European Journal of Pharmacology, 134 (1987) 83-88
Elsevier EJP 00645
Opposite effects of a D1 and a D2 agonist on oral movements in rats Per Johansson, Edward Levin, Lars Gunne and Gaylord Ellison * Psychology Department, UCLA, 405 Hilgard Ave, Los Angeles, CA 90024, U.S.A.
Received 16 June 1986, revised MS received 4 September 1986, accepted 11 November 1986
Oral movements in rats administered one of three doses of either a D1 agonist (SK&F 38393) or a D2 agonist (LY171555) were observed via closed'circuit television and simultaneously recorded using a computerized video analysis system which measured the distance between two fluorescent dots painted above and below the rat's mouth. The D1 agonist SK&F 38393 induced a dose-dependent increase in tremorous oral movements, tongue protrusions, and, at the highest dose, increased repetitive chewing movements. Conversely, the D2 agonist LY171555 produced an inhibition or oral activity at all dose levels. At the lowest dose tested this appeared to reflect a non-specific decrease in activity, for there was an inhibition of all categories of behavior measured, as well as of all amplitudes of computer-scored movements and slow, sluggish movements were recorded. But higher doses of LY171555 induced hyperactivity and stereotyped, repetitive head movements whereas chewing movements, tremorous oral movements, and tongue protrusions were still decreased. D1 and D2 dopamine receptors appear to have opposite effects on oral movements. D1 agonist; D2 agonist; Oral movements; Tardive dyskinesia
One of the most severe side-effects of antipsychotic drugs is tardive dyskinesia, which gradually develops in approximately 25% of schizophrenics administered chronic neuroleptics. The primary symptom of tardive dyskinesia involves abnormal oral movements involving the 'bucco-linguo-masticatory triad'. Because of notable characteristic of neuroleptics is their ability to block dopamine receptors (Snyder, 1981), it has been hypothesized that tardive dyskinesia is due at least in part to alterations in dopaminergic systems. Although the simple notion that it merely reflects supersensitivity of dopamine receptors is untenable (Fibiger and Lloyd, 1984), tardive dyskinesia often responds pharmacologically as though it represented in-
* To whom all correspondence should be addressed.
creased activity at dopamine receptors (i.e. it can be suppressed by dopamine antagonists and exacerbated by dopamine agonists). This suggests that it is important to determine how dopamine agonists influence the rate of oral movements. The discovery of two different classes of dopamine receptors (Kebabian and Calne, 1979) led to studies indicating that D2 receptors are important for m a n y of the classical actions attributed to dopamine agonists, such as the induction of motor stereotypies, and that D2 antagonist correlates well with antipsychotic activities of neuroleptics (Joyce, 1983). However, the role of D1 receptors has remained less clear. Relatively selective dopamine agonists such as the D1 agonist S K & F 38393 and the D2 agonist LY 171555 have been developed, but few studies using these compounds have focused upon oral activity. Several animal models of tardive dyskinesia have been proposed, including reports that rats administered chronic neuroleptics show exag-
0014-2999/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
84 gerated oral activity (Gunne and Haggstrom, 1983; Waddington et al., 1983; Sant and Ellison, 1984). We have been developing procedures (Ellison et al., in press) by which oral movements in rats administered chronic neuroleptics can be more precisely quantified using close-up television images of rats while they are restrained in tubes. We now report on the effects of two selective dopamine agonists using a much more previse quantification of oral movements which employs direct computerized measurements of mouth movements.
