This research was supported by a Discovery Grant to AAW from the Natural Sciences and Engineering Research Council of Canada.

1. Pre-requisites for Biomechanical Locomotion Analyses
Prior to embarking upon purchasing expensive locomotor analysis equipment, and planning experiments where kinematics and/or kinetic locomotor assessment will be performed, it is imperative that the experimenter be familiar with the technical and practical aspects of biomechanical analyses, sensorimotor behavior, operant conditioning of animals, and handling/storing/managing large amounts of digitized data. Though these pre-requisites seem obvious to many, it is only after embarking upon these types of experiments where trainees realize the technical and practical complexities of performing relatively detailed locomotor analysis. The authors recommend that experimenters enroll in a course dealing with introductory biomechanics, be familiar with or hire someone familiar with a programming language required for data management, and of equal importance, spend substantial time interacting, handling, and working with laboratory animals. For understanding locomotion analysis and sensorimotor behavioral analysis in the neurosciences, experimenters are referred to several important references (see 1, 4-7).
2. Kinematic and Kinetic Testing Apparatus
A kinematic and kinetic testing apparatus, useful for collecting bilateral data, is comprised of the following components (see "Table of Specific Reagents and Equipment" for more detail):
<ul>
	<li>Quiet and sufficiently-sized room (not necessarily sound-proofed, though located in a low-traffic area)</li>
	<li>Radio to supply background "noise" to limit distracting noises from outside the room to animals</li>
	<li>Clear plexiglass, enclosed, flat-surface runway (180 cm long X 20 cm wide X 60 cm high) with a square hole (11 cm X 11 cm) located within the centre of the runway equidistant from either end</li>
	<li>Piece of plexi-glass (10.5 cm X 10.5 cm)</li>
	<li>Double-sided adhesive scotch tape</li>
	<li>3M reflective adhesive tape for skin markers</li>
	<li>Hole punch for creating skin markers</li>
	<li>Isofluorane anesthesia machine required for anesthetizing animals for shaving prior to placement of skin markers</li>
	<li>Cheerios for encouraging movement of the animal along the length of the runway</li>
	<li>AMTI HE6X6 forceplate</li>
	<li>Vicon Motus customized system (software, computer, etc)</li>
	<li>4 video cameras (at least 60 Hz sampling) and tripods with appropriate connector cables</li>
	<li>4 camera-mounted halogen lights provides optimal lighting for illumination of the animal subject and the reflective skin markers</li>
	<li>Appropriate sized calibration frame or volume necessary for Vicon Motus system to calibrate x, y, z positions into meaningful distance measures</li>
</ul>
3. Animal Training
Prior to collecting data, each animal must be trained to cross a flat-surface, enclosed runway. Upon receiving rats from an appropriate animal supplier, animals should be acclimated to their new home for 1 week. During this acclimation time, several cheerios are placed daily into the rat's cage. Animals are food restricted to their maintenance energy requirements to prevent obesity and ensure motivation to perform this task. Thereafter, each animal is handled by the experimenter for 10-15 minutes daily for 1 week. During this same time period, each animal is placed into the runway with cheerios located at either end. Once the animal becomes familiar with their environment, they will begin eating the cheerios. Once the animal is comfortable and eating cheerios within the runway, the experimenter must then operantly condition the animal to run the length of the runway for a food reward. This is accomplished by tossing &frac14; cheerio to the opposite end of the runway where the rat is positioned. Once the rat eats this cheerio, another &frac14; cheerio is placed at the other end of the runway. This is done for 15-20 minutes daily until the rat consistently (&gt;90% of tosses) moves along the runway at a constant velocity (i.e. without starting, stopping, exploring, or without changing gait) to eat the cheerio without galloping/bounding. The rat should only be employing a trotting gait. Over-conditioning of the animals to this task can lead to animals galloping and bounding these gaits are indicative of animals traveling &gt;90 cm/s. Bounding and galloping gaits, biomechanically, are more difficult to interpret for a variety of reasons (e.g. leg lead inclusion criteria, etc). In our experience, once rats consistently employ galloping or bounding gaits, it is difficult, if not impossible to have them use a trotting gait while locomoting in the runway. Velocities &gt;90 cm/s are rarely seen after an animal has suffered from peripheral or central nervous system injury. Time to reach successful training is variable between strains and sexes of rats. Wistar, Lewis, Long-Evans, and Sprague-Dawley strains are able to consistently traverse the runway within 2 weeks from the onset of training 8. In our experience, Fischer (F-344) rats tend to take upwards of 4-6 weeks to learn this task 8.
4. Joint Position Marking
Forelimb kinematic analysis is unreliable due to skin movement artifact imposed by placing skin markers on the forelimbs which is exacerbated in species, like rats, that have a crouched posture 9. Instead, kinematics of the forelimbs must be achieved using x-ray cinematography or fluoroscopy 10-13. As such, hind limb joint position marker placement is only described herein.
Prior to data collection, each rat must be anesthetized at least 24 hours in advance of data collection using an appropriate inhalational anesthetic (e.g. isofluorane, 1.5-2% dialed on a precision vaporizor) and administered in oxygen via face mask, and key topographic anatomical landmarks must be marked. Given the brevity of the procedure, and because long-acting anesthetic agents are not used, use of an animal warming device need not be used to maintain the animal's body temperature. Once the animal is anesthetized, the hind limbs and the dorsum, to the level of the iliac crests are shaven. The animal is then placed in sternal recumbency and its hindlimbs are placed in an approximate standing position using firm packing foam to support it. The skin overlying the cranial-most portions of the iliac crests, the greater trochanter of the femur, the lateral tibial tuberosity, the tarsal joint, and the distal and lateral aspect of the 5th metatarsal is marked with a non-toxic permanent marker. The animal is recovered from anesthesia. For temporal studies, periodic anesthesia may be required to shave the hindlimbs thereby permitting subsequent reflective marker placement (see below). Also, daily highlighting of the previously marked anatomical landmarks (using the same non-toxic marker) will be required as rats will slowly remove the markers through natural grooming behavior.
5. Data Recording
All camera views are examined to ensure that their position is appropriate and capturing the same field of view. Each camera should be placed at approximately 60-80 degrees to each other. The field of view should include the forceplate in the centre and a length of runway sufficient to capture two strides.
