Tests Of Physiological Motor Behavior

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Investigators have applied many tests with success for evaluating physiological motor performance in 6-OHDA-lesioned rats. Qualitative assessments of motor behavior in 6-OHDA-lesioned rats have revealed motor patterns that are reminiscent of Parkinsonian motor features in humans (Miklyaeva et al. 1995; Whishaw et al. 2002), including tremor (for review see Cenci et al. 2002). Although qualitative observations are extremely important for interpreting the significance of one's models, such observations offer limited possibilities in the screening of new treatments, where quantitative measures of efficacy are needed. Some of the tests that provide quantitative measures of adaptive, sensorimotor behavior are listed in Table 1. Different tests evaluate different behavioral features and may differ greatly on several crucial aspects, for example, dynamic range, need for animal pretraining or aversive motivation, and feasibility of repeated testing during long-term experiments. The dynamic range is the range of numerical values that the test's outcome variable can have between the two end points represented by total motor impairment and a normal performance. A sufficiently large dynamic range is required to compare degrees of motor improvement (or motor disability) produced by different interventions. Most of the tests listed in Table 1 detect an altered motor performance even after pathological conditions that are different from PD, such as ischemic brain damage. Because of this potential lack of specificity, the researcher must verify the relevance of testing paradigms to PD by showing some relationship between the magnitude of the behavioral deficit in rats and the extent of striatal DA depletion and an improvement in the test's outcome measure(s) after L-dopa treatment.

In the following chapter we shall present the tests that are used in our laboratory for evaluating the therapeutic efficacy of antiparkinsonian drug treatment in the rat. We first provide methodological information and then discuss the range of applicability and the pros and cons of each test in the light of our experience. Although devised and optimized through research in the 6-OHDA lesion model, the tests described below are also suitable for application to other models of unilateral Parkinsonism in the rat (Kirik et al. 2002b).

A. The Stepping Test

Originally described by Schallert and collaborators (Schallert et al. 1992), the stepping test assesses a rat's ability to adjust its steps in response to experimenter-imposed lateral movements. The test does not require any special equipment other than a table surface (approx. 90-100 cm in length). This test has been renamed and modified in different studies, for example, the bracing test (Schallert and Tillerson 2000); akinesia test (Olsson et al. 1995; Lindner et al. 1996); forepaw adjusting steps (Chang et al. 1999). Here we describe the methodology that is used in our laboratory. The experimenter firmly holds a rat by lifting up its hindlimbs and one forelimb, but letting the unrestrained paw contact the table surface. Then, the experimenter moves the rat sideways (in the forehand direction) across the table at a predefined speed (0.9 m in five seconds), counting the number of adjusting steps that the animal performs to catch up with the translocation of the body. The animals are habituated to the handling associated with this test for a few days. The test is then applied twice daily on three consecutive days to reach a stable baseline performance before the experimenter evaluates the effect of different interventions.

The function specifically measured by this test is a rat's ability to use its forelimb to maintain center of gravity when rapid weight shifts are imposed. Parkinsonian-like motor features, such as akinesia and postural abnormalities, negatively affect this ability. Rats with unilateral 6-OHDA lesions show a dramatic deficit in this test, dragging the fore-limb contralateral to the lesion across the table and making either very few or no adjusting steps. A clear inverse relationship exists between the extent of DA-denervation on the side of the striatum ipsilateral to the lesion and the performance of the contralateral forelimb (Kirk et al. 2001). Investigators have obtained validation of this test as a method for antiparkinsonian drug screening by showing that

TABLE 1 Quantitative Tests Evaluating Sensorimotor Function in 6-OHDA Lesioned Rats

Test name

Task or function measured

Original description (or review)

Type of lesion

Improved by L-DOPA or DA agonists

Reaction time Reaction time Reactive capacity Sensorimotor orientation Disengage behaviour

Muscle tone (rigidity)

Tremor

Foot print analysis Rotarod test Videomonitored rotarod

The staircase test

The pasta matrix test

Forelimb akinesia test

Stepping (bracing) test

Postural adjustment

Cylinder test

Lever pressing after visual cue

Head orientation to eccentric visual cues

Release of a lever to escape footshock

Head onentation to body touch

Switching between eating behavior and orientation to somatosensory stim.

