Protocols in Current Issue
Protocols in Past Issues
0 Q&A 1559 Views Apr 5, 2022

Thermotaxis behaviors in C. elegans exhibit experience-dependent plasticity of thermal preference memory. This behavior can be assayed either at population level, on linear temperature gradients, or at the individual animal level, by radial isothermal or microfluidic tracking of orientation. These behaviors are low-throughput as well as variable, due to the inherent sensitivity to environmental perturbations. To facilitate reproducible studies, we describe an updated apparatus design that enables simultaneous runs of three thermal preference assays, instead of single-run assays described previously. By enabling parallel runs of control and experimental conditions, this set-up enables more throughput and rigorous assessment of behavioral variability.

0 Q&A 1377 Views Mar 5, 2022

The search for safe and efficient chronic pain treatments is dampened by the lack of reliable models that faithfully reproduce current pharmacological treatments for chronic spontaneous pain in humans. Preclinical models often assess the antinociceptive efficacy of non-contingent pharmacological treatments evaluated in the short-term. Here, we provide a protocol of contingent operant self-medication in mice, which allows the estimation of spontaneous pain relief and drug abuse liability in models of persistent pain. This paradigm requires preliminary habituation and animal handling, followed by training of mice in operant conditioning boxes, to allow subsequent analgesic drug self-administration. After the initial acquisition of food-maintained operant behavior, a chronic pain sensitization is induced. Posterior intravenous jugular catheterization and coupling of operant conditioning boxes to perfusion pumps allow quantification of operant responding for intravenous drug self-administration. All mice show an initial operant drug self-administration behavior associated with the previous food-maintained operant training. This initial operant responding is extinguished after administration of ineffective treatments, but continues when the compounds have analgesic efficacy or intrinsic reinforcing properties. The identification of a significant drug self-administration selectively expressed in mice exposed to the chronic pain condition is indicative of analgesic drug effects, whereas persistent self-administration in control mice is indicative of abuse liability. The present protocol provides the behavioral and surgical procedures needed to assess spontaneous pain relief and potential for abuse of pharmacological treatments, through contingent analgesic self-medication in mice.

Graphic abstract:

Experimental design. Animals are subjected to a 5-day food self-administration protocol with a fixed ratio of reinforcement of 1 (FR1, 1 interaction with the active nose-poke causes the release of 1 reinforcer/infusion), to acquire the operant behavior. After this training, mice are subjected to the chronic pain or sham procedure, and four days later an intravenous (i.v.) catheterization is performed, to allow self-administration with the selected compound or its vehicle. Three days after the catheterization, animals start the drug/vehicle self-administration protocol at FR1. The patency of the catheter is evaluated with the thiopental test after the last self-administration session. Adapted from Bura et al. (2018).

0 Q&A 2483 Views Nov 5, 2021

The neuromuscular junction (NMJ) is a specialized synapse that connects the terminal end of a motor neuron and a skeletal muscle fiber. Defects in NMJ cause abnormalities of neuromuscular transmission, leading to NMJ disorders. The mammalian diaphragm muscle is essential for respiration and has been widely used to study NMJ formation. Here, we provide a simple and straightforward protocol for preparing diaphragms from embryonic, neonatal, and adult mice and for subsequent NMJ staining.

0 Q&A 2730 Views Jul 5, 2021

Rodent cages equipped with access to a voluntary running wheel are commonly used to study the effects of aerobic physical activity on physiology and behavior. Notable discoveries in exercise neurobiology, including the key role of brain-derived neurotrophic factor (BDNF) in neural plasticity and cognition, have been made using rodents housed with voluntary running wheels. A major advantage of using home-cage running wheels over treadmills is the elimination of stress potentially associated with forced running. In addition, voluntary wheel running may simulate a more natural running pattern in laboratory mice. Singly housing mice with voluntary running wheels is traditionally employed to obtain exact quantitation of the distance ran; however, social isolation stress is often ignored to obtain precise running distances. Moreover, voluntary exercise studies in adolescent mice must consider the neurodevelopmental implications of isolation stress. In this protocol, we wean 21-day-old mouse pups directly into running wheel-equipped cages and pair-house them to reduce the impact of social isolation and other developmentally specific factors that could adversely affect their behavior or development. Individual running distances are obtained from each mouse in the cage using a radio-frequency identification (RFID) ear tag and a hidden antenna placed directly under the running wheel. We have demonstrated that voluntary running during a specific juvenile-adolescent developmental period can improve hippocampal memory when tested during adolescence (Ivy et al., 2020). Individual exercise tracking of group-housed mice can enable future studies to precisely correlate the amount of exercise with readouts such as cell-specific gene expression, epigenetic mechanisms, serum biomarkers, and behavior, in an intra-individual manner.