The calibration volume is placed within the pre-determined area of the runway. A single frame of the calibration volume within the runway, from each of the cameras, is captured. All calibrated marks along the length of each of the poles are digitized. Only once a satisfactory error in digitizing is accomplished, can the experimenter proceed to collecting locomotor data. This calibration step is critical prior to collecting data. If calibration is not performed accurately, or if calibration does not occur immediately prior to a recording session, all resulting data will be inaccurate and unusable. Importantly, if any of the cameras are touched or moved, it is safest to assume that calibration of the system needs to be repeated.
Immediately prior to placing the animal in the runway, its weight is recorded and pre-made conical reflective skin markers (using 3M reflective tape, see table) are adhered to the pre-determined felt marks made on the hind limb topographical landmarks. Recording the animal's weight will permit retrospective normalization of ground reaction forces to body weight - an important aspect if one wishes to make comparisons between groups. Additionally, body weight measurement facilitates monitoring of the animal's overall health for the duration of the experiment. Marker placement only requires appropriate animal handling and does not require anesthesia of the animal. If the adhesive on the reflective tape is insufficient to adhere to the animal's skin, a very small amount of non-toxic glue (e.g. 3M VetBond Tissue Adhesive) can be used to facilitate adherence of the marker on the animal's body. Once the markers are placed on the hindlimbs, the experimenter should be positioned comfortably near the keyboard of the computer and have in-hand the event marker attached through the Vicon Motus system. Using the calibrated file as a template, several files are made in advance of recording. Typically, 25 to 30 files need to be saved. Each file should be named uniquely. Each file will represent one recorded run of the animal being recorded. Twenty-five to 30 files are required to ensure that sufficient numbers of runs meeting the inclusion criterion velocity (i.e. 60-90 cm/s) are collected. After sufficient numbers of files are created the experimenter can begin collecting data. The experimenter must prompt the rat to shuttle within the runway by throwing &frac14; pieces of cheerios at either end of the runway. With careful coordination and timing, the event marker is triggered at initiation and just prior to completion of the rat successfully completing a pass along the runway. After examining the crude ground reaction force tracing, and after recording whether the left or the right limbs hit the force plate, the file is saved and closed. Equal numbers of left and right limb forceplate hits should be recorded. The process of recording the data from a given run is repeated until sufficient numbers of runs have been recorded.
6. Data Analysis
Upon completing data collection for kinematics and ground reaction forces, each run from every animal needs to be evaluated for speed. Using two relatively fixed markers (e.g. wings of the iliums) one can evaluate a virtual point between the markers (done prior when creating a Vicon Motus file template). Before calculating the speed of this "virtual" point, markers for the iliac markers must be digitized. Using Vicon motus software, velocity of this virtual point in the X-direction (horizontal direction of movement) is calculated. In so doing, only runs within a given range speeds (determined a priori) are used in the final analyses. We find that animals moving between 60-90 cm/s are using consistent trotting gaits. A minimum of 10 runs (5 runs where the left limb makes contact with the force platform and 5 runs where the right limb makes contact with the force platform) are required. Once the acceptable runs are identified for each animal, digitization of the remaining skin markers must be completed.
To compensate for skin movement artifact over the knee, estimation of the knee position is calculated using triangulation (intersection between two circles 2D kinematics; or intersection between 2 spheres 3D kinematics), as has been previously described. Hip, knee, and hock joint angles, velocities, and accelerations can now be determined. Stance and swing times can also be evaluated, though their accuracy is limited based upon the sampling speed of the cameras being employed. These and other calculations can be performed directly (i.e. without export) using Vicon Motus KineCalc software, or data can be exported as ASCII data and analyzed using customizable routines in software such as MatLab.
Ground reaction force data is measured and amplified by the AMTI force platform and collected at 1200 Hz by Vicon Motus. As such, once the ground reaction force data is collected, an appropriate digital filter is applied to the data using Vicon Motus. Given that the experimenter has already determined the speed of travel and identified acceptable runs after digitizing appropriate skin markers, ground reaction force data that was collected simultaneously as the kinematic data, can be analyzed using Vicon Motus KineCalc directly, or indirectly using some other customizable software routine. A variety of variables for forces, in each of the three orthogonal directions, can be calculated. Such variables include peak force, area und the curve (i.e. impulse), etc. Importantly, however, the experimenter must keep right and left limb data for each run of each animal separate. Data extracted from right or left limbs is averaged for each animal and used as the representative data for that animal. Data is then analyzed using appropriate statistical procedures.
7. Representative Results
To represent the utility of this form of locomotor analysis, kinematics and ground reaction forces were determined for young, middle-aged, and geriatric female Wistar rats. From this analysis, age-related differences were found for female Wistar rats. In particular, ground reaction force analysis demonstrates that geriatric rats have reduced forelimb braking ability and tend to use their hindlimbs more for lateral stabilization compared to the other groups of animals (Figure 1). Kinematic analysis did not reveal any statistical differences between each group, though demonstrates that kinematics can be readily recorded from virtually any age of rat (Figure 2).
<img src="/files/ftp_upload/2138/2138fig1tmb.jpg" alt="FIgure 1" /> Figure 1. Ground reaction force tracing taken from the left limbs of young (4 month old; n=7), middle-aged (13-14 months old; n=7), and geriatric (24 months old; n=5) female Wistar rats. Right limbs were similar. It is readily apparent that geriatric rats use their forelimbs less for braking (* = p&lt;0.05) compared to young and middle-aged rats, and geriatric rats tend to use their hindlimbs more for lateral stabilization compared to young rats (**). Solid lines represent mean, dotted lines represent mean + SE; dashed lines represent mean SE.<a href="https://www.jove.com/files/ftp_upload/2138/2138fig1.jpg">Please click here to see a larger figure.</a>
<img src="/files/ftp_upload/2138/2138fig2tmb.jpg" alt="FIgure 2" /> Figure 2. Kinematic stick figures for the left limb of young, middle-aged, and geriatric rats. Stick figures represent the mean movement of the left limb for each of the groups. There were no significant differences between the groups of animals for joint angle during the step cycle.<a href="https://www.jove.com/files/ftp_upload/2138/2138fig2.jpg">Please click here to see a larger figure.</a>