Hinding resistance to passive movement

EMG recordings Tremulous jaw movements Gait pattern and stride length General locomotor coordination

Rating scales of postures and movements during rotarod stepping

Independent limb use for reaching and grasping food pellets

Retrieving pieces of straight uncooked pasta arranged in a matrix Independent limb use of movement initiation

Adjusting steps induced by a passive, lateral translocation of the body Capacity to maintain balance when a rat is pushed laterally by the investigator

Independent forelimb use for weight shifting during vertical exploration

Amolric et al. (1995) Eur J Neurosci 7:972-980 Bilateral 6-OHDA

Carli et al. (1985) Nature 313:679-682 Unilateral 6-OHDA

Spirduse et al. (1985) Brain Res 335:45-54 Unilateral 6-OHDA

Marshall et al. (1980) Comp Phys Psy 94:833-846 Unilateral 6-OHDA

Schallert and Hall (1988) Behav Brain Res 30:15-24 Unilateral 6-OHDA

Wolfarth et al. (1996) Neuroscience 74:985-996 Bilateral 6-OHDA

Salamone et al. (1998) Prog Neurohiol 56:591-611 Bilateral 6-OHDA

Schallert et al. (1978) Science 199:1461-1463 Bilateral 6-OHDA

Rozas et al. (1997) Brain Res Protoc 2:75-84 Unilateral 6-OHDA

Whishaw et al. (2003) J Neurosci Meth 126:13-23 Unilateral 6-OHDA

Montoya et al. (1991) J Neurosci Meth 36:219-228 Unilateral 6-OHDA

Ballermann et al. (2001) J Neurosci Meth 106:39-45 Unilateral 6-OHDA

Schallert et al. (1992) 3:332-333 Unilateral 6-OHDA

Olsson et al. (1995) J Neurosci 15:3863-3875 Unilateral 6-OHDA

Rodter et al. (2000) Cell Transplant 9:197-214 Bilateral 6-OHDA

Rodter et al. (2000) Cell Transplant 9:197-214 Bilateral 6-OHDA

Yes (chronic treatment) Not known Not known

Not known Not known

Not known

Not known

Not known Not known

Schallert et al. (2000) Neuropharmacology 39:777-787 Unilateral 6-OHDA Yes the performance of the affected forelimb is significantly improved after treatment with L-dopa and DA-receptor agonists (Olsson et al. 1995; Lindner et al. 1996; Chang et al. 1999; Lundblad et al. 2002). The improvement induced by these treatments is maintained even when the rats develop dyskinesia (Figure 1). Indeed, the fact that the rat is firmly held by the investigator during the test counteracts the disabling effect of dyskinesia on the coordination of limb movements (Winkler et al. 2002). This issue constitutes a potential problem for drug-screening studies, because it implies that the test could not distinguish between truly beneficial treatments and treatments that relieve akinesia at the expense of producing abnormal movements and postures. Another potential caveat of the test lies in the need for direct, close, and continuous physical interaction between the animal and the investigator. This requirement implies that the experimenter's proficiency conditions the reproducibility of the test. In our experience, experimenters must handle the rat as gently and consistently as possible among all testing sessions. Any methodological inconsistency may cause a rat to perceive stress or fear, and thus attempt to escape from the hands of the experimenter. Rapid, self-initiated steps may cause the experimenter to overestimate the effects of the tested treatment (or to underestimate the effects of the lesion). A stable baseline performance must therefore be established in each rat prior to testing any intervention. The test lends itself to repeated applications during long-term experiments, although experimenters should check regularly for changes in baseline performance during the course of the experiment. For this purpose, a blind crossover design is recommended, whereby experimenters examine rats after the injection of either the tested drug or its vehicle on consecutive days (see, for example, Winkler et al. 2002).

