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The behavioural effects of manipulating GABA function in the globus pallidus
Brain Research, 116 (1976) 353-359 © Elsevier/North-Holland Biomedical Press, Amsterdam - Printed in The Netherlands
353
The behavioural effects of manipulating GABA function in the globus pallidus
C. PYCOCK, R. W. H O R T O N and C. D. M A R S D E N
University Department of Neurology, Institute of Psychiatry and King's College Hospital Medical School, Denmark Hill, London SE5 8AF (Great Britain) (Accepted July 27th, 1976)
There is now good evidence to suggest that ~,-aminobutyric acid (GABA) acts as a neurotransmitter substance in the brain 25. However, its exact functional role and interactions with other neurone systems of the brain are still the subject of much research. GABA is found widely distributed in mammalian brain 2,6, and although the direct, anatomical demonstration of gabaminergic pathways is still lacking, much indirect evidence, based on physiological, biochemical and pharmacological experiments, points to, for example, GABA-mediated descending pathways between the caudate nucleus-putamen (neostriatum) and substantia nigra~4,is, and globus pallidus and substantia nigra11,17. Such pathways are thought to exert an inhibitory action on the cells of the substantia nigra, thereby controlling activity of the ascending nigro-neostriatal dopaminergic pathway. Further interest in this putative neurotransmitter has recently been aroused by the suggestion that certain neurological disorders may involve abnormalities of cerebral GABA zS. In particular, patients dying from Huntington's chorea have low concentrations of GABA in regions of the basal ganglia3, 2a. Similarly, GABA may play an important role in certain aspects of epilepsy ~9. Unfortunately, there are few agents available to specifically manipulate GABA mechanisms for research and clinical purposes. GABA is synthesized from the amino acid L-glutamate by the enzyme glutamic acid decarboxylase (GAD) and is then metabolized by the enzyme GABA-transaminase (GABA-T) to succinic semialdehyde. Perhaps one of the best tools currently available for research purposes is the GABA-T inhibitor, ethanolamine-O-sulphate (EOS), which has been shown to specifically inhibit this enzyme and thereby elevate cerebral GABA concentrations7,8. Other agents that have been used to manipulate the GABA system are the GAD inhibitor, allylglycine, which decreases cerebral GABA concentrations 12, and the GABA receptor blocking drug, picrotoxin 22. One of the major projections of the neostriatum is to the globus pallidus, which is the principle source of output from the basal ganglia1~,2°, and therefore serves an important role in the control of motor behaviour. The globus pallidus is an area of the brain particularly rich in GABA 2 and GADL Synaptosomal preparations from
354 this region show a high uptake o f G A B A and a high sodium-independent binding of G A B A , which is t h o u g h t to represent interaction o f G A B A with the postsynaptic receptorL There is some evidence to suggest that the strio-pallidal efferent p a t h w a y is mediated by G A B A neurones 11. We have therefore chosen the globus pallidas as an area in which to investigate the effects o f agents which modify G A B A function and to observe the resultant response on m o t o r behaviour. Ethanolamine-O-sulphate (100/~g in 1.5 ul saline) or saline (1.5/zl) was injected bilaterally into the globus pallidus o f male and female S p r a g u e - D a w l e y rats (180-200 g), anaesthetised with chloral hydrate (300 mg/kg) using stereotaxic coordinates derived f r o m the atlas of K 6 n i g and Klippe115. Spontaneous and amphetamineinduced l o c o m o t o r activity after EOS or saline was assessed in a holeboard apparatus where the n u m b e r o f lines crossed were counted in an initial 3-min period. In another series o f animals, bilateral injection guide cannulae were m o u n t e d on the skull vertically over each globus pallidus in rats previously injected with either EOS or saline into that region. The m o t o r effects o f injecting either allylglycine (50200 #g) or picrotoxin (0.1-I #g) bilaterally into the globus pallidus t h r o u g h the injection guide cannulae were evaluated at various times after EOS or saline pretreatment, and the results were converted to a numerical