91 (1975) 331-335 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

Brain Research,


Evidence for glutamic acid decarboxylase-containing interneurons in the neostriatum

P. L. M c G E E R AND E. G. M c G E E R

Kinsmen Laboratory of Neurological Research, Department of Psychiatry, University t~f Brittsh Columbia, Vancouver, B.C. V6T 1 W5 (Canada) (Accepted March 18th, 1975)

The neostriatum contains reasonably high concentrations of GABA and its synthetic enzyme glutamic acid decarboxylase (GAD) 4,~,1s. However, the organization of' the GABA-containing neurons in the extrapyramidal system is not yet known. In this communication we report evidence favoring the majority of these neurons being interneurons of the neostriatum. Several possibilities exist for GABA-containing pathways in the extrapyramidal system based on well defined neuronal tracts. Afferents to the neostriatum are from the ventral tegmentum, cerebral cortex (particularly the anterior half) and certain thalamic nuclei 25. The biochemical nature of the cortical and thalamic input is unknown, although the tegmental input is believed to be largely dopamine- (ref. 1) and serotonin-containing 7. Lesioning studies have established that at least some of the neostriatal interneurons are cholinergic3,17. However, recent immunohistochemical studies 15,'2 have shown that a considerable proportion of these interneurons do not stain positively for choline acetyltransferase (CAT) indicating that there must be at least one other population of interneurons. Efferent projections from the neostriatum are to the globus pallidus and substantia nigra 25. Kim et al. 1~ and Fonnum et al. 6 have reported decreased G A D in the substantia nigra following neostriatal lesions and have concluded that at least some of the neostriatal efferent projections are gabaminergic. To explore further the possible localization of extrapyramidal gabaminergic neurons, we placed unilateral cortical or thalamic lesions in the brains of rats. Large areas of the frontal and parietal cortex were removed by suction of the tissue as indicated in Fig. I. Thalamic lesions were placed by electrocoagulation using stereotaxic methods, covering the region shown in Fig. 2. As the figures indicate, the lesions did not completely destroy either the cortex or thalamus, but were nevertheless large enough to interrupt the majority of efferents from these structures to the neostriatum. The animals were sacrificed 1-3 weeks later by cervical dislocation. In the case of cortical lesions, sketches were made of the area suctioned prior to dissection of the brain. In the case of thalamic lesions, the diencephalon was preserved in formalin and


~s~'~" Fig. 1. Diagrammatic view of top and latelal aspects of rat brain. Hatching indicates area of cortex removed by suction. F, frontal; P, parietal, O, occipital lobes. the lesion verified by histological staining. Dissected tissues were weighed, homogenized in 10 vol. of isotonic sucrose and aliquo~s of the homogenates assayed for various enzymes. Glutamic acid decarboxylase activity was determined as previously reported by measuring the 14CO2 formed on incubation of 0.04 ml tissue homogenate with L-[1-14C]glutamic acid; tyrosine hydroxylase 14 and CAT 16 were measured by previously reported methods, using exogenous cofactor for tyrosine hydroxylase. Protein was measured by the Folin-Ciocalteau procedure. In confirmation of previous findings 17, neostriatal tyrosine hydroxylase and CAT levels were not significantly affected by these lesions. Mean G A D levels on the lesioned side were higher than those on the contralateral side but the differences were not significant (Table I). The non-lesioned side in each group gave values comparable to one another and to controls with no lesions. Thus, the major neostriatal inputs from the cortex and thalamus cannot consist of gabaminergic neurons. We have previously reported that neostriatal G A D is not reduced by transecting the brain at the level of the ventromedial nucleus of the hypothalamus in rats 9. K a t a o k a e t al. 11 subsequently obtained highly similar results after hemitransecting baboon brains at a comparable level. Since such transections would interrupt the input to the striatum

~CP Fig. 2. Cross-sectional diagram of rat brain at the level of the optic chiasm. Cross-hatching indicates area of thalamus destroyed by electrocoagulation. A, amygdala; CP, caudate-putamen; F, fornix; GP, globus pallidus; H, hippocampus; IC, internal capsule; OC, optic chiasm.