B. The Staircase Test

Originally introduced by Montoya and collaborators (Montoya et al. 1991), the staircase test evaluates a rat's ability to use its forelimbs to reach for small objects and pick them up. The test is also called the "paw-reaching test." The test apparatus consists of a narrow Plexiglas box (285 x 90 x 60 mm) accommodating a double staircase, which is positioned along the sides of a central platform. This platform provides a support for the rat's body. The rat must stretch its forelimbs down each side of the platform to retrieve food pellets that are loaded on each stair. The central platform is sufficiently high to prevent the forelimbs from crossing over between the two sides. In our test apparatus, the staircases are baited with ten sugar pellets per step (Noyes Inc., England), on four steps on each side. The outcome measures provided by the test are the number of pellets taken and the number of pellets eaten, which can amount to a maximum count of forty per paw. If the rat drops

FIGURE 1 Stepping test and staircase test: effects of 6-OHDA lesions and l-dopa treatment. These data were collected from a large (n = 52) group of rats with unilateral 6-OHDA lesions ("6-OHDA"). The lesioned rats or a group of non-lesioned controls ("Normal") had been treated chronically with a dose of l-dopa that was just above threshold to produce a significant improvement in the stepping test (6 mg/kg methyl l-dopa, combined with 15 mg/kg benserazide; single daily ipsilateral [ip] injections). The left-hand panel in A shows that the 6-OHDA lesion reduced the number of steps performed with the contralateral paw to about 25% of normal values, and that l-dopa treatment enhanced the level of performance to about 40% of normal. In the right-hand panel, the same 6-OHDA lesioned rats are divided in two subgroups based on the presence or absence of dyskinesia ("with AIMs" and "no AIMs," respectively). This analysis shows that the motor improvement produced by l-dopa in this test is not affected by the presence of dyskinesia. In B, the same rats were examined for their performance in the staircase test. The left-hand panel in B shows that the number of pellets taken with the paw contralateral to the lesion was reduced to approximately 40% of normal values in the 6-OHDA lesioned rats, and that it was not improved at all by l-dopa treatment. The right-hand panel in B shows that rats exhibiting dyskinesia in response to l-dopa ("with AIMs") actually performed worse in this test after the the drug was administered, compared to vehicle. These tests were performed between forty-five and sixty minutes after the injection of l-dopa. * p < 0.05 vs. normal controls; p < 0.05 vs performance of the same rats after vehicle; (in B) + p < 0.05 vs. 6-OHDA lesioned rats without AIMs sustaining the same (vehicle or l-dopa) treatment (from Winkler et al. 2002). (Reproduced by permission of Elsevier Publishing Ltd.)

a pellet after taking it up, the pellet will fall down in a compartment of the box that is not accessible to the rat. This allows the investigator to estimate the success rate in the test, as defined by the ratio between "pellets eaten" and "pellets taken." DA denervation affects all components of the performance of the contralateral paw, that is, attempt, motor coordination, and success (Montoya et al. 1991; Barneoud et al. 1995; Barneoud et al. 2000). There is a close relationship between the number of pellets taken and eaten on the side contralateral to the lesion and the degree of DA depletion in the lesioned striatum (Barneoud et al. 1995; Kirik et al. 1998). Moreover, the test is highly sensitive to even mild DA-depleting lesions. Indeed, partial DA dener-vating lesions that are targeted on the dorsal striatum and do not result in either apomorphine-induced rotation or stepping deficits cause a significant impairment in the staircase test (Barneoud et al. 2000). These features make the test suitable for monitoring the effects of manipulations that either deplete or restore DA terminals in the striatum in a graded way. Improved performance in the staircase test depends on a precise anatomical restoration of the DA input to the critical striatal region, as obtained by such approaches as intra-striatal delivery of GDNF (Kirik et al. 2001). Interventions that attain only a partial anatomical restoration of the nigrostriatal DA pathway, such as transplants of fetal ventral mes-