score (see legend to Fig. 2). In a further series o f animals, regional G A B A concentrations were determined on days 1, 3 and 8 after pallidal injections of either EOS or saline. Rats were killed by decapitation and brain areas from EOS-treated and saline controls were always dissected in the same order and placed directly into liquid nitrogen; the time interval f r o m decapitation to freezing the last sample did not exceed 60 sec. G A B A was determined enzymatically 9 on deproteinised extracts, with appropriate tissue blanks. A l t h o u g h brain G A B A concentration is k n o w n to increase post mortem, we felt TABLE I Regional brain GABA concentrations at various times following focal bilateral injection o l E O S ( 100 l~g) into the globus pallidus
Values represent the mean ± S.E.M. for the number of estimations denoted by N, together with the of control. Significance of difference of the means (Student's t test) is denoted by * P < 0.05 ; • * P < 0.01 ; *** P < 0.001. The 'pallidum' consisted of the globus pallidus with some posterior caudate nucleus; the 'mesolimbic' consisted of the nucleus accumbens and tuberculum olfactorium. Treatment
Control (saline) (N 8) EOS day 1 (N 5) EOS day 3 (N 5) EOS day 8 (N -- 5)
GABA concentrations (Izmoles/g) Cerebellum
Mesolimbic
Cortex
Pallidum
0.76 ± 0.08 3.87 ± 0.34 (509~)*** 1.55 ± 0.27 (204%)** 0.83 ± 0.07 (109 ~)
2.67 ± 0.34 12.91 ± 1.37 (483 ~)*** 9.06 ± 1.00 (339~)*** 3.39 ± 0.34 (l 27 ~)
1.15 ± 0.10 6.61 ± 0.51 (574%)*** 5.27 ± 0.57 (458 ~)*** 1.49 ± 0.10 (129 ~)*
2.21 z~ 0.20 15.91 ± 0.21 (720%)*** 7.45 ± 0.58 (337~)*** 2.13 ± 0.70 (96 ~)
355 40 >-
~- 3 0 >_ n, 2 0
§
,o
.J y,,-~
CONTROL
DAY I
DAY 3
DAY 5
DAY 7
Fig. I. Spontaneous (open columns) and amphetamine-induced (dotted columns) locomotor activity in rats previously injected with EOS (100 ,ug) or saline (control) bilaterally into the globus pallidus. Amphetamine-induced locomotor activity was tested 1 h after injection of dexamphetamine sulphate (0.5 mg/kg, i.p.). Observations were made on days 1, 3, 5, and 7 following saline or EOS injection. (The results of the saline (control) group did not differ significantly at the times tested and were therefore combined.) Each column from the EOS observations is the mean of 6; vertical bars representing the standard error of the mean. The significance of differences in spontaneous and amphetamineinduced locomotor activity was tested between EOS and control groups (Student's t test) and is denoted b y * P < 0.05; *~' P <-: 0.01.
justified in using the technique of rapid dissection followed by freezing in liquid nitrogen in this study since the post-mortem increases within 1 min are likely to be small (18 % at 2 rain 2) compared with the large pharmacological increase induced by EOS (Table l). Bilateral injection of EOS into the globus pallidus resulted in a complete lack of spontaneous movement, but with normal righting reflexes (akinetic state) on day I after injection 24. Spontaneous locomotor activity began to return by day 3 and was the same as in the saline-injected animals by day 7 (Fig. 1). On day 1 after EOS injection, the akinesia was not reversed with amphetamine (animals tested 1 h after 0.5 mg/kg amphetamine, i.p.): an indirectly acting dopamine agonist which stimulated locomotor activity in saline-injected rats (Fig. 1). On day 3, amphetamine induced locomotor activity in the EOS-injected rats, but did not induce the hyperactive response seen in normal animals. The hyperactive response to amphetamine in the EOS-pretreated rats returned by day 5 (Fig. l). The behavioural effects paralleled changes in brain GABA concentrations (Table I). On day 1 after injection of EOS into the g[obus pallidus, GABA levels were increased 7-fold in the globus pallidus and 4-6-fold in the frontal cortex, cerebellum and mesolimbic areas (nucleus accumbens and tuberculum olfactorium). By day 3, the GABA concentrations were still greatly elevated as compared to the control levels, but were now only increased 2-fold in the cerebellum and 3-4-fold in the other regions. On day 8, GABA concentrations were no different from those in the control group, except in the cortex (30 ~/o increased, P < 0.05). EOS-induced akinesia could be partially reversed by pharmacological blockade
