This project was supported by grants from the Natural Sciences and Engineering Research Council of Canada (355881) and the Canadian Foundation for Innovation.

*Most important steps.
1. Equipment and Software
<ol>
	<li>Training and testing is carried out in mouse operant chambers equipped with two ultra-light, retractable levers1 positioned on either side of a food receptacle (see Table 1). A stimulus light is located above each lever and there is a house light at the top of the cage. A pellet dispenser is located outside of the chamber. Each chamber is housed inside a sound-attenuating cubicle with ventilating fan. Chambers are connected to a PC computer with MedPC IV software via a "Smart Control" interface cabinet.</li>
</ol>
Note: When ultra-light levers are not available it is recommended that nose-poke be used as the operant response for mouse studies. However, with both options available we suggest selection of ultra-light levers as a more reliable measure of motivation given that nose-poke responses can be triggered by exploratory sniffing.
<ol start="2">
	<li>The MedPC programs used to run operant tasks are written in Medstate notation code which a user can learn with the help of the programming manual provided. Programming provides flexibility for the user to modify existing codes or generate unique experimental parameters. Certain codes can be obtained for free from Med Associates "Medstate Notation Repository" <a href="http://www.mednr.com" target="_blank">http://www.mednr.com</a>. Additional codes may be purchased from Med Associates.</li>
</ol>
2. Mouse Acclimation and Food Restriction
<ol>
	<li>Upon arrival mice are group-housed for at least 10 days to acclimatize to a reverse 12 h light/dark cycle and provided with ad libitum access to standard chow and water. In our case we use adult C57BL/6 mice purchased from Charles River (St. Constant, QC). All mouse manipulations and behavioral experiments are performed during the dark cycle. All procedures involving the use of animals were approved by the CRCHUM Animal Care Committee.</li>
	<li>Mice are subsequently singly-housed and food restricted (70% of their daily food intake) until they reach 90-95% free-feeding body weights in order to facilitate acquisition of lever-press responding. Mice were provided their daily quota of food in the home cage after the termination of session. Upon reaching 5-10% weight loss, the daily food allotted is adjusted to stabilize the lower body weights for the remainder of the training period. Once mice have attained acquisition criteria (see below) they are put back on an ad libitum feeding schedule. Mice regain lost body weight in about 3-4 days.</li>
	<li>Mice are handled in the test room for 3 consecutive days prior to the first training session. The day prior to the start of training 10-15 high-fat and high-sugar (HFHS) pellets are placed into the home cage to prevent the potential influence of food neophobia on operant performance. We use 20 mg HFHS, dust-free, precision food pellets (14 mg also available) containing 48.9% Kcal as fat (Bio Serv, Frenchtown, NJ).</li>
</ol>
3. Operant Training
<ol>
	<li>Mice are initially trained to press the lever on a fixed ratio (FR)-1 reinforcement schedule whereby a single lever press elicits the delivery of a food pellet to the receptacle. Only one lever is designated as "active" (triggering delivery of food reward) and the allocation of right and left levers is counterbalanced between mice. It is important to add at least a 5 second timeout (TO) to the FR1 schedule (FR1/TO-5), during which additional lever-pressing does not result in the delivery of a food pellet. This TO period allows time for mice to consume the food pellet, however longer time outs may be used if the experimenter feels it is necessary for their mouse model. Each FR training session lasts 1 hour or when 50 pellets have been delivered.</li>