encephalic neurons, have a limited ability to improve performance in this test (Abrous et al. 1993; Nikkhah et al. 2001). Likewise, pharmacological DA replacement with l-dopa does not significantly improve performance in this test, if results are expressed as the number of pellets taken or eaten (Winkler et al. 2002). L-dopa treatment may however increase the likelihood that the rat successfully retrieves pellets by the paw contralateral to the lesion, as reflected by the ratio between pellets eaten and pellets taken (Lundblad and Cenci unpublished). When treatment with L-dopa induces dyskinesia, the rat's performance in the staircase test is dramatically worsened compared to baseline levels (Figure 2). These results can easily be explained by the fact that dyskinesias disrupt the precision and coordination of limb movements (Hagell and Widner 1999).

The staircase test is usually referred to as a test of skilled forelimb use, although it also depends on the rat's ability to learn a new and complex motor pattern. Indeed, it may take ten consecutive days of testing for a normal rat to reach a stable and optimal performance in this test (Lundblad and Cenci unpublished). The staircase test provides several advantages: it has a good dynamic range and it assesses a physiological type of motor behavior that has a clear adaptive value for the animal. As stated above, the performance in this test is very sensitive to the effects of interventions

Dopa Test Muster

FIGURE 2 Effects of anti-Parkinsonian drug treatment on the rat's performance in the cylinder test. The cylinder test was executed both before ("baseline") and during a three-week course of treatment with either l-dopa (6 mg/kg/day combined with benserazide, 15mg/kg; n = 14), bromocriptine (3.5 mg/kg/day; n = 14), or vehicle (n = 8). Limb use asymmetry (i.e., the percentage of wall contacts performed with the Parkinsonian [left] paw) is shown in A, while the absolute number of wall contacts performed with this paw is shown in B. Animals affected by severe l-dopa-induced dyskinesia (grade 4 AIMs) performed zero wall contacts and were therefore excluded from the computation of a percentage value in A. The plot in C shows that an inverse relationship exists between the absolute number of wall contacts performed in the cylinder test and the rats'AIM scores. * p < 0.05 vs. vehicle (From Lundblad et al. 2002). (Reproduced by permission of Blackwell Publishing Ltd.)

FIGURE 2 Effects of anti-Parkinsonian drug treatment on the rat's performance in the cylinder test. The cylinder test was executed both before ("baseline") and during a three-week course of treatment with either l-dopa (6 mg/kg/day combined with benserazide, 15mg/kg; n = 14), bromocriptine (3.5 mg/kg/day; n = 14), or vehicle (n = 8). Limb use asymmetry (i.e., the percentage of wall contacts performed with the Parkinsonian [left] paw) is shown in A, while the absolute number of wall contacts performed with this paw is shown in B. Animals affected by severe l-dopa-induced dyskinesia (grade 4 AIMs) performed zero wall contacts and were therefore excluded from the computation of a percentage value in A. The plot in C shows that an inverse relationship exists between the absolute number of wall contacts performed in the cylinder test and the rats'AIM scores. * p < 0.05 vs. vehicle (From Lundblad et al. 2002). (Reproduced by permission of Blackwell Publishing Ltd.)

that cause depletion or restoration of striatal DA fiber terminals. A potential disadvantage of the test lies in its complexity. The performance in this test depends not only on the animals' fine motor skills but also on learning and motivational components. Indeed, rats that learn the test prior to the 6-OHDA lesion show a much milder degree of impairment post-lesion (Lundblad and Cenci unpublished).

A regimen of partial food deprivation lasting for a few days before and during the test is necessary in order for the rat to perform in the test. Researchers should consider the need for food deprivation of animals when planning complex experiments that require multiple testing with and without drug treatments. For example, we have found that a lower dose of L-dopa is required to produce appreciable dys-kinetic effects when the drug is administered to food-deprived rats. Indeed, L-dopa competes with dietary amino acids for transport across the blood-brain barrier (Pincus and Barry 1987).

