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Tremor production by intracaudate injections of morphine
European Journal of Pharmacology, 32 (1975) 45--51 © North-Holland Publishing Company, Amsterdam - - Printed in The Netherlands
TREMOR PRODUCTION BY INTRACAUDATE INJECTIONS OF MORPHINE* PETER M. LALLEY, G. VICTOR ROSSI and WALTER W. BAKER
Department of Neuropharmacology, Eastern Pennsylvania Psychiatric Institute, and Department of Pharmacology, Philadelphia College of Pharmacy and Science, Philadelphia, Pennsylvania, U.S.A. Received 12 November 197'4, revised MS received 4 February 1975, accepted 10 February 1975
P.M. LALLEY, G.V. ROSSI and W.W. BAKER, Tremor production by intracaudate injections of morphine, European J. Pharmacol. 32 (1975) 45--51. Pronounced resting tremor was produced in unanesthetized cats by injecting morphine (25--110 p g ) i n t o the caudate nucleus. The effects of morphine were antagonized by intracaudate (i.c.) injections of nalorphine (81--263 pg). Tremor activity was also inhibited by i.c. injections of dopamine (61--145 pg), Ca 2÷ (24--40 pg), scopolamine (88--121 pg) and hemicholinium-3 (HC-3; 73--129 pg) while serotonin (125 pg) was ineffective. Tremor inhibition by HC-3 was reversed by i.c. doses of acetylcholine (15--30 pg) which were subthreshold for tremor production in the absence of morphine. Morphine (55--110 pg) further increased the intensity of ongoing tremor activity induced by physostigmine (111 pg i.c.). I.c. injections of nalorphine antagonized the motor effects of morphine without affecting physostigmine tremor. Tremor production by morphine is attributed to a reduction in dopamine function which allows cholinergic activity in the caudate nucleus to predominate.
Cholinergic predominance Intracaudate injection
Morphine
Tremor
1. Introduction Iatrogenic tremors can be elicited by injections of various chemical substances into the caudate nucleus of unanesthetized cats. A number of tremorgenic agents appear to exert their effects either by mimicking or enhancing the synaptic actions of acetylcholine in the caudate nucleus, or by suppressing the inhibitory actions of endogenous dopamine (Connor et al., 1966; Lalley et al., 1973). It has been proposed that the underlying cause of these tremors is a neurochemical imbalance in the caudate resulting in a state of cholinergic predominance. This mechanism would explain why cholinomimetic * Address reprint requests to: Peter M. Lalley, Ph.D., Department of Pharmacology and Therapeutics, The University of Florida College of Medicine, Box 728, The J. Hillis Miller Medical Center, Gainesville, Florida 32610, U.S.A.
Caudate nucleus
Dopamine
substances such as carbachol or physostigmine, and agents such as tetrabenazine or bretylium which inhibit dopamine synthesis or release, elicit tremor activity following intracaudate injection (Lalley et al., 1973). There is evidence that morphine blocks the release of acetylcholine from peripheral nerve terminals (Paton, 1957; Schaumann, 1957}, from motoneuronal axon collaterals to tenshaw cells (Duggan and Curtis, 1972; Lodge et al., 1974), and from neurons in structures adjacent to the lateral cerebral ventricles (Beleslin and Polak, 1965). The caudate nucleus appears to be the predominant source of ventricular acetylcholine. We were therefore interested at the start of this study in determining if morphine might by an identical action inhibit the tremor which follows cholinesterase inhibition by physostigmine. In preliminary experiments, morphine was injected into the caudate nucleus during peak physostigmine-
46 induced tremor activity. The results were quite unexpected in that morphine enhanced ongoing tremor activity. Further experiments revealed t h a t morphine in the absence of physostigmine is a p o t e n t tremorgen. A partial account of these observations was recently published {Baker et al., 1974).
2. Materials and methods 2.1. Preparation o f cats for chronic studies 24 chronically prepared cats were utilized in the present investigation. The methods were similar to those described in detail in earlier studies (Connor et al., 1966; Lalley et al., 1970). An electrode-injection assembly ('injectrode') was permanently implanted during pentobarbital anesthesia into each caudate nucleus at coordinates A~ s, Ls and Rs, D+ s {Jasper and Ajmone-Marsan, 1953). Bipolar {twisted wire) recording electrodes were implanted in the substantia nigra (A6, L4 and R4, D_4), dorsal hippocampus (A2, Ls and Rs, D+ s)and positioned on the frontal (postcruciate) cortex. Insulated lead-off wires were terminated in an amphenol plug (series 223--1217) which was anchored to the skull with acrylic dental cement and hard sealing wax. 2.2. Tremor recording in unanesthetized cats After a recovery period at least two weeks, the unanesthetized cat was suspended comfortably in a canvas sling which permitted free movement of the left hindlimb, from which m o t o r activity was recorded with a lightweight piezoelectric stereo phonocartridge (Astatic 13TB) affixed to the dorsal {metatarsal) surface of the foot. Motor activity was recorded from only one hindlimb i n t h e present investigation. However, previous experiments established that iatrogenic tremors develop in all limbs w i t h o u t any distinct lateral predominance (Connor et al., 1966). Tremor and electrographic activities were continuously monitored on an oscilloscope and recorded on an
P.M. LALLEY ET AL. electroencephalograph at appropriate intervals. Tremor responses were analyzed and expressed in terms of the amplitude of the hindlimb t r e m o r excursions, tremor frequency, the number of tremor bursts per minute, and the percentage of time, measured over one minute epochs, during which tremor activity occurred. The two latter criteria were necessary since tremor activity was not continuous but occurred in bursts. Tremor amplitudes are expressed in terms of the measured o u t p u t of the phonocartridge, in microvolts. Increases in tremor amplitude produced by morphine during ongoing physostigmine tremor activity were analyzed for statistical significance by a two-sided comparison of paired data (Snedecor, 1956). 2. 3. Drug injections in to the caudate nucleus All microinjections were made into the head of the caudate nucleus by means of a 24 gauge delivery cannula coupled to a calibrated micrometer-driven hypodermic syringe. The injection cannula was inserted through the 20 gauge guide of the 'injectrode' assembly. Only one caudate nucleus was utilized in any given experiment. Concentrations of drug solutions were chosen so that microinjected volumes did not exceed 10 pl. Pilot experiments showed that alterations of pH and tonicity over a wide range produced no discernible effect on tremor activity or on other drug responses. Injections of distilled water, saline or isotonic dextrose (5--10 pl) prior to or during tremor activity were w i t h o u t effect. Thus fluid volume per se did not appear to influence motor-regulatory mechanisms. Solutions of the following compounds dissolved in distilled water were employed: morphine sulfate, physostigmine sulfate, acetylcholine chloride, calcium chloride, dopamine hydrochloride, hemicholinium-3 (HC-3) hydrobromide, scopolamine hydrochloride, serotonin creatine sulfate, and Nallylnormorphine (nalorphine) hydrochloride. Drug doses are expressed as the free base. Animals were rested at least one week between experiments.