No conflicts of interest declared.

The present paper provides methodology for evaluating locomotion using continuous quantitative kinematics and ground reaction force determination. Important for anyone interested in embarking upon this methodology is a strong background in biomechanics of locomotion, animal sensorimotor behavior, and data management and manipulation. Though kinematic and ground reaction force determination requires additional time and expertise, compared to some other forms of locomotor analysis (e.g. endpoint measures, ordinal rating scales), the data obtained is sensitive, objective and quantitative for a variety of orthopedic14-22 and neurologic1-3, 23-32 models of disease, in a variety of species.
We have provided data that describes differences in locomotion between various ages of strain-matched rats information that could not be gleaned using simple and less sensitive measures. Further, kinematic and kinetic analysis of locomotion has been used to describe locomotor alterations in a variety of nervous system conditions where other forms of evaluation would be unsuccessful.8, 23-25, 27 The use of sensitive measures becomes especially important when evaluating potential therapeutants for various models of disease. If a test is not sensitive enough to discern an effect of a potential therapeutant the experimenter runs the risk of committing a type-II statistical error (i.e. concluding there is no effect of a treatment when in fact there was an effect). Further, because endpoint measures and more subjective tests that evaluate locomotion, there exists a potential for bias. Kinematic and kinetic evaluation is purely objective in that, provided appropriate inclusion/exclusion criterion are made a priori, the experimenter simply collects, examines and applies appropriate statistics to the data (i.e. there is no subjective component to data determination). 
Kinematic and kinetic analysis also affords the ability to be used for a multitude of species. In fact, kinematics, ground reaction force determination, or both have been used in a variety of species such as elephants33-35, cattle36, horses37-40, dogs4, 41-45, cats21, 46-49, various rodents3, 8, 50, 51, birds4, 52-55, and fish56, 57 (this list is by no means exhaustive). In the authors' experience, however, the use of mice is problematic given that mice are not easy to operantly condition to travel along a runway. Given this, mice will not travel at a relatively constant speed and instead speed-up and slow-down when traversing the runway. This behavior can likely, in part, be overcome by running mice on a treadmill and video-taping the animal locomoting on the treadmill.58 If the experimental apparatus for ground reaction force determination was to be modified for treadmill usage, ground reaction force determination would likely only be easily obtained for vertical ground reaction force as the treadmill belt would interfere with fore-aft and medio-lateral force determination.
Altogether, kinematic and kinetic analysis of locomotion is a reliable, sensitive, and objective method that can be employed for various models of orthopedic and neurologic conditions. Furthermore, all of the equipment has become available for use in rodents, thus negating any related reason for not performing this form of sensorimotor behavioral analysis.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Low load miniature force platform</td>
<td>&nbsp;</td>
<td>AMTI</td>
<td>HE6X6</td>
<td>http://amti.biz/PDFDownloads/0908HE6x6.pdf</td>
</tr>
<tr>
<td>Vicon Motus</td>
<td>&nbsp;</td>
<td>Vicon</td>
<td>Vicon Motus software</td>
<td>http://www.vicon.com/applications/animal.html 