C. The Cylinder Test

The cylinder test, which was originally described by Schallert and Tillerson (2000), assesses the independent use of each forelimb in the context of a naturally occurring behavior. The test takes advantage of rats' innate drive to explore a novel environment by standing on their hind limbs and using their forelimbs to lean on the enclosing walls. To perform this test, rats are put individually in a glass cylinder (21 cm diameter, 34 cm height) and video recorded for five minutes. The rats are not habituated to the cylinder prior to filming. Investigators then use the video recordings to count the number of supporting wall contacts the rat executes with the right and the left paw. Only supporting wall contacts are counted, that is, appositions of the paw to the walls of the cylinder with fully extended digits. We usually count up to a maximum of twenty wall contacts per session. To stimulate rats that show little or no tendency to explore, we apply the following maneuvers in this given order: (1) turn the room light on and off two to three times, and then leave it off with only a red light bulb as a source of illumination, (2) mildly shake the cylinder for two to three seconds (red light on), and (3) take the rat out of the cylinder for less than thirty seconds and then put it back.

Researchers use the results from the cylinder test to compute a limb-use asymmetry score, either by expressing the performance of the contralateral limb as a percentage of the total performance (Lundblad et al. 2002), or by subtracting the percentage of wall contacts executed by the impaired limb from the percentage of the non-impaired limb (Picconi et al. 2003). A rat with unilateral 6-OHDA lesions uses the paw contralateral to the lesion in about 10-30% of all supporting wall contacts, whereas a normal rat uses the right and the left paw indifferently in this test (Figure 2). The dynamic range of this test is therefore relatively narrow

(from approximately 20% to 50%). Another potential disadvantage of the test lies in the fact that it cannot be repeated too often, or else the rat will lose interest in exploring the novel environment and will not perform at all. In our hands, a testing frequency of one session per week over two months may still be feasible, but additional repetitions of the tests will compromise its sensitivity (Lundblad et al. 2002).

Despite these potential caveats, the cylinder test offers some notable advantages: it is very rapid in its execution, it does not require any particular experience on the part of the investigator, nor does it require pretraining of the animals. The test provides a true measure of spontaneous forelimb use as the movements exhibited by the rat in the testing cylinder are identical to those performed in the home cage. The performance in the cylinder test is improved by treatment with L-dopa and bromocriptine (Figure 2A) and it is disrupted by the appearance of dyskinesia (Figure 2B). The dependence on an intact nigrostriatal system, the improvement produced by antiparkinsonian medications, and the sensitivity of the outcome measure to the disrupting effect of L-dopa-induced dyskinesia fully validate the cylinder test as a method for the preclinical screening of antiparkinson-ian treatments in the rat.

D. The Rotarod Test

The rotarod test is widely used to generally assess motor performance in rats and mice (Zausinger et al. 2000; Luesse et al. 2001; Jeong et al. 2003; Karl et al. 2003). The test measures a rat's ability to maintain itself on a rod that turns at accelerating speeds. Rozas and Labandeira Garcia (Rozas et al. 1997; Rozas and Labandeira Garcia 1997) were the first to report that performance in this test is disrupted after unilateral 6-OHDA lesions and improved after treatment with DA receptor agonists, a finding that was confirmed in our laboratory (Lundblad et al. 2003; Picconi et al. 2003). The same authors introduced a simple formula to express rotarod performance as the integral of time spent on the rod at different rotational speeds (Rozas et al. 1997). Thanks to this formula, the rotarod test has gained an extremely wide dynamic range, and is very sensitive to detecting graded improvements or graded deterioration of motor function after different types of interventions.

