The Wellcome Trust for providing Open Access funding to Oxford University. Robert Deacon is a member of Oxford OXION group, funded by Wellcome Trust grant WT084655MA.

1. Apparatus
For all tests, a soft padded surface is placed at the base of the apparatus to cushion any mice that fall off. NB This is not shown in the figures, in order not to obscure any part of the apparatus. During the testing of hundreds of mice, both normal and mutant, we have never observed any injury caused by falling from such apparatus, which is typically 30-50 cm high. Also, be sure to clean and sterilize the equipment between each mouse tested.
1.1 Rotarod
There are several commercial versions of this apparatus on the market, but some have disadvantages, such as failing to accelerate at an adequate speed to detect motor incoordination (rather than endurance). Another common fault is that the rod is not high enough above the base, resulting in mice tending to drop or jump off.
The rotarod shown in Figure 1 was made to the design of the author by the engineering sections of the department of Pharmacology, University of Oxford. The rod is 3 cm in diameter, supported 30 cm above the base of the apparatus. The surface is knurled in a series of parallel ridges along the longitudinal axis, enabling the mice to grip it effectively (Figure 2). NB The depth of the ridges is a vital detail; if the mouse cannot get a good grip the test will be very much harder; on the other hand, if the grip is too good the mouse will "cartwheel" around the rod by passively holding on to it. For "amateur rotarod makers" it may be useful to know that knurled wooden dowelling of an appropriate size for mice is available commercially; it would of course need to be treated with a good waterproof varnish before use. The start speed is adjusted to 4 rpm, the acceleration rate to 20 rpm/min. Maximum speed is 40 rpm.
Two flanges prevent the mouse from leaving the rod. They are 30 cm in diameter (this could probably be reduced to 20 cm). Their separation is set at 6 cm (maximum) but may need to be adjusted smaller for sub-adult mice if they tend to turn around on the rod.
1.2 Triple horizontal bars (Figure 3)
The bars are made of brass, 38 cm long, held 49 cm above the bench surface by a wooden support column at each end. The columns are secured to a heavy wooden base. Three bar diameters are available: 2, 4 and 6 mm. The 2 mm bar is the standard one we use, but many mice reach maximum scores on this. Therefore larger diameter bars have been included in an attempt to refine the test, as mice cannot grip these so well.
1.3 Static rods
Five wooden dowels or rods of varying thickness (35 (rod 1), 28, 22, 15 and 9 (rod 5) mm diameter) each 60 cm long are fixed by a G-clamp to a laboratory shelf such that the rods horizontally protrude into space (Figure 4). The end of the rod near the bench has a mark 10 cm from the end, to denote the finishing line. The height of the rods above the floor is 60 cm.
If a shorter and less detailed test is required, use only the largest, smallest and middle rods. Van Dellen et al.14 used only a single rod to assess the progress of Huntington's disease in mice.
1.4 Parallel bars
Two parallel steel bars 1 m in length and 4 mm in diameter are fixed 30 mm (on their centers) apart by wooden supporting columns at their ends (Figure 5). The bars are 60 cm above the floor.
2. Procedure
For all tests, bring mice to the experimental room 5-20 min before testing, to ensure they are fully awake. As a general rule, to allow recovery of muscular strength and a return to normal levels of arousal, rest the mice by a return to the home cage after each motor test.
2.1 Rotarod
Set the rotarod with a start speed of 4 rpm, acceleration rate 20 rpm/min. Holding the mouse by the tail, and place it on the rotating rod, facing away from the direction of rotation so it has to walk forward to stay upright. This is easiest to do if the mouse is brought up towards the rod at an angle of 45&deg; below horizontal; attempting to lower the mouse from above will result in it spreading its hind legs and grasping the edges of the flanges. Quickly release the mouse when it is almost touching the rod, just forward of and above the rods top dead center, enabling it to easily grip the rod. A (probably easier and more reliable) technique is to use a thin (10-15 mm) dowel. Holding the mouse in one hand, lower it on to the dowel, parallel to the long axis. The dowel is angled with the mouse's head downwards about 30&deg; to give it more time to grip the rotarod once it is released. Lower the mouse and dowel between the flanges of the rotarod, and once over the rotating rod the pull the dowel downwards away from the mouse, which will grasp the rod and walk forwards.
At 10 sec after placing the mouse on the rod, start acceleration (as long as the mouse is facing forward: if not, wait till it does face forward before starting acceleration), and note the speed at which the mouse falls off. If it falls off before 10 sec, note the time of fall and try again, up to three times in total, recording the speed at the first fall after the 10 sec point. However, falls before 5 sec which are due to poor placing by the experimenter should not be recorded. The mean speed at fall is the datum; rather than using the maximum speed, this corrects for the extra practice the mouse receives during the failed runs, which it is assumed would assist performance. Thus a mouse that falls before 10 sec once then stays on till 12 rpm the second time scores (4+12)/2 = 8 rpm. A mouse falling twice then staying on for 10 rpm scores (4+4+10)/3=6 rpm. If a mouse fails to grip in 10 sec three times assign it a score of 4 rpm.