356 o f G A B A function in the globus pallidus. Bilateral injections of either picrotoxin or allylglycine into the globus pallidus o f saline-pretreated rats resulted in a characteristic dose-dependent m o t o r response. The low dose o f picrotoxin (0.1 /zg) or allylglycine (50/~g) caused mild arousal with faint twitches increasing in intensity to rhythmic myoclonic jerks o f the trunk and hindlimbs (score 2 or 3) (Fig. 2). The intermediate doses produced disabling jerks with some hyperactivity (score 4 or 5), while the highest doses o f picrotoxin (l/~g) or allylglycine (200/~g) caused generalised seizures (score 6). In these n o r m a l animals the picrotoxin effect occurred after 4-10 rain and lasted about 55-70 min, while the allylglycine effect had a delayed onset o f 35-50 rain and remained up to 4.5 h. O n day 1 after EOS pretreatment, the low dose ofpicrotoxin and allylglycine had no effect, the intermediate doses produced only mild arousal while the high doses caused strong m y o c l o n i c j e r k s (Fig. 2). On day 3, the low doses induced mild arousal, the intermediate doses caused myoclonic jerking and the high doses p r o d u c e d hyperactivity and generalised seizures. By day 7, the arousal responses to all doses of picrotoxin and allylglycine were indistinguishable f r o m those in normal animals (Fig. 2). On days 1 and 3 after EOS pretreatment, the responses to picrotoxin and allylglycine occurred after a longer latent period and were for a shorter duration o f action than in the normal animals. We conclude that G A B A neurons m a y play an important role in the control o f certain aspects o f m o t o r behaviour. Elevation o f brain G A BA concentrations resulted PICROTOXIN
6 W
"H J
DOSE~ C~T~L
0'1 0 ' 5 I DAY I
0'1 0"5 DAY 3
O'I 0"5 DAY 7
rg'
H DOSE 5 0 1 0 0 2 0 0 J~] CONTROL.
50100200 DAY I
50100200 DAY 3
5OIOO~ DAY7
Fig. 2. Arousal and seizure responses following bilateral injections of either picrotoxin (0.1-1 /tg) or allylglycine (50-200/xg) into the globus pallidus of rats previously injected bilaterally with EOS (100/xg) or saline into the globus pallidus. Observations were made on days 1, 3 and 7 following saline or EOS injection. (The results of the saline (control) group did not differ significantly at the times tested and were therefore combined.) Each column from the EOS observations is the mean of 5; standard errors fell within the range of 10-15~ of means. Arousal score: 0 ~ akinetic state; 1 -normal animal; 2 -- mild active arousal with faint twitches in trunk and limbs; 3 -- rhythmic myoclonic jerks of trunk and limbs; 4 strong disabling jerks that upset animal's routine; 5 hyperactive response prior to seizure; 6 generalised seizure.
357 in akinesia, a syndrome not initially reversed by the indirectly acting dopamine agonist amphetamine. Return of spontaneous locomotor activity from the akinetic state was paralleled by falls in brain GABA concentrations. Decreased GABA transmission, following either picrotoxin or allylglycine, was accompanied by body jerks, hyperactivity and seizure responses in normal animals. Such agents antagonised the effect of EOS providing further support for a gabaminergic mechanism. Although EOS was injected focally into the globus pallidus, significant elevations of GABA occurred in all regions of the brain studied. Such an observation indicates the rapid diffusion of EOS away from the site of injection which may, unfortunately, limit its usefulness as a research tool. However, the fact that focal injections of picrotoxin or allylglycine into the globus pallidus antagonised the effect of EOS points to a probable gabaminergic synapse in the pallidum as the mediator of these behavioural responses. Further evidence for a pallidal site is provided from the observation that increases in GABA concentrations in the mesolimbic areas alone do not inhibit spontaneous locomotor activity in rats 23, and that electrolesions in the globus pallidus of rats result in an akinetic state 24, similar to that seen on day 1 after EOS injection into the globus pallidus. A striking observation in these studies was the similarity of animals injected bilaterally with EOS into the globus pallidus to rats treated with reserpine. Both types of animals sat hunched and motionless in the cage, showed few and sluggish responses to stimulation, exhibited limb rigidity and sometimes tremor. This suggests that increasing pallidal GABA concentrations (and, to a lesser extent those in other