Fig. 1.
(a) During a chronic treatment period with daily injections of l-DOPA at low doses, the rodents will gradually develop abnormal involuntary movements, reaching a plateau after 10–14 days of treatment. (b) Both the duration and the peak severity of the AIMs will gradually change after chronic l-DOPA administration. The amount of time the rodents show AIMs will decrease, while the peak severity increases as the treatment period progresses. (c) Subcutaneous (s.c. in the scruff) and intraperitoneal (i.p.) injections of l-DOPA result in a similar onset, time profile and severity grade of AIMs. Note that there may be a slight shift in the temporal profile of AIMs depending on where the injection is made.
When testing potential anti-dyskinetic drugs, animals should be pre-treated with l-DOPA (primed) until they reach a plateau in their AIMs development (usually around 10–14 days). Animals are allocated into matched experimental groups based on their AIMs scores and one group given l-DOPA together with the anti-dyskinetic drug for one to several days. The other group will receive l-DOPA plus the drug vehicle for the same period of time. Thereafter, the group allocation is switched and the animals are tested again. In general, 3 days of washout between different drugs and doses is sufficient, but this is of course dependent on the pharmacokinetics of the drug. The crossover design allows for within-animal comparisons and counter-acts the fact that both the severity and expression pattern of AIMs may differ between animals.
When testing anti-dyskinetic properties of a drug, it is crucial to include tests of sensorimotor performance in the experimental design (see Schallert, this volume for well-validated tasks for motor performance in rats with a 6-OHDA lesion) since the anti-dyskinetic property of the drug must not come at the expense of l-DOPA efficacy. It is also of great importance that an observer blinded to the treatment of the animals makes the AIMs ratings. In addition, it is essential to keep all testing parameters consistent in order to get reliable results and to fully comprehend the efficacy of the anti-dyskinetic drug.
3 Methods
3.1 Methodology
Using either translucent rotometers or transparent Perspex cages (see Note 4), the animal enclosures are positioned in such a way that the animals can be observed from all directions, since they will move around during the testing session. Rodents are placed individually in the cages and allowed to habituate for at least 15 min. During the habituation period, the l-DOPA and benserazide HCl are dissolved in saline (see Note 3) and the syringes and needles are prepared. The timer is started upon injection of the first animal, and thereafter one animal is injected per minute (for a group of up to 20 animals) until all animals have received their l-DOPA (or saline) injection (see Note 5 for route of administration). AIMs ratings are then started 20 min after the first animal was injected and each animal is rated for 1 min in turn (see Note 6). This continues for 180 min following drug administration (or until all dyskinetic movements are replaced by a normal motor pattern). For each animal and observation period, a score (and an amplitude if the complimentary amplitude rating scale is used, see below) is given for each AIM subtype, based on the duration and persistence of the abnormal movement during the 1-min observation period.
The description of the scales and classification of the different subtypes has been taken from the publication made by Cenci and Lundblad in Current Protocols in Neuroscience (17), in order to keep the conformity of the rating scale with the addition of hindlimb AIMs.
3.2 Basic Rating Scale
In the following description, five different classes of abnormal involuntary movement (axial, limb, orolingual, hindlimb and locomotor) are each rated, within each 1-min time bin, according to a five point scale:
0 = no AIMs.
1 = occasional AIMs, which are present during less than half of the observation time.
2 = frequent signs of AIMs, which are present more than half of the observation time.
3 = AIMs are present during the entire observations time, but can be interrupted by external stimuli (e.g. sudden, load opening of the lid of the cage).
4 = continuous AIMs that are not suppressible by external stimuli.
If the scores are summed for each rat across all AIM classes and time points, the theoretical maximum score in one test (nine monitoring periods over 180 min) is 180 if locomotion is included, 144 if it is considered separately, see specific comment below.
3.2.1 Axial AIMs
At the beginning of the treatment period when the AIMs are mild, the axial component can be described as a lateral flexion of the trunk or neck towards the side contralateral to the lesion. When the severity increases, the flexion will be of a more dystonic and choreiform-twisted character (dystonic refers to a slow forcing movement of a body part into an abnormal position).
