Psychopharmacology (1991) 103:462-466 003331589100052X

Psychopharmacology © Springer-Verlag 1991

Effects of MK-801, a non-competitive N M D A antagonist, on linguopharyngeal events in rats Luis A. Marco, 1 Rajani S. Joshi, 1 and Robert B. Chronister 2

Departments of i Psychiatry and z Neurology, University of South Alabama, 2451 Fillingim Street, Mobile, AL 36617, USA Received May 31, 1990 / Final version September 4, 1990 Abstract. The effects of MK-801 at doses from 0.005 to 1 mg/kg IP on linguopharyngeal events (protrusions, retrusions and swallows) were determined in rats to find out whether MK-801 resembles ketamine in its capacity to increase the frequency of recurrence of such events that we have demonstrated in previous studies. All rats receiving a dose of 0.05 mg/kg or higher showed an increase in linguopharyngeal event frequency within 5 min and this enhancement (3-fold from baseline level) was maintained tbr longer than 1 h. At the lowest dose of 5 gg/kg the effect lasted only very briefly. A general increase in motor behavior was also observed within 10 min of drug administration. More complex patterns of motor behavior, consisting of stereotypical head bobbing, paw movements reminiscent of walking activity, nystagmus, and ataxia were observed with doses of 0.25 mg/kg and higher. All rats showed a marked startle response at early stages post-injection and hypersensitivity to external stimuli such as noise or movement in the room. However, there was an absolute lack of coordinated avoidance responses normally associated with such startle responses or arousing stimuli. Key words: MK-801 - N M D A antagonism - Dyskinesia - Dissociative anesthetics - Ketamine - Linguopharyngeal events

MK-801 {(+)-5-methyl-10, 11-dihydro-5H-dibenzo[a,d] cyclohepten 5, 10-imine maleate} is a dibenzocycloheptenimine with potent noncompetitive antagonism of the N-methyl-D-aspartate (NMDA) receptor subtype (Kemp et al 1986, 1987; Wong et al. 1986). N M D A receptor antagonists have been shown to exhibit a neuroprotective effect in models of ischemic (Simon et al. 1984; Gerhardt et al. 1986), hypoglycemic (Wieloch 1985) and seizure-mediated (Labruyere et aL 1986) brain damage. Offprint requests to:

L. A. Marco

Most of these studies utilized competitive N M D A receptor antagonists such as 2-amino-7-phosphoheptanoic acid (APH). However, experiments have confirmed that the noncompetitive N M D A antagonist MK-801 also protects against neuronal damage following forebrain ischemia (Foster et al. 1987) and that it possesses anticonvulsive properties (Clineschmidt et al. 1982a). Additionally, MK-801 exhibits sympathomimetic and anxiolytic effects (Clineschmidt et al. 1982b, c). This unique combination of effects has also prompted the clinical evaluation of MK-801 for treatment of adults with a diagnosis of Attention Deficit Disorder, Residual Type (Riemherr et al. 1986). Its potential as a treatment for stroke and epilepsy is being evaluated in several centers. Phencyclidine (PCP) and ketamine, classified as "dissociative" anesthetics (Corssen and Domino 1966), also antagonize noncompetitively the actions of excitatory amino acids presumably by an interaction with N M D A receptor-associated ion channels (Harrison and Simmonds 1985; Martin and Lodge 1985; Aram et al. 1986). It has been proposed that N M D A antagonism by PCP and ketamine could lead to the "dissociative" anesthetic effects seen with these drugs (Anis et al. 1983). MK-801 and the dissociative anesthetics may share common sites of action in the brain, as PCP and ketamine displace [3H]MK-801 in binding assays (Wong et al. 1986) and autoradiographic studies have shown that [3H]MK-801 binds selectively and with high affinity to receptors with the same distribution as the PCP-receptor labeled by [3H]PCP or by its more potent analog [3H] thienylcyclohexylpiperidine (Maragos et al. 1986, 1988). Additionally, use of 2-deoxyglucose autoradiography has recently shown another similarity between PCP and MK-801 : the two drugs produce very similar patterns of alterations in metabolism in the caudate nucleus, accumbens, dendritic regions of the hippocampus, anterior thalamus, mamillary bodies and other structures of the circuit of Papez (Piercey et al. 1988). The functional similarity of the two drugs has led to the postulate that MK-801 may also have a PCP-like psychotomimetic potential. Behaviors like those induced by PCP have been