The force platform can be purchased as an integrated package with the Vicon Motus system.</td>
</tr>
<tr>
<td>Vicon Motus KineCalc</td>
<td>&nbsp;</td>
<td>Vicon</td>
<td>Vicon Motus KineCalc software</td>
<td>http://www.vicon.com/applications/animal.html Proprietary software compatible with Vicon Motus data, thus enabling direct and customizable data calculations.
</td>
</tr>
<tr>
<td>Video Cameras</td>
<td>&nbsp;</td>
<td>Vicon</td>
<td>Varies depending upon needs</td>
<td>http://www.vicon.com/applications/animal.html A minimum of 4 cameras are required for bilateral 2D or 3D imaging of hindlimb kinematics during rat locomotion. Cameras that sample at 60 Hz are sufficient for rats locomoting between 60 and 90 cm/s. High speed cameras will permit identification of very small, yet possibly important, differences in interlimb coordination.</td>
</tr>
<tr>
<td>Calibration volume or frame</td>
<td>&nbsp;</td>
<td>Vicon</td>
<td>Custom</td>
<td>http://www.vicon.com/applications/animal.html An appropriate calibration volume must be purchased and used for calibrating the coordinates prior to performing videography each and every time of new data collection or if any of the cameras are moved. This dimensions of this calibration volume are based upon the field of view being examined.
</td>
</tr>
<tr>
<td>Reflective tape</td>
<td>&nbsp;</td>
<td>3M</td>
<td>NA</td>
<td>Can be purchased bulk through a 3M distributor or on an as-needed basis from VICON
 http://solutions.3m.com/en_US/?WT.mc_id=www.3m.com/us http://www.vicon.com/applications/animal.html</td>
</tr>
<tr>
<td>Cheerios</td>
<td>&nbsp;</td>
<td>General Mills</td>
<td>Cheerios</td>
<td>http://www.cheerios.com/</td>
</tr>
<tr>
<td>Tissue adhesive</td>
<td>&nbsp;</td>
<td>3M</td>
<td>&nbsp;</td>
<td>Can be purchased through a 3M distributor 
http://solutions.3m.com/en_US/?WT.mc_id=www.3m.com/us</td>
</tr>		</tbody>
		</table>
		</div>

kinematics and ground reaction force determination: a demonstration quantifying locomotor abilities of young adult, middle-aged, and geriatric rats

Behavior, in its broadest definition, can be defined as the motor manifestation of physiologic processes. As such, all behaviors manifest through the motor system. In the fields of neuroscience and orthopedics, locomotion is a commonly evaluated behavior for a variety of disease models. For example, locomotor recovery after traumatic injury to the nervous system is one of the most commonly evaluated behaviors 1-3. Though locomotion can be evaluated using a variety of endpoint measurements (e.g. time taken to complete a locomotor task, etc), semiquantitative kinematic measures (e.g. ordinal rating scales (e.g. Basso Beattie and Bresnahan locomotor (BBB) rating scale, etc)) and surrogate measures of behaviour (e.g. muscle force, nerve conduction velocity, etc), only kinetics (force measurements) and kinematics (measurements of body segments in space) provide a detailed description of the strategy by which an animal is able to locomote 1. Though not new, kinematic and kinetic measurements of locomoting rodents is now more readily accessible due to the availability of commercially available equipment designed for this purpose. Importantly, however, experimenters need to be very familiar with theory of biomechanical analyses and understand the benefits and limitations of these forms of analyses prior to embarking on what will become a relatively labor-intensive study. The present paper aims to describe a method for collecting kinematic and ground reaction force data using commercially available equipment. Details of equipment and apparatus set-up, pre-training of animals, inclusion and exclusion criteria of acceptable runs, and methods for collecting the data are described. We illustrate the utility of this behavioral analysis technique by describing the kinematics and kinetics of strain-matched young adult, middle-aged, and geriatric rats.