brain regions) mimics the motor effects of reserpine. These are believed to be due to depletion of brain catecholamines, particularly dopamine 4, for they are reversed by L-DOPA 4,a6 and dopamine agonists such as apomorphine a. Specifically, the motor effects of reserpine are thought to be due primarily to depletion of striatal dopamine 4. Accordingly, it may be that loss of striatal dopaminergic action is mimicked by enhanced GABA activity in globus pallidus, which suggests that dopamine released from nigro-striatal nerve terminals finally modifies GABA activity in the pallidum. Nigro-striatal fibres appear to terminate on striatal cholinergic interneurones 10, which themselves might excite GABA neurones in striatum projecting to pallidum or, alternatively, might project to GABA interneurones in the pallidum. Further experiments are required to explore this hypothesis. These observations have important clinical implications. If GABA acts as a neurotransmitter in the globus pallidus, as is suggested, although not proven by the present studies, it may prove possible to manipulate the output of the basal ganglia by the use of drugs modifying pallidal GABA function. The globus pallidus could thus provide a new target at which to direct drug treatment of movement disorders such as Parkinson's disease and dyskinesias. As a working hypothesis it might be possible to remedy (a) Parkinson's disease which is characterised by striatal dopamine depletion, by preventing GABA action in pallidum, and (b) dyskinesias, which in general appear to result from overactivity of striatal dopamine mechanisms, by increasing pallidal GABA.
358 C . P . is a F e l l o w o f the P a r k i n s o n ' s D i s e a s e S o c i e t y ; R . W . H . a c k n o w l e d g e s the Wellcome Trust for financial support. Mr. Colin Brewer provided expert technical assistance.
1 And6n, N.-E., Str6mbom, U. and Svensson, T. H., Dopamine and noradrenaline receptor stimulation: reversal of reserpine-induced suppression of motor activity, Psychopharmacologia (Berl.), 29 (1973) 289-298. 2 Balcom, G. J., Lenox, R. H. and Meyerhoff, J. L., Regional 7-aminobutyric acid levels in rat brain determined by microwave fixation, J. Neurochem., 24 (1975) 609-613. 3 Bird, E. D. and Iversen, L. L., Huntington's chorea: post-mortem measurement of glutamic acid decarboxylase, choline acetyltransferase and dopamine in basal ganglia, Brain, 97 (1974) 457-472. 4 Carlsson, A., Lindqvist, M. and Magnusson, T., 3,4-Dihydroxyphenylalanine and 5-hydroxytryptophan as reserpine antagonists, Nature (Lond.), 180 (1957) 1200. 5 Enna, S. J., Kuhar, M. J. and Snyder, S. H., Regional distribution of postsynaptic receptor binding for gamma-aminobutyric acid (GABA) in monkey brain, Brain Research, 93 (1975) 168-174. 6 Fahn, S. and C6t6, L. J., Regional distribution of ;~-aminobutyric acid (GABA) in brain of the rhesus monkey, J. Neurochem., 15 (1968) 209-213. 7 Fowler, L. J., Analysis of the major amino acids of rat brain after in vivo inhibition of GABA transaminase by ethanolamine-O-sulphate, J. Neurochem., 21 (1973) 437440. 8 Fowler, L. J. and John, R. A., Active-site-directed irreversible inhibition of rat brain 4-aminobutyrate aminotransferase by ethanolamine-O-sulphate in vitro and in vivo, Biochem. J., 130 (1972) 569-573. 9 Graham, L.T. and Aprison, M. H., Fluorometric determination of aspartate, glutamate and ~-aminobutyrate in nerve tissue using enzymic methods, Anal, Biochem., 15 (1966) 487 497. 10 Guyenet, P. G., Agid, Y., Javoy, F., Beaujouan, J. C., Rossier, J. and Glowinski, J., Effects of dopaminergic receptor agonists and antagonists on the activity of the neo-striatal cholinergic system, Brain Research, 84 (1975) 227-244. 11 Hattori, T., McGeer, P. L., Fibiger, H. C. and McGeer, E. G., On the source of GABA-containing terminals in the substantia nigra. Electron microscopic, autoradiographic and biochemical studies, Brain Research, 54 (1973) 103-114. 12 Horton, R. W. and Meldrum, B. S., Seizures induced by allylglycine, 3-mercaptopropionic acid and 4-deoxypyridoxine in mice and photosensitive baboons, and different modes of inhibition of cerebral glutamic acid decarboxylase, Brit. J. Pharmacol., 49 (1973) 52 63. 13 Johnson, T. N. and Rosvold, H. E., Topographic projections on the g[obus pallidus and substantia nigra of selectively placed lesions in the precommissural caudate nucleus and putamen in the monkey, Exp. Neurol., 33 (1971) 584-596. 