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Brain Res'earch, 383 ( I c~8(~) 4112 4()7 l?ilsc~icl

BRE 21812

Saccadic eye movement deficits in the MPTP monkey model of Parkinson's disease BARBARA A. BROOKS 1, ALBERT F. FUCHS2 and DOM FINOCCHIO2 1Department of Physiology, University of Texas Health Science Center at San Antonio, TX," and 2Department of Physiology and Biophysics, and Regional Primate Research Center, University of Washington, Seattle, WA (U.S.A. ) (Accepted 17 June 1986) Key words: Saccade - - Parkinsonism - - N-methyl-4-phenyl- 1,2,3,6-tetrahydropyridine (MPTP) - - Monkey model

Saccadic eye tracking was studied in a monkey given i.v. injections of N-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP). The Parkinson-like symptoms which appeared in the animal's general motor behavior (akinesia, bradykinesia, hypokinesia) were also observed in its eye tracking. Similar oculomotor deficits are seen in patients with idiopathic Parkinsonism. The MPTP model offers excellent possibilities for studying the mechanisms underlying the motor disabilities of Parkinson's disease. Exposure to N-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (NMPTP, or MPTP) in human drug addicts causes a syndrome strongly resembling idiopathic Parkinson's disease 4'14. Both humans and monkeys affected by MPTP exhibit rigidity, akinesia and bradykinesia, with variable tremor 2'6~15. Pathological changes produced by MPTP in m o n k e y brains seem greatest in the dopaminergic cells of substantia nigra pars compacta; there appears to be little or no pathology in other brain areas, including potentially susceptible dopaminergic systems such as that originating in the ventral tegmental area 1'13. The selective action of MPTP and the similarity to human symptomatology have resulted in its increasingly popular use in monkey models of Parkinson's disease (P.D.) 2'15. When asked to perform rapid, self-paced saccadic eye movements between two stationary visual targets or to follow unpredictable step changes in a visual target, P.D. patients reveal oculomotor deficits which parallel some of their general skeletal motor symptoms3'5"]7-2°: latency to initiation of movement is often significantly longer than controls (akinesia); saccades frequently fall short of the target (undershoot or hypokinesia) and several small 'staircase' saccades occur to place the eye on targetlS'2°; the duration of saccades may be unusually long and their

peak velocities decreased (bradykinesia) 5'18'2°. In addition, the eye may have difficulty maintaining steady fixation of gaze during intersaccadic intervals (the P.D. patient may also have other oculomotor problems3'L8"2°). We were able to document similar phenomena in monkey eye tracking, following injection of MPTP. Data were obtained from a single adolescent (3 kg) rhesus monkey (M. mulatta) which furnished its own normal control measures prior to MPTP injection. The monkey was trained to track horizontal step changes in the position of a small red target as it moved across a dimly illuminated tangent screen. Accurate eye movements were directly rewarded by applesauce mixed with protein powder; the daily diet was consumed during training and testing sessions. The monkey was trained (1) to track periodic, horizontal step movements of the target spot of 5 °, 10°, 20 °, 30 °, and 40 ° and (2) to track the spot when the timing, amplitude and horizontal direction of its movement were varied according to a pseudo-random program. Eye movements were recorded by an electromagnetic technique (scleral search coil) developed by Fuchs and Robinson 7. Briefly, under general anesthesia a fine, teflon-coated, stranded stainless steel

Correspondence: B.A. Brooks-Eidelberg, Department of Physiology, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78284, U.S.A. 0006-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)