	<li>*Acquisition Criteria: The criteria used to determine acquisition of food-maintained operant responding includes: (1) a minimum number of active responses and rewards earned; (2) a measure of discrimination between active and inactive levers, and (3) between-session performance stability. Mice exhibiting discrimination of &ge;3:1 for the active versus inactive lever and obtaining &ge; 20 rewards per session over three consecutive sessions are considered to achieve acquisition criteria10. We have observed that the majority (~75%) of C57BL6 mice require about 7-10 days of training to achieve acquisition criteria. Mice that do not reach acquisition criteria by this time undergo further training for an additional 5-7 days. We exclude mice from testing (~5% of cases) when there is no progress by the extra 5th training day. It is important to keep in mind that impaired operant acquisition may be an outcome of certain pharmacological and genetic interventions and should therefore be documented.</li>
	<li>Following three successive sessions of obtaining &ge; 20 pellets, the schedule is increased to FR5/TO-5 seconds in which 5 active lever presses trigger the delivery of the food pellet. Training on the FR5 schedule lasts three days.</li>
	<li>*The mice are then trained in the progressive ratio (PR) schedule of reinforcement. The response ratio schedule during PR testing can be calculated as per Richardson and Roberts (1996)11 using the following formula (rounded to the nearest integer): 
	= [5e (R*0.2)] - 5 
	where R is equal to the number of food rewards already earned plus 1 (i.e., next reinforcer). Thus, the number of responses required to earn a food reward follow the order: 1, 2, 4, 6, 9, 12, 15, 20, 25, 32, 40, 50, 62, 77, 95 and so on. The final ratio completed is the breakpoint.</li>
	<li>A PR session lasts a maximum of 1 hour. Failure to press the lever in any 10 min period results in termination of the session. Performance on the PR schedule of reinforcement is considered stable when the number of rewards earned in a 1 hour session deviates by &le;10% for at least 3 consecutive days.</li>
</ol>
4. Progressive Ratio Testing and Validation
<ol>
	<li>To validate food-maintained operant responding in the PR task, one can assess breakpoint responding following manipulations known to modulate food reward. A relatively easy test to carry out is food deprivation which will increase the motivational state of the mouse for food and thereby increase breakpoints. Mice must first be tested in the PR task during a period of ad libitum feeding until stable, baseline performance is achieved. The day after the last "baseline" day food is removed from the home cage and mice are then tested in the PR task 24 hours after the start of the fast.</li>
	<li>We also investigated whether peripheral administration of the anorectic hormone leptin would have the opposite effect on breakpoint thresholds. Leptin (A.F. Parlow, National Hormone and Peptide Program, NIDDK) was dissolved in sterile PBS. PR testing was carried out following an IP injection of PBS on one day and then following injection of leptin (5 mg/kg) the following day. Leptin was injected one hour prior to PR testing. Breakpoints were compared using a paired t-test (GraphPad Prism).</li>
</ol>
5. Representative Results
When testing the ability of food deprivation to modulate breakpoint responding on a PR schedule of reinforcement, we expect to significantly increase breakpoints as shown in Figure 1. In this experiment a 24 h period of food deprivation produced a ~3.6-fold increase in breakpoints as compared to those obtained in the free-feeding baseline state thereby suggesting that food deprivation increases the rewarding effects of food. In contrast, peripheral administration of the anorectic hormone leptin (5 mg/kg, IP) one hour prior to testing should decrease breakpoint responding as shown in Figure 2.
<img alt="Figure 1" src="/files/ftp_upload/3754/3754fig1.jpg" /> 
Figure 1. Fasting increases the rewarding effects of food. A 24 h fast significantly increased breakpoints on a progressive ratio (PR) schedule of reinforcement in mice (n=4). Mean&plusmn;SEM; *p&lt;.05.
<img alt="Figure 2" src="/files/ftp_upload/3754/3754fig2.jpg" /> 
Figure 2. Leptin decreases food reward in mice. Peripheral leptin administration (5 mg/kg) decreased PR breakpoint response thresholds in mice (n=4). Mean&plusmn;SEM; *p&lt;0.05.