14 Kim, J. S., Bak, 1. J., Hassler, R. and Okada, Y., Role of ~,-aminobutyric acid (GABA) in the extrapyramidal motor system. 2. Some evidence for the existence of a type of GABA-rich strionigral neurons, Exp. Brain Res., 14 (1971) 95-104. 15 K6nig, J. F. R. and Klippel, R. A., The Rat Brain: A Stereotaxic Atlas of the Forebrahz and Lower Parts of the Brain Stem, Williams and Wilkins, Baltimore, Md., 1963. 16 Marsden, C. D., Dolphin, A., Duvoisin, R. C., Jenner, P. and Tarsy, D., Role of noradrenaline in levodopa reversal of reserpine akinesia, Brain Research, 77 (1974) 521 525. 17 McGeer, P. L., McGeer, E. G., Wada, J. A. and Jung, E., Effects of globus pallidus lesions and Parkinson's disease on brain glutamic acid decarboxylase, Brain Research, 32 (1971) 425 431. 18 McGeer, E. G., Fibiger, H. C., McGeer, P. L. and Brooke, S., Temporal changes in amine synthesizing enzymes of rat extrapyramidal structures after hemitransections or 6-hydroxydopamine administration, Brain Research, 52 (1973) 289 300. 19 Meldrum, B. S., Epilepsy and GABA-mediated inhibition, Int. Rev. Neurobiol., 17 (1975) 1-36. 20 Niimi, K., Ikeda, T., Kawamura, S. and lnoshita, M., Efferent projections of the head of the caudate nucleus in the cat, Brain Research, 21 (1970) 327-343. 21 Perry, T. L., Hansen, S. and Kloster, M., Huntington's chorea. Deficiency of T-aminobutyric acid in brain, New Engl. J. Med., 288 (1973) 337-342. 22 Precht, W. and Yoshida, M., Blockade of caudate-evoked inhibition of neurons in the substantia nigra by picrotoxin, Brain Research, 32 (1971) 229 233.
359 23 Pycock, C. J. and Horton, R. W., Possible GABA-mediated control of dopamine-dependent behavioural effects from the nucleus accumbens of the rat, Psychopharmacologia (BerL), (1976) in press. 24 Pycock, C. J. and Horton, R. W., Evidence for an accumbens-pallidal pathway in the rat and its possible gabaminergic control, Brain Research, I l0 (1976) 629-634. 25 Roberts, E., 7-Aminobutyric acid and nervous system function - - a perspective, Biochem. Pharmacol., 23 (1974) 2637-2649.
353
The behavioural effects of manipulating GABA function in the globus pallidus
C. PYCOCK, R. W. H O R T O N and C. D. M A R S D E N
University Department of Neurology, Institute of Psychiatry and King's College Hospital Medical School, Denmark Hill, London SE5 8AF (Great Britain) (Accepted July 27th, 1976)
There is now good evidence to suggest that ~,-aminobutyric acid (GABA) acts as a neurotransmitter substance in the brain 25. However, its exact functional role and interactions with other neurone systems of the brain are still the subject of much research. GABA is found widely distributed in mammalian brain 2,6, and although the direct, anatomical demonstration of gabaminergic pathways is still lacking, much indirect evidence, based on physiological, biochemical and pharmacological experiments, points to, for example, GABA-mediated descending pathways between the caudate nucleus-putamen (neostriatum) and substantia nigra~4,is, and globus pallidus and substantia nigra11,17. Such pathways are thought to exert an inhibitory action on the cells of the substantia nigra, thereby controlling activity of the ascending nigro-neostriatal dopaminergic pathway. Further interest in this putative neurotransmitter has recently been aroused by the suggestion that certain neurological disorders may involve abnormalities of cerebral GABA zS. In particular, patients dying from Huntington's chorea have low concentrations of GABA in regions of the basal ganglia3, 2a. Similarly, GABA may play an important role in certain aspects of epilepsy ~9. Unfortunately, there are few agents available to specifically manipulate GABA mechanisms for research and clinical purposes. GABA is synthesized from the amino acid L-glutamate by the enzyme glutamic acid decarboxylase (GAD) and is then metabolized by the enzyme GABA-transaminase (GABA-T) to succinic semialdehyde. Perhaps one of the best tools currently available for research purposes is the GABA-T inhibitor, ethanolamine-O-sulphate (EOS), which has been shown to specifically inhibit this enzyme and thereby elevate cerebral GABA concentrations7,8. Other agents that have been used to manipulate the GABA system are the GAD inhibitor, allylglycine, which decreases cerebral GABA concentrations 12, and the GABA receptor blocking drug, picrotoxin 22. One of the major projections of the neostriatum is to the globus pallidus, which is the principle source of output from the basal ganglia1~,2°, and therefore serves an important role in the control of motor behaviour. The globus pallidus is an area of the brain particularly rich in GABA 2 and GADL Synaptosomal preparations from
