The authors would like to thank Dr. DeAnna Adkins, Dr. Marty Woodlee, Cole Husbands, M&oacute;nica A. Maldonado, Jackie Kane, and Dr. Tim Schallert for assistance in developing the Vermicelli Handling Test, and Lu Chou for assistance in developing the Capellini Handling Test. Supported by grants from the NIH-NINDS (NS05689) and NIH-NIA (1F31AG034032-01).

1. Preparing for the onset of testing
<ol>
 <li>Uncooked pasta pieces are cut to length using a razor blade. For rats, Skinner brand vermicelli pasta (1.5 mm diameter) is cut to 7 cm lengths. For mice, DeCecco brand capellini pasta (1.0 mm diameter) is cut to 2.6 cm lengths (Fig. 1C). Because pasta brands vary in texture and diameter, which could influence handling manner, it is recommended that a single source of pasta be used for repeated testing.</li>
 <li>To overcome neophobic responses and to establish skill in handling, animals are exposed to the pasta pieces in their home cage several times in the weeks prior to testing. (Typically, 5-10 days of exposure, about 4 pieces each time, is sufficient.)</li>
 <li>If the test will be administered in a chamber other than the home cage, or if the home cage is moved for testing, habituation to eating in the testing environment is provided. (It typically takes 3-5 sessions until animals are readily eating the pasta in this environment.)</li>
 <li>Even after animals eagerly accept pasta, they can suddenly become reticent when a large camera is moved next to the testing area. It is helpful to place the camera in front of the testing environment for one or more of the habituation sessions.</li>
 <li>Because most animals act as if the pasta is highly palatable, it is not essential to food restrict them prior to testing. However, removal of food a few hours before or overnight may facilitate eating in front of the experimenter and camera and ensure that they are not sated at the test time.</li>
 <li>Breaking the pasta piece during eating invalidates a trial. We have found this habit is largely avoided if animals are never given pieces longer than the ones used in the tests.</li>
 <li>For the testing sessions, pasta pieces are marked with an ultrafine tip marker at specific intervals using a cardstock template (1.75 cm increments on vermicelli and 0.86 cm increments on capellini strands). This facilitates visualization of the movement of the pasta strand during eating.</li>
</ol>
2. Test set up
<ol>
 <li>Animals should be tested in either the home cage or another apparatus with unmarred, clear walls, and should be isolated from their cagemates during the test session.</li>
 <li>Though rats can be tested in real time by a practiced experimenter (Allred et al., 2008), quantification is facilitated by video recording so that data can be collected during slow-motion video playback. We have not found it feasible to reliably quantify mouse forepaw movements without slow motion video playback due to the small size of their paws and fast movements.</li>
 <li>A video camera with a high frame rate and adequate zoom is used. We use a Canon XL1S camera with a 16X zoom lens set to a shutter speed of at least 420 s-1 in normal room lighting conditions. The use of a wheeled tripod with a panning and tilting head facilitates camera positioning.</li>
 <li>Rats typically sit upright on their haunches to eat pasta and the paws are most easily viewed with the camera positioned at approximately chamber floor level with a slight upward angle (Fig. 1A). If testing in a cage with shavings, removal of some shavings may be useful to optimize the view.</li>
 <li>Mice tend to bend forward during pasta eating, holding the piece at more of an angle than rats do. Thus, a testing chamber with an open floor is placed on top of a mirror. The camera is placed at a slightly higher level than the floor, and angled downward to focus on the reflection of the paws in the mirror (Fig 1B). The use of a first surface mirror greatly improves the clarity of the paws for video playback.</li>
 <li>Each bite of the pasta piece is audible and, thus, audio recording is useful for verifying eating.</li>
</ol>
3. Administering pasta handling trials
<ol>
 <li>Test sessions consist of sequential trials, with one pasta piece eaten per trial. We typically record data from 4-5 trials per rat and 3-4 trials per mouse.</li>
 <li>Prior to videotaping, a piece of pasta is placed into the chamber to initiate eating and to move the animal into an appropriate camera view.</li>
 <li>It is necessary for the animal to face the camera and to focus and zoom sufficiently (~10-12X) to view discrete adjustments with both forepaws and all digits. The knuckles of both paws should be viewable. At least with this camera, we find it useful to use autofocus features only prior to trial onset, to narrow in on the region of interest. We subsequently switch to manual focus.</li>