<ol>
	<li>Fulton, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Appetite+and+reward.">Appetite and reward.</a> Front. Neuroendocrinol. 31, 85-103 (2010).</li><li>Zheng, H., Lenard, N. R., Shin, A. C., Berthoud, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Appetite+control+and+energy+balance+regulation+in+the+modern+world:+reward-driven+brain+overrides+repletion+signals.">Appetite control and energy balance regulation in the modern world: reward-driven brain overrides repletion signals.</a> International journal of Obesity. 33, S8-S13 (2009).</li><li>Skinner, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Behavior of Organisms: An Experimental Analysis. , (1938).</li><li>Hodos, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progressive+ratio+as+a+measure+of+reward+strength.">Progressive ratio as a measure of reward strength.</a> Science. 134, 943-944 (1961).</li><li>Hodos, W., Kalman, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+increment+size+and+reinforcer+volume+on+progressive+ratio+performance.">Effects of increment size and reinforcer volume on progressive ratio performance.</a> J. Exp. Anal. Behav. 6, 387-392 (1963).</li><li>Stafford, D., LeSage, M. G., Glowa, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progressive-ratio+schedules+of+drug+delivery+in+the+analysis+of+drug+self-administration:+a+review.">Progressive-ratio schedules of drug delivery in the analysis of drug self-administration: a review.</a> Psychopharmacology. , 139-169 (1998).</li><li>Glass, M. J., O'Hare, E., Cleary, J. P., Billington, C. J., Levine, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+naloxone+on+food-motivated+behavior+in+the+obese+Zucker+rat.">The effect of naloxone on food-motivated behavior in the obese Zucker rat.</a> Psychopharmacology (Berl). 141, 378-384 (1999).</li><li>Brennan, K., Roberts, D. C., Anisman, H., Merali, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Individual+differences+in+sucrose+consumption+in+the+rat:+motivational+and+neurochemical+correlates+of+hedonia.">Individual differences in sucrose consumption in the rat: motivational and neurochemical correlates of hedonia.</a> Psychopharmacology (Berl). 157, 269-276 (2001).</li><li>Vaughan, C., Moore, M., Haskell-Luevano, C., Rowland, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Food+motivated+behavior+of+melanocortin-4+receptor+knockout+mice+under+a+progressive+ratio+schedule.">Food motivated behavior of melanocortin-4 receptor knockout mice under a progressive ratio schedule.</a> Peptides. 27, 2829-2835 (2006).</li><li>Haluk, D. M., Wickman, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+study+design+variables+and+their+impact+on+food-maintained+operant+responding+in+mice.">Evaluation of study design variables and their impact on food-maintained operant responding in mice.</a> Behav. Brain Res. 207, 394-401 (2010).</li><li>Richardson, N. R., Roberts, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progressive+ratio+schedules+in+drug+self-administration+studies+in+rats:+a+method+to+evaluate+reinforcing+efficacy.">Progressive ratio schedules in drug self-administration studies in rats: a method to evaluate reinforcing efficacy.</a> J. Neurosci. Methods. 66, 1-11 (1996).</li><li>Jewett, D. C., Cleary, J., Levine, A. S., Schaal, D. W., Thompson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+neuropeptide+Y,+insulin,+2-deoxyglucose,+and+food+deprivation+on+food-motivated+behavior.">Effects of neuropeptide Y, insulin, 2-deoxyglucose, and food deprivation on food-motivated behavior.</a> Psychopharmacology (Berl). 120, 267-271 (1995).</li><li>Fulton, S., Woodside, B., Shizgal, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+brain+reward+circuitry+by+leptin.">Modulation of brain reward circuitry by leptin.</a> Science. 287, 125-128 (2000).</li><li>Figlewicz, D. P., Bennett, J. L., Naleid, A. M., Davis, C., Grimm, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraventricular+insulin+and+leptin+decrease+sucrose+self-administration+in+rats.">Intraventricular insulin and leptin decrease sucrose self-administration in rats.</a> Physiol. Behav. 89, 611-616 (2006).</li></ol>