2. Materials and methods
2.1. Subjects The subjects were 21 female Sprague-Dawley rats (initial weight 200-230 g). All rats, initially drug-naive, were first habituated to being placed in plastic tubes (5.7 cm diameter, 15.5 cm length) which rested inside a soundproof chamber. At one end of the tube was a 3.3 cm hole through which the rats head could protrude. Each animal was given at least five 6 rain habituation sessions over a period of 3 weeks. Then the animals were split into seven subgroups matched for equal mean body weight. Each subgroup was tested first with a different drug (i.e. either saline or S K & F 38393 at a dose of 0.3, 1.0 or 3.0 mg/kg, or LY 171555 at a dose of 0.03, 0.1 or 0.3 mg/kg). All drugs were dissolved in saline and injected i.p. 20 min before testing. Each animal was then given a 3 or 4 day recovery period, and then tested with the next drug in the ordered sequence described above until all rats had received all drug doses. This was done so that order effects could be assessed by comparing, for each given drug dose level, the effects in animals receiving the drug for the first time with effects induced in animals receiving the drug after experience with the other dosages.
2.2. Procedures 2.2.1. Human observer During each 6 rain testing session the animal
was placed in the tube which rested inside a testing chamber. This chamber was illuminated only by a 6 W black-light bulb placed in front of and under the rat's muzzle. On the right side of the animal, a closed circuit TV camera with a close-up lens provided a picture of the animal's muzzle; this was displayed on a large television screen. The observer watched this image and recorded the presence of four behaviors upon each occurrence by pressing keys on a computer-linked keyboard. The four behaviors recorded, with their behavioral descriptions, were: 'Tremor', this consisted of rapid oscillations of the masseter muscles. 'Chewing', this was repetitive openings and closing of the mouth. Directed oral movements, such as licking or biting at surfaces, were excluded and in fact did not occur during the experiment because of the physical location of the tube. 'Head movement', movements of the entire head, often accompanied by sniffing movements of the whiskers. 'Tongue protrusion', the tongue could be observed to protrude from the oral cavity.
2.2.2. Computerized recordings On the upper and lower jaws of the rat small spots were painted using a UV sensitive dye (' Black-Ray Swimming-pool readmission ink' from UVP, Inc., P.O. Box 1501, San Gabriel, CA 91778). A second closed circuit TV camera with a close-up lens and a UV filter was positioned 22 cm in front of the rat. The output from this camera was fed to a computer with a movement detection circuit (the ' M M ' board from Biotronic Designs, Tarzana, CA). This circuit (cf. Ellison et al., in press) calculated the distance (number of TV rasters) between the upper and the lower spot, and stored these data, together with the human observer's reports, in computer memory 60 times each second. The computer records of the direct measurements of the amount of mouth openings were analyzed by first detecting individual 'movelets' which were defined as individual openings or closures of the mouth as reflected by progressive increases or decreases in distance between the two spots. A movelet was defined as at least two rasters of change in the size of mouth opening (i.e. 0.6 ram), and each movelet terminated when the direction of movement reversed. Each raster of
change represented 0.3 mm of movement. The distribution of individual movelet amplitudes (change in distance between the two spots) and the spacing between movelets was calculated. The movelets were divided into five different amplitude categories according to how many rasters were covered by the movelet (2, 3, 4-5, 6-9, > 10). In addition, the average slope of the movelets (amplitude/duration) in each amplitude category was calculated. 2.3. Statistics
All data were analyzed using repeated measures analyses of variance. For hand-scored data, four analyses were conducted for each of the four behaviors discussed using drug dose as a repeated measure. Specific contrasts were made between the drug doses and saline. For machine-scored data, repeated measures of drug dose and amplitude of movelet were used; again, specific contrasts between drug doses and saline were made.
