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Behavioral and Electromyographic Characterization of the Local Frequency of Tacrine-induced Tremulous Jaw Movements
Physiology & Behavior, Vol. 64, No. 2, pp. 153–158, 1998 © 1998 Elsevier Science Inc. All rights reserved. Printed in the U.S.A. 0031-9384/98 $19.00 1 .00
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Behavioral and Electromyographic Characterization of the Local Frequency of Tacrine-induced Tremulous Jaw Movements M. S. COUSINS, A. ATHERTON AND J. D. SALAMONE1 Department of Psychology, University of Connecticut, Storrs, CT 06269-1020, USA Received 20 March 1997; Accepted 7 January 1998 COUSINS, M. S., A. ATHERTON AND J. D. SALAMONE. Behavioral and electromyographic characterization of the local frequency of tacrine-induced tremulous jaw movements. PHYSIOL BEHAV 64(2) 153–158, 1998.—Rats were implanted with fine-wire electromyograph (EMG) electrodes and were videotaped to identify the local frequency characteristics and muscle activity associated with tacrine-induced tremulous jaw movements. All rats received intraperitoneal injections of 2.5 mg/kg tacrine. The videotape sessions were played back in slow motion (i.e., one-sixth normal speed), and an observer entered each jaw movement into a computer program that recalculated the interresponse time and the local frequency (in hertz) for each movement within a burst. Analyses of the distribution of frequencies showed that the peak frequency of jaw movements was in the 3- to 5-Hz frequency range, with an average frequency of 4.0 Hz. EMG electrodes were implanted into three jaw muscles: temporalis, anterior belly of digastricus, and masseter. Tremulous jaw movements were not accompanied by consistent changes in masseter activity. The anterior belly of digastricus showed bursts of EMG activity during some jaw movements, although the temporal relation between jaw movements and EMG activity was somewhat inconsistent. The muscle that showed activity most closely related to tremulous jaw movements was the temporalis. During bursts of jaw movements, temporalis muscles across several different rats showed bursts of EMG activity. Sections of videotape corresponding to bursts of EMG activity were reanalyzed by freeze-frame examination of the tape; typically, the temporalis showed a burst for each jaw movement, with the burst of activity occurring during the jaw-closing phase and the transition between jaw closing and opening. These results indicate that the local frequency of tremulous jaw movements is within the 3- to 7-Hz frequency that is typically associated with parkinsonian tremor. Moreover, the EMG data suggest that temporalis is a major contributor to the muscle activity that underlies tremulous jaw movements. © 1998 Elsevier Science Inc. Tremor
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showed that most of the interresponse times were within the range of 142–333 ms, with a peak in the range of 200 –250 ms (42). Similar temporal characteristics are shown with the jaw movements induced by ventrolateral striatal dopamine depletion, the muscarinic agonist pilocarpine, and 5.0 mg/kg of the anticholinesterase tacrine (16,33). Based upon analyses of interresponse times, it was calculated that the local frequency of jaw movement activity induced by dopamine depletion and cholinergic stimulation was in the range 3.0 – 6.6 Hz. These data are consistent with the report of See and Chapman (45), who used different methods but nevertheless observed that physostigmine and pilocarpine increased jaw movement activity in the 4- to 6-Hz frequency range. Thus, it has been suggested that the vacuous, or nondirected, jaw movements induced by dopamine depletion or cholinomimetics are appropriately referred to as “tremulous” (16,31,42). Although the clinical significance of drug-induced perioral behaviors remains uncertain (14,41,47), it has been suggested that tremulous jaw movements share characteristics with parkinsonian tremor (23,43,47). For example, the acetylcholine– dopamine in-
IN RATS, perioral movements are induced by a wide variety of neurochemical and pharmacological conditions (4,5,14,23,39, 40,54). One of the most commonly studied of these movements is known as vacuous jaw movements (also known as tremulous jaw movements or vacuous chewing); these movements are defined as rapid, vertical deflections of the lower jaw that resemble chewing but are not directed at any particular stimulus (4,5,8,10,42). The term “vacuous” refers to the nondirected nature of the these movements. Recently, it has been suggested that these movements also are “tremulous;” i.e., they are characterized by a periodic oscillation of the jaw (15). It has been observed for several years that drug-induced jaw movements occur mostly in rapid “bursts” of activity (44). In recent studies, slow-motion videotape analysis has been used to characterize the temporal features of vacuous, or tremulous, chewing-like movements induced under a number of different conditions. Coadministration of reserpine with a low, presynaptic dose of apomorphine resulted in jaw movement activity that was mostly in rapid bursts, with interresponse times less than 1 s (42). Analysis of the distribution of interresponse times 1
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teraction that is shown with parkinsonian symptoms is similar to that shown for vacuous jaw movements (3,12,13,16,18,23,30,32, 34,47–50). The local frequency of movement is another characteristic that is worth considering. For example, tardive dyskinesia in humans shows activity largely within the 1- to 2-Hz frequency range (2,56). In contrast, parkinsonian tremor is generally reported to be in the 3- to 7-Hz frequency range (1,21). Although parkinsonian tremor usually involves the hand, it can involve an up-anddown movement of the lower jaw (1); one recent study of parkinsonian jaw tremor showed peak activity in the 3- to 5-Hz frequency range (21). Thus, tremulous jaw movements in rats appear to have frequency characteristics that are similar to parkinsonian tremor. In this study, tacrine-induced tremulous jaw movements in rats were simultaneously analyzed by slow-motion video and electromyography (EMG). Tacrine (Cognex) is an anticholinesterase that is used to treat Alzhemier’s disease; this drug has been shown to produce parkinsonian side effects, including tremor (36,37). Tacrine was administered to rats at a dose of 2.5 mg/kg, because this dose produces a robust jaw movement response (7,12,31) and also because the jaw movements induced by 2.5 mg/kg tacrine are reversed by several antiparkinsonian drugs, including L-DOPA, apomorphine, bromocriptine, amantadine, and benztropine (12). Computer-aided slow-motion video observations and EMG recordings were performed to characterize the local frequency of tacrine-induced jaw movements. Previous research using slowmotion video analysis of tremulous jaw movements partitioned each jaw movement into interresponse times [i.e., the time between each jaw movement in milliseconds (31)]. In this study, the precise frequency of tacrine-induced tremulous jaw movements, in hertz, was directly determined. Although videotape analyses can yield important information about the frequency characteristics of jaw movements, such observational methods do not identify which muscles are active during cholinomimetic-induced jaw movements, nor do they determine the temporal pattern of muscle activity. Thus, EMG methods were also employed. EMG methods have been used previously to study drug-induced oral movements (20,26,28,29,33,38), and previous EMG studies have focused upon digastricus and masseter muscle activity during cholinomimeticinduced oral movements (11,25). Digastricus EMG activity was reported to increase during pilocarpine-induced jaw movements (25). Reports of masseter activity have been somewhat equivocal, with one paper reporting small increases in activity during chewing-like movements (25) and another failing to find consistent masseter activity during pilocarpine-induced chewing movements (11). The time scale used in some previous studies [e.g., (25)] did not permit one to determine the local frequency of jaw movements within bursts based on EMG activity. In addition, the temporalis muscle, which accounts for approximately 24% of the mass of all masticatory muscles in the rat (19,20,55), has yet to be characterized with EMG during cholinomimetic-induced tremulous jaw movements. Therefore, videotape and EMG methods in conscious, mobile rats were used in the present study to determine the local frequency of tacrine-induced tremulous jaw movements and to study the involvement of digastricus, masseter, and temporalis muscles in these jaw movements.