Production and Free Access of this video-article is sponsored by Med Associates, Inc.

Operant conditioning tasks provide an effective means to evaluate changes in the motivational properties of food. The experimental procedures detailed in the present article permit training mice to lever press for food rewards and stably respond on a PR schedule of reinforcement.
Measuring the amount of food consumed does not provide the same readout as effort-based response measures since not all factors that affect food intake modulate the rewarding effects of food. For example, depleting utilizable glucose by peripheral 2-deoxyglucose administration in rats increases the amount of freely available food consumed but fails to alter break points in a PR task for food12. PR breakpoint responding can also reflect changes in the quality of food rewards. For example, break points are positively correlated with sucrose concentration demonstrating that motivation for sucrose increases in a manner that follows sweet taste8. Here we demonstrate that 24h food deprivation substantially increases PR breakpoints for HFHS food in mice. In contrast, we found that peripheral administration of the anorectic hormone leptin significantly decreases breakpoints for HFHS food in mice. These findings are consistent with previously reported actions of food deprivation and leptin on reward thresholds in rats12-14.
When using this protocol it is important to keep in mind that acquisition of food-maintained operant responding is significantly enhanced by regular training (every 1-2 days) and by increasing the motivational state of the mouse by implementing a food restriction regimen. In addition, ensuring that mice exhibit relatively stable responding on one reinforcement schedule before moving on to more complex schedules (e.g, FR1 to FR5) is key to minimizing variable performance during testing. Despite the application of these steps, it is not unusual to obtain some variability especially between different mice. For this reason, it is highly recommended to use a within-subjects (repeated measures) design when possible to remove across subject variability. Although within-subject testing is not possible for investigating stable genetic modifications (i.e., knockout versus control mice) it can be used to examine the influence of drugs, lesions or acute genetic (e.g., optogenetic) manipulations.
Obesity and obesity-related illnesses are a growing problem that has increased the urgency to identify the molecules and neural pathways involved. Accumulating evidence highlights the important role of brain reward circuitry in food craving, increased caloric intake and weight gain. The PR task is a valuable tool for measuring the impact of neurobiological, genetic or pharmacological manipulations on the rewarding effects of food.

<table border="1">
 <tbody>
 <tr>
 <td>Item</td>
 <td>Catalogue Number</td>
 <td>Company</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Mouse operant chambers</td>
 <td>ENV-009A</td>
 <td><a href="http://www.med-associates.com/" target="_blank">Med Associates, Inc.</a></td>
 <td>Med Associates Inc. PO Box 319 St. Albans, Vermont 05478, USA Phone: (802) 527-2343</td>
 </tr>
 <tr>
 <td>8 inputs/16 inputs Smart 
 Control Connection Panel</td>
 <td>SG-716B</td>
 <td><a href="http://www.med-associates.com/" target="_blank">Med Associates, Inc.</a></td>
 <td></td>
 </tr>
 <tr>
 <td>Ultra-light retractable mouse lever</td>
 <td>ENV-312</td>
 <td><a href="http://www.med-associates.com/" target="_blank">Med Associates, Inc.</a></td>
 <td></td>
 </tr>
 <tr>
 <td>Pellet Dispenser</td>
 <td>ENV-203</td>
 <td><a href="http://www.med-associates.com/" target="_blank">Med Associates, Inc.</a></td>
 <td></td>
 </tr>
 <tr>
 <td>House Light</td>
 <td>ENV-215</td>
 <td><a href="http://www.med-associates.com/" target="_blank">Med Associates, Inc.</a></td>
 <td></td>
 </tr>
 <tr>
 <td>Stimulus Light</td>
 <td>ENV-221</td>
 <td><a href="http://www.med-associates.com/" target="_blank">Med Associates, Inc.</a></td>
 <td></td>
 </tr>
 <tr>
 <td>Dust-free Precision 20 mg high-fat &amp; high-sugar pellets</td>
 <td>F-06649</td>
 <td>Bio-Serv</td>
 <td>Bio-Serv One 8th Street, Suite 1 Frenchtown, NJ 08825, USA Phone: (800)-996-9908</td>
 </tr>
 </tbody>
</table>
Table 1. Specific equipment and material.