47
TREMOR PRODUCTION BY MORPHINE
2. 4. Confirmation of electrode placements At the end of the study, electrolytic lesions were produced by passing direct current through the electrode tips. The electrode placement was confirmed by examination of coronal sections of the formalin-fixed brains.
TABLE 1 Facilitation of physostigmine tremor activity by intracaudate (i.c.) injections of morphine. Dose of morphine
n= 9
Average latency**
Control ampli° tude***
Amplitude after mor
-
phinet
3. Results
3.1. Additive tremor effects of physostigmine and morphine Injection of physostigmine into the caudate nucleus of unanesthetized cats was shown in earlier studies (Connor et al., 1966; Lalley et al., 1970) to elicit tremor activity. The most intense tremor responses were produced by 150 pg doses of physostigmine sulfate (equivalent to 111 tzg of the base). Further increases in tremor intensity were not observed at higher doses, nor was there any change in ongoing activity when acetylcholine (15--30 pg) was injected. In the present study, control tremor activity was induced in 9 different cats with 111/ag intracaudate (i.c.) injections of physostigmine. When tremor activity had reached peak, sustained intensities, morphine was injected into the caudate nucleus. Doses of 55 pg to 110 pg produced a further increase in tremor activity which was maximal within 5 min (table 1). The tremor amplitude showed the greatest increase (approximately two-fold), although the duration of individual tremor bursts also increased. In 5 experiments, nalorphine was administered in order to determine if the augmented motor activity could be selectively reversed. In all experiments, i.c. injections of nalorphine (67--268 #g) decreased the tremor intensity to control levels within 30 min. These findings suggested that morphine had independent tremorgenic actions.
3.2. Tremor induction by i.c. morphine The independent tremorgenic actions of morphine were confirmed in succeeding experi-
88 pg (55--110)*
~ S.E.
4.8 min + 0.8
256.9 pV -+ 55.2
573.2 pV -+ 138.4
* Range of morphine doses injected into homolateral caudate nucleus during physostigmine tremor activity. ** Latency for maximum increase in tremor amplitude after morphine injection. *** Amplitude of peak control tremors evoked by 111 pg physostigmine i.c. t Amplitude of enhanced tremor activity after i.c. morphine injection. Increase in tremor activity is statistically significant (0.05 > p > 0.025).
ments. In the first experiment of this series, a single 110/ag dose was injected. This dose of morphine was supramaximal since in 8 additional experiments, maximal tremor activity of approximately the same intensity was observed TABLE 2 Characteristics of tremor produced by i.c. injections of morphine*. 1. Dose of morphine 2. Latency***
53 pg (25--110)** 4.7 min -+
1.3 (S.E.)t
3. Characteristics of peak tremor activity: (a) onset of peak tremor activity
12.9 rain +- 2.3
(b) duration of peak activity
175.3 min -+ 9.9
(c) amplitude
339.2 pV -+ 42.5
(d) frequency
25.2 cps +- 0.7
(e) tremor bursts/min
22.0
-+ 0.9
(f) tremor time (%)
69.7
-+ 2.7
* ** *** t
Values obtained from 9 trials on 9 different cats. Mean dose; range of doses in parentheses. Time t o appearance of earliest tremor activity. Standard error.
48
P.M. LALLEY ET AL.
within a d o s e range o f 2 5 - - 5 5 pg. In t h e s e e x p e r i m e n t s doses were increased in 10 or 15 pg i n c r e m e n t s f r o m an initial i n j e c t i o n o f 15 pg t o p r o d u c e m a x i m a l t r e m o r activity. T h e characteristics o f t r e m o r a c t i v i t y o b s e r v e d in all 9 e x p e r i m e n t s are p r e s e n t e d in t a b l e 2. T r e m o r a c t i v i t y was n o t i c e a b l e w i t h i n 5 m i n on t h e average. T h e single 1 1 0 pg d o s e in t h e initial e x p e r i m e n t p r o d u c e d t h e first d e t e c t a b l e tremor within 1 min. In t h e o t h e r e x p e r i m e n t s t h e latencies r a n g e d f r o m 1 t o 9 m i n f o r t r e m o r o n s e t a n d 7 t o 24 m i n f o r full d e v e l o p m e n t o f m o t o r activity. F o r all e x p e r i m e n t s t h e durat i o n o f p e a k t r e m o r a c t i v i t y r a n g e d f r o m 143 to 2 1 0 min ( m e a n , 1 7 5 m i n ) b e f o r e declining to l o w e r intensities. T h e r e w e r e n o d e t e c t a b l e changes in gross b e h a v i o r , e l e c t r o g r a p h i c activit y or p u p i l d i a m e t e r p r i o r to, or d u r i n g t r e m o r activity.
3.3. Pharmacological alterations of morphine tremor An analysis o f t h e m o d e o f t r e m o r p r o d u c t i o n b y m o r p h i n e was a t t e m p t e d b y injecting v a r i o u s drugs i n t o t h e c a u d a t e n u c l e u s d u r i n g m a x i m a l t r e m o r activity and o b s e r v i n g t h e i r effects. The results o f t h e s e e x p e r i m e n t s are s u m m a r i z e d in t a b l e 3.
3.3.1. Tremor antagonism by nalorphine The opiate antagonist nalorphine (81--263 pg) r e d u c e d o n g o i n g t r e m o r a c t i v i t y t o v e r y
l o w levels within 25 t o 35 min, a f t e r an initial increase in i n t e n s i t y . R e c o v e r y o f t r e m o r activit y was n e v e r o b s e r v e d .
3.3.2. Inhibition by dopamine I n t r a c a u d a t e injections o f d o p a m i n e abolished all signs o f t r e m o r within 2 t o 6 min in 6 e x p e r i m e n t s (fig. 1 A). In 5 e x p e r i m e n t s , doses ranging f r o m 61 pg t o 101 pg a b o l i s h e d t r e m o r within 2 to 4 min. T h e d u r a t i o n o f i n h i b i t i o n , f r o m t o t a l c e s s a t i o n to c o m p l e t e r e c o v e r y , r a n g e d f r o m 54 to 71 min. In o n e e x p e r i m e n t , a t o t a l o f 1 4 5 p g was r e q u i r e d t o p r o d u c e c o m p l e t e i n h i b i t i o n o f t r e m o r activity, w h i c h lasted 30 min.