One problem is that mice sometimes stop walking forward and grip the rod firmly instead, so that they passively somersault round, although most eventually fall off. It might therefore be useful to also note the speed when the first such inversion occurs. A possible solution might be to use a rod with a larger diameter or less pronounced ridges.
2.2 Horizontal bars
As mice find it easiest to grasp the narrow 2 mm bar, it is advisable to test them first on this one. Holding the mouse by the tail, place it on the bench in front of the apparatus, slide it quickly backwards about 20 cm (this aligns it perpendicular to the bar), rapidly raise it and let it grasp the horizontal bar at the central point with its forepaws only, and release the tail, simultaneously starting the stopclock. This can be difficult to achieve efficiently; some mice grip better if the tail is released suddenly; if they feel they are still supported they may fail to grip. The criterion point is either a fall from the bar before the mouse reaches one of the end columns of the bar, or the time till one forepaw touches a column. Maximum test time (cut-off time) is 30 sec.
If the mouse fails to grasp the bar properly first time and this appears to be attributable to the experimenter's technique, rather than the mouse, try again (after a brief rest while another one or two mice are tested) and do not record this fall. If the mouse falls before 5 sec and this is apparently not due to poor placing by the experimenter, repeat up to three times in an attempt to get a &gt;5 sec score. If &gt;5 sec on the second attempt, do not do a third trial. Take the best score as the datum. A mouse that repeatedly fails to support itself for &gt;5 sec scores only 1.
If the triple-bar version is being used, if the mouse scores 5 on this first 2 mm bar it can then be tested on thicker bars, after a brief rest period while another one or two mice are tested. The scoring system for the 4 and 6 mm bars is the same as for the 2 mm bar, and the final score is the cumulated total. Thus a mouse that scores 5 on the 2 mm bar but falls from the 4 mm bar after 13 sec scores 5+3 = 8.
2.2.1
Scoring the horizontal bars: the first two intervals are less than the last two as once the mice have initially mastered the task they are less likely to fall: 
Falling between 1-5 sec = 1 
Falling between 6-10 sec = 2 
Falling between 11-20 sec = 3 
Falling between 21-30 sec = 4 
Falling after 30 sec = 5 
Placing one forepaw on a bar support without falling = 5
NB as the optional use of 4 and 6 mm bars is a new addition to our protocol we do not yet have extensive experience with them.
2.3 Static rods
Place the mouse at the far end of the widest rod (nose tip one head's length from the end is ideal). Take two measures: orientation time (time taken to orientate 180&deg; from the starting position towards the shelf) and transit time (the time taken to travel to the shelf end (nose beyond the 10 cm mark from the shelf end of the rod).
Orientation is dependent on the mouse staying upright; if it turns upside down and clings below the rod, arbitrarily assign it (for statistical purposes) the maximum orientation score of 120 sec. Do not test it on smaller rods; also the transit time becomes the maximum value as a mouse that transits upright is obviously better coordinated than one which transits while hanging on upside down.
If, after orienting, the mouse falls or it reaches the maximum test time (arbitrarily set at 120 sec) do not test it on smaller rods. For statistical purposes, assign mice falling are assigned 120 for that particular rod and for subsequent ones, as it is assumed they would again fall from a narrower rod. If the mouse turns upside down, also note this event; for successful transit as well as orientation it must stay upright on the rod. Otherwise assign it the maximum score. Remove the mouse after it reaches the end of the rod or falls.
After testing on one rod return the mouse to the home cage to rest while you test another mouse. Then place it on the next smaller size rod and test it in the same way again. Stop testing if the mouse falls off a rod after being on it for more than 5 sec. If it fell off in less than 5 sec replace it and allow another attempt (as falling within 5 sec could be due to faulty placing by the experimenter), for a maximum of three trials, and use the best result.
2.4 Parallel bars
Place the mouse in the center of the two bars with its longitudinal axis perpendicular to that of the bars; both front paws should be on one bar, both hind paws on the other bar. Scoring is similar to that for the static rods. Take two measures: the time taken until the mouse orients 90&deg; to the start position, and the time until it subsequently reaches one of the end supports. If the mouse turns upside down, also note this event. As for the static rods test, orientation and transit must be achieved without it turning upside down, otherwise the maximum time is allotted. It is rare for reasonably healthy mice to fall in less than 5 sec; if this does happen it is probably due to poor placing. Therefore discard the result and retest the mouse.