Watch this Scientific Journal Video about Kinematics and Ground Reaction Force Determination: A Demonstration Quantifying Locomotor Abilities of Young Adult, Middle-aged, and Geriatric Rats at JoVE.co

Kinematics and Ground Reaction Force Determination: A Demonstration Quantifying Locomotor Abilities of Young Adult, Middle-aged, and Geriatric Rats

Locomotion is often examined as a behavioural outcome in various models of disease in fields such as neuroscience and orthopedics. This video paper intends to describe a method for collecting ground reaction forces and kinematics from rats during unrestrained locomotion.

Behavior, in its broadest definition, can be defined as the motor manifestation of physiologic processes.  As such, all behaviors manifest through the ...

kinematics-ground-reaction-force-determination-demonstration

Research

JoVE Journal

Neuroscience

19.6K Views.  Riverview, NB. Locomozione è spesso esaminato come un risultato di comportamento in vari modelli di malattie in campi quali le neuroscienze e ortopedia. In questo documento il video propone di descrivere un metodo per raccogliere le forze di reazione del terreno e la cinematica di ratti durante la locomozione sfrenato.

Video: Kinematics and Ground Reaction Force Determination: A Demonstration Quantifying Locomotor Abilities of Young Adult, Middle-aged, and Geriatric Rats

Growth and Differentiation of Adult Hippocampal Arctic Ground Squirrel Neural Stem Cells

Arctic ground squirrels (Urocitellus parryii, AGS) are unique in their ability to hibernate with a core body temperature near or below freezing 1. These animals also resist ischemic injury to the brain in vivo 2,3 and oxygen-glucose deprivation in vitro 4,5. These unique qualities provided the impetus to isolate AGS neurons to examine inherent neuronal characteristics that could account for the capacity of AGS neurons to resist injury and cell death caused by ischemia and extremely cold temperatures. Identifying proteins or gene targets that allow for the distinctive properties of these cells could aid in the discovery of effective therapies for a number of ischemic indications and for the study of cold tolerance. Adult AGS hippocampus contains neural stem cells that continue to proliferate, allowing for easy expansion of these stem cells in culture. We describe here methods by which researchers can utilize these stem cells and differentiated neurons for any number of purposes. By closely following these steps the AGS neural stem cells can be expanded through two passages or more and then differentiated to a culture high in TUJ1-positive neurons (~50%) without utilizing toxic chemicals to minimize the number of dividing cells. Ischemia induces neurogenesis 6 and neurogenesis which proceeds via MEK/ERK and PI3K/Akt survival signaling pathways contributes to ischemia resistance in vivo7 and in vitro 8 (Kelleher-Anderson, Drew et al., in preparation). Further characterization of these unique neural cells can advance on many fronts, using some or all of these methods.

Neural stem cells were prepared from the hippocampus of adult non-hibernating yearling Arctic ground squirrels (AGS). These neural stem cells can be expanded through numerous passages, differentiated and maintained as a nearly 50:50 neuron to glial culture.

Arctic ground squirrels (Urocitellus parryii, AGS) are unique in their ability to hibernate with a core body temperature near or below freezing 1. These ...

Cerebrovascular Casting of the Adult Mouse for 3D Imaging and Morphological Analysis

Vascular imaging is crucial in the clinical diagnosis and management of cerebrovascular diseases, such as brain arteriovenous malformations (BAVMs). Animal models are necessary for studying the etiopathology and potential therapies of cerebrovascular diseases. Imaging the vasculature in large animals is relatively easy. However, developing vessel imaging methods of murine brain disease models is desirable due to the cost and availability of genetically-modified mouse lines. Imaging the murine cerebral vascular tree is a challenge. In humans and larger animals, the gold standard for assessing the angioarchitecture at the macrovascular (conductance) level is x-ray catheter contrast-based angiography, a method not suited for small rodents.
In this article, we present a method of cerebrovascular casting that produces a durable skeleton of the entire vascular bed, including arteries, veins, and capillaries that may be analyzed using many different modalities. Complete casting of the microvessels of the mouse cerebrovasculature can be difficult; however, these challenges are addressed in this step-by-step protocol. Through intracardial perfusion of the vascular casting material, all vessels of the body are casted. The brain can then be removed and clarified using the organic solvent methyl salicylate. Three dimensional imaging of the brain blood vessels can be visualized simply and inexpensively with any conventional brightfield microscope or dissecting microscope. The casted cerebrovasculature can also be imaged and quantified using micro-computed tomography (micro-CT)1. In addition, after being imaged, the casted brain can be embedded in paraffin for histological analysis.
The benefit of this vascular casting method as compared to other techniques is its broad adaptation to various analytic tools, including brightfield microscopic analysis, CT scanning due to the radiopaque characteristic of the material, as well as histological and immunohistochemical analysis. This efficient use of tissue can save animal usage and reduce costs. We have recently demonstrated application of this method to visualize the irregular blood vessels in a mouse model of adult BAVM at a microscopic level2, and provide additional images of the malformed vessels imaged by micro-CT scan. Although this method has drawbacks and may not be ideal for all types of analyses, it is a simple, practical technique that can be easily learned and widely applied to vascular casting of blood vessels throughout the body.