3. Results 3.1. H u m a n obsercer
Figure 1 presents the results from the analysis of the observer's reports. Analyses of variance of drug effects for all four behaviors recorded were highly significant (in all cases P < 0.001). The D1 agonist S K & F 38393 induced dose-dependent increases in all three oral movements observed, with the highest dose producing significant increases (P < 0.01; F(1,120) in the total duration of tremorous movements of the masseter muscles, in the observed frequency of tongue protrusions, and in the frequency of repetitive chewing movements. These repetitive oral movements were non-directed, consisting of repetitive vacuous openings and closings of the mouth. Head movements were not significantly elevated by S K & F 38393 at any dose (F(1,120), P > 0.05). On the other hand, the lowest dose of the D2 agonist LY171555 induced significantly decreased (P < 0.005) head movements but this same drug produced increased head movements at the highest
~J 80 Z 60 " ~ ~o w ~ 2o 0 l
800 400 0
~ +o w c~ LL
LOW MED DOSE
LOW NED DOSE
Fig. 1. Average duration of tremor throughout the 6 min session (in 60ths of a second) and average frequency of occurrence of each of three behaviors (vacuous chewing movements, head movements and tongue protrusions). (Ill) Saline; (O) SK&F 38393; (@) LY 171555. The low, medium and high doses, respectively, are 0.3, 1.0 and 3.0 mg/kg for SK&F 38393 and 0.03, 0.1 and 0.3 mg/kg for LY 171555.
dose (P < 0.001). Different mechanisms appear to be involved at these two doses, for at the lowest dose level of LY171555 there was a significant decrease in all observed behaviors, whereas at the highest dose these animals appeared extremely hyperactive, engaging in stereotyped, repetitive headmovements typically accompanied by sniffing of the air. However, in spite of this extreme hyperactivity, all three forms of oral movements were significantly decreased at all doses of LY171555 (in each case P < 0.05), F(1,120). At all three dose levels this took the form of a complete suppression of mouth tremors and tongue protrusions accompanied by a significant decrease in chewing movements. Because all animals in the experiment were tested with all drugs in a balanced design, a further analysis was conducted in order to determine whether order effects were present in any of these effects. While all of the above effects were also significant when only the first two testing sessions were analyzed, tremor and tongue protrusions were maximal in animals tested for the first time with higher doses of S K & F 38393, whereas head movements also began to appear when animals were injected with S K & F 38393 following previous experience with LY171555. The converse was not
86 true, however, for prior injections of S K & F 38393 did not alter the response to LY171555.
3.2. Computerized recordings Figure 2 shows that the two drugs induced distinctively different patterns in alterations in the amplitude of the distance recorded between the two spots on the mouth. Because small amplitude movelets are so much more prevalent than larger ones, in this figure the number of movelets of each amplitude are plotted as a percent of control (saline) frequency. S K & F 38393 tended to increase the frequency of small amplitude movelets whereas LY171555, at the higher doses, principally increased large amplitude movelets. This figure also shows the sedative properties of low doses of LY 171555, where all amplitudes of movelets were decreased. Because these computer files of distance between the two spots on the mouth were collected simultaneously with the observer files, the two recordings could be compared by correlating the 160
5K+ F 38393
140 O rr" I-Z O C.)
;e U3 i--
W ILl > O ~r
LL O 140
z LJJ 0 UJ CE LL
Fig. 2. Frequency of computer-scored 'movelets' of various amplitudes expressed as a percentage of control (saline) levels. One raster = 0.3 mm, so a movelet of amplitude 2 represents- a mouth opening of 0.45-0.75 mm. (O) Low dose; ( O ) middle dose; (zx) high dose of the two drugs as described in the text.