333 TABLE 1 GAD ACTIVITY(counts/min/mg protein-h)* ~NRATNEos'rR,AThLTISSUE(mean _4_S.E. ; 6 animals per group)

Thalamic lesions Cortical lesions

Lesioned side

Control side

t Jbr difference

9.78 ~ 0.70 9.56 ~ 0.76

8.12 ~_ 0.84 8.36 -- 0.64

1.55 (P > 0.1) 1.23 (P > 0.1)

* The incubation was done for 0.5 h with 3.6 mg of tissue at 3.2 mM L-glutamate (spec. act. 45 : 10a counts/min/llmole).

from the ventral tegmentum, this source must also have little or no gabaminergic content. The GAD-containing neurons must, therefore, originate in the neostriatum. They could be projecting neurons to the pallidum and/or the substantia nigra, Golgi type l l interneurons, or both. Evidence from a variety of sources suggests the latter two possibilities as being the most likely. We have found that extensive lesioning of the globus pallidus in cats does not produce a significant decrease in caudate G A D 19 while hemitransecting the brain in rats between the globus pallidus and caudateputamen produces less than a 15~o decrease 9. If the GAD-containing cells in the neostriatum were exclusively efferent cells to the pallidum and substantia nigra then such lesions should have caused a total disappearance of G A D due to retrograde degeneration. The fact that most of the G A D was retained indicates that the GABA cells must be largely Golgi type II interneurons. The slight drop in G A D in the rats hemitransected between the neostriatum and globus pallidus is compatible with some projecting efferents to the pallidum and substantia nigra. Fonnum et al. 6 reported decreases of G A D in the cat substantia nigra following caudate and putamen lesions as did Kim et al. for rats la. Both these groups, as well as Kataoka et al. it, have also reported data similar to our own, in that substantially larger drops in substantia nigral G A D are found following pallidal as opposed to neostriatal lesions. While there is now strong neuroanatomical evidence for a pallido-nigral tract TM, which most probably has a substantial GABA-containing component 2°, the possibility cannot be excluded that there may be strio-pallidal and strio-nigral gabaminergic pathways as well. Thus, more than one type of GAD-containing neuron may exist in the neostriatum although interneurons must be the dominant type. lnterneurons make up over 95 ~o of the neuronal cell population of the neostriatum 12. Other evidence also supports the concept that one population of these are GABA-containing. Uptake studies of [3H]GABA into extrapyramidal structures showed that while the substantia nigra had uptake primarily into nerve endings, the caudate-putamen had a substantial uptake into cell somata and dendrites as well as into nerve endings 9. Huntington's chorea is a disease which involves widespread cellular loss in the basal ganglia and cortex, but in which the most conspicuous lesion is a dropout of interneurons in the caudate and putamen s. Perry et al. 2a and Bird and iversen 2 re-

334 ported average decreases ~ l• 40/o " and 41 '",ii respectively in caudate G A B A in H u n t i n g t o n ' s chorea. McGeer et a/. ~J, Stahl and S w a n s o n 24 a n d Bird and Iversen" reported average decreases of 59 o , 81 ?i; and 77 *~i'in neostriatal G A D in this condition. These o/' O / groups also found decreases in neostriatal C A T of 50/o, 56/o and 55 o ,. respectively, but in each series some normal C A T values were f o u n d in established cases. Thus, the d r o p o u t of G A B A cells seems to be more p r o n o u n c e d a n d more consistent than that