progressive-ratio responding for palatable high-fat and high-sugar food in mice

Foods that are rich in fat and sugar significantly contribute to over-eating and escalating rates of obesity. The consumption of palatable foods can produce a rewarding effect that strengthens action-outcome associations and reinforces future behavior directed at obtaining these foods. Increasing evidence that the rewarding effects of energy-dense foods play a profound role in overeating and the development of obesity has heightened interest in studying the genes, molecules and neural circuitry that modulate food reward1,2. The rewarding impact of different stimuli can be studied by measuring the willingness to work to obtain them, such as in operant conditioning tasks3. Operant models of food reward measure acquired and voluntary behavioral responses that are directed at obtaining food. A commonly used measure of reward strength is an operant procedure known as the progressive ratio (PR) schedule of reinforcement.4,5 In the PR task, the subject is required to make an increasing number of operant responses for each successive reward. The pioneering study of Hodos (1961) demonstrated that the number of responses made to obtain the last reward, termed the breakpoint, serves as an index of reward strength4. While operant procedures that measure changes in response rate alone cannot separate changes in reward strength from alterations in performance capacity, the breakpoint derived from the PR schedule is a well-validated measure of the rewarding effects of food. The PR task has been used extensively to assess the rewarding impact of drugs of abuse and food in rats (e.g.,6-8), but to a lesser extent in mice9. The increased use of genetically engineered mice and diet-induced obese mouse models has heightened demands for behavioral measures of food reward in mice. In the present article we detail the materials and procedures used to train mice to respond (lever-press) for a high-fat and high-sugar food pellets on a PR schedule of reinforcement. We show that breakpoint response thresholds increase following acute food deprivation and decrease with peripheral administration of the anorectic hormone leptin and thereby validate the use of this food-operant paradigm in mice.

Watch this Scientific Journal Video about Progressive-ratio Responding for Palatable High-fat and High-sugar Food in Mice at JoVE.com

Progressive-ratio Responding for Palatable High-fat and High-sugar Food in Mice

The present report details the protocol employed to measure the rewarding effects of high-fat food in mice using a progressive ratio operant conditioning task.

Foods that are rich in fat and sugar significantly contribute to over-eating and escalating rates of obesity. The consumption of palatable foods can ...

progressive-ratio-responding-for-palatable-high-fat-high-sugar-food

Research

JoVE Journal

Neuroscience

21.9K Views.  University of Montreal. The present report details the protocol employed to measure the rewarding effects of high-fat food in mice using a progressive ratio operant conditioning task.

Video: Progressive-ratio Responding for Palatable High-fat and High-sugar Food in Mice

Measuring Oral Fatty Acid Thresholds, Fat Perception, Fatty Food Liking, and Papillae Density in Humans

Emerging evidence from a number of laboratories indicates that humans have the ability to identify fatty acids in the oral cavity, presumably via fatty acid receptors housed on taste cells. Previous research has shown that an individual&#39;s oral sensitivity to fatty acid, specifically oleic acid (C18:1) is associated with body mass index (BMI), dietary fat consumption, and the ability to identify fat in foods. We have developed a reliable and reproducible method to assess oral chemoreception of fatty acids, using a milk and C18:1 emulsion, together with an ascending forced choice triangle procedure. In parallel, a food matrix has been developed to assess an individual&#39;s ability to perceive fat, in addition to a simple method to assess fatty food liking. As an added measure tongue photography is used to assess papillae density, with higher density often being associated with increased taste sensitivity.

Oral chemoreception of fatty acids and the association with diet and fatty food preferences may enable the identification of mechanisms involved with the development of obesity and why dietary changes may be difficult for many individuals.

Emerging evidence from a number of laboratories indicates that humans have the ability to identify fatty acids in the oral cavity, presumably via fatty ...

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 ...

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

For most animals, feeding is an essential behavior for securing survival, and it influences development, locomotion, health and reproduction. Ingestion of the right type and quantity of food therefore has a major influence on quality of life. Research on feeding behavior focuses on the underlying processes that ensure actual feeding and unravels the role of factors regulating internal energy homeostasis and the neuronal bases of decision-making. The model organism Drosophila melanogaster, with its great variety of genetically traceable tools for labeling and manipulating single neurons, allows mapping of neuronal networks and identification of molecular signaling cascades involved in the regulation of food intake. This report demonstrates the CApillary FEeder assay (CAFE) and shows how to measure food intake in a group of flies for time spans ranging from hours to days. This easy-to-use assay consists of glass capillaries filled with liquid food that flies can freely access and feed on. Food consumption in the assay is accurately determined using simple measurement tools. Herein we describe step-by-step the method from setup to successful execution of the CAFE assay, and provide practical examples to analyze the food intake of a group of flies under controlled conditions. The reader is guided through possible limitations of the assay, and advantages and disadvantages of the method compared to other feeding assays in D. melanogaster are evaluated.