403 wire is passed under the 4 rectus muscles to form a coil which rotates with the eye. The coil leads are led under the skin to a Winchester plug, which is permanently anchored to the skull with dental cement. When the animal's head is fixed within horizontal and vertical alternating magnetic fields, a voltage is induced in the coil which is a function of the angle between the coil and the magnetic field. The frequency response, DC to 1 kHz (-3 dB), is adequate to follow the fastest saccade; the sensitivity is 15 min of arc. The voltage output is linearly related to the angular deviation of the eyeball within +20 ° of visual angle around the primary direction of gaze (eyes straight ahead). All testing took place within an electrically shielded room. MPTP was purchased as the HC1 salt from ChemServices (East Hanover, N J). Its purity and stability (dry) were assayed by HPLC, reversed phase, in ODS columns with a 1:4 0.2 M ammonium acetatemethanol solvent. The drug was freshly dissolved in Ringer's solution and injected into the monkey's leg vein at 0.4 mg/kg twice daily on two consecutive days, with the intent to produce clear Parkinsonian symptoms within 5-6 days after the first injection 1'2. Eye tracking movements were recorded on the day before drug administration, on the two days of injection and on one day after the last injection, after which the monkey refused to track until treated with a mixture of 100 mg L-DOPA and 10 mg of a DOPA decarboxylase inhibitor, carbidopa (Sinemet 100/10; Merck, Sharp and Dohme). Parkinson symptoms in gross behavior were semi-quantified with a modified version of the Hoehn and Yahr human P.D. rating scale 6. The signs of akinesia, bradykinesia and hypokinesia were quite marked by the 4th day after the first injection. Acute effects immediately following each injection and lasting less than 20 min included skin flushing, pupillary dilation, piloerection, vocalization, tremor and a dazed appearance, and were similar to those noted by other authors 1"2. Fig. 1 gives an overview of eye tracking data before treatment with MPTP (Pre-drug, top panel), on the two days during which the drug was administered (Days 1 and 2), on the last day the animal was willing to work (Day 3), and on day 6 when he was given Sinemet. All tracking was recorded monocularly with the implanted eye. As seen in the top panel, a target step to the right (upward deflection) before MPTP

PREDRUG

DAY 1

DAY 2

SINEMET

Fig. 1, Saccadic tracking of the target spot during horizontal displacements of 10°, 20*, 30° or 40° in pseudo-random order of timing and direction. Upper trace in each panel shows vertical eye movement channel. Middle trace depicts target movements; rightward movements are up and leftward are down. The lowest trace showing horizontal eye movement record is displaced downward to facilitate viewing. Each panel samples approximately 36 s of tracking. Pre-drug: horizontal saccadic tracking on the day before the first dose of MPTP. Leftward movements in this animal frequently produced an undershoot followed by a normal correction saccade. Day 1: records obtained approximately 3 h following the first injection with MPTP. Day 2: two h after the third injection with MPTP and 27 h after the first injection. Day 3: approximately 20 h after the 4th and final MPTP injection. On this day the animal ceased working before the session was completed and did not resume until treated with Sinemet. Sinemet: example of eye tracking on Day 6 following tube ingestion of one tablet of 100/10 Sinemet.

regularly elicited a single saccade whose amplitude, duration, and average velocity were within the normal range s. In response to a leftward target step (downward deflection) the eye frequently required a

4(14 corrective saccade, but both saccades had normal amplitude-duration relations. The Day 1 samples of tracking, taken just after the animal had received the second MPTP injection, are not appreciably different from pre-drug conditions. The data of Day 2 however, taken immediately after the final MPTP injection, reveal some indications of saccadic abnormality (increased hypometria, irregular trajectories). The first, most noticeable deficit was a fixational instability which appeared during intersaccadic intervals in both the horizontal and vertical eye movement records. On Day 2 the monkey also showed mild akinesia and bradykinesia and was listless and inactive in his home cage, although he ate normally and worked during the entire test session. By Day 3 (20 h after the final MPTP injection) instability during eye fixation was even more obvious, many saccadic movements were bradykinetic and hypometric, and sometimes the animal failed altogether to respond to a target movement. Akinesia and bradykinesia were also marked in the animal's other movements, and his appetite was failing. By the 5th day following the first injection of MPTP the animal displayed severe parkinsonian signs, required tube feeding, and was largely immobile in the home cage. Spontaneous eye movements occurred infrequently, with prolonged periods of staring into space. He did not protest when his limbs were extended (rigidity was apparent, although we saw little cogwheeling), and tremor was observed sporadically, especially on the few occasions of self-initiated movement. On the 6th day, one tablet of Sinemet was crushed, mixed with water, and administered by stomach tube, following which the animal was placed in his primate testing chair. The change in his overall behavior following Sinemet was dramatic; the monkey became hyperactive within half an hour, twisting and turning in his chair, pulling and pushing at its structure. Spontaneous saccadic eye movements were far more frequent than before treatment with MPTP; they often interfered with tracking behavior in the post-Sinemet testing. When the animal finally settled to eye tracking under good stimulus control, saccade metrics were qualitatively normal as seen in the lowest panel of Fig. 1. (They were also quantitatively normal as shown in the Sinemet data of Fig. 3.) PostSinemet records of Fig. 1 indicate that fixational stability also returned to the pre-MPTP state, for both