Animal care procedures were approved by the University’s Institutional Animal Care and Use Committee. EMG Electrode Implantation and Recording Rats were injected with 1.0 mg/kg methylscopolamine and anesthetized with 50.0 mg/kg sodium pentobarbital. Lidocaine was injected into all incisions for local anesthesia. Bipolar wire-hook electrodes (50 mM diameter wire with 1.0 mm of insulation exposed at the tip; electrode tips separated by 2.0 mm) were implanted under direct vision with a 27-gauge needle approximately 1.0 mm deep into the left digastricus (anterior belly), temporalis, or masseter muscles (temporalis and digastricus, n 5 4; temporalis and masseter, n 5 2). The wires led subcutaneously to the skull and were attached to a six-pole connector (Amphenol). The rostral skull screw served as ground. The assembly was fastened to the skull with two additional screws and cranioplastic cement. The six-pole connector was attached to an electrical commutator that led to a preamplifier and amplifier (Knight differential amplifier; 300-Hz low-pass and 500-Hz high-pass filter). The signal was converted to analog (Coulbourn Instruments) and relayed to an IBM-type microcomputer operating a two-channel data recorder (Coulbourn AT L19-69). At the end of the experiment, each muscle was contracted with a constant-current generator (Grass CCU 1A; 0.1– 0.3 mA, 100 pulses with 1.0-ms duration at 100 Hz) to confirm the correct placement of electrodes. In addition, after the experiment, rats were sacrificed with sodium pentobarbital and the muscles were dissected to confirm placement of the recording electrodes. Computerized Slow-Motion Videotape Analysis A video camera (Panasonic AG-180) was used to simultaneously record the facial region of each rat while the EMG signal appeared on the computer monitor. To quantify the temporal characteristics of tremulous jaw movements, videos were played back (Panasonic AG-1730) at one-sixth normal speed, using a procedure similar to that reported previously (16,31,42). Tremulous jaw movements were defined as a small deflection of the lower jaw that resembled chewing but was not directed at any apparent stimulus. These did not include yawns or gapes. Gapes were defined as a maximal opening of the jaw. During slowmotion video playback each occurrence of a tremulous jaw movement or a gape was recorded by an observer on an IBM-type microcomputer running BASIC. The computer program converted the timing of the jaw movements back into normal speed and recorded the total number of jaw movements, the total number of single jaw movements (those preceded or followed by another jaw movement by more than 1 s), the total number of jaw movements occurring in bursts (at least two jaw movements separated by no more than 1 s), the total number of jaw movement bursts, average number of jaw movements in each burst (i.e., average burst size), and average frequency of jaw movements in bursts (expressed in hertz). In addition, the computer program placed each jaw movement into one of ten 1-Hz time bins between 1 and 10.999 Hz (i.e., 1–1.999 Hz, 2–2.999 Hz, 3–3.999 Hz, etc.). Drugs
METHODS
Subjects Six male Sprague–Dawley rats (Harlan Sprague–Dawley, Indianapolis, IN) were used in this experiment. All rats were individually housed in a colony room maintained at 23°C with a 12-h light– dark cycle (lights on 0700 hours). Animals were tested between 2 and 5 h after lights on. Water was available ad lib.
Tacrine hydrochloride (Sigma) was dissolved in 0.9% saline. All injections were intraperitoneal (i.p.), in a total volume of 1.0 mL/kg. Procedure Following i.p. injection of 2.5 mg/kg tacrine, animals were placed in a clear PlexiglasTM chamber (20 3 15 3 28 cm) so that
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FIG. 1. Frequency histogram showing the percentage of all jaw movements (mean 6 SEM; n 5 6) occurring in each 1-Hz time bin between 1 and 11 Hz.
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FIG. 2. A lateral (top) and ventral (bottom) view of the masticatory musculature of the rat showing the placement of the electrodes into the temporalis (T), masseter (M), and anterior belly of the digastricus (D).
each rat was oriented perpendicular to the video camera. Rats were videotaped during the EMG recording. After some of the tacrine recording sessions, additional sessions were conducted in which food was placed in the PlexiglasTM chamber. Slow-motion videotape analysis was conducted off-line. Tremulous jaw movements observed by videotape were later examined during EMG playback.
ANOVA revealed that there was a significant overall difference across the time bins [F(9, 45) 5 13.84, p , 0.001]. There was also a significant quadratic trend [F(1, 5) 5 16.7, p , 0.05], which indicates that the overall tendency to peak in the middle of the frequency distribution (i.e., 3–5 Hz) was statistically significant.
Data Analysis
EMG Recordings
All videotapes were analyzed, and data are only reported for those segments in which clear EMG traces and jaw movements were both visible from examination of the tape. Descriptive statistics were used to characterize the parameters of tacrine-induced tremulous jaw movements (see above). The average local frequency of jaw movements within bursts was calculated, and the jaw movement distribution was analyzed by simple ANOVA. Tremulous jaw movements that occurred during the segments of EMG shown in the figures were visually confirmed by freezeframe analysis. The local frequency of each EMG trace shown in the figures was directly determined by slow-motion examination of the videotape.