3.2.2 Limb AIMs
In mild cases, limb AIMs are expressed as hyperkinetic, purposeless, jerky stepping movements of the forelimb contralateral to the lesion. Another common feature is small circular movements around the snout. As the severity increases, AIMs may be of a more hyperkinetic and/or dystonic character with prolonged circular movements involving the full arm and shoulder.
3.2.3 Orolingual AIMs
The facial, tongue and masticatory muscles are involved in this type of AIMs and are recognized as empty jaw movement, twisting of facial muscles and occasionally tongue protrusion on the side contralateral to the lesion. When this subtype reaches substantial severity, it may involve self-mutilating biting of the contralateral forepaw, easily detectable as a round bald patch on the forelimb. In extraordinary circumstances with chronic testing, this can lead to sores, since these animals are typically severely dyskinetic, refraining from injecting on non-test days will allow the skin to heal between sessions without adversely affecting behaviour.
3.2.4 Hindlimb AIMs
The contralateral hindlimb may also be taken into consideration. Scoring of the hindlimb was not included in the original Cenci scale, but some researchers have begun to use this as an additional measure (18). In some animals following l-DOPA administration, the hindlimb is abnormally postured, twisted but still supporting weight or elevated and non-weight bearing.
3.2.5 Locomotive AIMs
The locomotive AIM subtype is described as increased locomotion with contralateral side bias, i.e. the animal will turn towards the side contralateral to the lesion. Locomotive activity requires tactile contact of at least three paws on the floor, which rules out scoring this subtype of AIMs when rodents are sitting or standing on their hindlimbs, thus keeping their forelimbs off the floor.
It has been repeatedly shown that the amount of contralateral rotation induced by l-DOPA is directly related to the locomotive AIM score and is a less representative AIM than the other three subtypes. Locomotive AIMs are included in the ratings in conformity with the first description of the rodent AIMs scale, but it generally accepted that this is excluded when analysing the combined AIMs score, and considered as a separate measure. It is useful to maintain this measure so that a score can be obtained of locomotor activity if rotometers are not used to assess locomotion independently.
3.3 Ratings of AIMs Using the Amplitude Scale
As the original rating scale only takes into account the amount of time that the rodents are displaying abnormal movements and not that the quality of these movements may change with chronic l-DOPA administration, Winkler and co-authors implemented a complementary amplitude scale (14) to further expand the dynamic range of the ratings. This scale is very sensitive to variations in the amplitude of the dyskinetic movements between different phases of the l-DOPA treatment and/or between individual animals.
Amplitude scores are given to axial, limb and orolingual AIMs, and the score should reflect the maximum amplitude of the AIM observed during the 1-min observation period.
3.3.1 Amplitude of Limb AIMs
Score L1: Tiny movements of the paw around a fixed position, either as lateral or circular movements around the snout or as a repetitive tapping of the forepaw on the floor (as if the rodent was about to start walking, but could not).
Score L2: Movements resulting in visible displacement of the whole limb, e.g. the paw looses contact with the snout, and reaches halfway to the floor.
Score L3: Large displacement of the whole limb with visible contraction of shoulder muscles.
Score L4: Vigorous limb displacement of maximal proximal amplitude, with conspicuous contraction of both shoulder groups and extensor muscles.
Extensor muscles: those on the backside of the paw.
Vigorous: the movement is very energetic.
Maximal proximal amplitude: if the movement is circular, the limb is displaced around approximately half of the circumference around the body, if the movement is sagittal and/or frontal, the limb is lifted up to reach an angle of >90° with respect to the body.
3.3.2 Amplitude of Axial AIMs
Score A1: Sustained deviation of the head and neck at ∼30° angle.
Score A2: Sustained deviation of the head and neck at angle ≤60°.
Score A3: Sustained of the head, neck and upper trunk at an angle >60° but ≤90°.
Score A4: Sustained twisting of the head, neck and upper trunk at an angle >90° (maximal amplitude), causing the animal to loose balance (from a bipedal position).
Angle is calculated as a deviation from the longitudinal axis of the body.
In A1 and A2, the rodent can still maintain a quadrupedal position, whereas A3 and A4 refer to torsion of the upper body forcing the rat to assume a bipedal position.