 <li>As soon as the animal and camera are in position, the first trial is initiated. A piece is dropped or dangled directly in front of the animal and recording is initiated prior to eating onset.</li>
 <li>Thereafter, the trials are given in immediate succession. It is advisable to minimize delays between trials so that animals do not move away from the optimal viewing position.</li>
 <li>Trials in which the paws are viewed to the side or in which focus is lost are not scored.</li>
 <li>The test sessions can be repeated over days, e.g., to document pre- and post-treatment temporal patterns (e.g., recovery after CNS injury; Allred et al., 2008).</li>
 <li>A challenge can be getting animals to eat in desired positions relative to the camera. It may be necessary to habituate camera-shy animals to the presence of the camera for a few minutes prior to testing. Animals can often be coaxed into the position by dropping or dangling pasta in the ideal region. It may require a few pieces to accomplish this desired position, though the number of pieces given should be minimized to avoid satiety.</li>
 <li>For sequential testing of several rats, we have found it convenient to configure a few of their cages at a time in a circle, with the camera on a wheeled tripod in the middle. A piece is dropped into an ideal region of each cage and the camera then moved to the rat that begins eating in an appropriate position first. This allows more time for the slower (to eat in position) rats to become habituated to the camera and experimenter presence and they may also be stimulated to eat by the biting sounds of the faster rats.</li>
</ol>
4. Quantitative video analysis of forepaw adjustments and eating time
<ol>
 <li>The analysis of forepaw adjustments during eating is a simple quantitative measure that is sensitive to lateralized impairments after CNS injury in rats (Allred et al., 2008; Whishaw &amp; Coles, 1996; Whishaw et al., 1997) and to aging-related changes in mice (Tennant et al., 2009).</li>
 <li>The quantified portion of the trial begins when the pasta piece is first placed into the mouth and ends when the last of the pasta is released from the paws and disappears into the mouth.&nbsp;</li>
 <li>Video playback is slowed (~ 50% of real-time), or frame-by-frame advance is used, as needed, to identify adjustments.</li>
 <li>An adjustment is counted each time the paw releases and re-contacts the pasta piece, when there is reformation of the paw hold on the piece, or when there is an extension-flexion or abduction-adduction of the digits (Fig. 2). Each adjustment is recorded separately for the left and right paw.</li>
 <li>Adjustments are not counted when the paw slides across the pasta piece with no movement of the digits or repositioning of the paw hold. Movements of the paw when it is not in contact with the pasta piece (i.e., movements next to the piece) are also not counted.</li>
 <li>The time to eat is also recorded for each trial, starting with eating onset and ending when the pasta disappears into the mouth. We record time using video playback display features, but it can also be recorded with a stopwatch during normal speed playback.</li>
 <li>If the animal pauses in eating, but maintains a hold on the piece, the time paused is subtracted from the total time. A pause in eating is evident by either movement of the piece out of the mouth or a cessation in audible biting.</li>
 <li>The trial is omitted from the analysis if during any part of the eating, the view of the paws is obscured, or if the animal breaks the piece, because the shorter pieces alter handling patterns.</li>
 <li>Some rats and mice consistently make more adjustments with one paw than the other. For unilateral CNS injury models, lateralized impairments are more sensitively detected if the injuries affect the paw that made more adjustments in pre-injury testing (Allred et al., 2008).</li>
 <li>Mice sometimes drop the pasta piece in the middle of eating, and, after a short break, pick it up and continue eating. This is most common in the last trial of the day, possibly due to partial satiety and loss of focus on eating. Because this is common even in intact mice, we do not omit these trials. However, the time that the piece is dropped is subtracted from the total eating time. </li>
 <li>In intact rats, dropping pieces is atypical and resumption in eating is often very delayed or absent. Such trials are omitted from the analysis of adjustments and eating time. </li>
 <li>Adjustments prior to the eating onset are not counted. Pre-eating handling behaviors are affected by CNS injuries (e.g., Whishaw et al., 1992; Whishaw et al., 1986), but we have not yet extended the quantitative methods for forepaw adjustments to this period.</li>
