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. 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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.

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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 ...

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22.0K 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

T-maze Forced Alternation and Left-right Discrimination Tasks for Assessing Working and Reference Memory in Mice

Forced alternation and left-right discrimination tasks using the T-maze have been widely used to assess working and reference memory, respectively, in rodents. In our laboratory, we evaluated the two types of memory in more than 30 strains of genetically engineered mice using the automated version of this apparatus. Here, we present the modified T-maze apparatus operated by a computer with a video-tracking system and our protocols in a movie format. The T-maze apparatus consists of runways partitioned off by sliding doors that can automatically open downward, each with a start box, a T-shaped alley, two boxes with automatic pellet dispensers at one side of the box, and two L-shaped alleys. Each L-shaped alley is connected to the start box so that mice can return to the start box, which excludes the effects of experimenter handling on mouse behavior. This apparatus also has an advantage that in vivo microdialysis, in vivo electrophysiology, and optogenetics techniques can be performed during T-maze performance because the doors are designed to go down into the floor. In this movie article, we describe T-maze tasks using the automated apparatus and the T-maze performance of &alpha;-CaMKII+/- mice, which are reported to show working memory deficits in the eight-arm radial maze task. Our data indicated that &alpha;-CaMKII+/- mice showed a working memory deficit, but no impairment of reference memory, and are consistent with previous findings using the eight-arm radial maze task, which supports the validity of our protocol. In addition, our data indicate that mutants tended to exhibit reversal learning deficits, suggesting that &alpha;-CaMKII deficiency causes reduced behavioral flexibility. Thus, the T-maze test using the modified automatic apparatus is useful for assessing working and reference memory and behavioral flexibility in mice.

This article presents the protocol of T-maze tests using a modified automated apparatus for assessing the learning and memory functions in mice.

Forced alternation and left-right discrimination tasks using the T-maze have been widely used to assess working and reference memory, respectively, in ...

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 ...

Assessment of Dopaminergic Homeostasis in Mice by Use of High-performance Liquid Chromatography Analysis and Synaptosomal Dopamine Uptake

Dopamine (DA) is a modulatory neurotransmitter controlling motor activity, reward processes and cognitive function. Impairment of dopaminergic (DAergic) neurotransmission is strongly associated with several central nervous system-associated diseases such as Parkinson&#39;s disease, attention-deficit-hyperactivity disorder and drug addiction1,2,3,4. Delineating disease mechanisms involving DA imbalance is critically dependent on animal models to mimic aspects of the diseases, and thus protocols that assess specific parts of the DA homeostasis are important to provide novel insights and possible therapeutic targets for these diseases.
Here, we present two useful experimental protocols that when combined provide a functional read-out of the DAergic system in mice. Biochemical and functional parameters on DA homeostasis are obtained through assessment of DA levels and dopamine transporter (DAT) functionality5. When investigating the DA system, the ability to reliably measure endogenous levels of DA from adult brain is essential. Therefore, we present how to perform high-performance liquid chromatography (HPLC) on brain tissue from mice to determine levels of DA. We perform the experiment on tissue from dorsal striatum (dStr) and nucleus accumbens (NAc), but the method is also suitable for other DA-innervated brain areas.
DAT is essential for reuptake of DA into the presynaptic terminal, thereby controlling the temporal and spatial activity of released DA. Knowing the levels and functionality of DAT in the striatum is of major importance when assessing DA homeostasis. Here, we provide a protocol that allows to simultaneously deduce information on surface levels and function using a synaptosomal6 DA uptake assay.
Current methods combined with standard immunoblotting protocols provide the researcher with relevant tools to characterize the DAergic system.

Synaptosomal dopamine uptake and high-performance liquid chromatography analysis represent experimental tools to investigate dopamine homeostasis in mice by assessing the function of the dopamine transporter and levels of dopamine in striatal tissue, respectively. Here we present protocols to measure dopamine tissue content and assess the functionality of the dopamine transporter.

Dopamine (DA) is a modulatory neurotransmitter controlling motor activity, reward processes and cognitive function. Impairment of dopaminergic (DAergic) ...

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 ...

Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools to selectively manipulate brain rhythms. Here we describe an approach combining projection-specific optogenetics with extracellular electrophysiology for high-fidelity control of hippocampal theta oscillations (5-10 Hz) in behaving mice. The specificity of the optogenetic entrainment is achieved by targeting channelrhodopsin-2 (ChR2) to the GABAergic population of medial septal cells, crucially involved in the generation of hippocampal theta oscillations, and a local synchronized activation of a subset of inhibitory septal afferents in the hippocampus. The efficacy of the optogenetic rhythm control is verified by a simultaneous monitoring of the local field potential (LFP) across lamina of the CA1 area and/or of neuronal discharge. Using this readily implementable preparation we show efficacy of various optogenetic stimulation protocols for induction of theta oscillations and for the manipulation of their frequency and regularity. Finally, a combination of the theta rhythm control with projection-specific inhibition addresses the readout of particular aspects of the hippocampal synchronization by efferent regions.

We describe the use of optogenetics and electrophysiological recordings for selective manipulations of hippocampal theta oscillations (5-10 Hz) in behaving mice. The efficacy of the rhythm entrainment is monitored using local field potential. A combination of opto- and pharmacogenetic inhibition addresses the efferent readout of hippocampal synchronization.

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools ...

A Protocol for Transcranial Photobiomodulation Therapy in Mice

Transcranial photobiomodulation is a potential innovative noninvasive therapeutic approach for improving brain bioenergetics, brain function in a wide range of neurological and psychiatric disorders, and memory enhancement in age-related cognitive decline and neurodegenerative diseases. We describe a laboratory protocol for transcranial photobiomodulation therapy (PBMT) in mice. Aged BALB/c mice (18 months old) are treated with a 660 nm laser transcranially, once daily for 2 weeks. Laser transmittance data shows that approximately 1% of the incident red light on the scalp reaches a 1 mm depth from the cortical surface, penetrating the dorsal hippocampus. Treatment outcomes are assessed by two methods: a Barnes maze test, which is a hippocampus-dependent spatial learning and memory task evaluation, and measuring hippocampal ATP levels, which is used as a bioenergetics index. The results from the Barnes task show an enhancement of the spatial memory in laser-treated aged mice when compared with age-matched controls. Biochemical analysis after laser treatment indicates increased hippocampal ATP levels. We postulate that the enhancement of memory performance is potentially due to an improvement in hippocampal energy metabolism induced by the red laser treatment. The observations in mice could be extended to other animal models since this protocol could potentially be adapted to other species frequently used in translational neuroscience, such as rabbit, cat, dog, or monkey. Transcranial photobiomodulation is a safe and cost-effective modality which may be a promising therapeutic approach in age-related cognitive impairment.

Photobiomodulation therapy is an innovative noninvasive modality for the treatment of a wide range of neurological and psychiatric disorders and can also improve healthy brain function. This protocol includes a step-by-step guide to performing brain photobiomodulation in mice by transcranial light delivery, which can be adapted for use in other laboratory rodents.

Transcranial photobiomodulation is a potential innovative noninvasive therapeutic approach for improving brain bioenergetics, brain function in a wide ...

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 ...

Quantitative Measurement of Intrathecally Synthesized Proteins in Mice

Cerebrospinal fluid (CSF), a fluid found in the brain and the spinal cord, is of great importance to both basic and clinical science. The analysis of the CSF protein composition delivers crucial information in basic neuroscience research as well as neurological diseases. One caveat is that proteins measured in CSF may derive from both intrathecal synthesis and transudation from serum, and protein analysis of CSF can only determine the sum of these two components. To discriminate between protein transudation from the blood and intrathecally produced proteins in animal models as well as in humans, CSF protein profiling measurements using conventional protein analysis tools must include the calculation of the albumin CSF/serum quotient (Qalbumin), a marker of the integrity of the blood-brain interface (BBI), and the protein index (Qprotein/Qalbumin), an estimate of intrathecal protein synthesis. This protocol illustrates the entire procedure, from CSF and blood collection to quotients and indices calculations, for the quantitative measurement of intrathecal protein synthesis and BBI impairment in mouse models of neurological disorders.

Elevated spinal fluid protein levels can either be the result of diffusion of plasma protein across an altered blood-brain barrier or intrathecal synthesis. An optimized testing protocol is presented in this article that helps to discriminate both cases and provides quantitative measurements of intrathecally synthesized proteins.

Cerebrospinal fluid (CSF), a fluid found in the brain and the spinal cord, is of great importance to both basic and clinical science. The analysis of the ...

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

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Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice

A Protocol for Transcranial Photobiomodulation Therapy in Mice

Data Acquisition and Analysis In Brainstem Evoked Response Audiometry In Mice

Quantitative Measurement of Intrathecally Synthesized Proteins in Mice