luciferin injection in the anesthetized live P7 pup by in vivo bioluminescence, using an IVIS Spectrum device with 3D quantification software. 
 Area definition by bioluminescence could clearly differentiate between cortical and hippocampal electroporations and detect a signal longitudinally over time up to 5 weeks after birth. This imaging technique allowed us to select pups with a sufficient number of transfected cells assumed necessary for triggering biological effects and, subsequently, to perform behavioral investigations at 3 month of age. As an example, this study demonstrates that IUE with the human full length DISC1 gene into the rat cortex led to amphetamine hypersensitivity. Co-transfected GFP could be detected in neurons by post mortem fluorescence microscopy in cryosections indicating gene expression present at &ge;6 months after birth.
 We conclude that postnatal bioluminescence imaging allows evaluating the success of transient transfections with IUE in rats. Investigations on the influence of topical gene manipulations during neurodevelopment on the adult brain and its connectivity are greatly facilitated. For many scientific questions, this technique can supplement or even replace the use of transgenic rats and provide a novel technology for behavioral neuroscience.

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

Genes can be manipulated during development of the cortex or the hippocampus of the rat via in utero electroporation (IUE) at E16, to enable fast and targeted modifications in neuronal connectivity for later studies of behavior or neuropathology in adult animals. Postnatal in vivo imaging for control of IUE success is performed by bioluminescence of activating co-transfected luciferase.

 In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the ...

Preparation of Synaptic Plasma Membrane and Postsynaptic Density Proteins Using a Discontinuous Sucrose Gradient

Neuronal subcellular fractionation techniques allow the quantification of proteins that are trafficked to and from the synapse. As originally described in the late 1960&#8217;s, proteins associated with the synaptic plasma membrane can be isolated by ultracentrifugation on a sucrose density gradient. Once synaptic membranes are isolated, the macromolecular complex known as the post-synaptic density can be subsequently isolated due to its detergent insolubility. The techniques used to isolate synaptic plasma membranes and post-synaptic density proteins remain essentially the same after 40 years, and are widely used in current neuroscience research. This article details the fractionation of proteins associated with the synaptic plasma membrane and post-synaptic density using a discontinuous sucrose gradient. Resulting protein preparations are suitable for western blotting or 2D DIGE analysis.

This article details the enrichment of proteins associated with the synaptic plasma membrane by ultracentrifugation on a discontinuous sucrose gradient. The subsequent preparation of post-synaptic density proteins is also described. Protein preparations are suitable for western blotting or 2D DIGE analysis.

Neuronal subcellular fractionation techniques allow the quantification of proteins that are trafficked to and from the synapse. As originally described in ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the experimental time, and increases the number of animals that are utilized per study. This limitation specifically impacts protocols such as calcium imaging that require prolonged pre-incubation with bath-applied dyes. Exponential bacterial growth within 3 - 4 h after slicing is tightly correlated with a decrease in tissue health. This study describes a method for&#160;limiting the proliferation of bacteria in acute preparations to maintain viable neuronal tissue for prolonged periods of time (&#62;24 h) without the need for antibiotics, sterile procedures, or tissue culture media containing growth factors. By cycling the extracellular fluid through UV irradiation and keeping the tissue in a custom holding chamber at 15 - 16 &#176;C, the tissue shows no difference in electrophysiological properties, or calcium signaling through intracellular calcium dyes at &#62;24 h postdissection. These methods will not only extend experimental time for those using acute neuronal tissue, but will reduce the number of animals required to complete experimental goals, and will set a gold standard for acute neuronal tissue incubation.

Once removed from the body, neuronal tissue is greatly affected by environmental conditions, leading to eventual degradation of the tissue after 6 - 8 h. Using a unique incubation method, which closely monitors and regulates the extracellular environment of the tissue, tissue viability can be significantly extended for &#62;24 h.

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the ...

Recording EEG in Freely Moving Neonatal Rats Using a Novel Method

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such as epilepsy and neurodegenerative disorders. However, it is technically difficult to obtain EEG recordings in neonates as it requires specialized handling and great care. Here, we present a novel method to record EEG in neonatal rat pups (P8-P15). We designed a simple and reliable electrode using computer pin loci; it can be easily implanted into the skull of a rat pup to record high-quality EEG signals in the normal and epileptic brain. Pups were given an intraperitoneal (i.p.) injection of the neurotoxin kainic acid (KA) to induce epileptic seizures. The surgical implantation performed in this procedure is less expensive than other EEG procedures for neonates. This method allows one to record high-quality and stable EEG signals for more than 1 week. Furthermore, this procedure can also be applied to adult rats and mice to study epilepsy or other neurological disorders.

Here, we introduce a novel technique designed to record electroencephalography (EEG) in freely moving neonatal epileptic pups and describe its procedures, features, and applications. This method allows one to record EEG for more than 1 week.

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such ...

Touchscreen Sustained Attention Task (SAT) for Rats

Sustained attention is the ability to monitor intermittent and unpredictable events over a prolonged period of time. This attentional process subserves other aspects of cognition and is disrupted in certain neurodevelopmental, neuropsychiatric, and neurodegenerative disorders. Thus, it is clinically important to identify mechanisms that impair and improve sustained attention. Such mechanisms are often first discovered using rodent models. Therefore, several behavior procedures for testing aspects of sustained attention have been developed for rodents. One, first described by McGaughy and Sarter (1995), called the sustained attention task (SAT), trains rats to distinguish between signal (i.e., brief light presentation) and non-signal trials. The signals are short and thus require careful attention to be perceived. Attentional demands can be increased further by introducing a distractor (e.g., flashing houselight). We have modified this task for touchscreen operant chambers, which are configured with a touchscreen on one wall that can present stimuli and record responses. Here we detail our protocol for SAT in touchscreen chambers. Additionally, we present standard measures of performance in male and female Sprague-Dawley rats. Comparable performance on this task in both sexes highlights its use for attention studies, especially as more researchers are including female rodents in their experimental design. Moreover, the easy implementation of SAT for the increasingly popular touchscreen chambers increases its utility.

Touchscreen Sustained Attention Task SAT for Rats

Sustained attention, or continuously monitoring situations for intermittent and unpredictable events, is a critical aspect of cognition. Here we detail how to test sustained attention in rats using touchscreen operant chambers. We demonstrate comparable performance in male and female rats, making this task useful for studying attention in both sexes.

Sustained attention is the ability to monitor intermittent and unpredictable events over a prolonged period of time. This attentional process subserves ...