In this article, we present a simple, practical technique for cerebrovascular casting that is easy to perform and can be utilized to image the vascular tree of the adult mouse brain.

Vascular imaging is crucial in the clinical diagnosis and management of cerebrovascular diseases, such as brain arteriovenous malformations (BAVMs). ...

Rapid Determination of the Thermal Nociceptive Threshold in Diabetic Rats

Painful diabetic neuropathy (PDN) is characterized by hyperalgesia i.e., increased sensitivity to noxious stimulus, and allodynia i.e., hypersensitivity to normally innocuous stimuli1. Hyperalgesia and allodynia have been studied in many different rodent models of diabetes mellitus2. However, as stated by B&ouml;lcskei et al, determination of "pain" in animal models is challenging due to its subjective nature3. Moreover, the traditional methods used to determine behavioral responses to noxious thermal stimuli usually lack reproducibility and pharmacological sensitivity3. For instance, by using the hot-plate method of Ankier4, flinch, withdrawal and/or licking of either hind- and/or fore-paws is quantified as reflex latencies at constant high thermal stimuli (52-55 &deg;C). However, animals that are hyperalgesic to thermal stimulus do not reproducibly show differences in reflex latencies using those supra-threshold temperatures3,5. As the recently described method of B&ouml;lcskei et al.6, the procedures described here allows for the rapid, sensitive and reproducible determination of thermal nociceptive thresholds (TNTs) in mice and rats. The method uses slowly increasing thermal stimulus applied mostly to the skin of mouse/rat plantar surface. The method is particularly sensitive to study anti-nociception during hyperalgesic states such as PDN. The procedures described bellow are based on the ones published in detail by Alm&aacute;si et al 5 and B&ouml;lcskei et al 3. The procedures described here have been approved the Laboratory Animal Care and Use Committee (LACUC), Wright State University.

Here, we describe a rapid reliable and simple procedure to determine the lowest temperature at which rats or mice show nocifensive behavior, i.e. the thermal nociceptive threshold (TNT). This method applies a slowly increasing thermal stimulus allowing precise and reproducible estimation of TNTs with minimum, if any, stress to the animals.

Painful diabetic neuropathy (PDN) is characterized by hyperalgesia i.e., increased sensitivity to noxious stimulus, and allodynia i.e., hypersensitivity ...

Microdialysis of Ethanol During Operant Ethanol Self-administration and Ethanol Determination by Gas Chromatography

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol. However, ethanol is typically self-administered orally, rather than intravenously like many other drugs of abuse. The pharmacokinetics of orally administered drugs are more complex than intravenously administered drugs. Because understanding the relationship between the pharmacological and behavioral effects of ethanol requires knowledge of the time course of ethanol reaching the brain during and after drinking, we use in vivo microdialysis and gas chromatography with flame ionization detection to monitor brain dialysate ethanol concentrations over time. 
Combined microdialysis-behavioral experiments involve the use of several techniques. In this article, stereotaxic surgery, behavioral training and microdialysis, which can be adapted to test a multitude of self-administration and neurochemical centered hypotheses, are included only to illustrate how they relate to the subsequent phases of sample collection and dialysate ethanol analysis. Dialysate ethanol concentration analysis via gas chromatography with flame-ionization detection, which is specific to ethanol studies, is described in detail. Data produced by these methods reveal the pattern of ethanol reaching the brain during the self-administration procedure, and when paired with neurochemical analysis of the same dialysate samples, allows conclusions to be made regarding the pharmacological and behavioral effects of ethanol.

A method to determine the time course of ethanol concentration in the brains of rats during operant ethanol self-administration is described. Gas chromatography with flame ionization detection is used to quantify ethanol in the dialysate samples, because it has the sensitivity required for the small volumes that are generated.

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol.  ...