3.3.3. Effects of hemicholinium, scopolamine, and acetylcholine T h e e f f e c t o f a r e d u c t i o n in cholinergic a c t i v i t y o n m o r p h i n e t r e m o r was t e s t e d in o t h e r e x p e r i m e n t s b y injecting e i t h e r HC-3 or s c o p o l a m i n e . I n j e c t i o n s o f HC-3 ( 7 3 - - 1 2 9 pg) a b o l i s h e d t r e m o r within 38 t o 48 m i n (fig. 1B, c o l u m n 2). T r e m o r i n h i b i t i o n was a c c o m p a nied b y h i g h - a m p l i t u d e s y n c h r o n o u s slow waves w h i c h in m o s t e x p e r i m e n t s were observed o n l y in r e c o r d i n g s f r o m t h e i n j e c t i o n site, a n d bilaterally f r o m the m o t o r c o r t e x . In 5 e x p e r i m e n t s , t r e m o r i n h i b i t i o n b y HC-3 was o b s e r v e d f o r at least 30 rain, w h e r e u p o n acetylc h o l i n e was i n j e c t e d (1 or 2 injections, 15 pg p e r dose, i.c.). In all eases m a x i m a l t r e m o r a c t i v i t y was r e s t o r e d w i t h i n 8 to 20 min b y
TABLE 3 Effects of locally injected drugs on peak tremor activity elicited by intracaudate morphine. Locally injected drug
Effect on tremor*
Activity parameters . . . . . . . . . . . . . . . . . . . . . . . . . . Dose (pg) Latency (min) Activity ratio**
Nalorphine Dopamine Hemicholinium Scopolamine Ca 2÷
($) (--) (--) (--) (--)
130 67 102 100 32
(81--263) (61--145) (73--129) (81--121) (24-- 40)
27.8 4.0 42.2 14.5 5.2
-+ 6.1 + 0.5 ± 1.5 -+ 2.0 ± 1.1
5/5 6/6 6/6 6/6 6/6
* (~) = tremor activity decreased to low level; tremor could not be completely eliminated. (--) = tremor activity abolished; values in parentheses represent range of doses required for complete tremor inhibition * * Activity ratio = (no. of experiments where drug altered tremor)/( total no. of experiments).
TREMOR PRODUCTION BY MORPHINE
49
4 l I N . AFTER DA
I0 MIN. MrlrER ~
(soj, a I.¢.)
3.
( |1 kilH.)
ACTIVITY
( 3S kilfL )
36 M,N. AFTER HC-3 (ISOj, G I.e.)
(SS~S Lc.)
(iOHIN.)
-"
15 MIN. AFTER AClk (,~)~S I.e.)
B ( ,4s kiIN.)
ACTIVITY I
2.
| 170 kilN. )
CALCIUM SUPPRESSION. 8 kilN: AFTER CACLz ZH~O
TREMOR
RECOVERY
24 G CA++ I~¢ i
IL C.AUDAI'E
( aS U ~ )
z.
Fig. 1. Inhibition of maximal morphine tremor activity by intracaudate (i.c.) injections of dopamine (A), hemicholinium-3 (B) and calcium (C). In each panel, upper tracings are caudate electrograms and lower tracings are tremor recordings. Numbers in parentheses below tremor tracings indicate the time sequence in minutes, continuous from the injection of morphine. Columns I and 2 in (A) show the onset of tremor and its progression to peak activity, respectively. In (B) and (C), control tremors in column I are at peak intensities. Tremor activity was reinstated in (B), column 3 by i.c. acetylcholine. The dose of HC-3 in (B), column 2 is expressed as the salt, equivalent to 129 pg of the base. Calibrations are 50 p V and i sec for all tracings.
acetylcholine (fig. 1B, column 3). The re-established tremor activity persisted on the average for 30 min before declining again. In the one experiment in which tremor activity was allowed to recover completely without injecting acetylcholine, the duration of inhibition following injection of HC-3 was 104 min. Scopolamine (81--121 pg i.c.) abolished tremor activity within 8 to 21 min. Tremor inhibition was not reversible with acetylcholine and no a t t e m p t was made to determine the duration of inhibition. Intracaudate injections of acetylcholine (32 pg) had no effect during the maximal tremor
activity elicited by morphine, nor was there any change in electrographic patterns. 3.3.4. Effects of calcium and sero tonin Injection of calcium chloride (24---40 #g of Ca ~÷) resulted in complete cessation of tremor activity within 1 to 9 min (table 3; fig. 1C, column 2). The duration of inhibition ranged from 40 to 51 min in 6 experiments. Results from previous studies indicate that the acetate and chloride salts of calcium are equally effective in depressing tremor activity (Lalley et al., 1971). Cumulative doses of serotonin up to 125 pg
5o had no effect on tremor activity in 6 experiments.
4. Discussion Intracaudate injections of morphine in the present investigation produced further increases in tremor intensity, above the control activity elicited by physostigmine. The increased activity generated by morphine was unrelated to an action at cholinergic synapses, since the morphine c o m p o n e n t was selectively antagonized by nalorphine, w i t h o u t altering the tremor produced by physostigmine. In an earlier study (Lalley et al., 1973) we demonstrated that i.c. injections of bretylium or tetrabenazine, whose respective actions in blocking catecholamine release or depleting neuronal stores of monoamines are known, produced tremors that were abolished by dopamine, Ca 2÷, scopolamine and HC-3, b u t were unaffected by serotonin. We concluded that the tremors were the result of a functional deficit in dopamine in the caudate nucleus which allowed cholinergic activity to predominate. Morphine might be acting in a similar fashion in the present study. Tremor activity was abolished by dopamine, scopolamine, and in a reversible manner by HC-3, b u t was unaffected b y serotonin. In addition, other investigators have shown that morphine, along with several psychoactive drugs including haloperidol, chlorpromazine and oxypertine, increase dopamine turnover in the striatum (Carlsson, 1967; Gunne and Jonsson, 1969; Clouet and Ratnor, 1970; Anddn et al., 1971}. Haloperidol and chlorpromazine are known for their tremorgenic effects which are attributed to a blockade of striatal dopamine receptors. Both drugs also increase acetylcholine release in the cat caudate nucleus (Stadler et al., 1973), which might be expected to further enhance cholinergic predominance, and therefore tremor production. The increase in dopamine turnover which these agents produce is evidently secondary to receptor blockade, by virtue of a feedback mechanism that regulates dopamine re-
