The rotarod performance test was first developed by Dunham and Miya (1975) in order to test the neurologic effects of various drugs in rats. The task involved rodents trying to maintain their equilibrium on a rod (rotating cylinder), rotating at a constant speed.  Later, the sensitivity of the task was increased by gradually increasing the speed of the rod while the rodent was walking. Both methods of accelerating and constant speed can be used for different purposes. In more detail, the rod is suspended above a cage floor, placed low enough to not injure the rodent, but high enough to avoid falling. The length of time that a given rodent stays on the rotarod is a measure of their endurance, balance coordination, physical condition, and motor-planning.  The diameter of the rod depends on the type of rodent and the variable of interest.
Due to the fact that this test does not require a subjective judgment of the rodent’s ability, but that it gives a measurable, continuous variable instead, it is considered a reliable assay for quantifying the effects of drugs, conditions and procedures. However, for inter-lab reliability, it is recommended to replicate various parameters: e.g. size of the cylinder, speed of the cylinder, composition material of the surface, and amount of practice/pre-training given before the experiment.  These parameters can be adjusted according to the condition of interest. Details for different factors of the test that can influence the outcome for some conditions will be mentioned below. Other advantages include the use of inexpensive equipment, simple validation protocols and consistent results. 
Hamm et al. (1994) were the first to use the task to assess motor deficits in mild to moderate traumatic brain injuries. They found that the rotarod task is more sensitive for measuring balance and motor function/coordination impairment than the beam-walk and beam-balance tasks.  However, in some cases like determining benzodiazepine-induced motor coordination deficits, the beam walking task seemed to be a better predictor for clinical sedation.  The rotarod task is also frequently used in testing the early stages of drug development in order to eliminate drugs that might cause subtle motor impairments.  For example, alcohol is known to markedly impair the performance of mice in the rotarod test.  Moreover, the test is used to specifically determine which components of neurons mediate the effects of various chemical agonists and antagonists.  Using the rotarod task with genetic knockout rodents can help determinate the genes that are responsible for maintaining balance and coordination. One study, for example, found that vitamin D receptor-deficient KO mice have decreased balance function.  Also, brain lesion studies interested in mapping which structures are critical for maintaining balance can use the rotarod task to compare performances of mice with different brain lesions. 
Factors influencing the rotarod
When there are no training sessions preceding repeated testing on the rotarod, results could be confounded by longer latencies to fall due to learning during the experiment, rather than by drug treatment or other factors of interest. Pre-training until a plateau of performance is reached is recommended to avoid increased performance during testing.  It is also important that pre-training is done on an accelerating rotarod in order to analyze the learning curve. 
Genotype of rodent
Some studies using the rotarod test on heterogeneous mice saw no changes in performance across days of testing some of the mice. However, some found that certain inbred or transgenic mice may demonstrate increased performance with repeated testing. This is an important consideration if repeated testing with manipulated mice is planned. Rustay et al. (2003) tested eight inbred strains and found differences in acquisition, peak performance, and carry-over performance among the strains. This might suggest that there are differences in the underlying structure and/or function of the brain regions involved in rotarod performance among inbreeds. 
The rod diameter is important for the initial performance of the animals and makes the learning curve steeper. Rozas et al. (1998) argue that the use of larger rods with mice may reflect their ability to walk or run, while this does not seem to be true for rats. Rustay et al. (2003) state that the use of a larger diameter rod is recommended due to the fact that smaller sizes (e.g. ≤ 5.1 cm) allow for the rodent to cling to the rod and passively rotate at least once around the rod (i.e. passive rotating). While this kind of passive rotating could be another measure of coordination or muscle strength, it should not be a confound in measuring the rodent’s ability to balance and walk on top of the apparatus. In other words, passive rotating should be analyzed separately from balance and walking performance. Using rods with a diameter of 6.3 cm or greater the proportion of mice passively rotating should be negligible. On the other hand, Shiotsuki et al. (2010) noticed that untrained mice showed poor performance staying on a large-diameter drum (i.e. 9 cm), originally designed for rat experiments. On the top of the large-diameter drum, the mice were unstable and easily slipped off unless they used a lot of effort.  To summarize, when testing mice the recommended diameter of the rod should be larger than 6.3 cm (to avoid passive rotation) and smaller than 9 cm (to avoid poor performance). Pre-training is recommended to establish a stable performance on larger rods.
As one would expect, faster acceleration rates lead to decreased latencies to fall. Another factor that should be taken into account is that when testing the effects of drug treatments on the rotarod, fast acceleration rates suppresses possible effects of the drug on the performance that are at lower rates significantly detectable. For example, different doses of ethanol cause different performance scores at a rate of 20 rpm/min, while at an acceleration rate of 60 rpm/min those differences are suppressed. This means that certain effects could be underestimated at a too high acceleration rate. Shiotsuki et al. (2010) argue that in order to prevent individual differences in maximal gait and cardiopulmonary endurance influencing the task, low rotation speed is recommended.
Multiple drug doses
When testing on a rotarod it is recommended to test multiple drug doses. When there is a lack of variety in doses, the effect of the acceleration rate on the post-drug-induced performance might be missed. For example, some lower doses of ethanol can enhance performance at low acceleration rates.
Some forms of mutation in mice, like cerebellar mutants with severe ataxia, will likely make the mice not able to perform on the rotarod. Also, there are likely certain mutant mice with severe ataxia (e.g. some cerebellar mutants) which will not be able to perform on the rotarod. Less demanding tasks to test ataxia like gait analyses are advisable.
Graber et al. (2013) found that age is negatively correlated with rotarod performance. Using age groups; adults (6-7 months), old (24-26 months) and elderly (> 28 months), they found that there was a 40% reduction in rotarod performance with increasing age. They did, however, found a small dip in performance around the ages 16 – 20 months, which might be due to a survivor effect. Meaning that the strongest and healthiest mice live until the oldest age which explains that in some cases the older individuals exceed their younger counterparts in performance.
Sex-specific differences in rotarod performance were observed in a study testing on 5-month old mice. Males fell off the rotarod at significantly lower speeds than females. This sex-specific difference was negligible for 1 month-old mice.
Stress and handling
A study found that chronic stress impairs the rotarod performance regardless of muscle relaxation or motor dysfunction. They state that this performance impairment may imply a behaviorally depressive state mediated by a serotonergic mechanism. In order to prevent rodents from experiencing chronic or acute stress, habituation to handling is often part of the pre-training sessions.
In order to avoid any confounds using the rotarod performance test, a few factors have to be taken into account. Pre-training should be conducted to avoid increased learning during testing and individual learning ability differences. When repeated testing is done on manipulated mice it is important to keep in mind that there are differences in performance between different strains. An important factor to avoid is passive rotating. Therefore, an optimal rod diameter is needed and this varies for different types of rodent: for mice, it is advised not to use too small a diameter due to the chance of passive rotating, while not using a too large diameter (e.g. used in rat experiments) due to poor performance scores. Generally, low rotation speed is recommended to avoid factors like individual maximal gait and cardiopulmonary endurance differences influencing the task. In addition, depending on which rotation speed is used gender can influence performance.
If the aim is to detect the effect of drugs on the rotarod, one should take into account that at too fast acceleration rates, possible effects might be suppressed. In addition, testing multiple drug doses prevents missing a possible interaction effect of dose and rotation speed. Using too old mice or mice that are too stressed, either by handling or other factors is not advisable, as this might decrease performance. Lastly, when testing mice with severe ataxia, it is advisable to use a milder test due to the chance that they might not be able to perform on the rotarod.
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