Graphic abstract:

Figure 1. Illustration of the dual RFID and Vital View system for individual mouse running in a pair-housed cage

0 Q&A 2176 Views Jul 5, 2021

Chronic pain is a complex disease that affects a large proportion of the population. With little to no effective treatments currently available for patients, this malady presents a large burden to society. Drosophila melanogaster has been previously used to describe conserved molecular components of nociception in larvae and adults. However, adult assays tend to rely on avoidance behaviours, and whilst larval acute thermal avoidance assays exist, larvae are not best suited to a chronic pain scenario as the condition must be long-term. Therefore, an adult thermal nociception response assay was required to study injury-evoked changes in heat nociception threshold (allodynia and hyperalgesia) over time, and we describe such a protocol here. Following leg amputation, flies display increased thermal sensitivity (allodynia) to innocuous temperatures but not an increase in magnitude of response (hyperalgesia) to noxious heat. Our method allows for individualised analysis of both allodynia and hyperalgesia.

0 Q&A 2584 Views Jun 5, 2021

In vivo sweat quantitation assays are required for the development of drugs for the management of focal hyperhidrosis before clinical trials; however, in vivo assays, particularly mouse models, are rare. Even in sweat assays using mice, sweating is quantitated by manually counting the number of sweating spots, which can contribute to various errors owing to arbitrary judgment. In this study, we developed a mouse sweat-assay model and a method for quantitating the amount of sweating to remove possible errors. The use of the iodine–starch test in the castor oil-covered hind footpad skin of anesthetized mice resulted in the sweating area being stained blue-black. After the anesthesia and treatment with drugs (pilocarpine, glycopyrrolate, botulinum neurotoxin, myricetin, and myricetin-loaded lipid nanoparticles), the remaining area of the footpad skin was eliminated from the acquired footpad images using ImageJ. Blue pixels extracted from the footpad image are automatically adjusted using the Phansalkar method, where the percentage of the blue area was determined based on the whole hind footpad skin area, finally indicating the percentage of the sweating area. Using this mouse model and analysis for sweat assays, a clear difference between the control group and antiperspirant-administered group was observed with respect to the sweating area % with no error. In conclusion, this assay can be used as a preclinical tool to screen potential antiperspirant drugs.

Graphic abstract:

Overview of the mouse-model sweat assay and objective quantitation of the focal sweating area

0 Q&A 3100 Views Sep 20, 2020
Songbirds, such as the zebra finch, are a popular animal model for studying the neural basis of vocal and complex skill learning. Adult male zebra finches produce courtship song toward females (referred to as ‘directed song’) and recording and analyzing sounds of directed song along with underlying neural activity is important for investigating behavioral and neural mechanisms of song production and learning. However, recording of directed song is easily contaminated by calls that are often as loud as directed songs and frequently produced by a female bird is presented in the same sound-recording chamber to elicit directed song. We developed a new surgical procedure to relatively easily and almost completely devocalize female zebra finches semi-permanently, without affecting other behaviors. This procedure enables researchers to record directed songs with almost no contamination by female calls. The procedure can also be used to devocalize male birds as well and, thus, has great potential for a variety of experimental purposes, such as long-term elimination of auditory feedback during singing in male birds.
0 Q&A 2558 Views Aug 20, 2020
The ability to perform a sequence of movements is a key component of motor skills, such as typing or playing a musical instrument. How the brain binds elementary movements together into meaningful actions has been a topic of much interest. Here, we describe two sequential reaching tasks that we use to investigate the neural substrate of skilled sequential movements in monkeys after long-term practice. The movement elements performed in these tasks are essentially identical, but are generated in two different contexts. In one task, monkeys perform reaching movements that are instructed by visual cues. In the other, the monkeys perform reaching movements that are generated from memory after extended practice. With this behavioral paradigm, we can dissociate the neural processes related to the acquisition and retention of motor skills from those related to movement execution.
1 Q&A 4503 Views Aug 20, 2020
The quantitative measurement of water flow-induced swimming of fish species using a swimmill is a powerful method to evaluate motor ability of individual fish. Zebrafish is a commonly used vertebrate that enables the study of morphological, physiological and behavioral characteristics associated with genes. We here established a reproducible method that allows to measure the body length and the critical swimming speed of adult zebrafish using a swimmill.
0 Q&A 2417 Views Jun 5, 2020
In the mammalian visual system, early stages of visual form perception begin with orientation selective neurons in primary visual cortex (V1). In many species (including humans, monkeys, tree shrews, cats, and ferrets), these neurons are organized in pinwheel-like orientation columns. To study the functional organization within orientation pinwheels, it is important to target pinwheel subdomains precisely. We therefore developed a technique to provide a quantitative determination of the location of pinwheel centers (PCs). Previous studies relied solely on blood vessel images of the cortical surface to guide electrode penetrations to PCs in orientation maps. However, considerable spatial error remained using this method. In the present study, we improved the accuracy of targeting PCs by ensuring perpendicularity of electrodes and by utilizing the orientation tuning of local field potentials (LFP) recorded at or near the optically determined positions.

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