463 reported in pigeons (Koek et al. 1986a, b) and in rats (Koek et al. 1987) following administration of the competitive N M D A antagonist 2-amino-5-phosphonovalerate (AP5), but little attention has been given to the behavioral effects o f MK-801. In the present experiments, we report that MK-801 produces stereotypical movements of the head, neck and paws and dyskinetic linguopharyngeal movements in rats which are identical to those observed with PCP (Marco et al. in preparation) and ketamine (Aldes et al. 1988; M a r c o et al. 1988, 1989). Materials and methods Twenty-six naive female SpragueDawtey rats (Charles River Breeding Laboratories, Raleigh, NC) of approximately 250 g body weight were used in this study. MK-801 was provided by Dr. G. T. Shearman (Merck Sharp and Dohme Research Laboratories, West Point, PA). Solutions of MK-801 were prepared in sterile water and injections were given by the intraperitoneal route. Counts of linguopharyngeal events (LPE) such as protrusions, retrusions and swallows were made after placing the rat in a Broome-styte rodent restrainer (Plas-Labs, Lansing, Michigan) which prevents locomotion and allows inspection of other movements. After positioning the rat in the restrainer, 5 min were allowed for habituation before any behavioral observations were noted. Two observers recorded the LPE and other behaviors such as grooming, paw movement, nystagmus, etc. The criterion set by the observers for labeling an oral movement as a LPE was that the action should either be a distinct swallow or include a visible tongue protrusion or retrusion. Mandible tremors and fibrillations of the tongue were also noted but not counted as discrete LPE. The baseline of LPE was determined for each rat before administration of MK-801 by counting LPE occurring spontaneously during three 5-min intervals separated from each other by 5 min. In this manner, each animal served as its own control. After estabilishing the baseline activity, MK-801 was administered at one of the following doses: 1, 0.5, 0.25, 0.1 0.05, or 0.005 mg/kg. Motor behaviors (see Results) and the appearance of the rat were monitored continuously during the testing period. Counts of LPE were made at 5 min intervals for the first 90-120 min following MK-801 injection and then were taken periodically over the next 2 3 h to determine duration of effect. For all doses tested, the LPE observed at each time interval were pooled and averaged for use in bar-graphs and statistical analysis. Some animals were utilized for polygraphic recordings of LPE and those were prepared as described in previous reports (Aldes et al. I988; Marco, et al. 1988). Essentially, after becoming listless and unresponsive to paw and pinna pinch, the tongue was pierced at about 2 mm from the tip with a curved suturing needle and a piece of nylon thread (4-0) attached to it. The animal was placed in a Kopf stereotaxic apparatus and the nylon thread was tied to a rigid arm extending from the beam shaft of the cantilever of a Grass force displacement transducer (FDT). The output from the FDT was fed to a pen recorder after amplification. The position of the FDTtongue tip junction was adjusted such that the tongue was free from both upper and lower teeth, lying as normally as possible, and stretched not more than about 2 mm beyond the insertion of the upper teeth. Swallows were identified by means of a pressure transducer placed in the soft palate (Marco et al. 1988).