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The author has no conflicts of interest to disclose.

Behavior is made manifest by action, and action requires motor skills, including coordination of the body. One of the first experiments in the history of animal behavior was performed by Colin Stewart, a doctoral student at Clark University in the US11. He measured the activity of rats by putting them into a drum and counted the number of times it turned. This early apparatus was probably the forerunner of running wheels and the presently used rotarod test, albeit in the Stewart apparatus the rat was inside the drum, the movement of which was driven by the rat, whereas in the rotarod the animal walks on the outside of a rotating drum or rod which is motor-driven. 
The original rod used by Dunham and Miya (1957)7 had a set speed, but nowadays accelerating versions (Jones and Roberts 1968)9 are available. The disadvantage of a set speed rotarod is that some animals with poor coordination will fall off at the start, whereas for those that do stay on, the test will soon start to measure endurance rather than coordination per se. The resulting bimodal distribution of data points may be sufficiently abnormal as to preclude parametric analysis. When an accelerating rotarod is used, particularly if it is accelerated only when a set criterion, such as staying on for 10 sec, is achieved, data distribution is more likely to be Gaussian. The rate of acceleration can be adjusted so that motor fatigue does not play a significant role in performance; a mouse is unlikely to be markedly fatigued after just 2-3 min time on the rotarod.
The "string test"1, "coat hanger test" or horizontal bar measures forelimb strength and coordination. The two epithets quoted refer to its development; originally a string was used, but the authors noted that performance depended very much on how taut the string was, hence future experimenters used a metal bar, often a coat hanger. We have found that a mouse's ability to grip the bar is inversely proportional to its diameter, and have standardly used a 2 mm bar. In the weights test (see JoVE protocol "Measuring the strength of mice") the very fine wire of a kettle fur/scale collector allows the strongest animals to secure such a good grip that they can lift more than their own body weight. However, using larger diameter horizontal bars can be an advantage as the sensitivity of the test improves; with only a 2 mm bar most normal C57BL/6 mice reach ceiling performance. Hence we have recently adopted a three-bar apparatus; the bars can be readily interchanged as they fit into notches cut into the tops of the supporting columns. The bars are held under tension (especially important to make the 2 mm bar rigid) as they only fit the notches when the supports are slightly drawn together (like a longbow and its string) (see Figure 3).
Static rods or beams have often been used to measure coordination, but additional motivation was always used to persuade the animals to traverse the rod. They negotiated a horizontal beam to reach a desired goal, such as the home cage, refuge from a bright light, or food (if they has been made hungry). However, in the course of investigating this type of test, I discovered that mice would spontaneously turn around when placed on the "open" end of a cantilevered rod, and then traverse it until they reached the supported end. This technique eliminates the need for training (necessary in the previously mentioned test variants) as the innate reaction of a mouse to being placed near the end of an elevated rod is to attempt to reach the supported end. This does not, of course, eliminate the problem that motivational changes might affect performance, which is always a possibility with this type of procedure. 
Cardboard tunnels are presently much used for home cage environmental enrichment, but their disadvantage in the present context is that animals will inevitably be practiced in balancing on cylinders that can wobble about their long axis, mimicking the skills necessary to balance on the rotarod. Thus home cage enrichment could potentially mask a behavioral phenotype; an example is amelioration of a Huntington's disease phenotype by enrichment14. Also animals often walk along the long axis of these tunnels, as when walking along an elevated static beam. Therefore I constructed a pair of elevated parallel bars, and placed the mice in the middle with the forepaws on one bar and the hindpaws on the other, a situation which they would never have encountered before. Mice had to reach the end supports of the parallel bars while remaining upright to pass the test. A somewhat similar device was a series of parallel bars placed in a mouse cage to assess the development of scrapie-induced motor dysfunction13.
We have now tested several hundred mice for motor functioning at Oxford. These had received various treatments including pharmacological agents, or passive transfer of tissue (plasma) from humans, but mostly genetically modified animals. Some animals were more impaired on strength than coordination tasks, others vice versa. Some mice were more impaired on the rotarod than on static substrates such as the static rods and parallel bars, others vice versa. So the present article and the accompanying JoVE protocol "Measuring strength in mice") describe how to characterize the way different treatments and mutations can differentially affect motor function in mice. Apart from the rotarod the apparatus described here is simple, cheap and particularly suitable for laboratories not specialized for testing motor skills. For fine-scale gait analysis, the Catwalk system is a much more sophisticated system than older methods such as persuading a mouse to walk through an ink bath and then down a piece of paper, but it is an expensive piece of apparatus. 
All of these tests are designed to measure coordination rather than strength, although obviously a minimal degree of muscle tone is necessary simply for the mouse to use its limbs. For strength assessment, the inverted screen procedure10 is more suitable, or, if a more graded measurement is required, the weights test or its automated bar-pull equivalent (described in JoVE protocol "Measuring strength in mice"). The exception is the horizontal bar test, which requires both strength and coordination for successful performance.