The placements of the electrodes are shown in Fig. 2. Bipolar EMG recordings were made of the anterior belly of the digastricus, masseter, and temporalis muscles during tacrine treatment. EMG data showed that all three muscles were quiescent when the jaw was not moving. Representative samples of EMG activity during tacrine-induced jaw movements are shown in Fig. 3. These traces were all obtained from 1-s sweeps during which the local frequency of jaw movements was 4.0 Hz, which was the average frequency as noted above. The muscle that showed activity most closely related to tremulous jaw movements was the temporalis. During bursts of jaw movements, temporalis muscles across several different rats consistently showed EMG activity, usually in the form of rhythmic bursts of EMG activity. Freeze-frame analysis of the videotape demonstrated that temporalis EMG activity was primarily initiated during jaw closing. However, this muscle was also sometimes observed to be active during the transition from jaw closing to jaw opening. The anterior belly of the digastricus showed bursts of EMG activity during some jaw movements, although the temporal relation between jaw movements and EMG activity was somewhat inconsistent (Fig. 3). In most cases, the anterior belly of the diagastricus was active along with the temporalis during jaw closing. Yet EMG activity in the digastricus could also be observed occasionally during the jaw-opening phase. Although slight variations in the placement of the electrodes could have substantial effects upon the amplitude of the response (51),
RESULTS
Computerized Slow-Motion Videotape Analysis Tacrine-induced tremulous jaw movements were analyzed by computerized slow-motion videotape analysis. Of the 903 chews detected in the six rats, the majority occurred in bursts [mean (6SEM) 94.5% (61.0)] with an average frequency within bursts of 4.0 Hz (6SEM 5 60.12 Hz). Figure 1 is a frequency histogram of the tacrine-induced jaw movements showing the percentage of chews in each 1-Hz time bin between 1 and 11 Hz. The majority of jaw movements (approximately 60.7%) were in the 3- to 5-Hz range, and each animal peaked within this frequency range.
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FIG. 3. One-second traces of EMG activity during bursts of jaw movements (four jaw movements for each trace) induced by 2.5 mg/kg tacrine (calibration: vertical 5 1.0 mV). A) Two traces of temporalis EMG during jaw movements (each trace is a different rat). B) Two traces from the same individual rat: top trace, masseter EMG; bottom trace, temporalis EMG. C) Two traces from the same individual rat: top trace, digastricus EMG; bottom trace, temporalis EMG.
the amplitude of the digastricus response was generally lower than that of the temporalis. The masseter showed no consistent EMG activity during tacrine-induced tremulous jaw movements (Fig. 3). In addition to being active during tacrine-induced tremulous jaw movements, the digastricus, temporalis, and masseter muscles were also active during grooming, gaping, licking, and facial tremor. After the tacrine sessions were completed, some of the rats were food-deprived for 24 h and fed in the chamber so that EMG activity could be recorded during feeding. All three muscles showed high-amplitude rhythmic activity during feeding (Fig. 4), which tended to occur during the jaw-closing phase. Electrical stimulation of the temporalis muscle in anesthetized animals resulted in jaw closing. However, during electrical stimulation of the anterior belly of the digastricus, the jaw did not open, and instead appeared to stiffen. No EMG activity was detected in any jaw muscles during passive movements of the jaw in rats under anesthesia. DISCUSSION
As reported previously, tacrine induces vacuous jaw movements, which occur as rapid vertical deflections of the lower jaw (31). Slow-motion videotape analysis of the jaw movement activity demonstrated that the movements occurred mostly in the 3- to 5-Hz frequency range, with a mean frequency of 4.0 Hz. Together with other studies, the present data indicate that tacrine, pilocarpine, and dopamine depletion all generate jaw movements that occur maximally in the 3- to 6.6-Hz frequency range (16,31,42). Analysis of the EMG data indicated that the temporalis muscle showed rhythmic bursts of activity that corresponded to the production of vacuous jaw movements; typically, this activity was also in the frequency range 3–5 Hz (e.g., 4 Hz is observed in Fig. 3). The frequency of tacrine-induced jaw movement activity was lower than the postural tremor that is observed in rats and mice after injections of cholinomimetics [.10 Hz; see (6)]. In addition, the local frequency of jaw movement activity observed in the present study did not peak within the 1- to 2-Hz frequency range that is characteristic of tardive dyskinesia (2,56). Instead, the frequency pattern of the jaw movement activity observed in the present study is consistent with the 3- to 7-Hz resting tremor
FIG. 4. One-second traces of EMG activity during bursts of jaw movements that occurred during feeding on laboratory chow (calibration: vertical 5 1.0 mV). The two traces are from the same individual rat: top trace, digastricus EMG; bottom trace, masseter EMG. The temporalis also showed high-amplitude activity during feeding (data not shown).
observed in parkinsonism (1,21,46). In particular, jaw tremors with a frequency of 4 Hz have been observed in parkinsonian patients (21). Thus, as noted in previous studies (16,31,42), the jaw movements induced by cholinomimetics not only are vacuous (i.e., nondirected) but also are tremulous (i.e., characterized by a periodic oscillation of a body member). The present work is the first to assess the role of the temporalis muscle in cholinomimetic-induced tremulous jaw movements, and the results indicate that the temporalis is a major contributor to the muscle activity shown during tremulous jaw movements. The temporalis was the muscle that was most closely related to the production of tremulous jaw movements. During bursts of jaw movements, temporalis muscles across several different rats showed rhythmic bursting EMG activity. Sections of videotape corresponding to bursts of EMG activity were reanalyzed by freeze-frame examination of the tape; typically, the temporalis showed EMG activity during each jaw movement, with bursts of activity occurring during the jaw-closing phase and the transition between jaw closing and opening. The involvement of the temporalis muscle in the jaw-closing and transitional phases of tacrineinduced jaw movements is consistent with previous reports of the activity of this muscle during feeding (9,51,53,55). Moreover, the identification of a particular muscle that is rhythmically activated during tremulous jaw movements provides important information about the anatomical organization of this response. There is a myotopic organization of the enervation of jaw muscles, and in the rat the motor neurons enervating the temporalis muscle originate within a well-defined subregion of the dorsolateral motor trigeminal nucleus (53). In view of the previous reports indicating that striatal mechanisms are involved in the generation of cholinomimetic-induced jaw movements (24,31,43), future research should focus on the pathways through which striatal output ultimately influences dorsolateral motor trigeminal neurons to instigate tremulous jaw movement activity.