P.M. LALLEY ET AL. lease as a function of post-synaptic receptor activation. A similar mode of action would explain the rapid appearance of morphineinduced tremor activity in the present study, and would also account for the increase in dopamine turnover reported by others. The inhibition of tremor by Ca 2÷ is notew o r t h y in view of recent discoveries. The analgesic effects of morphine are depressed by intracisternal injections of Ca 2+ whereas the chelating agent, Na2 EDTA, increases analgesic efficacy (Kakunaga et al., 1966). Morphine inhibits the in vitro binding of Ca 2÷ to phospholipids, depresses the facilitated transport of Ca 2÷ by phospholipids (Mule, 1969) and reduces Ca 2÷ levels in the corpus striatum (Ross et al., 1974). Although these actions may contribute to tremorgenesis, we are inclined to dismiss them as major components to the tremorgeni c action of morphine. A reduction of Ca 2÷ levels in the caudate nucleus does indeed lead to tremor activity. I.c. injections of Na2 EDTA were tremorgenic in an earlier study (Lalley et al., 1971}, whereas Na2 CaEDTA was not. However, when Ca 2÷ (32--67 #g) was injected during peak activity, the tremor was abolished and did not reappear during observation periods of 2 to 3 hours. In the present investigation, the inhibition of morphine tremor activity by Ca 2÷ was relatively short-lived (40--50 min). Furthermore, Ca 2÷ also inhibits the tremors elicited by bretylium and tetrabenazine for comparable durations. The inhibition therefore appears to be relatively nonspecific and might be related to an action which stabilized neuronal membranes (Goldman and Blaustein, 1966; Ritchie et al., 1965). Additional actions have been cited in explaining the various effects of morphine. These include a direct excitant action on neurons (Duggan and Curtis, 1972}, blockade of inhibitory amino acid receptors (Curtis and Duggan, 1969) and possibly a release of serotonin from nerve terminals (Mayer and Liebeskind, 1974). None of these mechanisms adequately explain the tremorgenic effects of morphine in this investigation. Injection of excitatory amino acids such as N-methyl-D,L-aspartic acid elicits
TREMOR
PRODUCTION
BY MORPHINE
rage behavior and mydriasis in unanesthetized cats (Baker, 1972). The GABA antagonist, picrotoxin, produces tremor only at doses which lead to seizures and postictal depression (Lalley, unpublished observations). I.c. injections of serotonin evoke tremor, but the tremor is totally resistant to HC-3 and scopolamine (Malseed and Baker, 1973). None of these features are characteristics of morphine tremor activity, whereas similarities between the tremorgenic psychoactive drugs and morphine suggest similar actions in blocking dopamine receptors in the caudate nucleus.
Acknowledgements This research was supported by grant MH-08833 from the National Institute of Mental Health, U.S. Public Health Service. The authors express their appreciation to Mr. Ron Mahoney for assistance with the photography, and to Mrs. Eileen Forsythe for assistance in the preparation of the manuscript.
References And~n, N.E., H. Corrodi, K. Fuxe and U. Ungerstedt, 1971, Importance of nervous impulse flow for the neuroleptic induced increase in amine turnover in central dopamine neurons, European J. Pharmacol. 15, 193. Baker, W.W., 1972, Excitatory responses following intracaudate injection of N-methyl-d,l-aspartic acid, Arch. Intern. Pharmacodyn. 196, 226. Baker, W.W., P.M. Lalley and R.L. Young, 1974, Biphasic effects of intracaudate morphine on caudate functioning, Federation Proc. Abstr. 293. Beleslin, P. and R.L. Polak, 1965, Depression by morphine and chloralose of acetylcholine release from the cat's brain, J. Physiol. 177, 411. Carlsson, A., 1967, Basic actions of psychoactive drugs, Intern. J. Neurobiol. 6, 27. Clouet, D.H. and M. Ratnor, 1970, Catecholamine biosynthesis in brains of rats treated with morphine, Science 163,854. Connor, J.D., G.V. Rossi and W.W. Baker, 1966, Characteristics of tremor following injections of carbachol into the caudate nucleus, Exptl. Neurol. 14, 371. Curtis, D.R. and A.W. Duggan, 1969, Depression of spinal inhibition by morphine, Agents and Actions 1, 14. Duggan, A.W. and D.R. Curtis, 1972, Morphine and the synaptic activation of renshaw cells, Neuropharmacol. 11,189.
51
Goldman, D.E. and M.P. Blaustein, 1966, Ions, drugs and the axon membrane, Ann. N.Y. Acad. Sei. 137, 967. Gunne, L.M. and J. Jonsson, 1969, Effects of morphine intoxication on brain cateeholamine neurons, European J. Pharmacol. 5, 338. Jasper, H.H. and C. Ajmone-Marson, 1953, A stereotoxic atlas of the diencephalon of the cat, National Research Council of Canada, Ottowa. Kakunaga, T., H. Kaneta and K. Hano, 1966, Pharmacologic studies on analgesics-VII. Significance of the calcium ion in morphine analgesia, J. Pharmacol. Exptl. Therap. 153,134. l.~lley, P.M., G.V. Rossi and W.W. Baker, 1970, Analysis of local cholinergic tremor mechanisms following selective neurochemical lesions, Exptl. Neurol. 27, 358. Lalley, P.M., G.V. Rossi and W.W. Baker, 1971, Alterations in tremor regulation after intracaudate injections of calcium ions or disodium edetate, Neuropharmacol. 10, 613. Lalley, P.M., G.V. Rossi and W.Wo Baker, 1973, Tremor induction by intracaudate injections of bretylium tetrabenazine or mescaline: Functional deficits in caudate dopamine, J. Pharm. Sci. 62, 1302. Lodge, D., M. Headley, A.W. Duggan and T.J. Biscoe, 1974, The effects of morphine, etorphine and sinomenine on the chemical sensitivity and synaptic responses of renshaw cells and other spinal neurones in the cat, European J. Pharmacoi. 26, 277. Malseed, R.T. and W.W. Baker, 1973, Analysis of tre morgenic effects of intracaudate serotonin, Proc. Soc. Exptl. Biol. Med. 143, 1088. Mayer, D.J. and J.C. Liebeskind, 1974, Pain reduction by focal electrical stimulation of the brain: an anatomical and behavioral analysis, Brain Res. 68, 73. Mul~, S.J., 1969, Inhibition of phospholipid-facilitated calcium transport by central nervous system-acting drugs, Biochem. Pharmacol. 18, 339. Paton, W.D.M., 1957, The action of morphine and related substances on contraction and on acetylcholine o u t p u t of coaxially stimulated guinea pig ileum, Brit. J. Pharmacol. 12, 119. Ross, D.W., M.A. Medina and H.L. Cardenas, 1974, Morphine and ethanol: Selective depletion of regional brain calcium, Science 186, 63. Ritchie, J.M., B. Ritchie and P. Greengard, 1965, The active structure of local anesthetics, J. Pharmacol. Exptl. Therap. 150, 152. Sehaumann, W., 1957, Inhibition by morphine of the release of acetylcholine from the intestine of the guinea-pig, Brit. J. Pharmacol. 12, 115. Snedecor, G.W., 1956, Statistical Methods (The Iowa State University Press, Ames, Iowa, U.S.A. ). Stadler, H., K.G. Lloyd, M. Gadea-Ciria and G. Bartholini, 1973, Enhanced striatal acetylcholine release by chlorpromazine and its reversal by apomorphine, Brain Res. 55,476.