Results

General observations The most immediate effect of MK-801 injection at all doses other than the lowest (5 gg/kg) was an increase in

general m o t o r behavior within 10 min of drug administration. During this period the animals appeared very restless and demonstrated stereotypical behaviors including repetitious front paw m o v e m e n t reminiscent o f walking or running, rotation o f the b o d y in the restrainer around the oro-caudal axis, and rapid side-to-side movement of the snout and m o u t h area at a b o u t two movements per second. A few rats were removed f r o m the restrainer immediately after injection to note changes in locomotion; these rats demonstrated continuous ataxic movement. Other behaviors frequently noted included intermittent lateral nystagmus and jerks or twitches o f the entire b o d y and a loss o f righting reflexes. As the test period progressed, the animals became more subdued and often allowed removal from the Broome restrainer and direct observation of movements, righting attempts, and LPE. After the first 10-15 rain post-injection most animals did not attempt locomotion and, if outside the restrainer, they would remain prone or on one side. Those that did attempt locomotion were ataxic and unable to effectively support their weight and some demonstrated circling behavior. Animals receiving >_ 0.005 mg/ kg typically developed a limp bodily tone and disappearance of defensive or avoidance behavior such that they could be held by an observer for an extended period of time without struggle. Despite the lack of locomotion and defensive behavior, all rats showed a m a r k e d startle response and hypersensitivity to external stimuli such as sudden noises and m o v e m e n t in the observation r o o m ; however, the animals appeared unable to respond with the avoidance behavior normally associated with such stimuli. These two characteristics, increased startle response and decreased avoidance behavior, persisted in mitigated form for 24-36 h following the injection in the animals receiving the higher doses. Emergence from the drug effects produced lateral nystagmus, recovery of righting reflexes, attempts at crawling and a gradual return to the standing, alert, and drug-free state. A majority of the rats receiving the 1 mg/kg dose would not eat or drink spontaneously during the 24-36 h following MK-801 injection. This group necessitated close supervision and a subcutaneous injection o f Ringer's lactate for hydration and volume replenishment after completion of the test period. The rats returned to normal feeding behavior within 2 days of MK-801 injection.

Effect of MK~801 on linguopharyngeal events" Rats with a low level of spontaneous LPE ( < 10 L P E in a 5-min period) showed a more dramatic increase in LPE following MK-801 injection than those rats displaying a high level of spontaneous LPE ( > 10 LPE per 5 rain). Plots of the mean data points revealed a dramatic effect o f MK-801 on L P E (see Fig. 1). Inspection of the mean data revealed a m a r k e d elevation of the LPE approximately 30 min after the injection. This elevation was most dramatic for the two highest doses; however, the results from the lower doses were m o r e variable in appearance. F o r this reason an A N O V A was run on the

464 P 14o ¢-

¢. 120

Fig. 1. A graphic representation of the mean number of LPE for a given dose of MK-801. The X-axis represents the time in 5-min intervals from injection, while the Y-axis is the mean number of LPE. Inspection of the figure shows that the highest number of LPE was obtained with the two highest doses of MK-801. In both of these cases, the peak responses were obtained in the 5-rain period from 25 to 30 min postinjection. E:1 0.05 mg/kg; 15~ 0.10 mg/kg; 0.25 mg/kg; INN0.50 mg/kg; N2t 1.00 mg/kg

E m~ loo LL! O_ 8O -J

0 =..

60

(D

-0

E

40

ct-

2o

:~

o

I

I

I

pre-drug

I

5-10 mins

injection

25-30 mins

45-50 mins 3 5 4 0 mins

15-20 rains

55-60 mins

" ~ 120

P

.~ 10o ¢E t o 8o I.kl [1. -I 60 0

•.#2

"'<;,_

$ e~

40

/'/

E

//

¢..

-O

..... [] . . . . . . . . . . . . . . ~ ...... . .D ........

O_

-® ........

® .....

20

ET'::':-:-:~/:::""

"O

- - -

0 pre-dmg

injection

_A 5-10rains

15-20rains

I 25-a0mins

i_ 35-40rains

i ,kS-50mins

I 55-60rains

Fig. 2. The data plotted from a regression analysis performed in SAS (general linear methods procedure). As in the data represented by Fig. 1, the highest number of LPe were found in the highest doses of MK-801. Inspection of the figure shows that the highest peaks for these two doses occurred between 25 and 50 rain afler injection and then dropped off appreciably. Comparable curves are found for all other doses, with the exception of the 0.25 mg/kg dose. The reason for this discrepancy is unknown. - . - 0.05 mg/kg; ---©--- 0.25 rag/ kg; - - A - - 0.50 mg/kg; . . . . . . . 1.00 mg/kg