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The involvement of the masseter muscle (52) in cholinomimetic-induced jaw movement activity has been somewhat uncertain. By using the cholinesterase inhibitor physostigmine, Collins et al. (11) found that the masseter was active during facial tremor, yawning, and gaping but not during vacuous, or “purposeless,” jaw movements. However, the masseter was found to be somewhat active during pilocarpine-induced chewing movements by Kikuchi de Beltran et al. (25). In the present study, there was no evidence of masseter activity during tacrine-induced tremulous jaw movements, which is similar to the results reported by Collins et al. (11). The reason for the discrepancies between studies is unclear. Different cholinomimetic drugs were used in each study, and there may be some differences between the effects of anticholinesterases such as tacrine and physostigmine and the effects of a muscarinic agonist such as pilocarpine. Differences in the placement of the electrode, the amount of muscle sampled, or the recording parameters may also be responsible. However, the present results, together with the findings previously reported (11,25), suggest that the major jaw-closing muscle active during vacuous jaw movements is the temporalis, and not the masseter. The present data confirm and extend the findings that the digastricus muscle can be active during cholinomimetic-induced jaw movements (25). The anterior belly of the digastricus showed bursts of EMG activity during some jaw movements, although the temporal relation between jaw movements and EMG activity was somewhat inconsistent. The digastricus is usually described as a jaw-opening muscle (53,55). However, there also are reports indicating that the digastricus can be active during jaw closing (17,51,55). Thus, the digastricus may be involved in stabilizing the jaw when it is in the closing phase (17,51,55). In this regard, it is interesting to note that in this experiment the anterior belly of the digastricus also cocontracted with the masseter and temporalis during feeding (Fig. 4). The complex role of the digastricus muscle in jaw movements, together with the observation that stimulation of the digastricus did not open the jaw, suggests that characterization of other portions of the digastricus (e.g., the posterior belly) or other jaw-opening muscles (e.g., the mylohyoid and geniohyoid) may be necessary for identifying the muscle that opens the jaw during cholinomimetic-induced jaw movements.
The purpose of the present study was to examine the jaw movements induced by tacrine. Nevertheless, jaw movement activity during feeding also was observed. It is interesting to note that the jaw movement activity induced by tacrine was similar to, but not identical with, jaw EMG during feeding. When the rats chewed laboratory chow, all three muscles studied showed high-amplitude rhythmic bursts of EMG activity. In contrast, the temporalis muscle was the only muscle that consistently showed EMG activity during tacrine-induced jaw movements. Although the jaw movements induced by cholinomimetics have been called “vacuous chewing” or referred to as “chewing-like,” it is not certain that these movements are identical to chewing that occurs during feeding. Future research should focus on detailed comparisons between normal mastication and cholinomimetic-induced movements. In summary, the temporalis muscle appears to be a major contributor to the generation of the nondirected jaw movements that are induced by tacrine. The temporalis consistently showed EMG activity during jaw movements, often in the form of rhythmic bursts. Examination of EMG data and slow-motion videotape analysis indicated that the jaw movements occurred largely in the frequency range 3–5 Hz, with a mean of about 4 Hz. Together with other studies, these data indicate that the jaw movements induced by cholinomimetics are tremulous, with a frequency in the same range as parkinsonian resting tremor. This conclusion is consistent with the well-established finding that cholinomimetic drugs are tremorogenic agents (6,22,27,35). Tacrine has been shown to induce parkinsonian symptoms, including tremor, in Alzheimer’s patients and normal volunteers (36,37). In addition, the present results are consistent with the recent finding that a variety of antiparkinsonian drugs, including apomorphine, bromocriptine, amantadine, benztropine, and L-DOPA, significantly reduce tacrine-induced tremulous jaw movements (12). ACKNOWLEDGEMENTS
This work was supported by a grant from the National Institutes of Health (NINDS). The authors appreciate the technical assistance of Dr. Harvey Swadlow.
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muscarinic agonist evoked tremor and salivation in rat and mouse. Psychopharmacology. 86:258 –264; 1985. Ott, B. R.; Lannon, M. C. Exacerbation of Parkinsonism by tacrine. Clin. Neuropharmacol. 15:322–325; 1992. Physicians’ Desk Reference. Montville: Medical Economics; 1995. Prinssen, E. P. M.; Koene, P.; Heeren, D.; Cools, A. R. Jaw muscle activity, the nucleus accumbens, and dopaminergic agonists: A new approach. Brain Res. Bull. 28:775–780; 1992. Rupniak, N. M. J.; Jenner, P.; Marsden, C. D. Cholinergic modulation of perioral behavior induced by chronic neuroleptic administration to rats. Psychopharmacology. 79:226 –230; 1983. Rupniak, N. M. J.; Jenner, P.; Marsden, C. D. Pharmacological characterization of spontaneous or drug-induced purposeless chewing movements in rats. Psychopharmacology. 85:71–79; 1985. Rupniak, N. M. J.; Jenner, P.; Marsden, C. D. Acute dystonia induced by neuroleptic drugs. Psychopharmacology. 88:403– 419; 1986. Salamone, J. D.; Baskin, P. B. Vacuous jaw movements induced by reserpine and low-dose apomorphine: Possible model of parkinsonian tremor. Pharmacol. Biochem. Behav. 53:179 –183; 1996. Salamone, J. D.; Johnson, C. J.; McCullough, L. D.; Steinpreis, R. E. Lateral striatal cholinergic mechanisms involved in oral motor activities in the rat. Psychopharmacology. 102:529 –534; 1990. Salamone, J. D.; Lalies, M. D.; Channell, S. L.; Iversen, S. D. Behavioral and pharmacological characterization of the mouth movements induced by muscarinic agonists in the rat. Psychopharmacology. 88: 467– 471; 1986. See, R.; Chapman, M. Cholinergic modulation of oral activity in drug-naive and chronic haloperidol-treated rats. Pharmacol. Biochem. Behav. 39:49 –54; 1991. Staude, G.; Wolf, W.; Ott, M.; Oertel, W. H.; Dengler, R. Tremor as a factor in prolonged reaction times of parkinsonian patients. Mov. Dis. 10:153–162; 1995. Steinpreis, R. E.; Baskin, P. P.; Salamone, J. D. Vacuous jaw movements induced by sub-chronic administration of haloperidol: Interactions with scopolamine. Psychopharmacology. 