TREMOR PRODUCTION BY INTRACAUDATE INJECTIONS OF MORPHINE* PETER M. LALLEY, G. VICTOR ROSSI and WALTER W. BAKER
Department of Neuropharmacology, Eastern Pennsylvania Psychiatric Institute, and Department of Pharmacology, Philadelphia College of Pharmacy and Science, Philadelphia, Pennsylvania, U.S.A. Received 12 November 197'4, revised MS received 4 February 1975, accepted 10 February 1975
P.M. LALLEY, G.V. ROSSI and W.W. BAKER, Tremor production by intracaudate injections of morphine, European J. Pharmacol. 32 (1975) 45--51. Pronounced resting tremor was produced in unanesthetized cats by injecting morphine (25--110 p g ) i n t o the caudate nucleus. The effects of morphine were antagonized by intracaudate (i.c.) injections of nalorphine (81--263 pg). Tremor activity was also inhibited by i.c. injections of dopamine (61--145 pg), Ca 2÷ (24--40 pg), scopolamine (88--121 pg) and hemicholinium-3 (HC-3; 73--129 pg) while serotonin (125 pg) was ineffective. Tremor inhibition by HC-3 was reversed by i.c. doses of acetylcholine (15--30 pg) which were subthreshold for tremor production in the absence of morphine. Morphine (55--110 pg) further increased the intensity of ongoing tremor activity induced by physostigmine (111 pg i.c.). I.c. injections of nalorphine antagonized the motor effects of morphine without affecting physostigmine tremor. Tremor production by morphine is attributed to a reduction in dopamine function which allows cholinergic activity in the caudate nucleus to predominate.
Cholinergic predominance Intracaudate injection
Morphine
Tremor
1. Introduction Iatrogenic tremors can be elicited by injections of various chemical substances into the caudate nucleus of unanesthetized cats. A number of tremorgenic agents appear to exert their effects either by mimicking or enhancing the synaptic actions of acetylcholine in the caudate nucleus, or by suppressing the inhibitory actions of endogenous dopamine (Connor et al., 1966; Lalley et al., 1973). It has been proposed that the underlying cause of these tremors is a neurochemical imbalance in the caudate resulting in a state of cholinergic predominance. This mechanism would explain why cholinomimetic * Address reprint requests to: Peter M. Lalley, Ph.D., Department of Pharmacology and Therapeutics, The University of Florida College of Medicine, Box 728, The J. Hillis Miller Medical Center, Gainesville, Florida 32610, U.S.A.
Caudate nucleus
Dopamine
substances such as carbachol or physostigmine, and agents such as tetrabenazine or bretylium which inhibit dopamine synthesis or release, elicit tremor activity following intracaudate injection (Lalley et al., 1973). There is evidence that morphine blocks the release of acetylcholine from peripheral nerve terminals (Paton, 1957; Schaumann, 1957}, from motoneuronal axon collaterals to tenshaw cells (Duggan and Curtis, 1972; Lodge et al., 1974), and from neurons in structures adjacent to the lateral cerebral ventricles (Beleslin and Polak, 1965). The caudate nucleus appears to be the predominant source of ventricular acetylcholine. We were therefore interested at the start of this study in determining if morphine might by an identical action inhibit the tremor which follows cholinesterase inhibition by physostigmine. In preliminary experiments, morphine was injected into the caudate nucleus during peak physostigmine-
46 induced tremor activity. The results were quite unexpected in that morphine enhanced ongoing tremor activity. Further experiments revealed t h a t morphine in the absence of physostigmine is a p o t e n t tremorgen. A partial account of these observations was recently published {Baker et al., 1974).
2. Materials and methods 2.1. Preparation o f cats for chronic studies 24 chronically prepared cats were utilized in the present investigation. The methods were similar to those described in detail in earlier studies (Connor et al., 1966; Lalley et al., 1970). An electrode-injection assembly ('injectrode') was permanently implanted during pentobarbital anesthesia into each caudate nucleus at coordinates A~ s, Ls and Rs, D+ s {Jasper and Ajmone-Marsan, 1953). Bipolar {twisted wire) recording electrodes were implanted in the substantia nigra (A6, L4 and R4, D_4), dorsal hippocampus (A2, Ls and Rs, D+ s)and positioned on the frontal (postcruciate) cortex. Insulated lead-off wires were terminated in an amphenol plug (series 223--1217) which was anchored to the skull with acrylic dental cement and hard sealing wax. 2.2. Tremor recording in unanesthetized cats After a recovery period at least two weeks, the unanesthetized cat was suspended comfortably in a canvas sling which permitted free movement of the left hindlimb, from which m o t o r activity was recorded with a lightweight piezoelectric stereo phonocartridge (Astatic 13TB) affixed to the dorsal {metatarsal) surface of the foot. Motor activity was recorded from only one hindlimb i n t h e present investigation. However, previous experiments established that iatrogenic tremors develop in all limbs w i t h o u t any distinct lateral predominance (Connor et al., 1966). Tremor and electrographic activities were continuously monitored on an oscilloscope and recorded on an
P.M. LALLEY ET AL. electroencephalograph at appropriate intervals. Tremor responses were analyzed and expressed in terms of the amplitude of the hindlimb t r e m o r excursions, tremor frequency, the number of tremor bursts per minute, and the percentage of time, measured over one minute epochs, during which tremor activity occurred. The two latter criteria were necessary since tremor activity was not continuous but occurred in bursts. Tremor amplitudes are expressed in terms of the measured o u t p u t of the phonocartridge, in microvolts. Increases in tremor amplitude produced by morphine during ongoing physostigmine tremor activity were analyzed for statistical significance by a two-sided comparison of paired data (Snedecor, 1956). 2. 3. Drug injections in to the caudate nucleus All microinjections were made into the head of the caudate nucleus by means of a 24 gauge delivery cannula coupled to a calibrated micrometer-driven hypodermic syringe. The injection cannula was inserted through the 20 gauge guide of the 'injectrode' assembly. Only one caudate nucleus was utilized in any given experiment. Concentrations of drug solutions were chosen so that microinjected volumes did not exceed 10 pl. Pilot experiments showed that alterations of pH and tonicity over a wide range produced no discernible effect on tremor activity or on other drug responses. Injections of distilled water, saline or isotonic dextrose (5--10 pl) prior to or during tremor activity were w i t h o u t effect. Thus fluid volume per se did not appear to influence motor-regulatory mechanisms. Solutions of the following compounds dissolved in distilled water were employed: morphine sulfate, physostigmine sulfate, acetylcholine chloride, calcium chloride, dopamine hydrochloride, hemicholinium-3 (HC-3) hydrobromide, scopolamine hydrochloride, serotonin creatine sulfate, and Nallylnormorphine (nalorphine) hydrochloride. Drug doses are expressed as the free base. Animals were rested at least one week between experiments.