354 this region show a high uptake o f G A B A and a high sodium-independent binding of G A B A , which is t h o u g h t to represent interaction o f G A B A with the postsynaptic receptorL There is some evidence to suggest that the strio-pallidal efferent p a t h w a y is mediated by G A B A neurones 11. We have therefore chosen the globus pallidas as an area in which to investigate the effects o f agents which modify G A B A function and to observe the resultant response on m o t o r behaviour. Ethanolamine-O-sulphate (100/~g in 1.5 ul saline) or saline (1.5/zl) was injected bilaterally into the globus pallidus o f male and female S p r a g u e - D a w l e y rats (180-200 g), anaesthetised with chloral hydrate (300 mg/kg) using stereotaxic coordinates derived f r o m the atlas of K 6 n i g and Klippe115. Spontaneous and amphetamineinduced l o c o m o t o r activity after EOS or saline was assessed in a holeboard apparatus where the n u m b e r o f lines crossed were counted in an initial 3-min period. In another series o f animals, bilateral injection guide cannulae were m o u n t e d on the skull vertically over each globus pallidus in rats previously injected with either EOS or saline into that region. The m o t o r effects o f injecting either allylglycine (50200 #g) or picrotoxin (0.1-I #g) bilaterally into the globus pallidus t h r o u g h the injection guide cannulae were evaluated at various times after EOS or saline pretreatment, and the results were converted to a numerical score (see legend to Fig. 2). In a further series o f animals, regional G A B A concentrations were determined on days 1, 3 and 8 after pallidal injections of either EOS or saline. Rats were killed by decapitation and brain areas from EOS-treated and saline controls were always dissected in the same order and placed directly into liquid nitrogen; the time interval f r o m decapitation to freezing the last sample did not exceed 60 sec. G A B A was determined enzymatically 9 on deproteinised extracts, with appropriate tissue blanks. A l t h o u g h brain G A B A concentration is k n o w n to increase post mortem, we felt TABLE I Regional brain GABA concentrations at various times following focal bilateral injection o l E O S ( 100 l~g) into the globus pallidus
Values represent the mean ± S.E.M. for the number of estimations denoted by N, together with the of control. Significance of difference of the means (Student's t test) is denoted by * P < 0.05 ; • * P < 0.01 ; *** P < 0.001. The 'pallidum' consisted of the globus pallidus with some posterior caudate nucleus; the 'mesolimbic' consisted of the nucleus accumbens and tuberculum olfactorium. Treatment
Control (saline) (N 8) EOS day 1 (N 5) EOS day 3 (N 5) EOS day 8 (N -- 5)
GABA concentrations (Izmoles/g) Cerebellum
Mesolimbic
Cortex
Pallidum
0.76 ± 0.08 3.87 ± 0.34 (509~)*** 1.55 ± 0.27 (204%)** 0.83 ± 0.07 (109 ~)
2.67 ± 0.34 12.91 ± 1.37 (483 ~)*** 9.06 ± 1.00 (339~)*** 3.39 ± 0.34 (l 27 ~)
1.15 ± 0.10 6.61 ± 0.51 (574%)*** 5.27 ± 0.57 (458 ~)*** 1.49 ± 0.10 (129 ~)*
2.21 z~ 0.20 15.91 ± 0.21 (720%)*** 7.45 ± 0.58 (337~)*** 2.13 ± 0.70 (96 ~)
355 40 >-
~- 3 0 >_ n, 2 0
§
,o
.J y,,-~
CONTROL
DAY I
DAY 3
DAY 5
DAY 7
Fig. I. Spontaneous (open columns) and amphetamine-induced (dotted columns) locomotor activity in rats previously injected with EOS (100 ,ug) or saline (control) bilaterally into the globus pallidus. Amphetamine-induced locomotor activity was tested 1 h after injection of dexamphetamine sulphate (0.5 mg/kg, i.p.). Observations were made on days 1, 3, 5, and 7 following saline or EOS injection. (The results of the saline (control) group did not differ significantly at the times tested and were therefore combined.) Each column from the EOS observations is the mean of 6; vertical bars representing the standard error of the mean. The significance of differences in spontaneous and amphetamineinduced locomotor activity was tested between EOS and control groups (Student's t test) and is denoted b y * P < 0.05; *~' P <-: 0.01.