two outputs. The reports of head movements by the observer were generally correlated with large changes in movelet amplitude (10 rasters or greater, corresponding to increases of at least 3.0 mm between the two spots). The differences between groups in movelets 10 or greater were highly significant (F(6,120) = 13.2, P < 0.0001), and the general pattern was similar to that reported for head movements by the observer. Compared to controls (for whom the mean number of movelets > 10 was l l . 8 / m i n ) , movelets of 10 or greater were decreased at the low dose of LYl715555 (mean = 3.7/min; F(1,20) = 13.3, P < 0.002) but increased at the high dose LY171555 (mean = 26.4/min; F(1,20) = 24.4, P < 0.001). Changes in large movelets for doses of S K & F 38393 were much smaller and statistically insignificant. On the other hand, oral movement reports by the observer were best correlated with changes in smaller movelet amplitudes. The differences between groups in movelets of two and three rasters were highly significant (F(6,120)= 22.5 and 16.6, respectively, both P < 0.0001), and in each case these small amplitude movelets were elevated in a dose-dependent manner by S K & F 38393 injections (both P < 0.01) and decreased by the LY171555 injections. There were two distinctive changes in the computerized movelet data for the animals administered the lowest dose of LY171555. In only these animals was there a decrease in frequency of movelets of all six amplitudes measured (P < 0.01 in all cases). Movelets were also analyzed for slope, or rate of change of distance between the two spots during openings or closings (i.e. amplitude of each movelet divided by time from beginning to end of movelet). It was found that there were generally opposite effects on slope induced by the two drugs. With the medium and high doses of S K & F 38393 the slopes of movelets in the categories of 2, 3 and 4-5 were all significantly increased (i.e. more brisk mouth movements; all P < 0.05), while with the lowest dose of S K & F 38393 only the movelets in amplitude category 3 were significantly increased (F(1,20)= 5.49, P < 0.05). Conversely, average movelet slope was significantly lower for all five categories of movelet amplitude after administration of the lowest dose
87 of LY171555 (F(1,20) = 28.36, P < 0.0001) and for the all but the largest amplitudes at the medium dose (F(1,20) = 6.81, P < 0.025). For the highest dose of LY 171555 the slopes were raised for the smallest-sized movelets (F(1,20)= 13.41, P < 0.005) but not significantly for the movelets of other sizes.
There were very different effects on oral movements produced by the two drugs investigated in this study. The human observer files indicated that the D1 agonist S K & F 38393 induced increases in all types of oral behaviors observed, and this was correlated with the computerized recordings, which indicated that this drug produced dose-dependent increases in the smallest amplitudes of mouth movements measured. This was a selective effect on oral activity, for observer-reported head movements were not significantly increased by S K & F 38393. Conversely, the D2 agonist LY171555 actively suppressed oral activity, but at the highest dose it induced a dramatic increase in both head movements and large amplitude movelets. The effect of LY171555 in suppressing oral movements appeared to reflect two different processes. At the lowest dose administered, this suppressive effect on oral activity of LY171555 can be interpreted as reflecting a non-selective, decreased arousal phenomenon, perhaps related to its actions at autoreceptors. Thus, at this low dose of LY171555 all activity appeared to be suppressed; this includes all behavioral categories observed as well as several general indices or arousal as measured by the computerized scoring system, such as the frequency and the slopes of all amplitudes of movelets. But at higher doses, LY171555 appeared to selectively facilitate sniffing and head movements while simultaneously suppressing oral activity. At the highest dose given, the large number of head movements in the animals administered LY171555 might be argued to have led to an inability of the human observer to observe small mouth movements. However, this suppression was also present at all lower dose levels, and, most importantly, it