47
TREMOR PRODUCTION BY MORPHINE
2. 4. Confirmation of electrode placements At the end of the study, electrolytic lesions were produced by passing direct current through the electrode tips. The electrode placement was confirmed by examination of coronal sections of the formalin-fixed brains.
TABLE 1 Facilitation of physostigmine tremor activity by intracaudate (i.c.) injections of morphine. Dose of morphine
n= 9
Average latency**
Control ampli° tude***
Amplitude after mor
-
phinet
3. Results
3.1. Additive tremor effects of physostigmine and morphine Injection of physostigmine into the caudate nucleus of unanesthetized cats was shown in earlier studies (Connor et al., 1966; Lalley et al., 1970) to elicit tremor activity. The most intense tremor responses were produced by 150 pg doses of physostigmine sulfate (equivalent to 111 tzg of the base). Further increases in tremor intensity were not observed at higher doses, nor was there any change in ongoing activity when acetylcholine (15--30 pg) was injected. In the present study, control tremor activity was induced in 9 different cats with 111/ag intracaudate (i.c.) injections of physostigmine. When tremor activity had reached peak, sustained intensities, morphine was injected into the caudate nucleus. Doses of 55 pg to 110 pg produced a further increase in tremor activity which was maximal within 5 min (table 1). The tremor amplitude showed the greatest increase (approximately two-fold), although the duration of individual tremor bursts also increased. In 5 experiments, nalorphine was administered in order to determine if the augmented motor activity could be selectively reversed. In all experiments, i.c. injections of nalorphine (67--268 #g) decreased the tremor intensity to control levels within 30 min. These findings suggested that morphine had independent tremorgenic actions.
3.2. Tremor induction by i.c. morphine The independent tremorgenic actions of morphine were confirmed in succeeding experi-
88 pg (55--110)*
~ S.E.
4.8 min + 0.8
256.9 pV -+ 55.2
573.2 pV -+ 138.4
* Range of morphine doses injected into homolateral caudate nucleus during physostigmine tremor activity. ** Latency for maximum increase in tremor amplitude after morphine injection. *** Amplitude of peak control tremors evoked by 111 pg physostigmine i.c. t Amplitude of enhanced tremor activity after i.c. morphine injection. Increase in tremor activity is statistically significant (0.05 > p > 0.025).
ments. In the first experiment of this series, a single 110/ag dose was injected. This dose of morphine was supramaximal since in 8 additional experiments, maximal tremor activity of approximately the same intensity was observed TABLE 2 Characteristics of tremor produced by i.c. injections of morphine*. 1. Dose of morphine 2. Latency***
53 pg (25--110)** 4.7 min -+
1.3 (S.E.)t
3. Characteristics of peak tremor activity: (a) onset of peak tremor activity
12.9 rain +- 2.3
(b) duration of peak activity
175.3 min -+ 9.9
(c) amplitude
339.2 pV -+ 42.5
(d) frequency
25.2 cps +- 0.7
(e) tremor bursts/min
22.0
-+ 0.9
(f) tremor time (%)
69.7
-+ 2.7
* ** *** t
Values obtained from 9 trials on 9 different cats. Mean dose; range of doses in parentheses. Time t o appearance of earliest tremor activity. Standard error.
48
P.M. LALLEY ET AL.
within a d o s e range o f 2 5 - - 5 5 pg. In t h e s e e x p e r i m e n t s doses were increased in 10 or 15 pg i n c r e m e n t s f r o m an initial i n j e c t i o n o f 15 pg t o p r o d u c e m a x i m a l t r e m o r activity. T h e characteristics o f t r e m o r a c t i v i t y o b s e r v e d in all 9 e x p e r i m e n t s are p r e s e n t e d in t a b l e 2. T r e m o r a c t i v i t y was n o t i c e a b l e w i t h i n 5 m i n on t h e average. T h e single 1 1 0 pg d o s e in t h e initial e x p e r i m e n t p r o d u c e d t h e first d e t e c t a b l e tremor within 1 min. In t h e o t h e r e x p e r i m e n t s t h e latencies r a n g e d f r o m 1 t o 9 m i n f o r t r e m o r o n s e t a n d 7 t o 24 m i n f o r full d e v e l o p m e n t o f m o t o r activity. F o r all e x p e r i m e n t s t h e durat i o n o f p e a k t r e m o r a c t i v i t y r a n g e d f r o m 143 to 2 1 0 min ( m e a n , 1 7 5 m i n ) b e f o r e declining to l o w e r intensities. T h e r e w e r e n o d e t e c t a b l e changes in gross b e h a v i o r , e l e c t r o g r a p h i c activit y or p u p i l d i a m e t e r p r i o r to, or d u r i n g t r e m o r activity.
3.3. Pharmacological alterations of morphine tremor An analysis o f t h e m o d e o f t r e m o r p r o d u c t i o n b y m o r p h i n e was a t t e m p t e d b y injecting v a r i o u s drugs i n t o t h e c a u d a t e n u c l e u s d u r i n g m a x i m a l t r e m o r activity and o b s e r v i n g t h e i r effects. The results o f t h e s e e x p e r i m e n t s are s u m m a r i z e d in t a b l e 3.
3.3.1. Tremor antagonism by nalorphine The opiate antagonist nalorphine (81--263 pg) r e d u c e d o n g o i n g t r e m o r a c t i v i t y t o v e r y
l o w levels within 25 t o 35 min, a f t e r an initial increase in i n t e n s i t y . R e c o v e r y o f t r e m o r activit y was n e v e r o b s e r v e d .
3.3.2. Inhibition by dopamine I n t r a c a u d a t e injections o f d o p a m i n e abolished all signs o f t r e m o r within 2 t o 6 min in 6 e x p e r i m e n t s (fig. 1 A). In 5 e x p e r i m e n t s , doses ranging f r o m 61 pg t o 101 pg a b o l i s h e d t r e m o r within 2 to 4 min. T h e d u r a t i o n o f i n h i b i t i o n , f r o m t o t a l c e s s a t i o n to c o m p l e t e r e c o v e r y , r a n g e d f r o m 54 to 71 min. In o n e e x p e r i m e n t , a t o t a l o f 1 4 5 p g was r e q u i r e d t o p r o d u c e c o m p l e t e i n h i b i t i o n o f t r e m o r activity, w h i c h lasted 30 min.