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

The CApillary FEeder (CAFE) assay is a simple, budget-friendly, highly reliable method for investigating mechanisms underlying food intake. Used with the highly versatile genetic model organism Drosophila melanogaster, it provides a powerful means of gaining new insights into regulatory mechanisms of food intake.

For most animals, feeding is an essential behavior for securing survival, and it influences development, locomotion, health and reproduction. Ingestion of ...

An In Ovo Model for Testing Insulin-mimetic Compounds

Elevated blood glucose levels in type 2 diabetes mellitus (T2DM), a complex and multifactorial metabolic disease, are caused by insulin resistance and &#946;-cell failure. Various strategies, including the injection of insulin or the usage of insulin-sensitizing drugs, were pursued to treat T2DM or at least reduce the symptoms. In addition, the application of herbal compounds has attracted increasing attention. Thus, it is necessary to find efficient test systems to identify and characterize insulin-mimetic compounds. Here we developed a modified chick embryo model, which enables testing of synthetic compounds and herbal extracts with insulin-mimetic properties. Using a fluorescence microscopy-based primary screen, which quantifies the translocation of Glucose transporter 4 (Glut4) to the plasma membrane, we were able to identify compounds, mainly herbal extracts, which lead to an increase of intracellular glucose concentrations in adipocytes. However, the efficacy of these substances requires further verification in a living organism. Thus, we used an in-ovo approach to identify their blood glucose-reducing properties. The approval by an ethics committee is not needed since the use of chicken embryos during the first two-thirds of embryonic development is not considered an animal experiment. Here, the application of this model is described in detail.

An In Ovo Model for Testing Insulin-mimetic Compounds

In this study, we describe an in-ovo system as a promising tool to test insulin-mimetic compounds in a living organism. The main advantages include adequate throughput rates and acceptable costs, which enable the identification of phytochemicals with insulin-mimetic properties.

Elevated blood glucose levels in type 2 diabetes mellitus (T2DM), a complex and multifactorial metabolic disease, are caused by insulin resistance and ...

Data Acquisition and Analysis In Brainstem Evoked Response Audiometry In Mice

Brainstem evoked response audiometry (BERA) is of central relevance in the clinical neurophysiology. As other evoked potential (EP) techniques, such as visually evoked potentials (VEPs) or somatosensory evoked potentials (SEPs), the auditory evoked potentials (AEPs) are triggered by the repetitive presentation of identical stimuli, the electroencephalographic (EEG) response of which is subsequently averaged resulting in distinct positive (p) and negative (n) deflections. In humans, both the amplitude and the latency of individual peaks can be used to characterize alterations in synchronization and conduction velocity in the underlying neuronal circuitries. Importantly, AEPs are also applied in basic and preclinical science to identify and characterize the auditory function in pharmacological and genetic animal models. Even more, animal models in combination with pharmacological testing are utilized to investigate for potential benefits in the treatment of sensorineural hearing loss (e.g., age- or noise-induced hearing deficits). Here we provide a detailed and integrative description of how to record auditory brainstem-evoked responses (ABRs) in mice using click and tone-burst application. A specific focus of this protocol is on pre-experimental animal housing, anesthesia, ABR recording, ABR filtering processes, automated wavelet-based amplitude growth function analysis, and latency detection.

Brainstem evoked response audiometry is an important tool in clinical neurophysiology. Nowadays, brainstem evoked response audiometry is also applied in the basic science and preclinical studies involving both pharmacological and genetic animal models. Here we provide a detailed description of how auditory brainstem responses can be successfully recorded and analyzed in mice.

Brainstem evoked response audiometry (BERA) is of central relevance in the clinical neurophysiology. As other evoked potential (EP) techniques, such as ...