was very precisely detected by the computerized measurements, which would not contain this observer artifact. Other investigators, employing completely different measures, have also found opposite effects of D1 and D2 receptor agonists (Sonsalla et al., 1984). The present results with oral activities are also consistent with other reports from the literature. It has been reported that when non-restrained rats are tested in open cages, whereas at extremely high doses the D1 agonist SK& F 38393 increases general activity (Molloy and Waddington, 1985), at doses comparable to those employed in the present study it selectively induces grooming activity using the mouth (Molloy and Waddington, 1984). In our testing apparatus, the rats were partially restrained in a tube with their heads free, so that they were unable to mouth or lick any physical surfaces. The appearance of various chewing movements in our animals administered S K & F 38393 suggests that rather than inducing grooming per se, the primary effect of this D1 agonist is on oral activity, with grooming representing an outlet for this oral activity in the free-behaving animal. Other investigators (Rosengarten et al., 1983) have also reported increased oral movements induced by S K & F 38393 and that they can be blocked by the dopamine antagonist cis-flupenthixol. It has also been reported that non-restrained rats administered the D2 agonist RU24213 engage in downward sniffing and locomotion (Pugh et al., 1985). The animals in our experiment, although administered a different D2 agonist, also tended to lower their heads, but with the close-up observation facilities and computerized recordings it was further observed that oral activities were simultaneously inhibited in a dose-related manner even though, at higher doses, head movements indicated that the animals were highly active. Furthermore, these higher-dose effects were clearly dissociated from the general reduction in activity seen with the lowest dose, which may have been due to effects on autoreceptors. A cautionary note is necessary in interpreting this growing literature, however, it is well-known that repeated administration of the dopamine agonist amphetamine induces an 'inverse toler-
88 a n c e ' p h e n o m e n o n , such that s u b s e q u e n t injections of the s a m e dose of the drug can i n d u c e h e i g h t e n e d b e h a v i o r a l responsivity, especially for certain c o m p o n e n t s of stereotypies. E a c h of the a n i m a l s in our e x p e r i m e n t was initially drug-naive, b u t was then tested with drugs six times. W h i l e m o s t of these tests involved very low doses of a selective d o p a m i n e agonist, nevertheless, previous experience with the highest dose of LY171555 was f o u n d to c h a n g e the response profile to S K & F 38393. It is i m p o r t a n t for a u t h o r s r e p o r t i n g exp e r i m e n t s o n the effects of d o p a m i n e agonists on b e h a v i o r to include i n f o r m a t i o n on the extent to which their a n i m a l s were d r u g naive at the b e g i n ning as well as d u r i n g the course of the experiment, for r e p e a t e d d r u g testing can p r o b a b l y lead to highly different results. T h e p r e s e n t results indicate that oral activity, at least for the two drugs tested here, is m o r e related to D1 d o p a m i n e r e c e p t o r activation than to D2 r e c e p t o r activity, O t h e r evidence that D1 agonistic m e c h a n i s m s m a y p l a y a special role in p r o d u c i n g these oral m o v e m e n t s is that aging, which d r a m a t i c a l l y increases the incidence of tardive dyskinesia, has b e e n r e p o r t e d to increase the ratio o f D1 to D2 sites ( O ' B o y l e a n d W a d d i n g t o n , 1984). These conclusions are the o p p o s i t e f r o m the n o t i o n that tardive d y s k i n e s i a m a y b e related to the D 2 r e c e p t o r a u g m e n t a t i o n which develops after p r o l o n g e d b l o c k a d e of r e c e p t o r s b y high doses of n e u r o l e p t i c s (Snyder, 1984), b u t they suggest several alternative possibilities. A b l o c k a d e of D 2 receptors m i g h t l e a d to increased oral m o v e m e n t s b e c a u s e the resulting i n c r e a s e d synthesis of d o p a m i n e w o u l d n o w p r o d u c e an o v e r s t i m u l a t i o n at D1 receptors; in this case the s y n d r o m e of h e i g h t e n e d oral activity should be worse with neuroleptics which act relatively selectively at the D2 site a n d should a p p e a r relatively r a p i d l y after i n i t i a t i o n of d r u g treatment. Alternatively, increased oral m o v e m e n t s m a y be due to the develo p m e n t of supersensitive D1 receptors p r o d u c e d b y p a r t i a l b l o c k a d e of D1 receptors; in this case the s y n d r o m e w o u l d develop m o r e g r a d u a l l y a n d be m o r e a p p a r e n t with neuroleptics which act at b o t h d o p a m i n e r e c e p t o r sites. These alternatives are clearly testable using a n i m a l m o d e l s with chronic a d m i n i s t r a t i o n of selective D1 a n d D 2 antagonists a n d m a y lead to the d e v e l o p m e n t o f safer neuroleptics.
Acknowledgements Research supported by MH39961 and The Swedish Medical Research Council. E.D.L was supported by NIMH training Grant No. MH15795. P.J. was supported by Swedish Academy of Pharmaceutical Science.
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