 <li>These data can be summarized as: (a) the average number of adjustments per paw per piece, (b) the average eating time per piece and (c) the rate of adjustments (adjustments/eating time).</li>
</ol>
5. Video analysis of atypical handling patterns
<ol>
 <li>Atypical handling patterns can distinguish between different CNS injury types in rats (Allred et al., 2008) and recording these behaviors may be useful as a supplemental analysis.</li>
 <li>Handling patterns are considered atypical if they are infrequently observed in intact animals. Intact animals typically initiate eating with one paw, the "grasp paw", placed lower on the pasta, and the "guide paw" is then placed near the mouth (Fig. 3A,C). The same paw is usually the grasp paw in all trials. Rats often position paws far apart along the length of the pasta at first, and move them together as the piece shortens. Mice typically position both paws close to the mouth, with the grasp and guide limbs held close to one another, even at the beginning of eating. In both species, the paws move into more symmetrical positions, side-by-side or with digits interposed, when the piece is very short (Fig. 3B,D). Both paws are in use continuously for guiding, holding or advancing the pasta during eating.</li>
 <li>Atypical patterns in paw position in rats and mice include: (a.) placing the paws together, in a symmetrical hold, when the pasta piece is long (more than half the original length), (b.) failing to move the paws into a symmetrical position before the piece is completely consumed and (c.) switching positions of the guide and grasp limbs in the midst of eating.</li>
 <li>Two additional atypical behaviors in rats and mice are: (a.) failing to contact the pasta piece with one paw for an extended time during eating ("failure to contact") and (b.) using the mouth to pull the pasta through the paws to reposition them ("mouth pulling").</li>
 <li>Two behaviors are considered atypical for rats, but are typical for mice: (a.) hunched posture and (b.) dropping the pasta piece after eating begins. However, the frequency of drops in mice may increase after CNS damage. Thus, we record drop frequency in both species.</li>
 <li>Other atypical behaviors have been seen in a rat unilateral striatal dopamine depletion model: (a.) an "iron grip", where the grasp limb is held in an unmoving, rigid position during eating, (b.) repeated movement of the guide paw around the rigid grasp paw, and (c.) and extreme tilting of the head as the pasta is angled into the mouth (Allred et al., 2008; see also Whishaw et al. 1997).</li>
 <li>We have observed that aged mice often make adjustments that are ineffective for moving the pasta into the mouth. Thus, adjustments that do not result in a visible movement of the pasta piece into the mouth are recorded separately.</li>
 <li>Switching the guide and grasp limbs, failures to contact, drops, and mouth pulling are recorded per occurrence, while all other atypical behaviors are recorded once per trial.</li>
</ol>
6. Representative Results
The results from the video analysis should include the number of adjustments of each paw per trial and the time to eat each pasta piece. Results from rats with sensorimotor cortical lesions or cerebral ischemia indicate that animals make fewer adjustments with the impaired (contra-to-lesion) limb, an effect that endures for at least a month after the injuries. Unilateral dopamine depletion results in both a decrease in adjustments with the contralateral limb and an increase in adjustments with the ipsilateral limb (Allred et al., 2008). Results from mice show that aged animals take longer to eat the pasta pieces, and make more adjustments that do not result in advancement of the pasta into the mouth, compared to young adult mice (Tennant et al., 2009). The analysis of these tests may also include observations of atypical handling behaviors. Striatal damage and middle cerebral artery occlusion additionally resulted in increased frequency of abnormal eating behaviors (Allred et al., 2008).
<img src="/files/ftp_upload/2076/2076fig1.jpg" alt="Figure 1" /> 
 Figure 1. Pasta handling posture of (A) rats and (B) mice. Note that the rats sit back on their haunches while the mice hunch over the pasta, necessitating the placement of a mirror below the mouse in order to view paw movements. (C) Examples of pasta pieces prepared for testing.
<img src="/files/ftp_upload/2076/2076fig2.jpg" alt="Figure 2" /> 
 Figure 2. An example forepaw adjustment of (A-C) a rat and (D-F) a mouse. The first panel shows the animal before the adjustment, the center panel shows the forepaw adjusting, with an asterisk indicating the adjusting paw(s), and the last panel shows the replacement on the pasta piece.
<img src="/files/ftp_upload/2076/2076fig3.jpg" alt="Figure 3" /> 
 Figure 3. Guide and grasp limb positions of rats and mice when the pasta is (A,C) long and (B,D) short.