Simultaneous Intracellular Recording of a Lumbar Motoneuron and the Force Produced by its Motor Unit in the Adult Mouse In vivo

The spinal motoneuron has long been a good model system for studying neural function because it is a neuron of the central nervous system with the unique properties of (1) having readily identifiable targets (the muscle fibers) and therefore having a very well-known function (to control muscle contraction); (2) being the convergent target of many spinal and descending networks, hence the name of "final common pathway"; and (3) having a large soma which makes it possible to penetrate them with sharp intracellular electrodes. Furthermore, when studied in vivo, it is possible to record simultaneously the electrical activity of the motoneurons and the force developed by their muscle targets. Performing intracellular recordings of motoneurons in vivo therefore put the experimentalist in the unique position of being able to study, at the same time, all the compartments of the "motor unit" (the name given to the motoneuron, its axon, and the muscle fibers it innervates1): the inputs impinging on the motoneuron, the electrophysiological properties of the motoneuron, and the impact of these properties on the physiological function of the motoneurons, i.e. the force produced by its motor unit. However, this approach is very challenging because the preparation cannot be paralyzed and thus the mechanical stability for the intracellular recording is reduced. Thus, this kind of experiments has only been achieved in cats and in rats. However, the study of spinal motor systems could make a formidable leap if it was possible to perform similar experiments in normal and genetically modified mice.
For technical reasons, the study of the spinal networks in mice has mostly been limited to neonatal in vitro preparations, where the motoneurons and the spinal networks are immature, the motoneurons are separated from their targets, and when studied in slices, the motoneurons are separated from most of their inputs. Until recently, only a few groups had managed to perform intracellular recordings of motoneurons in vivo2-4 , including our team who published a new preparation which allowed us to obtain very stable recordings of motoneurons in vivo in adult mice5,6. However, these recordings were obtained in paralyzed animals, i.e. without the possibility to record the force output of these motoneurons. Here we present an extension of this original preparation in which we were able to obtain simultaneous recordings of the electrophysiological properties of the motoneurons and of the force developed by their motor unit. This is an important achievement, as it allows us to identify the different types of motoneurons based on their force profile, and thereby revealing their function. Coupled with genetic models disturbing spinal segmental circuitry7-9, or reproducting human disease10,11, we expect this technique to be an essential tool for the study of spinal motor system.

Simultaneous Intracellular Recording of a Lumbar Motoneuron and the Force Produced by its Motor Unit in the Adult Mouse In vivo

This new method permits the simultaneous intracellular recording of a single adult mouse motoneuron and the measurement of the force produced by its muscle fibers. The combined investigation of the electrical and mechanical properties of motor units in normal and genetically modified animals is a breakthrough for the study of the neuromuscular system.

The  spinal motoneuron has long been a good model system for studying neural  function because it is a neuron of the central nervous system with the ...

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such behavior is spontaneous locomotor activity. Here we describe our protocol that utilizes Drosophila population monitors and a tracking system that allows continuous monitoring of the spontaneous locomotor activity of flies for several days at a time. This method is simple, reliable, and objective and can be used to examine the effects of aging, sex, changes in caloric content of food, addition of drugs, or genetic manipulations that mimic human diseases.

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster are useful in studying genetic or environmental manipulations that affect behaviors such as spontaneous locomotor activity.&#160; Here we describe a protocol that utilizes monitors with infrared beams and data analysis software to quantify spontaneous locomotor activity.&#160;

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such ...

Quantifying Microglia Morphology from Photomicrographs of Immunohistochemistry Prepared Tissue Using ImageJ

Microglia are brain phagocytes that participate in brain homeostasis and continuously survey their environment for dysfunction, injury, and disease. As the first responders, microglia have important functions to mitigate neuron and glia dysfunction, and in this process, they undergo a broad range of morphologic changes. Microglia morphologies can be categorized descriptively or, alternatively, can be quantified as a continuous variable for parameters such as cell ramification, complexity, and shape. While methods for quantifying microglia are applied to single cells, few techniques apply to multiple microglia in an entire photomicrograph. The purpose of this method is to quantify multiple and single cells using readily available ImageJ protocols. This protocol is a summary of the steps and ImageJ plugins recommended to convert fluorescence and bright-field photomicrographs into representative binary and skeletonized images and to analyze them using software plugins AnalyzeSkeleton (2D/3D) and FracLac for morphology data collection. The outputs of these plugins summarize cell morphology in terms of process endpoints, junctions, and length as well as complexity, cell shape, and size descriptors. The skeleton analysis protocol described herein is well suited for a regional analysis of multiple microglia within an entire photomicrograph or region of interest (ROI) whereas FracLac provides a complementary individual cell analysis. Combined, the protocol provides an objective, sensitive, and comprehensive assessment tool that can be used to stratify between diverse microglia morphologies present in the healthy and injured brain.

Microglia are brain immune cells that survey and react to altered brain physiology through morphologic changes which may be evaluated quantitatively. This protocol outlines an ImageJ based analysis protocol to represent microglia morphology as continuous data according to metrics such as cell ramification, complexity, and shape.