111:99 –105; 1993. Stewart, B. R.; Jenner, P.; Marsden, C. D. The pharmacological characterization of pilocarpine-induced chewing in the rat. Psychopharmacology. 97:228 –234; 1988. Stewart, B. R.; Jenner, P.; Marsden, C. D. Assessment of the muscarinic receptor subtype involved in the mediation of pilocarpine-induced purposeless chewing behaviour. Psychopharmacology. 97:228 – 234; 1089. Tarsy, D. Neuroleptic-induced extrapyramidal reactions: Classification, description and diagnosis. Clin. Neuropharmacol. 6:S9 –S26; 1983. Thomas, N. R.; Peyton, S. C. An electromyographic study of mastication in the freely-moving rat. Arch. Oral Biol. 28:939 –945; 1983. Thomas, N. R.; Peyton, S. C. Relationship of masseter electromyographic activity to mandible position in the freely-moving rat. Arch. Oral Biol. 28:1043–1046; 1983. Travers, J. B. Oromotor nuclei. In: Paxinos, G., ed. The rat nervous system. New York: Academic Press, 1995:239 –255. Waddington, J. L. Spontaneous orofacial movements induced in rodents by very long-term neuroleptic drug administration: Phenomenology, pathophysiology and putative relationship to tardive dyskinesia. Psychopharmacology. 101:431– 447; 1990. Weijs, W. A.; Dantuma, R. Electromyography and mechanics of mastication in the albino rat. J. Morphol. 146:1–34; 1975. Wirshing, W. C.; Cummings, J. L.; Dencker, S. J.; May, P. R. Electromechanical characteristics of tardive dyskinesia. Schizophren. Res. 2:40; 1989.
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Behavioral and Electromyographic Characterization of the Local Frequency of Tacrine-induced Tremulous Jaw Movements M. S. COUSINS, A. ATHERTON AND J. D. SALAMONE1 Department of Psychology, University of Connecticut, Storrs, CT 06269-1020, USA Received 20 March 1997; Accepted 7 January 1998 COUSINS, M. S., A. ATHERTON AND J. D. SALAMONE. Behavioral and electromyographic characterization of the local frequency of tacrine-induced tremulous jaw movements. PHYSIOL BEHAV 64(2) 153–158, 1998.—Rats were implanted with fine-wire electromyograph (EMG) electrodes and were videotaped to identify the local frequency characteristics and muscle activity associated with tacrine-induced tremulous jaw movements. All rats received intraperitoneal injections of 2.5 mg/kg tacrine. The videotape sessions were played back in slow motion (i.e., one-sixth normal speed), and an observer entered each jaw movement into a computer program that recalculated the interresponse time and the local frequency (in hertz) for each movement within a burst. Analyses of the distribution of frequencies showed that the peak frequency of jaw movements was in the 3- to 5-Hz frequency range, with an average frequency of 4.0 Hz. EMG electrodes were implanted into three jaw muscles: temporalis, anterior belly of digastricus, and masseter. Tremulous jaw movements were not accompanied by consistent changes in masseter activity. The anterior belly of digastricus showed bursts of EMG activity during some jaw movements, although the temporal relation between jaw movements and EMG activity was somewhat inconsistent. The muscle that showed activity most closely related to tremulous jaw movements was the temporalis. During bursts of jaw movements, temporalis muscles across several different rats showed bursts of EMG activity. Sections of videotape corresponding to bursts of EMG activity were reanalyzed by freeze-frame examination of the tape; typically, the temporalis showed a burst for each jaw movement, with the burst of activity occurring during the jaw-closing phase and the transition between jaw closing and opening. These results indicate that the local frequency of tremulous jaw movements is within the 3- to 7-Hz frequency that is typically associated with parkinsonian tremor. Moreover, the EMG data suggest that temporalis is a major contributor to the muscle activity that underlies tremulous jaw movements. © 1998 Elsevier Science Inc. Tremor
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showed that most of the interresponse times were within the range of 142–333 ms, with a peak in the range of 200 –250 ms (42). Similar temporal characteristics are shown with the jaw movements induced by ventrolateral striatal dopamine depletion, the muscarinic agonist pilocarpine, and 5.0 mg/kg of the anticholinesterase tacrine (16,33). Based upon analyses of interresponse times, it was calculated that the local frequency of jaw movement activity induced by dopamine depletion and cholinergic stimulation was in the range 3.0 – 6.6 Hz. These data are consistent with the report of See and Chapman (45), who used different methods but nevertheless observed that physostigmine and pilocarpine increased jaw movement activity in the 4- to 6-Hz frequency range. Thus, it has been suggested that the vacuous, or nondirected, jaw movements induced by dopamine depletion or cholinomimetics are appropriately referred to as “tremulous” (16,31,42). Although the clinical significance of drug-induced perioral behaviors remains uncertain (14,41,47), it has been suggested that tremulous jaw movements share characteristics with parkinsonian tremor (23,43,47). For example, the acetylcholine– dopamine in-
IN RATS, perioral movements are induced by a wide variety of neurochemical and pharmacological conditions (4,5,14,23,39, 40,54). One of the most commonly studied of these movements is known as vacuous jaw movements (also known as tremulous jaw movements or vacuous chewing); these movements are defined as rapid, vertical deflections of the lower jaw that resemble chewing but are not directed at any particular stimulus (4,5,8,10,42). The term “vacuous” refers to the nondirected nature of the these movements. Recently, it has been suggested that these movements also are “tremulous;” i.e., they are characterized by a periodic oscillation of the jaw (15). It has been observed for several years that drug-induced jaw movements occur mostly in rapid “bursts” of activity (44). In recent studies, slow-motion videotape analysis has been used to characterize the temporal features of vacuous, or tremulous, chewing-like movements induced under a number of different conditions. Coadministration of reserpine with a low, presynaptic dose of apomorphine resulted in jaw movement activity that was mostly in rapid bursts, with interresponse times less than 1 s (42). Analysis of the distribution of interresponse times 1
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To whom requests for reprints should be addressed. E-mail: [email protected]