justified in using the technique of rapid dissection followed by freezing in liquid nitrogen in this study since the post-mortem increases within 1 min are likely to be small (18 % at 2 rain 2) compared with the large pharmacological increase induced by EOS (Table l). Bilateral injection of EOS into the globus pallidus resulted in a complete lack of spontaneous movement, but with normal righting reflexes (akinetic state) on day I after injection 24. Spontaneous locomotor activity began to return by day 3 and was the same as in the saline-injected animals by day 7 (Fig. 1). On day 1 after EOS injection, the akinesia was not reversed with amphetamine (animals tested 1 h after 0.5 mg/kg amphetamine, i.p.): an indirectly acting dopamine agonist which stimulated locomotor activity in saline-injected rats (Fig. 1). On day 3, amphetamine induced locomotor activity in the EOS-injected rats, but did not induce the hyperactive response seen in normal animals. The hyperactive response to amphetamine in the EOS-pretreated rats returned by day 5 (Fig. l). The behavioural effects paralleled changes in brain GABA concentrations (Table I). On day 1 after injection of EOS into the g[obus pallidus, GABA levels were increased 7-fold in the globus pallidus and 4-6-fold in the frontal cortex, cerebellum and mesolimbic areas (nucleus accumbens and tuberculum olfactorium). By day 3, the GABA concentrations were still greatly elevated as compared to the control levels, but were now only increased 2-fold in the cerebellum and 3-4-fold in the other regions. On day 8, GABA concentrations were no different from those in the control group, except in the cortex (30 ~/o increased, P < 0.05). EOS-induced akinesia could be partially reversed by pharmacological blockade
356 o f G A B A function in the globus pallidus. Bilateral injections of either picrotoxin or allylglycine into the globus pallidus o f saline-pretreated rats resulted in a characteristic dose-dependent m o t o r response. The low dose o f picrotoxin (0.1 /zg) or allylglycine (50/~g) caused mild arousal with faint twitches increasing in intensity to rhythmic myoclonic jerks o f the trunk and hindlimbs (score 2 or 3) (Fig. 2). The intermediate doses produced disabling jerks with some hyperactivity (score 4 or 5), while the highest doses o f picrotoxin (l/~g) or allylglycine (200/~g) caused generalised seizures (score 6). In these n o r m a l animals the picrotoxin effect occurred after 4-10 rain and lasted about 55-70 min, while the allylglycine effect had a delayed onset o f 35-50 rain and remained up to 4.5 h. O n day 1 after EOS pretreatment, the low dose ofpicrotoxin and allylglycine had no effect, the intermediate doses produced only mild arousal while the high doses caused strong m y o c l o n i c j e r k s (Fig. 2). On day 3, the low doses induced mild arousal, the intermediate doses caused myoclonic jerking and the high doses p r o d u c e d hyperactivity and generalised seizures. By day 7, the arousal responses to all doses of picrotoxin and allylglycine were indistinguishable f r o m those in normal animals (Fig. 2). On days 1 and 3 after EOS pretreatment, the responses to picrotoxin and allylglycine occurred after a longer latent period and were for a shorter duration o f action than in the normal animals. We conclude that G A B A neurons m a y play an important role in the control o f certain aspects o f m o t o r behaviour. Elevation o f brain G A BA concentrations resulted PICROTOXIN
6 W
"H J
DOSE~ C~T~L
0'1 0 ' 5 I DAY I
0'1 0"5 DAY 3
O'I 0"5 DAY 7
rg'
H DOSE 5 0 1 0 0 2 0 0 J~] CONTROL.