Time from injection

total data with individual A N O V A ' s and mutiple t-test comparisons done where appropriate. All analyses were done using the general linear models procedure test available on the SAS programs. Statistical analysis o f the total LPE data revealed that the number of LPE were significantly different both by group and by time. The lowest dose given (0.05 mg/kg) did not show significant changes from baseline with respect to time. The remaining doses all caused significant changes from baseline. Subsequent A N O V A analysis of the group data demonstrated that the groups differed significantly at all time intervals except at pre-drug baseline ( F = 1.75, df4, 15, P < 0 . 1 9 ) and at 15-20 min (F=2.57, df4, 15, P < 0 . 0 8 ) . Analysis of the time data demonstrated that all groups differed with respect to time with the exception o f the 0.05 mg/kg, the lowest dose ( F = 1.39, dr6, 0.21, P < 0 . 2 5 ) . Multiple comparisons o f the data by group consistently showed that the two lower doses were significantly less effective in producing changes in LPE than were the two highest doses (P<0.05). The intermediate dose (0.25 mg/kg) was mixed in difference. Comparable comparisons of the time data revealed a complex interaction of time post-injection with respect to dose. These comparisons suggested a curvilinear nature of the data and subsequently, a regression analysis was performed using the general linear models method and normalized,

predicted points respected for each dose across time (see Fig. 2). As expected, the predicted points showed that the higher doses had their major effects 25-45 min from injection and then diminished. The lower doses had a less dramatic effect but did not diminish as rapidly (detailed statistical manipulations and results are available upon request). Dramatic m o t o r behavior alterations (increased LPE, stereotypical head and paw movement) were observed in all animals at the three highest doses. At the 0.05 mg/kg dose, the effects were greatly reduced in one-half of the rats tested and all the rats receiving 0.005 mg/kg failed to show stereotypical m o t o r behaviors or an alteration in alertness. Only one rat at this dose showed an increase in LPE which was attributable to the MK-801.

Discussion

The most interesting finding of this study is that MK-801, a more potent non-competitive N M D A antagonist than ketamine and phencyclidine, is also a more powerful enhancer o f LPE than these two agents. The protrusions, retrusions, and swallows caused by MK-801 resemble in configuration those generated by ketamine or phencyclidine and cannot be distinguished from them,

465 whether perceived visually or recorded in a polygraph. We have proposed elsewhere (Aldes et al. 1988; M a r c o et al. 1988, 1989) that these LPE resemble the dyskinetic protrusions, retrusions and swallows observed in h u m a n tardive dyskinesia. Although MK-801 has been stated to cause disturbances in both sensory and m o t o r function (Wozniak et al. 1990), our findings represent the only available evidence that MK-801 is capable o f causing LPE of dyskinetic configuration similar to those caused by ketamine or phencyclidine. This is of interest from two perspectives: a) it suggest that the mechanism may involve the PCP receptor to which MK-801 binds m o r e potently than phencyctidine itself (Lodge and Anis 1982; W o n g et al. 1986; Olney et al. 1989) and b) it also suggests the possibility o f an endogenous PCP receptor tigand as a causative factor in schizophrenia (Snyder 1980: O ' D o n o h u e et al. 1983). The latter thesis would derive support from preliminary studies suggesting the existence of such endogenous ligand to the PCP receptor (Quirion et al. 1984). Such endogenous agents could explain the descriptions o f dyskinetic activity in chronic schizophrenics long before the advent o f neuroleptics (Kraeplin 1919; Bleuler 1950). One last point is of concern. Much and growing interest has been generated in recent years a b o u t the capabilities o f MK-801 as a neuroprotective agent in the treatment of various degenerative neurological disorders and ischemic tissue (Labruyere et al. 1986; Olney et al. 1986) but only recently have its neurotoxic and pathomorphological canges begun to be addressed (Olney et al. 1989). Although these morphological changes appear reversible under light microscopy, absence of irreversible changes at the ultrastructural level has yet to be demonstrated. The changes we have described in the results would warrant caution in its use against various types o f brain damage.