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teraction that is shown with parkinsonian symptoms is similar to that shown for vacuous jaw movements (3,12,13,16,18,23,30,32, 34,47–50). The local frequency of movement is another characteristic that is worth considering. For example, tardive dyskinesia in humans shows activity largely within the 1- to 2-Hz frequency range (2,56). In contrast, parkinsonian tremor is generally reported to be in the 3- to 7-Hz frequency range (1,21). Although parkinsonian tremor usually involves the hand, it can involve an up-anddown movement of the lower jaw (1); one recent study of parkinsonian jaw tremor showed peak activity in the 3- to 5-Hz frequency range (21). Thus, tremulous jaw movements in rats appear to have frequency characteristics that are similar to parkinsonian tremor. In this study, tacrine-induced tremulous jaw movements in rats were simultaneously analyzed by slow-motion video and electromyography (EMG). Tacrine (Cognex) is an anticholinesterase that is used to treat Alzhemier’s disease; this drug has been shown to produce parkinsonian side effects, including tremor (36,37). Tacrine was administered to rats at a dose of 2.5 mg/kg, because this dose produces a robust jaw movement response (7,12,31) and also because the jaw movements induced by 2.5 mg/kg tacrine are reversed by several antiparkinsonian drugs, including L-DOPA, apomorphine, bromocriptine, amantadine, and benztropine (12). Computer-aided slow-motion video observations and EMG recordings were performed to characterize the local frequency of tacrine-induced jaw movements. Previous research using slowmotion video analysis of tremulous jaw movements partitioned each jaw movement into interresponse times [i.e., the time between each jaw movement in milliseconds (31)]. In this study, the precise frequency of tacrine-induced tremulous jaw movements, in hertz, was directly determined. Although videotape analyses can yield important information about the frequency characteristics of jaw movements, such observational methods do not identify which muscles are active during cholinomimetic-induced jaw movements, nor do they determine the temporal pattern of muscle activity. Thus, EMG methods were also employed. EMG methods have been used previously to study drug-induced oral movements (20,26,28,29,33,38), and previous EMG studies have focused upon digastricus and masseter muscle activity during cholinomimeticinduced oral movements (11,25). Digastricus EMG activity was reported to increase during pilocarpine-induced jaw movements (25). Reports of masseter activity have been somewhat equivocal, with one paper reporting small increases in activity during chewing-like movements (25) and another failing to find consistent masseter activity during pilocarpine-induced chewing movements (11). The time scale used in some previous studies [e.g., (25)] did not permit one to determine the local frequency of jaw movements within bursts based on EMG activity. In addition, the temporalis muscle, which accounts for approximately 24% of the mass of all masticatory muscles in the rat (19,20,55), has yet to be characterized with EMG during cholinomimetic-induced tremulous jaw movements. Therefore, videotape and EMG methods in conscious, mobile rats were used in the present study to determine the local frequency of tacrine-induced tremulous jaw movements and to study the involvement of digastricus, masseter, and temporalis muscles in these jaw movements.
Animal care procedures were approved by the University’s Institutional Animal Care and Use Committee. EMG Electrode Implantation and Recording Rats were injected with 1.0 mg/kg methylscopolamine and anesthetized with 50.0 mg/kg sodium pentobarbital. Lidocaine was injected into all incisions for local anesthesia. Bipolar wire-hook electrodes (50 mM diameter wire with 1.0 mm of insulation exposed at the tip; electrode tips separated by 2.0 mm) were implanted under direct vision with a 27-gauge needle approximately 1.0 mm deep into the left digastricus (anterior belly), temporalis, or masseter muscles (temporalis and digastricus, n 5 4; temporalis and masseter, n 5 2). The wires led subcutaneously to the skull and were attached to a six-pole connector (Amphenol). The rostral skull screw served as ground. The assembly was fastened to the skull with two additional screws and cranioplastic cement. The six-pole connector was attached to an electrical commutator that led to a preamplifier and amplifier (Knight differential amplifier; 300-Hz low-pass and 500-Hz high-pass filter). The signal was converted to analog (Coulbourn Instruments) and relayed to an IBM-type microcomputer operating a two-channel data recorder (Coulbourn AT L19-69). At the end of the experiment, each muscle was contracted with a constant-current generator (Grass CCU 1A; 0.1– 0.3 mA, 100 pulses with 1.0-ms duration at 100 Hz) to confirm the correct placement of electrodes. In addition, after the experiment, rats were sacrificed with sodium pentobarbital and the muscles were dissected to confirm placement of the recording electrodes. Computerized Slow-Motion Videotape Analysis A video camera (Panasonic AG-180) was used to simultaneously record the facial region of each rat while the EMG signal appeared on the computer monitor. To quantify the temporal characteristics of tremulous jaw movements, videos were played back (Panasonic AG-1730) at one-sixth normal speed, using a procedure similar to that reported previously (16,31,42). Tremulous jaw movements were defined as a small deflection of the lower jaw that resembled chewing but was not directed at any apparent stimulus. These did not include yawns or gapes. Gapes were defined as a maximal opening of the jaw. During slowmotion video playback each occurrence of a tremulous jaw movement or a gape was recorded by an observer on an IBM-type microcomputer running BASIC. The computer program converted the timing of the jaw movements back into normal speed and recorded the total number of jaw movements, the total number of single jaw movements (those preceded or followed by another jaw movement by more than 1 s), the total number of jaw movements occurring in bursts (at least two jaw movements separated by no more than 1 s), the total number of jaw movement bursts, average number of jaw movements in each burst (i.e., average burst size), and average frequency of jaw movements in bursts (expressed in hertz). In addition, the computer program placed each jaw movement into one of ten 1-Hz time bins between 1 and 10.999 Hz (i.e., 1–1.999 Hz, 2–2.999 Hz, 3–3.999 Hz, etc.). Drugs
METHODS
Subjects Six male Sprague–Dawley rats (Harlan Sprague–Dawley, Indianapolis, IN) were used in this experiment. All rats were individually housed in a colony room maintained at 23°C with a 12-h light– dark cycle (lights on 0700 hours). Animals were tested between 2 and 5 h after lights on. Water was available ad lib.