3.3.3. Effects of hemicholinium, scopolamine, and acetylcholine T h e e f f e c t o f a r e d u c t i o n in cholinergic a c t i v i t y o n m o r p h i n e t r e m o r was t e s t e d in o t h e r e x p e r i m e n t s b y injecting e i t h e r HC-3 or s c o p o l a m i n e . I n j e c t i o n s o f HC-3 ( 7 3 - - 1 2 9 pg) a b o l i s h e d t r e m o r within 38 t o 48 m i n (fig. 1B, c o l u m n 2). T r e m o r i n h i b i t i o n was a c c o m p a nied b y h i g h - a m p l i t u d e s y n c h r o n o u s slow waves w h i c h in m o s t e x p e r i m e n t s were observed o n l y in r e c o r d i n g s f r o m t h e i n j e c t i o n site, a n d bilaterally f r o m the m o t o r c o r t e x . In 5 e x p e r i m e n t s , t r e m o r i n h i b i t i o n b y HC-3 was o b s e r v e d f o r at least 30 rain, w h e r e u p o n acetylc h o l i n e was i n j e c t e d (1 or 2 injections, 15 pg p e r dose, i.c.). In all eases m a x i m a l t r e m o r a c t i v i t y was r e s t o r e d w i t h i n 8 to 20 min b y
TABLE 3 Effects of locally injected drugs on peak tremor activity elicited by intracaudate morphine. Locally injected drug
Effect on tremor*
Activity parameters . . . . . . . . . . . . . . . . . . . . . . . . . . Dose (pg) Latency (min) Activity ratio**
Nalorphine Dopamine Hemicholinium Scopolamine Ca 2÷
($) (--) (--) (--) (--)
130 67 102 100 32
(81--263) (61--145) (73--129) (81--121) (24-- 40)
27.8 4.0 42.2 14.5 5.2
-+ 6.1 + 0.5 ± 1.5 -+ 2.0 ± 1.1
5/5 6/6 6/6 6/6 6/6
* (~) = tremor activity decreased to low level; tremor could not be completely eliminated. (--) = tremor activity abolished; values in parentheses represent range of doses required for complete tremor inhibition * * Activity ratio = (no. of experiments where drug altered tremor)/( total no. of experiments).
TREMOR PRODUCTION BY MORPHINE
49
4 l I N . AFTER DA
I0 MIN. MrlrER ~
(soj, a I.¢.)
3.
( |1 kilH.)
ACTIVITY
( 3S kilfL )
36 M,N. AFTER HC-3 (ISOj, G I.e.)
(SS~S Lc.)
(iOHIN.)
-"
15 MIN. AFTER AClk (,~)~S I.e.)
B ( ,4s kiIN.)
ACTIVITY I
2.
| 170 kilN. )
CALCIUM SUPPRESSION. 8 kilN: AFTER CACLz ZH~O
TREMOR
RECOVERY
24 G CA++ I~¢ i
IL C.AUDAI'E
( aS U ~ )
z.
Fig. 1. Inhibition of maximal morphine tremor activity by intracaudate (i.c.) injections of dopamine (A), hemicholinium-3 (B) and calcium (C). In each panel, upper tracings are caudate electrograms and lower tracings are tremor recordings. Numbers in parentheses below tremor tracings indicate the time sequence in minutes, continuous from the injection of morphine. Columns I and 2 in (A) show the onset of tremor and its progression to peak activity, respectively. In (B) and (C), control tremors in column I are at peak intensities. Tremor activity was reinstated in (B), column 3 by i.c. acetylcholine. The dose of HC-3 in (B), column 2 is expressed as the salt, equivalent to 129 pg of the base. Calibrations are 50 p V and i sec for all tracings.
acetylcholine (fig. 1B, column 3). The re-established tremor activity persisted on the average for 30 min before declining again. In the one experiment in which tremor activity was allowed to recover completely without injecting acetylcholine, the duration of inhibition following injection of HC-3 was 104 min. Scopolamine (81--121 pg i.c.) abolished tremor activity within 8 to 21 min. Tremor inhibition was not reversible with acetylcholine and no a t t e m p t was made to determine the duration of inhibition. Intracaudate injections of acetylcholine (32 pg) had no effect during the maximal tremor
activity elicited by morphine, nor was there any change in electrographic patterns. 3.3.4. Effects of calcium and sero tonin Injection of calcium chloride (24---40 #g of Ca ~÷) resulted in complete cessation of tremor activity within 1 to 9 min (table 3; fig. 1C, column 2). The duration of inhibition ranged from 40 to 51 min in 6 experiments. Results from previous studies indicate that the acetate and chloride salts of calcium are equally effective in depressing tremor activity (Lalley et al., 1971). Cumulative doses of serotonin up to 125 pg
5o had no effect on tremor activity in 6 experiments.
4. Discussion Intracaudate injections of morphine in the present investigation produced further increases in tremor intensity, above the control activity elicited by physostigmine. The increased activity generated by morphine was unrelated to an action at cholinergic synapses, since the morphine c o m p o n e n t was selectively antagonized by nalorphine, w i t h o u t altering the tremor produced by physostigmine. In an earlier study (Lalley et al., 1973) we demonstrated that i.c. injections of bretylium or tetrabenazine, whose respective actions in blocking catecholamine release or depleting neuronal stores of monoamines are known, produced tremors that were abolished by dopamine, Ca 2÷, scopolamine and HC-3, b u t were unaffected by serotonin. We concluded that the tremors were the result of a functional deficit in dopamine in the caudate nucleus which allowed cholinergic activity to predominate. Morphine might be acting in a similar fashion in the present study. Tremor activity was abolished by dopamine, scopolamine, and in a reversible manner by HC-3, b u t was unaffected b y serotonin. In addition, other investigators have shown that morphine, along with several psychoactive drugs including haloperidol, chlorpromazine and oxypertine, increase dopamine turnover in the striatum (Carlsson, 1967; Gunne and Jonsson, 1969; Clouet and Ratnor, 1970; Anddn et al., 1971}. Haloperidol and chlorpromazine are known for their tremorgenic effects which are attributed to a blockade of striatal dopamine receptors. Both drugs also increase acetylcholine release in the cat caudate nucleus (Stadler et al., 1973), which might be expected to further enhance cholinergic predominance, and therefore tremor production. The increase in dopamine turnover which these agents produce is evidently secondary to receptor blockade, by virtue of a feedback mechanism that regulates dopamine re-
P.M. LALLEY ET AL. lease as a function of post-synaptic receptor activation. A similar mode of action would explain the rapid appearance of morphineinduced tremor activity in the present study, and would also account for the increase in dopamine turnover reported by others. The inhibition of tremor by Ca 2÷ is notew o r t h y in view of recent discoveries. The analgesic effects of morphine are depressed by intracisternal injections of Ca 2+ whereas the chelating agent, Na2 EDTA, increases analgesic efficacy (Kakunaga et al., 1966). Morphine inhibits the in vitro binding of Ca 2÷ to phospholipids, depresses the facilitated transport of Ca 2÷ by phospholipids (Mule, 1969) and reduces Ca 2÷ levels in the corpus striatum (Ross et al., 1974). Although these actions may contribute to tremorgenesis, we are inclined to dismiss them as major components to the tremorgeni c action of morphine. A reduction of Ca 2÷ levels in the caudate nucleus does indeed lead to tremor activity. I.c. injections of Na2 EDTA were tremorgenic in an earlier study (Lalley et al., 1971}, whereas Na2 CaEDTA was not. However, when Ca 2÷ (32--67 #g) was injected during peak activity, the tremor was abolished and did not reappear during observation periods of 2 to 3 hours. In the present investigation, the inhibition of morphine tremor activity by Ca 2÷ was relatively short-lived (40--50 min). Furthermore, Ca 2÷ also inhibits the tremors elicited by bretylium and tetrabenazine for comparable durations. The inhibition therefore appears to be relatively nonspecific and might be related to an action which stabilized neuronal membranes (Goldman and Blaustein, 1966; Ritchie et al., 1965). Additional actions have been cited in explaining the various effects of morphine. These include a direct excitant action on neurons (Duggan and Curtis, 1972}, blockade of inhibitory amino acid receptors (Curtis and Duggan, 1969) and possibly a release of serotonin from nerve terminals (Mayer and Liebeskind, 1974). None of these mechanisms adequately explain the tremorgenic effects of morphine in this investigation. Injection of excitatory amino acids such as N-methyl-D,L-aspartic acid elicits
TREMOR
PRODUCTION
BY MORPHINE
rage behavior and mydriasis in unanesthetized cats (Baker, 1972). The GABA antagonist, picrotoxin, produces tremor only at doses which lead to seizures and postictal depression (Lalley, unpublished observations). I.c. injections of serotonin evoke tremor, but the tremor is totally resistant to HC-3 and scopolamine (Malseed and Baker, 1973). None of these features are characteristics of morphine tremor activity, whereas similarities between the tremorgenic psychoactive drugs and morphine suggest similar actions in blocking dopamine receptors in the caudate nucleus.