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No conflicts of interest declared.

In this video, we demonstrate how The Vermicelli and Capellini Handling Tests are conducted in rats and mice, respectively. These tests are a simple, quantitative way to study paw and digit function in normal animals, after damage to motor systems of the brain, or during aging. Slow-motion video analysis of the paw and digit movements used to handle small pieces of uncooked pasta allow the researcher to quantify the adjustments made with each paw, as well as observe abnormal behaviors that may be seen following experimentally-induced CNS damage. Testing sessions can be done repeatedly on different days and most animals complete all trials of a session within a few minutes. Thus, the test is suitable for incorporation into a larger test battery or experimental design. The tests can also be used in studies of motor skill learning (Xu et al., 2009), but here we present a protocol to assay changes in an established skill over time.
Currently, skilled reaching tests are the most commonly used tests of dexterous forelimb function in rats (e.g., Whishaw et al., 1986; Ballermann et al., 2001; Montoya et al., 1991) and mice (Farr &amp; Whishaw, 2002; Tennant &amp; Jones, 2009; Baird et al., 2001). Reaching tests permit isolated testing of each limb, whereas pasta handling is a bimanual task. Nevertheless, the tests are similar in their sensitivity to impairments in skilled forepaw function and the acquisition of skill for both tests involves plasticity in the forelimb representation of the motor cortex (Xu et al., 2009). In our experience, the reaching tests are more complicated and laborious to perform and, for some experimental questions, the present tests may provide a more efficient alternative. However, unlike the tests described here, skilled reaching tests have been in use for decades (Peterson, 1934) and they now include a versatile set of highly sensitive measures and test parameters (Ballermann et al., 2001; Farr &amp; Whishaw, 2002; Montoya et al., 1991; Whishaw et al., 1992). In contrast, the Vermicelli and Capellini Handling Tests extend from relatively recent characterizations of the adroit food handling behavior of rats (Whishaw and Coles, 1996). In both administration and analysis, these tests could undoubtedly be further refined and extended, e.g., to more precisely quantify fine digit and bimanual movement patterns and to quantify the coordinated movements of the tongue, mouth and jaw.

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.

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

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 ...

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20.9K vistas.  University of Texas at Austin. El grano y pruebas de destreza de manipulación Capellini pata tomar ventaja de la inclinación natural de los roedores para manipular los alimentos con las patas delanteras y hábiles movimientos dígitos. Los animales se graban en vídeo durante la manipulación de cadenas cortas de pasta seca sin cocinar. Reproducción a cámara lenta de movimiento por vídeo permite la cuantificación de los ajustes de pata.

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

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

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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 ...

Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal cord of the majority of these fiber systems have not been thoroughly investigated in any species. Serotonergic fibers, which project to the spinal cord, have been studied in rats and opossums on histological sections and their functional significance has been deduced based on their fiber termination pattern in the spinal cord. With the development of CLARITY and CUBIC techniques, it is possible to investigate this fiber system and its distribution in the spinal cord, which is likely to reveal previously unknown features of serotonergic supraspinal pathways. Here, we provide a detailed protocol for imaging the serotonergic fibers in the mouse spinal cord using the combined CLARITY and CUBIC techniques. The method involves perfusion of a mouse with a hydrogel solution and clarification of the tissue with a combination of clearing reagents. Spinal cord tissue was cleared in just under two weeks, and the subsequent immunofluorescent staining against serotonin was completed in less than ten days. With a multi-photon fluorescent microscope, the tissue was scanned and a 3D image was reconstructed using Osirix software.

Supraspinal projections are important for pain perception and other behaviors, and serotonergic fibers are one of these fiber systems. The present study focused on the application of the combined CLARITY/CUBIC protocol to the mouse spinal cord in order to investigate the termination of these serotonergic fibers.

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal ...

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 ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

A Simple Approach to Induce Experimental Autoimmune Neuritis in C57BL/6 Mice for Functional and Neuropathological Assessments

Experimental autoimmune neuritis (EAN) is a well-appreciated experimental model of autoimmune peripheral demyelinating diseases. EAN disease is induced by immunizing mice with neurogenic peptides to direct an inflammatory attack toward components of the peripheral nervous system (PNS). Recent advances have enabled the induction of EAN in the relatively resistant C57BL/6 mouse line using myelin protein zero (P0)106-125 or P0180-199 peptides delivered in adjuvant combined with the injection of pertussis toxin. The ability to induce EAN in the C57BL/6 strain allows for the use of the numerous genetic tools that exist on this mouse background, and thus allows the sophisticated study of disease pathogenesis and interrogation of the mechanistic action of novel therapeutics in combination with transgenic approaches. In this study, we demonstrate a simple approach to successfully induce EAN using the P0180-199 peptide in C57BL/6 mice. We also outline a protocol for the assessment of functional deficits that occur in this model, accompanied by an array of neuropathological features. Thus, this model is a powerful experimental model to study the pathogenesis of human peripheral demyelinating neuropathies, and to determine the efficacy of potential therapies that aim to promote myelin repair and protect against nerve damage in autoimmune neuritis.

This report outlines a simple approach to successfully induce experimental autoimmune neuritis (EAN) using the myelin protein zero (P0)180-199 peptide in combination with Freund&#39;s complete adjuvant and pertussis toxin. We present a sophisticated paradigm capable of accurately assessing the extent of functional deficits and neuropathology that occur in this EAN.

Experimental autoimmune neuritis (EAN) is a well-appreciated experimental model of autoimmune peripheral demyelinating diseases. EAN disease is induced by ...

Intracerebroventricular Treatment with Resiniferatoxin and Pain Tests in Mice

The transient receptor potential vanilloid type 1 (TRPV1), a thermosensitive cation channel, is known to trigger pain in the peripheral nerves. In addition to its peripheral function, its involvement in brain functions has also been suggested. Resiniferatoxin (RTX), an ultrapotent TRPV1 agonist, has been known to induce long-term desensitization of TRPV1, and this desensitization has been an alternative approach for investigating the physiological relevance of TRPV1-expressing cells. Here we describe a protocol for intracerebroventricular (i.c.v.) treatment with RTX in mice. Procedures are described for testing nociception to peripheral TRPV1 stimulation (RTX test) and mechanical stimulation (tail pressure test) then follow. Although the nociceptive responses of mice that had been administered RTX i.c.v. were comparable to those of the control groups, RTX-i.c.v.-administered mice were insensitive to the analgesic effect of acetaminophen, suggesting that i.c.v. RTX treatment can induce supraspinal-selective TRPV1 desensitization. This mouse model can be used as a convenient experimental system for studying the role of TRPV1 in brain/supraspinal function. These techniques can also be applied to studies of the central actions of other drugs.

The transient receptor potential vanilloid type 1 (TRPV1) in the supraspinal region has been suggested to play some roles in the brain function. Described here is a protocol for intracerebroventricular injection of resiniferatoxin for supraspinal TRPV1 desensitization in mice. Procedures for some pain tests are also presented.