Tacrine hydrochloride (Sigma) was dissolved in 0.9% saline. All injections were intraperitoneal (i.p.), in a total volume of 1.0 mL/kg. Procedure Following i.p. injection of 2.5 mg/kg tacrine, animals were placed in a clear PlexiglasTM chamber (20 3 15 3 28 cm) so that
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FIG. 1. Frequency histogram showing the percentage of all jaw movements (mean 6 SEM; n 5 6) occurring in each 1-Hz time bin between 1 and 11 Hz.
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FIG. 2. A lateral (top) and ventral (bottom) view of the masticatory musculature of the rat showing the placement of the electrodes into the temporalis (T), masseter (M), and anterior belly of the digastricus (D).
each rat was oriented perpendicular to the video camera. Rats were videotaped during the EMG recording. After some of the tacrine recording sessions, additional sessions were conducted in which food was placed in the PlexiglasTM chamber. Slow-motion videotape analysis was conducted off-line. Tremulous jaw movements observed by videotape were later examined during EMG playback.
ANOVA revealed that there was a significant overall difference across the time bins [F(9, 45) 5 13.84, p , 0.001]. There was also a significant quadratic trend [F(1, 5) 5 16.7, p , 0.05], which indicates that the overall tendency to peak in the middle of the frequency distribution (i.e., 3–5 Hz) was statistically significant.
Data Analysis
EMG Recordings
All videotapes were analyzed, and data are only reported for those segments in which clear EMG traces and jaw movements were both visible from examination of the tape. Descriptive statistics were used to characterize the parameters of tacrine-induced tremulous jaw movements (see above). The average local frequency of jaw movements within bursts was calculated, and the jaw movement distribution was analyzed by simple ANOVA. Tremulous jaw movements that occurred during the segments of EMG shown in the figures were visually confirmed by freezeframe analysis. The local frequency of each EMG trace shown in the figures was directly determined by slow-motion examination of the videotape.
The placements of the electrodes are shown in Fig. 2. Bipolar EMG recordings were made of the anterior belly of the digastricus, masseter, and temporalis muscles during tacrine treatment. EMG data showed that all three muscles were quiescent when the jaw was not moving. Representative samples of EMG activity during tacrine-induced jaw movements are shown in Fig. 3. These traces were all obtained from 1-s sweeps during which the local frequency of jaw movements was 4.0 Hz, which was the average frequency as noted above. The muscle that showed activity most closely related to tremulous jaw movements was the temporalis. During bursts of jaw movements, temporalis muscles across several different rats consistently showed EMG activity, usually in the form of rhythmic bursts of EMG activity. Freeze-frame analysis of the videotape demonstrated that temporalis EMG activity was primarily initiated during jaw closing. However, this muscle was also sometimes observed to be active during the transition from jaw closing to jaw opening. The anterior belly of the digastricus showed bursts of EMG activity during some jaw movements, although the temporal relation between jaw movements and EMG activity was somewhat inconsistent (Fig. 3). In most cases, the anterior belly of the diagastricus was active along with the temporalis during jaw closing. Yet EMG activity in the digastricus could also be observed occasionally during the jaw-opening phase. Although slight variations in the placement of the electrodes could have substantial effects upon the amplitude of the response (51),
RESULTS
Computerized Slow-Motion Videotape Analysis Tacrine-induced tremulous jaw movements were analyzed by computerized slow-motion videotape analysis. Of the 903 chews detected in the six rats, the majority occurred in bursts [mean (6SEM) 94.5% (61.0)] with an average frequency within bursts of 4.0 Hz (6SEM 5 60.12 Hz). Figure 1 is a frequency histogram of the tacrine-induced jaw movements showing the percentage of chews in each 1-Hz time bin between 1 and 11 Hz. The majority of jaw movements (approximately 60.7%) were in the 3- to 5-Hz range, and each animal peaked within this frequency range.
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FIG. 3. One-second traces of EMG activity during bursts of jaw movements (four jaw movements for each trace) induced by 2.5 mg/kg tacrine (calibration: vertical 5 1.0 mV). A) Two traces of temporalis EMG during jaw movements (each trace is a different rat). B) Two traces from the same individual rat: top trace, masseter EMG; bottom trace, temporalis EMG. C) Two traces from the same individual rat: top trace, digastricus EMG; bottom trace, temporalis EMG.
the amplitude of the digastricus response was generally lower than that of the temporalis. The masseter showed no consistent EMG activity during tacrine-induced tremulous jaw movements (Fig. 3). In addition to being active during tacrine-induced tremulous jaw movements, the digastricus, temporalis, and masseter muscles were also active during grooming, gaping, licking, and facial tremor. After the tacrine sessions were completed, some of the rats were food-deprived for 24 h and fed in the chamber so that EMG activity could be recorded during feeding. All three muscles showed high-amplitude rhythmic activity during feeding (Fig. 4), which tended to occur during the jaw-closing phase. Electrical stimulation of the temporalis muscle in anesthetized animals resulted in jaw closing. However, during electrical stimulation of the anterior belly of the digastricus, the jaw did not open, and instead appeared to stiffen. No EMG activity was detected in any jaw muscles during passive movements of the jaw in rats under anesthesia. DISCUSSION
As reported previously, tacrine induces vacuous jaw movements, which occur as rapid vertical deflections of the lower jaw (31). Slow-motion videotape analysis of the jaw movement activity demonstrated that the movements occurred mostly in the 3- to 5-Hz frequency range, with a mean frequency of 4.0 Hz. Together with other studies, the present data indicate that tacrine, pilocarpine, and dopamine depletion all generate jaw movements that occur maximally in the 3- to 6.6-Hz frequency range (16,31,42). Analysis of the EMG data indicated that the temporalis muscle showed rhythmic bursts of activity that corresponded to the production of vacuous jaw movements; typically, this activity was also in the frequency range 3–5 Hz (e.g., 4 Hz is observed in Fig. 3). The frequency of tacrine-induced jaw movement activity was lower than the postural tremor that is observed in rats and mice after injections of cholinomimetics [.10 Hz; see (6)]. In addition, the local frequency of jaw movement activity observed in the present study did not peak within the 1- to 2-Hz frequency range that is characteristic of tardive dyskinesia (2,56). Instead, the frequency pattern of the jaw movement activity observed in the present study is consistent with the 3- to 7-Hz resting tremor
FIG. 4. One-second traces of EMG activity during bursts of jaw movements that occurred during feeding on laboratory chow (calibration: vertical 5 1.0 mV). The two traces are from the same individual rat: top trace, digastricus EMG; bottom trace, masseter EMG. The temporalis also showed high-amplitude activity during feeding (data not shown).