The following description explains how the test is applied in our laboratory. Our rotarod apparatus is a Rotamex 4/8 from Columbus Instruments (Ohio, USA). We pretrain the rats during three sessions on three consecutive days, where each session includes two separate testing trials. In the testing trials the animals are placed on the testing rod at an initial speed of 4 rotations per minute (rpm). Then the rod speed increased gradually to 44 rpm over ninety seconds. We tap the animals on their tails several times in each session, because we have found that this action helps them stay more alert in the test. The time spent on the rod is

Prevalence Atheltes Eating

J vehicle

] Amantadine (20 mgkg) □ KW-6002 (3 mg.'kg) ^ L-DOPA (6 nig/kg) w/o dyskinesia L-DOPA (6 mg/kg) with dyskinesia

J vehicle

] Amantadine (20 mgkg) □ KW-6002 (3 mg.'kg) ^ L-DOPA (6 nig/kg) w/o dyskinesia L-DOPA (6 mg/kg) with dyskinesia

FIGURE 3 Effects of anti-Parkinsonian drug treatment and dyskinesia on the performance of unilaterally 6-OHDA lesioned rats in the rotarod test. The first three pairs of bars from the left show that the overall performance in the rotarod test (area under the curve [AUC]; see text) is significantly improved after acute administration of the following anti-Parkinsonian drugs: amantadine (20 mg/kg ip., light gray), the A2a receptor antagonist KW-6002 (3 mg/kg p.o., dark gray), and l-dopa (6 mg/kg combined with 12 mg/kg benserazide ip, black solid bars). Investigators assess the improvement each drug produces by comparing the performance of the same animals after administering the corresponding vehicle (empty bars). The last pair of bars (to the right) shows the l-dopa-treated animals after several weeks of chronic treatment with this drug, causing severe dyskine-sia to develop (black striped bars). The dyskinetic movements interfere with the rats'ability to perform in the rotarod test, and the AUC value is now dramatically reduced after the injection of l-dopa compare to vehicle. All experiments were carried out using a randomized cross-over design, whereby the same rats were tested after the injection of a drug or its corresponding vehicle over two consecutive days. An experimentally blinded investigator tested animals. *p < 0.05 vs. vehicle.

recorded automatically for each animal, and the average performance in the two consecutive trials is used for within-animal comparisons. Between the two testing trials, all animals are allowed to remain on the rod over twenty-five seconds at a lower range of rotating speeds (from 4 rpm to 14 rpm). We have found that this intermediate session has a positive effect on the animals' willingness to perform in the test (probably because the rats learn that they can remain on the rod despite its accelerating rotation). By the last of these three pretraining sessions, all animals reach a stable baseline performance and they can be used to evaluate the effects of antiparkinsonian treatments. For this purpose, we recommend a blind cross-over design whereby rats are examined after the injection of either the tested drug or its vehicle on two consecutive days of testing (see Lundblad et al. 2003).

Rotarod performance is affected by virtually any abnormality of the motor system. However, the test has fulfilled all the essential criteria for validation in the field of pre-clinical Parkinson research. Indeed, performance in this test depends on an intact nigrostriatal DA system, and is improved by treatment with L-dopa and other antiparkin-sonian agents (Lundblad et al. 2003). Moreover, the treatment-induced improvement is compromised when the animal develops severe dyskinesia (Lundblad et al. 2003; Picconi et al. 2003) (see Figure 3). According to our experience, the rotarod test is the most sensitive method for screening drugs that aim to improve Parkinsonian disability without causing dyskinesia (Figure 3). The rotarod test also helps assess anti-dyskinetic treatments in the rat. Indeed, the test can be used to determine whether candidate anti-dyskinetic drugs attain a functionally meaningful and specific reduction of dyskinesia as opposed to a general motor depressant effect. Another advantage of the rotarod test lies in the fact that its sensitivity is not lost upon repeated application in long-term experiments. The test requires relatively high accuracy and consistency on the part of the investigator, perhaps not as high as in the stepping test, but certainly higher than in the cylinder and staircase tests. Indeed, any stressful situation severely compromises the rat's "willingness" to perform in the rotarod test.

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