Our Advice About The Dyskinesia Test

We are often approached by colleagues asking for practical advice about how to set up the rat dyskinesia test in their laboratories. A matter of concern pertains to the confidence with which the rat dyskinetic movements can be distinguished from naturally occurring behaviors. Overt dyskinesia that includes severe dystonic postures of the trunk and ample flexion-extension movements of the fore-limb are unequivocally abnormal, and do not require any particular familiarity with the rat behavioral repertoire to be detected and rated. Some mild, "borderline" forms of dys-kinetic movements, however, may require some experience to be detected and scored in a consistent way. Incipient dyskinesia of the forelimb may go unnoticed, as it may either appear to be an oscillatory movement of low amplitude (resembling tremor) or may manifest as a repeated stepping movement of the limb against the floor (A1 in box 1). However, ignoring this type of dyskinesia would not necessarily affect the experiment's outcome in a negative way, because we have noticed (Cenci and Lundblad unpublished data) that rats exhibiting these mild dyskinetic movements have not yet developed those molecular adaptations that constitute the hallmark of L-dopa-induced dyskinesia in both rodents and nonhuman primates, such as striatal upreg-ulation of FosB and opioid precursor mRNA (for review see Cenci et al. 2002). A more difficult case occurs with stereo-typic "gnawing-like" movements, which are by themselves not abnormal but may represent a rat equivalent of the bipha-sic dyskinesias caused by low-intermediate levels of DA in patients (see above). We are presently characterizing L-dopa-induced gnawing stereotypies with the specific aim of establishing their relationship to plasma and brain L-dopa levels. Thus far, when we encountered this type of behavior during the AIM ratings, we scored only the limb and orolingual components that showed abnormal, myoclonic, or jerky features and/or lack of symmetry.

Among the four AIM subtypes that we have described in the rat, orolingual dyskinesia is probably the most difficult subtype to rate, as it can be either overestimated or overlooked. Orolingual movements that lack specificity are movements that resemble swallowing or chewing without jaw opening in a rat that does not exhibit any other sign of dyskinesia. In particular, rats normally express these sorts of movements when they are either falling asleep or waking up. In these situations, the orolingual movements should never be scored as AIMs. Stress-induced orolingual

BOX 1 Dyskinesia Amplitude Scale

In most cases, rats display dyskinetic movements of varying amplitudes. We chose to give an amplitude score that reflects the most severe movements.

I. Amplitude of Forelimb Dyskinesia (Scale A1 to A4)

A1. Small Movements of the Paw Around a Fixed Position

There are two variants of A1: (1) while being apposed to the snout, the paw exhibits lateral translocations and circular movements. This type of behavior should be differentiated from tremor, which affects the paw contralateral to the lesion when it is at rest (i.e., in a non weight-bearing position; see video films published as an electronic link to Cenci, Whishaw, and Schallert 2002). Tremor is however not exacerbated but improved by L-dopa treatment. Moreover, a close apposition of the paw to the snout is not seen in the rat limb tremor. (2) The paw is engaged in fast and repetitive stepping-like movements with a fluttering character, that is, the paw is repetitively lifted up and down the cage floor, as if the rat were about to start going without being able to do it. Strictly speaking, this latter variant does not consist of "small movements of the paw," since the distal limb is obviously moved as well. However, we have found that this type movement represents an incipient form of forelimb dyskinesia in the same way as does the movement described above at (1).

A2. Low Amplitude Movements with Visible Translocation of the Distal Limb

Low-amplitude translocation of the limb either sideways or up-and-down (or in combination, resembling a circular movement). Practically, this means that the paw loses contact with the snout and reaches almost halfway to the floor.

A3. Notable Translocation of the Whole Limb (Both Distal and Proximal Limb)

Flexion-extension of the forelimb on the sagittal plane, or abduction-adduction on the frontal plane. More often, the movement is circular, and therefore implies both a sagittal and a frontal component. Like A4, grade A3 implies visible contraction of shoulder muscles, but the amplitude of the movement is not maximal.

A4. Vigorous Limb Movements of Maximal Amplitude and Speed with Conspicuous, Visible Contraction of Both Proximal Limb Muscles (at the Shoulder) and Extensor Muscles (Those on the Back Side of the Paw)

This practically means: (1) if the movement is circular, the limb is translocated around almost half of the circumference around the body (The movement is circular around the shoulder, but may be accompanied by flexion of the distal limb, and paw and digit movements). (2) If the movement is performed in the sagittal plane the limb is lifted up to form an angle >90° with respect to the body.

II. Amplitude of Axial Dystonia (Scale X1 to X4)

The amplitude of the axial subtype of dyskinesia is determined by the degree of deviation of the head and neck (X1, X2) or torsion of the upper trunk (X3, X4) with respect to the longitudinal axis of the rat's body.

X1. Sustained Deviation of the Head and Neck, at ~30° Angle

Make sure that the deviation is actively maintained (consistent and sustained), and exclude that the rat is simply looking at objects that are located on the side contralateral to the lesion.

X2. Sustained Deviation of the Head and Neck, Angle <60°

X3. Sustained Torsion of the Upper Trunk, at >60° Angle to <90° (Rat in Bipedal Position)

X4. Sustained Torsion of the Upper Trunk at >90° Angle, Causing the Rat to Lose Balance (from a Bipedal Position)

movements can also occur. We have noticed that even normal rats, when they are repeatedly exposed to stressful situations, can exhibit bursts of orolingual movements of considerable magnitude. To verify that a rat's orolingual movements have a dyskinetic character, we pay particular attention to the speed, magnitude, and perseverance of the movement and to its lack of symmetry. Facial grimaces should engage jaw muscles contralateral to the lesion more than the ipsilateral muscles. Tongue protrusion should be consistently directed toward the side contralateral to the lesion. One type of movement that is clearly abnormal is repetitive biting on the fur or the skin along the forelimb contralateral to the lesion. We have never seen this sort of movement in 6-OHDA rats injected with either saline or antiparkinsonian agents that do not cause dyskinesia (e.g., bromocriptine or A2a antagonists), nor have we seen it in normal rats injected with high doses of L-dopa.

In general, when the investigator is uncertain about the dyskinetic nature of a certain movement, it may help to remember that stress aggravates all forms of dyskinesia (Marsden et al. 1981; Luquin et al. 1992; Nutt 1992; Durif et al. 1999). To induce stress in a controlled way during the testing sessions we use the tail pinch stimulus, which is a well-established stressor in rodents (Antelman et al. 1975;

Rowland and Antelman 1976). We manually pinch the mid part of the rat's tail for ten seconds in a non-painful manner (i.e., the pinch does not cause the rat to squeak). This maneuver, however, should not be used indiscriminately and inconsistently. We would approve the use of tail pinch to verify or exclude the presence of dyskinesia in the following cases: (1) when the researcher is uncertain about whether or not gnawing- or grooming-like movements are part of a dyski-netic syndrome; (2) in rats that have previously shown dyskinesias but suddenly appear inactive on a certain monitoring period (however, one need not keep applying this maneuver if AIM scores have returned to 0 on two consecutive observation points at >100 minutes post L-dopa-injection); (3) in non-dyskinetic rats at the peak of the effect of L-dopa (i.e., at sixty and eighty minutes post injection). Tail pinch should not be applied to increase the AIM score in a rat that is already dyskinetic.

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