observed in parkinsonism (1,21,46). In particular, jaw tremors with a frequency of 4 Hz have been observed in parkinsonian patients (21). Thus, as noted in previous studies (16,31,42), the jaw movements induced by cholinomimetics not only are vacuous (i.e., nondirected) but also are tremulous (i.e., characterized by a periodic oscillation of a body member). The present work is the first to assess the role of the temporalis muscle in cholinomimetic-induced tremulous jaw movements, and the results indicate that the temporalis is a major contributor to the muscle activity shown during tremulous jaw movements. The temporalis was the muscle that was most closely related to the production of tremulous jaw movements. During bursts of jaw movements, temporalis muscles across several different rats showed rhythmic bursting EMG activity. Sections of videotape corresponding to bursts of EMG activity were reanalyzed by freeze-frame examination of the tape; typically, the temporalis showed EMG activity during each jaw movement, with bursts of activity occurring during the jaw-closing phase and the transition between jaw closing and opening. The involvement of the temporalis muscle in the jaw-closing and transitional phases of tacrineinduced jaw movements is consistent with previous reports of the activity of this muscle during feeding (9,51,53,55). Moreover, the identification of a particular muscle that is rhythmically activated during tremulous jaw movements provides important information about the anatomical organization of this response. There is a myotopic organization of the enervation of jaw muscles, and in the rat the motor neurons enervating the temporalis muscle originate within a well-defined subregion of the dorsolateral motor trigeminal nucleus (53). In view of the previous reports indicating that striatal mechanisms are involved in the generation of cholinomimetic-induced jaw movements (24,31,43), future research should focus on the pathways through which striatal output ultimately influences dorsolateral motor trigeminal neurons to instigate tremulous jaw movement activity.
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The involvement of the masseter muscle (52) in cholinomimetic-induced jaw movement activity has been somewhat uncertain. By using the cholinesterase inhibitor physostigmine, Collins et al. (11) found that the masseter was active during facial tremor, yawning, and gaping but not during vacuous, or “purposeless,” jaw movements. However, the masseter was found to be somewhat active during pilocarpine-induced chewing movements by Kikuchi de Beltran et al. (25). In the present study, there was no evidence of masseter activity during tacrine-induced tremulous jaw movements, which is similar to the results reported by Collins et al. (11). The reason for the discrepancies between studies is unclear. Different cholinomimetic drugs were used in each study, and there may be some differences between the effects of anticholinesterases such as tacrine and physostigmine and the effects of a muscarinic agonist such as pilocarpine. Differences in the placement of the electrode, the amount of muscle sampled, or the recording parameters may also be responsible. However, the present results, together with the findings previously reported (11,25), suggest that the major jaw-closing muscle active during vacuous jaw movements is the temporalis, and not the masseter. The present data confirm and extend the findings that the digastricus muscle can be active during cholinomimetic-induced jaw movements (25). The anterior belly of the digastricus showed bursts of EMG activity during some jaw movements, although the temporal relation between jaw movements and EMG activity was somewhat inconsistent. The digastricus is usually described as a jaw-opening muscle (53,55). However, there also are reports indicating that the digastricus can be active during jaw closing (17,51,55). Thus, the digastricus may be involved in stabilizing the jaw when it is in the closing phase (17,51,55). In this regard, it is interesting to note that in this experiment the anterior belly of the digastricus also cocontracted with the masseter and temporalis during feeding (Fig. 4). The complex role of the digastricus muscle in jaw movements, together with the observation that stimulation of the digastricus did not open the jaw, suggests that characterization of other portions of the digastricus (e.g., the posterior belly) or other jaw-opening muscles (e.g., the mylohyoid and geniohyoid) may be necessary for identifying the muscle that opens the jaw during cholinomimetic-induced jaw movements.
The purpose of the present study was to examine the jaw movements induced by tacrine. Nevertheless, jaw movement activity during feeding also was observed. It is interesting to note that the jaw movement activity induced by tacrine was similar to, but not identical with, jaw EMG during feeding. When the rats chewed laboratory chow, all three muscles studied showed high-amplitude rhythmic bursts of EMG activity. In contrast, the temporalis muscle was the only muscle that consistently showed EMG activity during tacrine-induced jaw movements. Although the jaw movements induced by cholinomimetics have been called “vacuous chewing” or referred to as “chewing-like,” it is not certain that these movements are identical to chewing that occurs during feeding. Future research should focus on detailed comparisons between normal mastication and cholinomimetic-induced movements. In summary, the temporalis muscle appears to be a major contributor to the generation of the nondirected jaw movements that are induced by tacrine. The temporalis consistently showed EMG activity during jaw movements, often in the form of rhythmic bursts. Examination of EMG data and slow-motion videotape analysis indicated that the jaw movements occurred largely in the frequency range 3–5 Hz, with a mean of about 4 Hz. Together with other studies, these data indicate that the jaw movements induced by cholinomimetics are tremulous, with a frequency in the same range as parkinsonian resting tremor. This conclusion is consistent with the well-established finding that cholinomimetic drugs are tremorogenic agents (6,22,27,35). Tacrine has been shown to induce parkinsonian symptoms, including tremor, in Alzheimer’s patients and normal volunteers (36,37). In addition, the present results are consistent with the recent finding that a variety of antiparkinsonian drugs, including apomorphine, bromocriptine, amantadine, benztropine, and L-DOPA, significantly reduce tacrine-induced tremulous jaw movements (12). ACKNOWLEDGEMENTS
This work was supported by a grant from the National Institutes of Health (NINDS). The authors appreciate the technical assistance of Dr. Harvey Swadlow.
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