measuring motor coordination in mice

Mice are increasingly being used in behavioral neuroscience, largely replacing rats as the behaviorist's animal of choice. Before aspects of behavior such as emotionality or cognition can be assessed, however, it is vital to determine whether the motor capabilities of e.g. a mutant or lesioned mouse allow such an assessment. Performance on a maze task requiring strength and coordination, such as the Morris water maze, might well be impaired in a mouse by motor, rather than cognitive, impairments, so it is essential to selectively dissect the latter from the former. For example, sensorimotor impairments caused by NMDA antagonists have been shown to impair water maze performance2. 
Motor coordination has traditionally been assessed in mice and rats by the rotarod test, in which the animal is placed on a horizontal rod that rotates about its long axis; the animal must walk forwards to remain upright and not fall off. Both set speed and accelerating versions of the rotarod are available.
The other three tests described in this article (horizontal bar, static rods and parallel bars) all measure coordination on static apparatus. The horizontal bar also requires strength for adequate performance, particularly of the forelimbs as the mouse initially grips the bar just with the front paws. 
Adult rats do not perform well on tests such as the static rods and parallel bars (personal observations); they appear less well coordinated than mice. I have only tested male rats, however, and male mice seem generally less well coordinated than females. Mice appear to have a higher strength:weight ratio than rats; the Latin name, Mus musculus, seems entirely appropriate. The rotarod, the variations of the foot fault test12 or the Catwalk (Noldus)15 apparatus are generally used to assess motor coordination in rats.

Expected Results
A static rod was originally used to monitor the progress of Huntington's disease in mutant mice, with or without environmental enrichment in their home cages14. The enrichment programme dramatically delayed the onset of motor dysfunction on the rod, which was paralleled by a delay in the onset of hind paw clasping, a general sign of neurological impairment.
Scrapie-infected mice showed a decrease in horizontal bar performance from week 16 post-injection, in rotarod testing from week 18, and static rods from at least week 198 (earlier testing was not performed). KATP channel subunit Kir6.2 KO mice were impaired on the static rods and horizontal bar, but not on the rotarod4. Hippocampal lesions had no effect on any measure of motor ability5, but 129S2/Sv mice were impaired relative to the C57BL/6 strain on the rotarod and static rods3. C57BL/10 mice also had worse motor performance than C57BL/6 mice6, although not to the same extent as 129S2/Sv mice.
 
<img alt="Figure 1" src="/files/ftp_upload/2609/2609fig1.jpg" /> 
Figure 1. The rotarod.
<img alt="Figure 2" src="/files/ftp_upload/2609/2609fig2.jpg" /> 
Figure 2. Detail of the knurled rod of the rotarod.
<img alt="Figure 3" src="/files/ftp_upload/2609/2609fig3.jpg" /> 
Figure 3. The triple horizontal bars. The bar held in the notches is the basic 2 mm one; tacked beneath it (for demonstration purposes only) are the 4 and 6 mm bars. In practice, only one bar at a time is slotted into the notched supports.
<img alt="Figure 4" src="/files/ftp_upload/2609/2609fig4.jpg" /> 
Figure 4. A mouse traversing a static rod.
<img alt="Figure 5" src="/files/ftp_upload/2609/2609fig5.jpg" /> 
Figure 5. A mouse on the parallel bars. Padding has been omitted for demonstration purposes only.

Watch this Scientific Journal Video about Measuring Motor Coordination in Mice at JoVE.com

Measuring Motor Coordination in Mice

Protocols are presented for two established motor coordination tasks, the accelerating rotarod and horizontal bar, also two tests developed in Oxford recently, the static rods and parallel bars. These tests can detect motor impairments potentially of interest in their own right, as well as being possible variables in tests of other areas of behavior.

Mice are increasingly being used in behavioral neuroscience, largely replacing rats as the behaviorist's animal of choice. Before aspects of behavior such ...

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JoVE Journal

Neuroscience

98.7K vistas.  University of Oxford. Los protocolos se presentan establecidas dos tareas de coordinación motora, la barra de rodillo giratorio horizontal y la aceleración, también dos pruebas desarrolladas en Oxford recientemente, las barras estáticas y barras paralelas. Estas pruebas pueden detectar deficiencias motoras potencialmente de interés en su propio derecho, así como que son posibles variables en las pruebas de otras áreas de la conducta.

Video: Measuring Motor Coordination in Mice

Protocols are presented for two established motor coordination tasks, the accelerating rotarod and horizontal bar, also two tests developed in Oxford recently, the static rods and parallel bars. These tests can detect motor impairments potentially of interest in their own right, as well as being possible variables in tests of other areas of...

assessment of motor balance and coordination in mice using the balance beam

Assessment of Motor Balance and Coordination in Mice using the Balance Beam

Deficits in fine motor coordination can be assessed with the balance beam test.  Performance on the beam is quantified by the speed at which the beam is traversed and the number of times the mouse slips on the...

assessing gait by footprint analysis: a method to test motor coordination in mice

In this video, we describe the footprint testing procedure and related analysis to assess gait and motor coordination in...