horizontal and vertical eye movement. The animal worked for the applesauce reinforcement for about 1.5-2 h. The effects of Sinemet were completely gone approximately 7 h following its administration and the animal once again was immobile in his home cage. Fig. 2 shows details of saccade waveforms made before and after the injections of MPTP, on an expanded time scale. Both rightward (top examples) and leftward (bottom) target movements elicited normal saccades before MPTP was administered. By 20 h after the last of the 4 MPTP injections, approximately 70% of all saccadic movements were hypometric (post-MPTP examples). Although the latency of the first of a hypometric (staircase or multi-step) series might be normal, the subsequent saccades, which could number 2-4, were separated by intervals as short as 20-30 ms or longer than 1 s. Such a multistep series almost always placed the eye ultimately on target. Durations of saccades achieving target, and of components of a multi-step series could be very long (see right post-MPTP saccades). Although most post-treatment saccades were altered in form and timing, the monkey was capable of an occasional normal movement. Similar mixtures of normal and abnormal saccades are observed in parkinsonian patients 18,

PRE-MPTP RIGHT SACCADES

POST-MPTP

2 0 0 ms I LEFT, PRE-MPTP

Fig. 2. Monkey horizontal saccadic eye movements (E) superimposed on single target movements (T) prior to MPTP treatment (pre-MPTP) and one day after the last of 4 MPTP injections (post-MPTP). Rightward target and eye movements are up while leftward movements are down. Post-treatment records show multiple step saccades (hypometria), increased saccadic duration (bradykinesia), and lengthened intervals (akinesia) between hypometric steps.

405 Fig. 3, upper panel, compares the duration of rightward saccades measured before and after MPTP treatment, and following oral ingestion of Sinemet. Duration was measured as the time from the onset of movement to the next zero velocity8. Each preMPTP mean and S.D. is based on 20 or more saccades that achieved at least 90% or more of the target amplitude (saccades with oblique components were not included in the analysis; neither were leftward

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Fig. 3. Upper panel: effects of MPTP on the duration of rightward saccadic eye movements of various amplitudes. Each triangular data point represents the duration of an individual saccade after treatment with MPTP; these data include single components of multiple step saccades and therefore are highly variable in amplitude. For comparison, the mean duration of normal pre-MPTP saccades at the various target-step amplitudes (filled circles) are connected with straight lines (minimum of 20 single saccades/data point; vertical bars indicate one S.D.). Means and S.D.s following the ingestion of Sinemet are based on at least 12 saccades/data point (open circles). Lower panel: the effects of MPTP on the latency of saccades of various amplitudes. Triangles show the latency of individual, initial saccades to target displacement (they do not include intervals between multiple hypometric steps). Means and S.D.s of normal pretreatment saccades are connected by straight lines; means and S.D.s for data collected after Sinemet are shown as open circles.

movements which frequently required a correction and had a long latency, probably because of a mild chronic irritation in the left outer canthus). The preMPTP data are within the normal range of monkey saccadic durations as described by Fuchs and Robinson 8. The durations of all the post-MPTP saccades that were measured on Day 3 of testing (20 h following the last injection) are plotted as triangles. The paucity of post-MPTP data is due to the fact that the monkey stopped tracking before the end of the session, and the large variety of durations and amplitudes reflect the inclusion of all saccades, including those in multi-step hypometric series. Approximately 65-70% of the post-MPTP durations significantly exceeded pre-MPTP durations; the large scatter in the data is also characteristic of the scatter of saccadic durations measured in Parkinson patients 5'18. The mean durations after treatment with Sinemet are based on 12 or more saccades; there were not enough target directed eye movements at the larger amplitudes to justify plotting mean values. The post-Sinemet data are well within the normal range for this monkey. Fig. 3, lower panel, compares the latency of rightward saccades before and after MPTP injection, and after ingestion of Sinemet. The results are from the same saccades whose durations were shown in the upper panel. Pre-MPTP latency means and S.D.s are within normal values for monkeys at the various movement amplitudes 8. Individual latencies of postMPTP saccades are seen as triangles. Only the latency of the first saccade in a hypometric series was counted; therefore, fewer data points are shown than for duration. There was a less dramatic effect of MPTP on initial latencies than on duration, the latencies of about 30% of movements exceeding this monkey's pre-MPTP range. Latencies following Sinemet were not significantly different from pre-MPTP performance. Despite meticulous nursing care and regular tube feeding, the animal expired from pneumonia 3 weeks after taking Sinemet, without being able to perform again in the eye tracking task. Histological reconstruction of the midbrain stained with Cresyl violet showed fewer than normal cells in the substantia nigra pars compacta. These results indicate that monkeys rendered parkinsonian by MPTP have saccadic abnormalities very