Acknowledgements This research was supported by grant MH-08833 from the National Institute of Mental Health, U.S. Public Health Service. The authors express their appreciation to Mr. Ron Mahoney for assistance with the photography, and to Mrs. Eileen Forsythe for assistance in the preparation of the manuscript.
References And~n, N.E., H. Corrodi, K. Fuxe and U. Ungerstedt, 1971, Importance of nervous impulse flow for the neuroleptic induced increase in amine turnover in central dopamine neurons, European J. Pharmacol. 15, 193. Baker, W.W., 1972, Excitatory responses following intracaudate injection of N-methyl-d,l-aspartic acid, Arch. Intern. Pharmacodyn. 196, 226. Baker, W.W., P.M. Lalley and R.L. Young, 1974, Biphasic effects of intracaudate morphine on caudate functioning, Federation Proc. Abstr. 293. Beleslin, P. and R.L. Polak, 1965, Depression by morphine and chloralose of acetylcholine release from the cat's brain, J. Physiol. 177, 411. Carlsson, A., 1967, Basic actions of psychoactive drugs, Intern. J. Neurobiol. 6, 27. Clouet, D.H. and M. Ratnor, 1970, Catecholamine biosynthesis in brains of rats treated with morphine, Science 163,854. Connor, J.D., G.V. Rossi and W.W. Baker, 1966, Characteristics of tremor following injections of carbachol into the caudate nucleus, Exptl. Neurol. 14, 371. Curtis, D.R. and A.W. Duggan, 1969, Depression of spinal inhibition by morphine, Agents and Actions 1, 14. Duggan, A.W. and D.R. Curtis, 1972, Morphine and the synaptic activation of renshaw cells, Neuropharmacol. 11,189.
51
Goldman, D.E. and M.P. Blaustein, 1966, Ions, drugs and the axon membrane, Ann. N.Y. Acad. Sei. 137, 967. Gunne, L.M. and J. Jonsson, 1969, Effects of morphine intoxication on brain cateeholamine neurons, European J. Pharmacol. 5, 338. Jasper, H.H. and C. Ajmone-Marson, 1953, A stereotoxic atlas of the diencephalon of the cat, National Research Council of Canada, Ottowa. Kakunaga, T., H. Kaneta and K. Hano, 1966, Pharmacologic studies on analgesics-VII. Significance of the calcium ion in morphine analgesia, J. Pharmacol. Exptl. Therap. 153,134. l.~lley, P.M., G.V. Rossi and W.W. Baker, 1970, Analysis of local cholinergic tremor mechanisms following selective neurochemical lesions, Exptl. Neurol. 27, 358. Lalley, P.M., G.V. Rossi and W.W. Baker, 1971, Alterations in tremor regulation after intracaudate injections of calcium ions or disodium edetate, Neuropharmacol. 10, 613. Lalley, P.M., G.V. Rossi and W.Wo Baker, 1973, Tremor induction by intracaudate injections of bretylium tetrabenazine or mescaline: Functional deficits in caudate dopamine, J. Pharm. Sci. 62, 1302. Lodge, D., M. Headley, A.W. Duggan and T.J. Biscoe, 1974, The effects of morphine, etorphine and sinomenine on the chemical sensitivity and synaptic responses of renshaw cells and other spinal neurones in the cat, European J. Pharmacoi. 26, 277. Malseed, R.T. and W.W. Baker, 1973, Analysis of tre morgenic effects of intracaudate serotonin, Proc. Soc. Exptl. Biol. Med. 143, 1088. Mayer, D.J. and J.C. Liebeskind, 1974, Pain reduction by focal electrical stimulation of the brain: an anatomical and behavioral analysis, Brain Res. 68, 73. Mul~, S.J., 1969, Inhibition of phospholipid-facilitated calcium transport by central nervous system-acting drugs, Biochem. Pharmacol. 18, 339. Paton, W.D.M., 1957, The action of morphine and related substances on contraction and on acetylcholine o u t p u t of coaxially stimulated guinea pig ileum, Brit. J. Pharmacol. 12, 119. Ross, D.W., M.A. Medina and H.L. Cardenas, 1974, Morphine and ethanol: Selective depletion of regional brain calcium, Science 186, 63. Ritchie, J.M., B. Ritchie and P. Greengard, 1965, The active structure of local anesthetics, J. Pharmacol. Exptl. Therap. 150, 152. Sehaumann, W., 1957, Inhibition by morphine of the release of acetylcholine from the intestine of the guinea-pig, Brit. J. Pharmacol. 12, 115. Snedecor, G.W., 1956, Statistical Methods (The Iowa State University Press, Ames, Iowa, U.S.A. ). Stadler, H., K.G. Lloyd, M. Gadea-Ciria and G. Bartholini, 1973, Enhanced striatal acetylcholine release by chlorpromazine and its reversal by apomorphine, Brain Res. 55,476.