3.3.3 Amplitude of Orolingual AIMs
Score O1: Twitching of facial muscles accompanied by small masticatory movements without jaw opening.
Score O2: Twitching of facial muscles accompanied by noticeable masticatory movements, occasionally leading to jaw opening.
Score O3: Movement with broad involvement of facial and masticatory muscles. Jaw opening is frequent, but tongue protrusion is occasional.
Score O4: All of the above muscle categories are involved to a maximal possible degree. Tongue protrusion is more frequent.
3.3.4 Amplitude of Hindlimb AIMs
Score H1: Twisting of the hindlimb but maintained under the body in proximity to the ipsilateral hindlimb.
Score H2: Extension of the hindlimb out from under the body, generally only partially weight bearing and may be twisted.
Score H3: (a) Hindlimb is elevated and non-weight bearing, but not fully dystonic (i.e. only partially extended) or (b) maximally dystonic but remaining in contact with the floor.
Score H4: The hindlimb is maximally dystonic and elevated, i.e. non-weight bearing.
In H3 and H4, the animal may be supporting itself against the wall of the observation cage or on the floor.
By using the amplitude scale, the dynamics of the AIMs ratings is expanded. For example, the maximum possible sum of axial, orolingual, limb and hindlimb AIMs increases from 16 to 64 and the theoretical maximum score in one test from 144 to 576 (see Note 7).
4 Analysis of the Data
Bearing in mind that the locomotive AIM subtype is more related to contralateral turning than the expression of AIMs, it is strongly advised to analyse this subtype separately from the other three subtypes.
The AIMs scores recorded from one testing session can be expressed in three different ways.
1.
As the sum of axial, orolingual and limb AIMs on all observation periods.
2.
As the integral of AIM scores over time (area under the curve, AUC, referred to as the “integrated AIMs score”).
3.
As the sum of the products (basic score × amplitude score) on each observation period (referred to as “global AIMs score”).
The most commonly used way of expressing data is as the sum of axial, orolingual and limb AIMs. More recently, global AIMs have become more popular in particular when comparing different lesion types and treatment groups as it expands the dynamic range of the AIMs ratings. In many studies, AIMs are observed during a chronic l-DOPA treatment period and in those studies, the development of AIMs is easily presented as a line graph over time (see Fig. 1a). Other interesting parameters may be the duration of the dyskinetic response (see Fig. 1b), and the peak severity of AIMs (see Fig. 1b).
The development of AIMs during the treatment period is often analysed using repeated-measures analysis of variance (ANOVA) even though non-parametric statistics are often more appropriate to use when handling rating scales which are considered ordinal data. However, the basic AIM scores reflect the time a specific movement is present unlike many other scales where a score often represent if a behaviour is present or not. It has also been shown that the AIM scores are strongly linearly correlated to parametric quantities such as the number of FosB-positive cells and levels of prodynorphin mRNA in the striatum (5, 6). Consequently, a basic score of 2 can be regarded as a quantity representing half as much as a basic score of 4. So using parametric statistics is not necessarily incorrect and it does have a descriptive power superior to non-parametric tests. We therefore suggest using parametric statistics (as long as basic statistical rules are followed, i.e. normal distribution of data) and in particular using repeated-measures ANOVA to analyse the development of AIMs over time. Repeated-measures ANOVA provides valuable information on the interaction between a time factor (testing session) and a group category (treatment), which is not readily available in non-parametric tests. However, it is recommended to corroborate key results with non-parametric statistics.
5 Experimental Considerations
Despite having full lesions, in every cohort of rats a proportion will not develop AIMs in response to therapeutic doses of l-DOPA. Typically, around 20% of the rats will remain “non-dyskinetic”, showing behavioural activation (contralateral rotations) and functional improvement in response to l-DOPA, but showing no or few AIMs with low amplitude. Non-dyskinetic animals have been defined as having a score of 0–1 on each AIM subtype on all time points for the duration of the l-DOPA challenge (19, 20). It has also been shown that animals with AIM severity grade ≤1 do not exhibit molecular markers of LID, such as the activation of extracellular signal-regulated kinases 1/2 and up-regulation of ΔFosB in striatal neurons (20, 21), and can thus be considered comparable to the animals showing absolutely no AIMs in response to l-DOPA.
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