Assessing Gait by Footprint Analysis: A Method to Test Motor Coordination in Mice

When the mouse reaches the box, gently scruff the animal tucking the back and tail between the ball of the thumb and the other fingers to limit the movement of the hindlimbs and immerse the bottoms of the forelimbs in red ink and the bottom of the hindlimbs in black ink. Then, immediately place the mouse back onto the proximal end of the paper facing the goal box and let the mouse walk from the proximal end to the goal box. After air drying the foot-printed paper, use a ruler to obtain three...

the accelerating rotating rod assay or rotarod test: a method to test motor coordination and learning in mice

Next, open the rotor rod software. Set the start settings to 4 rpm, acceleration settings to 5 minutes, and final speed settings to 40 rpm to provide a constant acceleration from 4 to 40 rpm over a period of 5...

The Accelerating Rotating Rod Assay or Rotarod Test: A Method to Test Motor Coordination and Learning in Mice

In this video, we perform the accelerating rotating rod or rotarod test to assess motor coordination and learning in mouse...

battery of behavioral tests assessing general locomotion, muscular strength, and coordination in mice

Battery of Behavioral Tests Assessing General Locomotion, Muscular Strength, and Coordination in Mice

Behavioral testing is used in pre-clinical trials to assess the phenotypic effects of a disease or treatment on an animal's wellbeing. In order to globally assess motor functioning, we selected tests for general locomotion, muscular strength, and coordination: the open field test, the mesh test, and the rotarod test, respectively.

measuring local anaphylaxis in mice

Measuring Local Anaphylaxis in Mice

Allergic responses, characterized by the activation of mast cells and basophils, are driven by the cross-linking of IgE and release of proinflammatory mediators. A quantitative assessment of allergic responses can be achieved by using Evans Blue dye to monitor changes in vascular permeability after allergen...

measuring the strength of mice

Measuring the Strength of Mice

Deficits in muscular strength occur in many clinical conditions such as motor neuron disease. The inverted screen and weight lifting tests described here measure strength in mice almost exclusively, with minimal influence of factors such as...

retrograde neuroanatomical tracing of phrenic motor neurons in mice

Retrograde Neuroanatomical Tracing of Phrenic Motor Neurons in Mice

Here, we describe a protocol for identifying phrenic motor neurons in mice after intrapleural delivery of fluorophore conjugated cholera toxin subunit beta. Two techniques are compared to inject the pleural cavity: transdiaphragmatic versus transthoracic...

gait analysis of age-dependent motor impairments in mice with neurodegeneration

Gait Analysis of Age-dependent Motor Impairments in Mice with Neurodegeneration

In this study, we demonstrate the use of kinematic gait analysis based on ventral plane imaging to monitor the subtle changes in motor coordination as well as the progression of neurodegeneration with advancing age in mouse models (e.g., endophilin mutant mouse...

monitoring fine and associative motor learning in mice using the erasmus ladder

Author Spotlight: Unveiling Neural Mechanisms Through Automated Evaluation of Motor Learning and Myelin Plasticity Studies Using the Erasmus Ladder 

This article presents a protocol that allows a non-invasive and automated assessment of fine motor performance, as well as adaptive and associative motor learning upon challenges, using a device called the Erasmus Ladder. Task difficulty can be titrated to detect motor impairment ranging from major to subtle degrees.

Monitoring Fine and Associative Motor Learning in Mice Using the Erasmus Ladder

polygraphic recording procedure for measuring sleep in mice

Polygraphic Recording Procedure for Measuring Sleep in Mice

The recording of electroencephalogram (EEG) and electromyogram (EMG) in freely behaving mice is a critical step to correlate behavior and physiology with sleep and wakefulness. The experimental protocol described herein provides a cable-based system for acquiring EEG and EMG recordings in...

measuring active and passive tameness separately in mice

Measuring Active and Passive Tameness Separately in Mice

Tameness in animals includes a reduction of avoidance responses to humans (passive tameness) and an increase in active approaches to humans (active tameness). Here, we describe detailed protocols for three behavioral tests (active tameness, passive tameness, and stay-on-hand tests) to separately measure the active and passive tameness of...

measuring skeletal muscle thermogenesis in mice and rats

Measuring Skeletal Muscle Thermogenesis in Mice and Rats

Mice and rats are surgically implanted with remote temperature transponders and then habituated to the testing environment and procedure. Changes in muscle temperature are measured in response to pharmacological or contextual stimuli in the home cage or during prescribed physical activity (i.e., treadmill walking at a constant speed).

study motor skill learning by single-pellet reaching tasks in mice

Study Motor Skill Learning by Single-pellet Reaching Tasks in Mice

Persistent practice improves the precision of coordinated movements.&#160;Here we introduce a single-pellet reaching task, which is designed to assess the learning and memory of forelimb skill in...

measuring ascending aortic stiffness in vivo in mice using ultrasound

Measuring Ascending Aortic Stiffness In Vivo in Mice Using Ultrasound

We describe a technique for measuring aortic stiffness from its pressure-diameter relationship in vivo in mice. Aortic diameter is recorded by ultrasound and aortic pressure is measured invasively with a solid-state pressure catheter. Blood pressure is changed incrementally and the resulting diameter is...