Electrocorticographic Recording of Cerebral Cortex Areas Manipulated Using an Adeno-Associated Virus Targeting Cofilin in Mice

The use of electrocorticographic (ECoG) recordings in rodents is relevant to sleep research and to the study of a wide range of neurological conditions. Adeno-associated viruses (AAVs) are increasingly used to improve understanding of brain circuits and their functions. The AAV-mediated manipulation of specific cell populations and/or of precise molecular components has been tremendously useful to identify new sleep regulatory circuits/molecules and key proteins contributing to the adverse effects of sleep loss. For instance, inhibiting activity of the filamentous actin-severing protein&#160;cofilin&#160;using AAV prevents sleep deprivation-induced memory impairment. Here, a protocol is&#160;described that combines the manipulation of cofilin function in a cerebral cortex area with the recording of ECoG activity to examine whether cortical cofilin modulates the wakefulness and sleep ECoG signals. AAV injection is performed during the same surgical procedure as the implantation of ECoG and electromyographic (EMG) electrodes in adult male and female mice. Mice are anesthetized, and their heads are shaved. After skin cleaning and incision, stereotaxic coordinates of the motor cortex are determined, and the skull is pierced at this location. A cannula prefilled with an AAV expressing cofilinS3D, an inactive form of cofilin, is slowly positioned in the cortical tissue. After AAV infusion, gold-covered screws (ECoG electrodes) are screwed through the skull and cemented to the skull with gold wires inserted in the neck muscles (EMG electrodes). The animals are allowed three weeks to recover and to ensure sufficient expression of cofilinS3D. The infected area and cell type are verified using immunohistochemistry, and the ECoG is analyzed using visual identification of vigilance states and spectral analysis. In summary, this combined methodological approach allows the investigation of the precise contribution of molecular components regulating neuronal morphology and connectivity to the regulation of synchronized cerebral cortex activity during wakefulness and sleep.

This article describes a protocol for the manipulation of molecular targets in the cerebral cortex using adeno-associated viruses and for monitoring the effects of this manipulation during wakefulness and sleep using electrocorticographic recordings.

The use of electrocorticographic (ECoG) recordings in rodents is relevant to sleep research and to the study of a wide range of neurological conditions. ...

A Rapid Food-Preference Assay in Drosophila

To select food with nutritional value while avoiding the consumption of harmful agents, animals need a sophisticated and robust taste system to evaluate their food environment. The fruit fly, Drosophila melanogaster, is a genetically tractable model organism that is widely used to decipher the molecular, cellular, and neural underpinnings of food preference. To analyze fly food preference, a robust feeding method is needed. Described here is a two-choice feeding assay, which is rigorous, cost-saving, and fast. The assay is Petri-dish-based and involves the addition of two different foods supplemented with blue or red dye to the two halves of the dish. Then, ~70 prestarved, 2-4-day-old flies are placed in the dish and&#160;allowed to choose between blue and red foods in the dark for about 90 min. Examination of the abdomen of each fly is followed by the calculation of the preference index. In contrast to multiwell plates, each Petri dish takes only ~20 s to fill and saves time and effort. This feeding assay can be employed to quickly determine whether flies like or dislike a particular food.

A Rapid Food-Preference Assay in Drosophila

We present a protocol for a two-choice feeding assay for flies. This feeding assay is fast and easy to run and is suitable not only for small-scale laboratory research, but also for high-throughput behavioral screens in flies.

To select food with nutritional value while avoiding the consumption of harmful agents, animals need a sophisticated and robust taste system to evaluate ...

Modeling Stroke in Mice: Focal Cortical Lesions by Photothrombosis

Stroke is a leading cause of death and acquired adult disability in developed countries. Despite extensive investigation for novel therapeutic strategies, there remain limited therapeutic options for stroke patients. Therefore, more research is needed for pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity, and regeneration. Given the inability of in vitro models to reproduce the complexity of the brain, experimental stroke models are essential for the analysis and subsequent evaluation of novel drug targets for these mechanisms. In addition, detailed standardized models for all procedures are urgently needed to overcome the so-called replication crisis. As an effort within the ImmunoStroke research consortium, a standardized photothrombotic mouse model using an intraperitoneal injection of Rose Bengal and the illumination of the intact skull with a 561 nm laser is described. This model allows the performance of stroke in mice with allocation to any cortical region of the brain without invasive surgery; thus, enabling the study of stroke in various areas of the brain. In this video, the surgical methods of stroke induction in the photothrombotic model along with&#160;histological analysis are demonstrated.