50100200 DAY I
50100200 DAY 3
5OIOO~ DAY7
Fig. 2. Arousal and seizure responses following bilateral injections of either picrotoxin (0.1-1 /tg) or allylglycine (50-200/xg) into the globus pallidus of rats previously injected bilaterally with EOS (100/xg) or saline into the globus pallidus. Observations were made on days 1, 3 and 7 following saline or EOS injection. (The results of the saline (control) group did not differ significantly at the times tested and were therefore combined.) Each column from the EOS observations is the mean of 5; standard errors fell within the range of 10-15~ of means. Arousal score: 0 ~ akinetic state; 1 -normal animal; 2 -- mild active arousal with faint twitches in trunk and limbs; 3 -- rhythmic myoclonic jerks of trunk and limbs; 4 strong disabling jerks that upset animal's routine; 5 hyperactive response prior to seizure; 6 generalised seizure.
357 in akinesia, a syndrome not initially reversed by the indirectly acting dopamine agonist amphetamine. Return of spontaneous locomotor activity from the akinetic state was paralleled by falls in brain GABA concentrations. Decreased GABA transmission, following either picrotoxin or allylglycine, was accompanied by body jerks, hyperactivity and seizure responses in normal animals. Such agents antagonised the effect of EOS providing further support for a gabaminergic mechanism. Although EOS was injected focally into the globus pallidus, significant elevations of GABA occurred in all regions of the brain studied. Such an observation indicates the rapid diffusion of EOS away from the site of injection which may, unfortunately, limit its usefulness as a research tool. However, the fact that focal injections of picrotoxin or allylglycine into the globus pallidus antagonised the effect of EOS points to a probable gabaminergic synapse in the pallidum as the mediator of these behavioural responses. Further evidence for a pallidal site is provided from the observation that increases in GABA concentrations in the mesolimbic areas alone do not inhibit spontaneous locomotor activity in rats 23, and that electrolesions in the globus pallidus of rats result in an akinetic state 24, similar to that seen on day 1 after EOS injection into the globus pallidus. A striking observation in these studies was the similarity of animals injected bilaterally with EOS into the globus pallidus to rats treated with reserpine. Both types of animals sat hunched and motionless in the cage, showed few and sluggish responses to stimulation, exhibited limb rigidity and sometimes tremor. This suggests that increasing pallidal GABA concentrations (and, to a lesser extent those in other brain regions) mimics the motor effects of reserpine. These are believed to be due to depletion of brain catecholamines, particularly dopamine 4, for they are reversed by L-DOPA 4,a6 and dopamine agonists such as apomorphine a. Specifically, the motor effects of reserpine are thought to be due primarily to depletion of striatal dopamine 4. Accordingly, it may be that loss of striatal dopaminergic action is mimicked by enhanced GABA activity in globus pallidus, which suggests that dopamine released from nigro-striatal nerve terminals finally modifies GABA activity in the pallidum. Nigro-striatal fibres appear to terminate on striatal cholinergic interneurones 10, which themselves might excite GABA neurones in striatum projecting to pallidum or, alternatively, might project to GABA interneurones in the pallidum. Further experiments are required to explore this hypothesis. These observations have important clinical implications. If GABA acts as a neurotransmitter in the globus pallidus, as is suggested, although not proven by the present studies, it may prove possible to manipulate the output of the basal ganglia by the use of drugs modifying pallidal GABA function. The globus pallidus could thus provide a new target at which to direct drug treatment of movement disorders such as Parkinson's disease and dyskinesias. As a working hypothesis it might be possible to remedy (a) Parkinson's disease which is characterised by striatal dopamine depletion, by preventing GABA action in pallidum, and (b) dyskinesias, which in general appear to result from overactivity of striatal dopamine mechanisms, by increasing pallidal GABA.
358 C . P . is a F e l l o w o f the P a r k i n s o n ' s D i s e a s e S o c i e t y ; R . W . H . a c k n o w l e d g e s the Wellcome Trust for financial support. Mr. Colin Brewer provided expert technical assistance.
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