Measuring Ascending Aortic Stiffness In Vivo in Mice Using Ultrasound

electrophysiological motor unit number estimation (mune) measuring compound muscle action potential (cmap) in mouse hindlimb muscles

Electrophysiological Motor Unit Number Estimation MUNE Measuring Compound Muscle Action Potential CMAP in Mouse Hindlimb Muscles

We present refined protocols that allow in vivo monitoring of motor unit function in the mouse. Techniques to measure compound muscle action potential (CMAP) and motor unit number estimation (MUNE) in the mouse hind limb muscles innervated by the sciatic nerve are...

Electrophysiological Motor Unit Number Estimation (MUNE) Measuring Compound Muscle Action Potential (CMAP) in Mouse Hindlimb Muscles

measuring left ventricular pressure in late embryonic and neonatal mice

Measuring Left Ventricular Pressure in Late Embryonic and Neonatal Mice

Measuring left ventricular pressure (LV) in embryonic and neonatal mice is described. Pressure is measured by inserting a needle connected to a fluid-filled transducer into the LV under ultrasound guidance. Care must be taken to maintain normal cardiac function during the experimental...

Brain injury, genetic manipulations, and pharmacological treatments can result in alterations of motor skills in mice. Fine motor coordination and balance can be assessed by the beam walking assay. The goal of this test is for the mouse to stay upright and walk across an elevated narrow beam to a safe platform. This test takes place over 3 consecutive days: 2 days of training and 1 day of testing. Performance on the beam is quantified by measuring the time it takes for the mouse to traverse the beam and the number of paw slips that occur in the process. Here we report the protocol used in our laboratory, and representative results from a cohort of C57BL/6 mice. This task is particularly useful for detecting subtle deficits in motor skills and balance that may not be detected by other motor tests, such as the Rotarod.

Deficits in fine motor coordination can be assessed with the balance beam test. Performance on the beam is quantified by the speed at which the beam is traversed and the number of times the mouse slips on the beam.

Brain injury, genetic manipulations, and pharmacological treatments can result in alterations of motor skills in mice.  Fine motor coordination and ...

Measuring Circadian and Acute Light Responses in Mice using Wheel Running Activity

Circadian rhythms are physiological functions that cycle over a period of approximately 24 hours (circadian- circa: approximate and diem: day)1, 2. They are responsible for timing our sleep/wake cycles and hormone secretion. Since this timing is not precisely 24-hours, it is synchronized to the solar day by light input. This is accomplished via photic input from the retina to the suprachiasmatic nucleus (SCN) which serves as the master pacemaker synchronizing peripheral clocks in other regions of the brain and peripheral tissues to the environmental light dark cycle3-7. The alignment of rhythms to this environmental light dark cycle organizes particular physiological events to the correct temporal niche, which is crucial for survival8. For example, mice sleep during the day and are active at night. This ability to consolidate activity to either the light or dark portion of the day is referred to as circadian photoentrainment and requires light input to the circadian clock9. Activity of mice at night is robust particularly in the presence of a running wheel. Measuring this behavior is a minimally invasive method that can be used to evaluate the functionality of the circadian system as well as light input to this system. Methods that will covered here are used to examine the circadian clock, light input to this system, as well as the direct influence of light on wheel running behavior.

This article will review methods that can be used to determine circadian function and light responsiveness in mice.

Circadian rhythms are physiological functions that cycle over a period of approximately 24 hours (circadian- circa: approximate and diem: day)1, 2.  They ...

Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica

In animals with large identified neurons (e.g. mollusks), analysis of motor pools is done using intracellular techniques1,2,3,4. Recently, we developed a technique to extracellularly stimulate and record individual neurons in Aplysia californica5. We now describe a protocol for using this technique to uniquely identify and characterize motor neurons within a motor pool.
This extracellular technique has advantages. First, extracellular electrodes can stimulate and record neurons through the sheath5, so it does not need to be removed. Thus, neurons will be healthier in extracellular experiments than in intracellular ones. Second, if ganglia are rotated by appropriate pinning of the sheath, extracellular electrodes can access neurons on both sides of the ganglion, which makes it easier and more efficient to identify multiple neurons in the same preparation. Third, extracellular electrodes do not need to penetrate cells, and thus can be easily moved back and forth among neurons, causing less damage to them. This is especially useful when one tries to record multiple neurons during repeating motor patterns that may only persist for minutes. Fourth, extracellular electrodes are more flexible than intracellular ones during muscle movements. Intracellular electrodes may pull out and damage neurons during muscle contractions. In contrast, since extracellular electrodes are gently pressed onto the sheath above neurons, they usually stay above the same neuron during muscle contractions, and thus can be used in more intact preparations.
To uniquely identify motor neurons for a motor pool (in particular, the I1/I3 muscle in Aplysia) using extracellular electrodes, one can use features that do not require intracellular measurements as criteria: soma size and location, axonal projection, and muscle innervation4,6,7. For the particular motor pool used to illustrate the technique, we recorded from buccal nerves 2 and 3 to measure axonal projections, and measured the contraction forces of the I1/I3 muscle to determine the pattern of muscle innervation for the individual motor neurons.
We demonstrate the complete process of first identifying motor neurons using muscle innervation, then characterizing their timing during motor patterns, creating a simplified diagnostic method for rapid identification. The simplified and more rapid diagnostic method is superior for more intact preparations, e.g. in the suspended buccal mass preparation8 or in vivo9. This process can also be applied in other motor pools10,11,12 in Aplysia or in other animal systems2,3,13,14.

Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica

In animals with large identified neurons (e.g. mollusks), analysis of motor pools is done using intracellular techniques1,2,3,4. Recently, we developed a technique to extracellularly stimulate and record individual neurons in Aplysia californica5. We now describe a protocol for using this technique to uniquely identify and characterize motor neurons within a motor pool.

In animals with large  identified neurons (e.g. mollusks), analysis of motor pools is done using  intracellular techniques1,2,3,4. Recently,  we developed ...

Measuring Changes in Tactile Sensitivity in the Hind Paw of Mice Using an Electronic von Frey Apparatus

Measuring inflammation-induced changes in thresholds of hind paw withdrawal from mechanical pressure is a useful technique to assess changes in pain perception in rodents. Withdrawal thresholds can be measured first at baseline and then following drug, venom, injury, allergen,&#160;or otherwise evoked inflammation by applying an accurate force on very specific areas of the skin. An electronic von Frey apparatus allows precise assessment of mouse hind paw withdrawal thresholds that are not limited by the available filament sizes in contrast to classical von Frey measurements. The ease and rapidity of measurements allow for incorporation of assessment of tactile sensitivity outcomes in diverse models of rapid-onset inflammatory and neuropathic pain as multiple measurements can be taken within a short time period. Experimental measurements for individual rodent subjects can be internally controlled against individual baseline responses and exclusion criteria easily established to standardize baseline responses within and across experimental groups. Thus, measurements using an electronic von Frey apparatus represent a useful modification of the well-established classical von Frey filament-based assays for rodent mechanical allodynia that may also be applied to other nonhuman mammalian models.

Electronic von Frey measurements are an effective and improved alternative to classical von Frey filaments, as they allow rapid and precise measurement of changes in rodent mechanical withdrawal thresholds to continuously applied pressure stimuli before and after an inflammatory event.

Measuring inflammation-induced changes in thresholds of hind paw withdrawal from mechanical pressure is a useful technique to assess changes in pain ...

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of primary afferent fibers mediated by GABAergic axo-axonal synapses (primary afferent depolarization). The strength of primary afferent depolarization can be measured by recording of volume-conducted potentials at the dorsal root (dorsal root potentials, DRP). Pathological changes of presynaptic inhibition are crucial in the abnormal central processing of certain pain conditions and in some disorders of motor hyperexcitability. Here, we describe a method of recording DRP in vivo in mice. The preparation of spinal cord dorsal roots in the anesthetized animal and the recording procedure using suction electrodes are explained. This method allows measuring GABAergic DRP and thereby estimating spinal presynaptic inhibition in the living mouse. In combination with transgenic mouse models, DRP recording may serve as a powerful tool to investigate disease-associated spinal pathophysiology. In vivo recording has several advantages compared to ex vivo isolated spinal cord preparations, e.g. the possibility of simultaneous recording or manipulation of supraspinal networks and induction of DRP by stimulation of peripheral nerves.

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

GABAergic presynaptic inhibition is a powerful inhibitory mechanism in the spinal cord important for motor and sensory signal integration in spinal cord networks. Underlying primary afferent depolarization can be measured by recording of dorsal root potentials (DRP). Here we demonstrate a method of in vivo recording of DRP in mice.

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of ...

The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. Structural changes at the NMJ can result in neurotransmission failure, resulting in weakness, atrophy and even death of the muscle fiber. Many studies have investigated how genetic modifications or disease can alter the structure of the mouse NMJ. Unfortunately, it can be difficult to directly compare findings from these studies because they often employed different parameters and analytical methods. Three protocols are described here. The first uses maximum intensity projection confocal images to measure the area of acetylcholine receptor (AChR)-rich postsynaptic membrane domains at the endplate and the area of synaptic vesicle staining in the overlying presynaptic nerve terminal. The second protocol compares the relative intensities of immunostaining for synaptic proteins in the postsynaptic membrane. The third protocol uses Fluorescence Resonance Energy Transfer (FRET) to detect changes in the packing of postsynaptic AChRs at the endplate. The protocols have been developed and refined over a series of studies. Factors that influence the quality and consistency of results are discussed and normative data are provided for NMJs in healthy young adult mice.