406 similar to those o b s e r v e d in patients with Parkinson's disease. The abnormalities include h y p o m e t r i a (the b r e a k d o w n of a single saccadic response into a staircase of smaller saccades), increase in saccade durations, and occasional long latency responses. It is likely that Parkinson-like deficits would also have been found in other classes of eye m o v e m e n t s , including smooth pursuit and the vestibulo-ocular reflex, had there been a d e q u a t e testing time 19'2°. Perhaps the most sensitive early indicator of the M P T P syndrome in our pilot animal was the a p p e a r a n c e of fixational instability during the intersaccadic intervals of tracking. A similar p h e n o m e n o n has been d o c u m e n t e d in parkinsonian patients as 'gaze impersistence '3 and gaze instability with frequent 'square wave jerks '19'2°. In the M P T P - t r e a t e d m o n k e y all of

dopaminergic insufficience in the nigro-striatal projection 13. D a m a g e to the dopaminergic leg of the nigro-striatal-nigral qoop' has been associated with serial changes in G A B A e r g i c transmission to targets ' d o w n s t r e a m ' from the striatum, including the substantia nigra pars reticulata and its own G A B A e r g i c input to the superior colliculus 1°16. D o p a m i n e deficiency may, therefore, be the first in a cascade of causally linked, neurotransmitter imbalances whose final m o t o r expression is due, at least partly, to chemical malfunction at sites rather far from the striatum. Thus, the M P T P m o n k e y model offers rich opportunities for the neurologic and pharmacologic investigation of the mechanisms underlying oculom o t o r and other m o t o r deficits in Parkinson's disease.

the symptoms were dramatically but transiently reversed by administration of Sinemet. It has been suggested 18'2° that some of the saccadic signs of parkinsonism may be due to defective supranuclear triggering of the saccadic event, possibly from the superior colliculus which is known to have a m a j o r role in the initiation of saccades and which is a m a j o r projection target of the substantia nigra pars reticulata 1~'12. It is d o c u m e n t e d that M P T P causes

W e acknowledge with thanks the excellent nursing and veterinary care provided by William M o r t o n , D V M , Glenn Knitter and Nancy W i n d s o r of the Primate Center Colony staff. S u p p o r t e d in part by N I H Biomedical Research Support Grant no. S07RR07187 to the U T H S C - S A , no. RR00166 ( N I H ) to the Regional Primate Research Center at Seattle, Washington, and EY00745 to A . F . F . (NIH).

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407 (Suppl. 1) 268. 16 Melis, R.M. and Gale, K., Effects of dopamine agonists on gamma-aminobutyric acid (GABA) turnover in the superior eolliculus: evidence that nigrotectal GABA projections are under the influence of dopaminergic transmission, J. Pharmacol., 226 (1983) 425-431. 17 Shibasaki, H., Tsuji, S. and Kuroiwa, Y., Oculomotor abnormalities in Parkinson's disease, Arch. Neurol., 36 (1979) 360-364. 18 Teravainen, H. and Calne, D.B., Studies of Parkinsonian

movement. I. Programming and execution of fast eye movements, Acta Neurol. Scand., 62 (1980) 137-148. 19 White, O.B., Saint-Cyr, J.A. and Sharpe, J.A., Ocular motor deficits in Parkinson's disease. I. The vestibulo-ocular reflex and its regulation, Brain, 106 (1983) 555-570. 20 White, O.B., Saint-Cyr, J.A., Tomlinson, R.D. and Sharpe, J.A., Ocular motor deficits in Parkinsbn's disease. II. Control of the saccadic and smooth pursuit systems, Brain, 106 (1983) 571-587.