The transient receptor potential vanilloid type 1 (TRPV1), a thermosensitive cation channel, is known to trigger pain in the peripheral nerves. In ...

Using Optical Coherence Tomography and Optokinetic Response As Structural and Functional Visual System Readouts in Mice and Rats

Optical coherence tomography (OCT) is a fast, non-invasive, interferometric technique allowing high-resolution retinal imaging. It is an ideal tool for the investigation of processes of neurodegeneration, neuroprotection and neuro-repair involving the visual system, as these often correlate well with retinal changes. As a functional readout, visually evoked compensatory eye and head movements are commonly used in experimental models involving the visual function. Combining both techniques allows a quantitative in vivo investigation of structure and function, which can be used to investigate the pathological conditions or to evaluate the potential of novel therapeutics. A great benefit of the presented techniques is the possibility to perform longitudinal analyses allowing the investigation of dynamic processes, reducing variability and cuts down the number of animals needed for the experiments. The protocol described aims to provide a manual for acquisition and analysis of high quality retinal scans of mice and rats using a low cost customized holder with an option to deliver inhalational anesthesia. Additionally, the proposed guide is intended as an instructional manual for researchers using optokinetic response (OKR) analysis in rodents, which can be adapted to their specific needs and interests.

A detailed protocol for the assessment of structural and visual readouts in rodents by optical coherence tomography and optokinetic response is presented. The results provide valuable insights for ophthalmologic as well as neurologic research.

Optical coherence tomography (OCT) is a fast, non-invasive, interferometric technique allowing high-resolution retinal imaging. It is an ideal tool for ...

Electrophysiological Investigations of Retinogeniculate and Corticogeniculate Synapse Function

The lateral geniculate nucleus is the first relay station for the visual information. Relay neurons of this thalamic nucleus integrate input from retinal ganglion cells and project it to the visual cortex. In addition, relay neurons receive top-down excitation from the cortex. The two main excitatory inputs to the relay neurons differ in several aspects. Each relay neuron receives input from only a few retinogeniculate synapses, which are large terminals with many release sites. This is reflected by the comparably strong excitation, the relay neurons receive, from retinal ganglion cells. Corticogeniculate synapses, in contrast, are simpler with few release sites and weaker synaptic strength. The two synapses also differ in their synaptic short-term plasticity. Retinogeniculate synapses have a high release probability and consequently display a short-term depression. In contrast, corticogeniculate synapses have a low release probability. Corticogeniculate fibers traverse the reticular thalamic nuclei before entering the lateral geniculate nucleus. The different locations of the reticular thalamic nucleus (rostrally from the lateral geniculate nucleus) and optic tract (ventro-laterally from the lateral geniculate nucleus) allow stimulating corticogeniculate or retinogeniculate synapses separately with extracellular stimulation electrodes. This makes the lateral geniculate nucleus an ideal brain area where two excitatory synapses with very different properties impinging onto the same cell type, can be studied simultaneously. Here, we describe a method to investigate the recording from relay neurons and to perform detailed analysis of the retinogeniculate and corticogeniculate synapse function in acute brain slices. The article contains a step-by-step protocol for the generation of acute brain slices of the lateral geniculate nucleus and steps for recording activity from relay neurons by stimulating the optic tract and corticogeniculate fibers separately.

Here, we present protocols for the preparation of acute brain slices containing the lateral geniculate nucleus and the electrophysiological investigation of retinogeniculate and corticogeniculate synapses function. This protocol provides an efficient way to study synapses with the high-release and low-release probability in the same acute brain slices.

The lateral geniculate nucleus is the first relay station for the visual information. Relay neurons of this thalamic nucleus integrate input from retinal ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

We introduce a kinematic analysis method that uses a three-dimensional motion capture apparatus containing four cameras and data processing software for performing functional evaluations during fundamental research involving rodent models.

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...

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 ...