The neuromuscular junction (NMJ) is altered in a variety of conditions that can sometimes culminate in synaptic failure. This report describes fluorescence microscope-based methods to quantify such structural changes.

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. ...

Recording of the epidural electroencephalogram (EEG) and electromyogram (EMG) in small animals, like mice and rats, has been pivotal to study the homeodynamics and circuitry of sleep-wake regulation. In many laboratories, a cable-based sleep recording system is used to monitor the EEG and EMG in freely behaving mice in combination with computer software for automatic scoring of the vigilance states on the basis of power spectrum analysis of EEG data. A description of this system is detailed herein. Steel screws are implanted over the frontal cortical area and the parietal area of 1 hemisphere for monitoring EEG signals. In addition, EMG activity is monitored by the bilateral placement of wires in both neck muscles. Non-rapid eye movement (Non-REM; NREM) sleep is characterized by large, slow brain waves with delta activity below 4 Hz in the EEG, whereas a shift from low-frequency delta activity to a rapid low-voltage EEG in the theta range between 6 and 10 Hz can be observed at the transition from NREM to REM sleep. By contrast, wakefulness is identified by low- to moderate-voltage brain waves in the EEG trace and significant EMG activity.

The recording of electroencephalogram (EEG) and electromyogram (EMG) in freely behaving mice is a critical step to correlate behavior and physiology with sleep and wakefulness. The experimental protocol described herein provides a cable-based system for acquiring EEG and EMG recordings in mice.

Recording of the epidural electroencephalogram (EEG) and electromyogram (EMG) in small animals, like mice and rats, has been pivotal to study the ...

Phrenic motor neurons are cervical motor neurons originating from C3 to C6 levels in most mammalian species. Axonal projections converge into phrenic nerves innervating the respiratory diaphragm. In spinal cord slices, phrenic motor neurons cannot be identified from other motor neurons on morphological or biochemical criteria. We provide the description of procedures for visualizing phrenic motor neuron cell bodies in mice, following intrapleural injections of cholera toxin subunit beta (CTB) conjugated to a fluorophore. This fluorescent neuroanatomical tracer has the ability to be caught up at the diaphragm neuromuscular junction, be carried retrogradely along the phrenic axons and reach the phrenic cell bodies. Two methodological approaches of intrapleural CTB delivery are compared: transdiaphragmatic versus transthoracic injections. Both approaches are successful and result in similar number of CTB-labeled phrenic motor neurons. In conclusion, these techniques can be applied to visualize or quantify the phrenic motor neurons in various experimental studies such as those focused on the diaphragm-phrenic circuitry.

Here, we describe a protocol for identifying phrenic motor neurons in mice after intrapleural delivery of fluorophore conjugated cholera toxin subunit beta. Two techniques are compared to inject the pleural cavity: transdiaphragmatic versus transthoracic approaches.

Phrenic motor neurons are cervical motor neurons originating from C3 to C6 levels in most mammalian species. Axonal projections converge into phrenic ...

Evaluation of Hemisphere Lateralization with Bilateral Local Field Potential Recording in Secondary Motor Cortex of Mice

This article demonstrates complete, detailed procedures for both in vivo bilateral recording and analysis of local field potential (LFP) in the cortical areas of mice, which are useful for evaluating possible laterality deficits, as well as for assessing brain connectivity and coupling of neural network activities in rodents. The pathological mechanisms underlying Alzheimer&#39;s disease (AD), a common neurodegenerative disease, remain largely unknown. Altered brain laterality has been demonstrated in aging people, but whether or not abnormal lateralization is one of the early signs of AD has not been determined. To investigate this, we recorded bilateral LFPs in 3-5-month-old AD model mice, APP/PS1, together with littermate wild type (WT) controls. The LFPs of the left and right secondary motor cortex (M2), specifically in the gamma band, were more synchronized in APP/PS1 mice than in WT controls, suggesting a declined hemispheric asymmetry of bilateral M2 in this AD mouse model. Notably, the recording and data analysis processes are flexible and easy to carry out, and can also be applied to other brain pathways when conducting experiments that focus on neuronal circuits.

We present in vivo electrophysiological recording of the local field potential (LFP) in bilateral secondary motor cortex (M2) of mice, which can be applied to evaluate hemisphere lateralization. The study revealed altered levels of synchronization between the left and right M2 in APP/PS1 mice compared to WT controls.

This article demonstrates complete, detailed procedures for both in vivo bilateral recording and analysis of local field potential (LFP) in the cortical ...

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 ...