of cholinergic cells. Since neostriatal i n t e r n e u r o n s are invariably diseased i n H u n t i n g t o n ' s chorea, the consistent decrease of G A B A and G A D is not only further evidence in favor of g a b a m i n e r g i c interneurons, but an i m p o r t a n t lead in seeking more effective forms of therapy. This research was supported by grants from the Medical Research Council of C a n a d a a n d the Province of British C o l u m b i a . The authors are grateful to Mr. H a r o l d Urstad a n d Mrs. K i m Searl for surgical p r e p a r a t i o n of the a n i m a l s used in this study. 1 ANDI~N, N. E., CARLSSON,A., DAHLSTROM,A., FUXE, K., HILLARP, N. A., AND LARSSON,P. R., Demonstration and mapping out of nigro-neostriatal dopamine neurons, Life Sci., 3 (1964) 523-530. 2 BIRD, E. D., AND 1VERSEN, L. L., Huntington's chorea: post mortem measurement of glutamic acid decarboxylase, choline acetyltransferase and dopamine in basal ganglia, Brain, 97 (1974) 457472. 3 BUTCHER,S. G., AND BUTCHER, L. L., Origin and modulation of acetylcholine activity in the neostriatum, Brain Research, 71 (1974) 167-171. 4 CHALMERS, A., M CGEER, E. G., WICKSON, V., AND MCGEER, P. L., Distribution of glutamic acid decarboxylase in the brains of various mammalian species, Comp. gen. Pharmacol., 1 (1970) 385-390. 5 FAUN, S., AND COTI~, L.J., Regional distribution of GABA in brain of the rhesus monkey, J. Neurochem., 15 (1968) 209-213. 6 FONNUM, F., GROFOV.~,l., R1NVIK, E., STORM-MATHISEN,J., AND WALBERG, F., Origin and distribution of glutamate decarboxylase in substantia nigra of the cat, Brain Research, 7t (1974) 77-92. 7 FUXE, K., AND JONSSON, G., Further mapping of central 5-hydroxytryptamine neurons: studies with neurotoxic dihydroxytryptamines. In E. COSTA, G. L. GESSA AND M. SANDLER (Eds.), Serotonin New Vistas, Advances in Biochemical Psychopharmacology, VoL I0, Raven Press, New York, 1974, pp. 1-12. 8 GREENFIELD,J. G., Neuro-pathology, Williams and Wilkins, London, 1958, pp. 502-507. 9 HATTORI, T., McGEER, P. L., FIBIGER, H. C., AND McGEER, E. G., On the source of GABAcontaining terminals in the substantia nigra. Electron microscopic autoradiographic and biochemical studies, Brain Research, 54 (1973) 103-114. 10 HATTORI, T., FIBIGER, H. C., AND McGEER, P. L., Demonstration of a pallidonigral projection innervating dopaminergic neurons, J. comp. Neurol., in press. 11 KATAOKA,K., BAK L J., HASSLER,R., KIM, J. S., AND WAGNER,A., L-Glutamate decarboxylase and choline acetyltransferase activity in the substantia nigra and the striatum after surgical interruption of the strio-nigral fibres of the baboon, Exp. Brain Res., 19 (1974) 217-227. 12 KEMP,J. M., AND POWELE, T. P. S., The structure of the caudate nucleus of the cat, Phil. Trans. B, 262 (1971) 383~01. 13 KtM, J. S., BAK, I. J., HASSLER, R., AND OKADA, Y., Role of y-aminobutyric acid (GABA) in the extrapyramidal motor system, Exp. Brain Res., 14 (1971) 95-104. 14 MCGEER, E. G., McGEER,P. L., AND WADA, J. A., Distribution of tyrosine hydroxylase in human and animal brain, J. Neurochem., 18 (t971) 1647-1658. 15 MCGEER,E. G., MeGEER, P. L., GREWAAL,O. S., AND SINGH, V. K., Striatal cholinergic interneurons and their relation to dopaminergic nerve endings, J. PharmacoL, (1975) in press.

335 16 MCGEER, P. L., AND MCGEER, E. G., Cholinergic enzyme systems in Parkinson's disease, Arch. Neurol. (Chic.), 25 (1971) 265-268. 17 McGEER, P. L., MCGEER, E. G., FIBIGER, H. C., AND WICKSON, V., Neostriatal choline acetylase and acetylcholinesterase following selective brain lesions, Brain Research, 35 (1971) 308-314. 18 MCGEER, P. L., MCGEER, E. G., AND WADA, J. A., Glutamic acid decarboxylase in Parkinson's disease and epilepsy, Neurology (Minneap.), 21 (1971) 1000~1007. 19 MCGEER, P. L., MCGEER, E. G., WADA, J. A., AND JUNG, E., Effect of globus pallidus lesions and Park inson's disease on brain glutamic acid decarboxylase, Brain Research, 32 (197 l) 425-43 I. 20 MCGEER, P. L., FIBIGER, H. C., MALER, L., HATTORI, T., AND McGEER, E. G., Evidence for descending pallidonigral GABA containing neurons. In F. H. McDOWELL ANDA. BARBEAU(Eds.), Advances in Neurology, Parkinson's Disease. Proceedings of the 2nd Canadian American Conference , Vol. 5, Raven Press, New York, 1974, pp. 153-160. 21 MCGEER, P.L., MCGEER, E . G . , AND FIBIGER, H . C . , Choline acetylase and glutamic acid decarboxylase in Huntington's chorea, Neurology (Minneap.), 23 0973) 912-917. 22 MCGEER, P. L., MCGEER, E. G., SINGH, V. K., AND CHASE, W. H., Choline acetyltransferase localization in the central nervous system by immunohistochemistry, Brain Research 81 0974) 373-379. 23 PERRY, T. L., HANSEN, S., AND KLOSTER, M., Huntington's chorea, deficiency of ?-aminobutyric acid in brain, New Engl. J. Med., 288 (1973) 337-342. 24 STAHL, W. L., AND SWANSON, P. O., Biochemical abnormalities in Huntington's chorea, Neurology (Minneap.), 24 (1974) 813-819. 25 TRUEX, R. C., AND CARPENTER, M. B., Human Neuroanatomy, 6th ed., Williams and Williams, New York, 1969, pp. 501-512.

Evidence for glutamic acid decarboxylase-containing interneurons in the neostriatum.

91 (1975) 331-335 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands Brain Research, 331 Evidence for glutamic acid d...
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