Microglia are brain phagocytes that participate in brain homeostasis and continuously survey their environment for dysfunction, injury, and disease. As ...

Quantifying Spontaneous Ca2+ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons

Parkinson&#8217;s disease (PD) is a devastating neurodegenerative disorder caused by the degeneration of dopaminergic (DA) neurons. Excessive Ca2+ influx due to the abnormal activation of glutamate receptors results in DA excitotoxicity and has been identified as an important mechanism for DA neuron loss. In this study, we isolate, dissociate, and culture midbrain neurons from the mouse ventral mesencephalon (VM) of ED14 mouse embryos. We then infect the long-term primary mouse midbrain cultures with an adeno-associated virus (AAV) expressing a genetically encoded calcium indicator, GCaMP6f under control of the human neuron-specific synapsin promoter, hSyn. Using live confocal imaging, we show that cultured mouse midbrain neurons display spontaneous Ca2+ fluxes detected by AAV-hSyn-GCaMP6f. Bath application of glutamate to midbrain cultures causes abnormal elevations in intracellular Ca2+ within neurons and this is accompanied by caspase-3 activation in DA neurons, as demonstrated by immunostaining. The techniques to identify glutamate-mediated apoptosis in primary mouse DA neurons have important applications for the high content screening of drugs that preserve DA neuron health.

Quantifying Spontaneous Ca2+ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons

Here we present a protocol to measure in vitro Ca2+ fluxes in midbrain neurons and their downstream effects on caspase-3 using primary mouse midbrain cultures. This model can be employed to study pathophysiologic changes related to abnormal Ca2+ activity in midbrain neurons, and to screen novel therapeutics for anti-apoptotic properties.

Parkinson&#8217;s disease (PD) is a devastating neurodegenerative disorder caused by the degeneration of dopaminergic (DA) neurons. Excessive Ca2+ influx ...

A Metric Test for Assessing Spatial Working Memory in Adult Rats Following Traumatic Brain Injury

Impairments to sensory, short-term, and long-term memory are common side effects after traumatic brain injury (TBI). Due to the ethical limitations of human studies, animal models provide suitable alternatives to test treatment methods, and to study the mechanisms and related complications of the condition. Experimental rodent models have historically been the most widely used due to their accessibility, low cost, reproducibility, and validated approaches. A metric test, which tests the ability to recall the placement of two objects at various distances and angles from one another, is a technique to study impairment in spatial working memory (SWM) after TBI. The significant advantages of metric tasks include the possibility of dynamic observation, low cost, reproducibility, relative ease of implementation, and low stress environment. Here, we present a metric test protocol to measure impairment of SWM in adult rats after TBI. This test provides a feasible way to evaluate physiology and pathophysiology of brain function more effectively.

Traumatic brain injury (TBI) is commonly associated with memory impairment. Here, we present a protocol to assess spatial working memory after TBI via a metric task. A metric test is a useful tool to study spatial working memory impairment after TBI.

Impairments to sensory, short-term, and long-term memory are common side effects after traumatic brain injury (TBI). Due to the ethical limitations of ...

A Scalable Model to Study the Effects of Blunt-Force Injury in Adult Zebrafish

Blunt-force traumatic brain injuries (TBI) are the most common form of head trauma, which spans a range of severities and results in complex and heterogenous secondary effects. While there is no mechanism to replace or regenerate the lost neurons following a TBI in humans, zebrafish possess the ability to regenerate neurons throughout their body, including the brain. To examine the breadth of pathologies exhibited in zebrafish following a blunt-force TBI and to study the mechanisms underlying the subsequent neuronal regenerative response, we modified the commonly used rodent Marmarou weight drop for the use in adult zebrafish. Our simple blunt-force TBI model is scalable, inducing a mild, moderate, or severe TBI, and recapitulates many of the phenotypes observed following human TBI, such as contact- and post-traumatic seizures, edema, subdural and intracerebral hematomas, and cognitive impairments, each displayed in an injury severity-dependent manner. TBI sequelae, which begin to appear within minutes of the injury, subside and return to near undamaged control levels within 7 days post-injury. The regenerative process begins as early as 48 hours post-injury (hpi), with the peak cell proliferation observed by 60 hpi. Thus, our zebrafish blunt-force TBI model produces characteristic primary and secondary injury TBI pathologies similar to human TBI, which allows for investigating disease onset and progression, along with the mechanisms of neuronal regeneration that is unique to zebrafish.

We modified the Marmarou weight drop model for adult zebrafish to examine a breadth of pathologies following blunt-force traumatic brain injury (TBI) and the mechanisms underlying subsequent neuronal regeneration. This blunt-force TBI model is scalable, induces a mild, moderate, or severe TBI, and recapitulates injury heterogeneity observed in human TBI.