Acknowledgements.Sam Clemens and Kathy Burnt ran some of the experiments. We appreciate Amelia Harrington's skills in typing the manuscript. Work was supported by NINCDS Grant No. 5-20430 to L. A. M.

References Aldes LD, Prater SR, Chronister RB, Marco LA (1988) Ketamine as a pharmacological model for tongue dyskinesia. Biol Psychiatry 23:159-168 Anis NA, Berry SC, Burton NR, Lodge D (1983) The dissociative anesthetics, ketamine and phencyclidine, selectively reduce excitation of central mamalian neurons by N-methyl-aspartate. Br J Pharmacol 79: 565-575 Aram J, Church J, Davies SN, Lodge D, Martin D (1986) Comparison of phencyclidine, thienylcyclohexylpiperidine and MK-801 an NMDA antagonists on rat spinal and cortical neurones. Br J Pharmacol [Proc Suppl] 89: 778P Bleuler E (I950) Dementia precox or the group of schizophrenias (Trans. Zukin J) International University Press, New York Clineschmidt BV, Martin GE, Bunting PR (1982a) Anticonvulsant activity of (+)-5-methyl-10, l l-dihydro-5H-dibenzo[a,d] cyclohepten-5, 10-imine (MK-801), a substance with potent anticonvulsant, central sympathomimetic, and apparent anxiolytie properties. Drug Dev Res 2:123-134

Clineschmidt BV, Martin GE, Bunting PR, Papp NL (1982b) Central sympathominetic activity of (+)-5-methyl-10, 11 dihydro5H-dibenzo[a,d]cyclohepten-5, 10-imine (MK-801), a substance with potent anticonvulsant, central sympathomimetie, and apparent anxiolytic properties. Drug Dev Res 2:135-145 Clineschmidt BV, Williams M, Witoslawski JJ, Bunting PR, Risley EA, Totaro JA (1982c) Restoration of shock-suppressed behavior by treatment with (+)-5methyl-10, 11-dihydro-5H-dibenzo [a,d]cyclohepten-5, 10-imine (MK-801), a substance with potent anticonvulsant, central sympathomimetic, and apparent anxiolytic properties. Drug Dev Res 2:147-163 Corssen G, Domino EF (1966) Dissocciative anesthesia: further pharmacologic studies and first clinical experience with the phencyclidine derivative CI-581. Anesth Analg 45:29-40 Foster AC, Gill R, Iversen LL, Woodruff GN (1987) Systemic administration of MK-801 protects against ischaemia-induced hippocampal neurodegeneration in the gerbil. Br J Pharmaeol [Proc Suppl] 90: 9P Gerhardt SC, Bernard P, Pastor G, Boast CA (1986) Effects of systemic administration of the NMDA antagonist, CPP, on ischemic brain damage in gerbils. Soc Nenrosci Abstr 12: 59 Harrison NL, Simmonds MA (1985) Quantitative studies on some antagonists of N-methyl-D-aspartate in slices of rat cerebral cortex. Br J Pharmacol 84:381-391 Kemp JA, Priestley T, Woodruff GN (1986) MK-80I, a novel, orally active anticonvulsant, is a potent, non-competitive N-methyl-D-aspartate receptor antagonist. Br J Pharmacol [Proc Suppl] 89: 535P Kemp JA, Foster AC, Wong EHF (1987) Non-competitive antagonists of excitatory amino acid receptors. Trends Neurosci 10:294-298 Koek W, Kleen E, Mudar PJ, Woods JH (1986a) Phencyclidine-like catalepsy induced by the excitatory amino acid antagonist DL-2-amino-5-phosphonovalerate. Behav Brain Res 19: 257259 Koek W, Woods JH, Ornstein P (1986b) Phencyclidine-like behavioral effects in pigeons induced by sylstemic administration of the excitatory amino acid, antagonist, 2-amino-5phosphonovalerate. Life Sci 39:973-978 Koek W, Woods JH, Ornstein P (1987) A simple and rapid method for assessing similarities among directly observable behaw ioral effects of drugs: PCP-like effects of 2-amino-5phosphonovalerate in rats. Psychopharrnacology 91:297304 Kraeplin E (1919) Signs of mental disorder. Alienist and Neurologist 40: 85 Labruyere J, Fuller TA, Olney JW, Price MT, Zorumski C, Clifford D (1986) Phencyclidine and ketamine protect against kainic acidqnduced seizures and seizure-related brain damage. Soc Neurosci Abstr 12:344 Lodge D, Anis NA (1982) Effects of phencyclidine on exitatory amino acid activation of spinal interneurons in the cats. Eur J Pharmacol 77: 203-204 Maragos WF, Chu DCM, Greenamyre JT, Penny JB, Young AB (1986) 3H-TCP binding and NMDA receptors. Eur J Pharmaeol 123:173 174 Maragos WF, Penney JB, Young AB (1988) Anatomic correlation of NMDA and ~tt-TCP-labeled receptors in rat brain. Neurosci 8:493-501 Marco LA, Reed TF, Aldes LD, Chronister RB (1988) An electrographic characterization of ketamine-induced linguopharyngeal motor activity. Clin Neuropharmacol 11 : 141-150 Marco LA, Pendleton R, Reed TF, Bringham TE, Chronister RB, Aldes LD (1989) Ketamine-induced tongue protrusions in rats. Comp Biochem Physiol 94C:29-33 Martin D, Lodge D (1985) Ketamine acts as a non-competitive N-methyl-D-aspartate antagonist on frog spinal cord in vitro. Neuropharmaccology 24: 999-10003 O'Donohue TL, Pest CB, French E, Pert A, DiMaggio DA, Everist H, Quirion R (1983) Evidence of an endogenous CNS Ligand