Described here is the photothrombotic stroke model, where a stroke is produced through the intact skull by inducing permanent microvascular occlusion using laser illumination after administration of a photosensitive dye.

Stroke is a leading cause of death and acquired adult disability in developed countries. Despite extensive investigation for novel therapeutic strategies, ...

Caenorhabditis elegans as a Model System for Discovering Bioactive Compounds Against Polyglutamine-Mediated Neurotoxicity

Age-related misfolding and aggregation of pathogenic proteins are responsible for several neurodegenerative diseases. For example, Huntington&#39;s disease (HD) is principally driven by a CAG nucleotide repeat that encodes an expanded glutamine tract in huntingtin protein. Thus, the inhibition of polyglutamine (polyQ) aggregation and, in particular, aggregation-associated neurotoxicity is a useful strategy for the prevention of HD and other polyQ-associated conditions. This paper introduces generalized experimental protocols to assess the neuroprotective capacity of test compounds against HD using established polyQ transgenic Caenorhabditis elegans models. The AM141 strain is chosen for the polyQ aggregation assay as an age-associated phenotype of discrete fluorescent aggregates can be easily observed in its body wall at the adult stage due to muscle-specific expression of polyQ::YFP fusion proteins. In contrast, the HA759 model with strong expression of polyQ-expanded tracts in ASH neurons is used to examine neuronal death and chemoavoidance behavior. To comprehensively evaluate the neuroprotective capacity of target compounds, the above test results are ultimately presented as a radar chart with profiling of multiple phenotypes in a manner of direct comparison and direct viewing.

Caenorhabditis elegans as a Model System for Discovering Bioactive Compounds Against Polyglutamine-Mediated Neurotoxicity

Here, we present a protocol to assess the neuroprotective activities of test compounds in Caenorhabditis elegans, including polyglutamine aggregation, neuronal death, and chemoavoidance behavior, as well as an exemplary integration of multiple phenotypes.

Age-related misfolding and aggregation of pathogenic proteins are responsible for several neurodegenerative diseases. For example, Huntington&#39;s ...

Constipation Assays in &lt;em&gt;Drosophila&lt;/em&gt; Flies

Measuring Constipation in a Drosophila Model of Parkinson's Disease

Non-motor symptoms in Parkinson's disease (PD) are common, difficult to treat, and significantly impair quality of life. One prevalent non-motor symptom is constipation, which can precede the diagnosis of PD by years or even decades. Constipation has been underexplored in animal models of PD and lacks specific therapies. This assay utilizes a Drosophila model of PD in which human alpha-synuclein is expressed under a pan-neuronal driver. Flies expressing alpha-synuclein develop the hallmark features of PD: the loss of dopaminergic neurons, motor impairment, and alpha-synuclein inclusions.
This protocol outlines a method for studying constipation in these flies. Flies are placed on fly food with a blue color additive overnight and then transferred to standard food the following day. They are subsequently moved to new vials with standard fly food every hour for 8 h. Before each transfer, the percentage of blue-colored fecal spots compared to the total fecal spots on the vial wall is calculated. Control flies that lack alpha-synuclein expel all the blue dye hours before flies expressing alpha-synuclein. Additionally, the passage of blue-colored food from the gut can be monitored with simple photography. The simplicity of this assay enables its use in forward genetic or chemical screens to identify modifiers of constipation in Drosophila.

Author Spotlight: Exploring Non-Motor Symptoms in Parkinson&#039;s Disease 

This protocol presents an assay for modeling constipation in an alpha-synuclein based Drosophila model of Parkinson's disease.

Non-motor symptoms in Parkinson's disease (PD) are common, difficult to treat, and significantly impair quality of life. One prevalent non-motor symptom ...