466 for the PCP receptor. In: Hruby D J, Rich DH (eds) Proceedings of the Eighth American Peptide Symposium. Pierce Chemical Company, Rockford IL, pp 433-436 Olney JW, Price MT, Fuller TA, Labruere J, Samson L, Carpenter M, Mahan K (1986) The anti-excitotoxic effects of certain anesthetics, analgesics and sedative-hypnotics. Neurosci Lett 68 : 29-34 Olney JW, Labruyere J, Price MT (1989) Pathological changes induced in cerebrocortical neurons by phencycildine and related drugs. Science 244:1360-1362 Quirion R, DiMaggio DA, Frendh FD, Contreras PC, Shiloach J, Pert CB, Everist II, Pert A, O'Donohue T (1984) Evidence for an endogenous peptide ligand for the phencyclidine receptor. Peptides 5: 967-973 Piercey MF, Hoffmann WE, Kaczkofsky P (1988) Functional evidence for PCP-like effects of the anti-stroke candidate MK-801. Psychopharmacology 96: 561-562

Reimherr FW, Wood DR, Wender PH (1986) The use of MK-801, a novel sympathomimetic, in adults with attention deficit disorder, residual type. Psychopharmacol Bull 22:237-242 Simon RP, Swan JH, Griffiths T, Meldrum BS (1984) Blockade of N-methyl-D-aspartate receptors may protect against ischemic damage in the brain. Science 226:850-852 Snyder SH (1980) Phencyclidine. Nature 285:355-356 Wieloch T (1985) Hypoglycemia-induced neuronal damage prevented by an N-methybD-aspartate antagonist. Science 230: 681-683 Wong EHF, Kemp JA, Priestley T, Knight AR, Woodruff GN, Iversen L L (1986) The anticonvulsant MK-801 is a potent N-methyl-D-aspartate antagonist. Proc Natl Acad Sci USA 83 : 7104-7108 Woznisk DF, Olney JW, Kettinger L III, Price M, Miller JP (1990) Behavioral effects of MK-801 in the rat. Psychopharmaccology 101:47-56