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Long descending direct projection from the basal ganglia to the spinal cord: a revival of the extrapyramidal concept
Brain Resew& Elsevier
436 ( 1987) 129- I.35
BRE 13116
M. Takada, 2X. Li and T. Department of Anatomy, Umiversity of Toronto, Toronto, Ont. (Canada) (Accepted 26 May 1987) Key words: Subthalamic nucleus; Spinal cord; Globus pallidus; Extrapyramidal system: Basal ganglia; Retrograde fluorescent labeling; Rat
Our retrograde fluorescent labeling study shows that a distinct cell group of the subthalamic nucleus. posited in the basal ganglia. directly sends long descending axons contralaterally to the upper cervical segments (C,-C,) of the spinal cord in the rat. A large population (60-70%) of these subthalamic cells projecting to contralateral spinal levels give off axonal branches innervating the ipsilateral globus pallidus. Now, the classical concept of the ‘extrapyramidal’ motor system needs to be reconsidered. Furthermore. our results may provide a morphological substrate for the onset of a violent form of dyskinesia. ‘hemiballism’. which occurs in the contratateral limbs both clinically and experimentally following discrete lesions in the subthalamic nucleus or its fiber connections with the globus pallidus.
INTRODUCTION
Since the basal ganglia represent large subcortical nuclear masses comprising the corpus striatum and its related brainstem nuclei6*‘*“, neurologists have found it expedient to use the term ‘extrapyramidal’ motor system, which groups together these classically telencephalic derivatives subserving somatic motor functions. This term, coined but not defined by Wilso#, has been utilized widely to parallel the corticospinal (pyramidzl) motor system and emphasize functional relationships between the basal ganglia and specific brainstem nuclei. However, there are many objections to the use of the term. The components of this so-called system. in addition to the corpus striatum (the striatum and the pallidal complex), include the subthalamic nucleus, substantia nigra. red nucleus, and brainstem reticular formation. Explicit neuroanatomical relationships wi‘th the corpus striatum are established only for the subthalamic nucleus and substantia nigra6.‘. Although the corpus Correspondence: M. Takada. Department Canada.
of Anatomy.
striatum and these two nuclei are considered to play a critical role in somatic motor functions, none of them have so far been shown to send their axons directly to spinal levels. Indeed. attempts by many neurologists to establish direct neuronal connections between the basal ganglia and spinal cord have been made in vain. On the other hand, both the red nucleus and braInstem reticular formation project to spinal levels. giving rise to part of a non-pyramidal motor pathway. However, relationships between these two regions and the corpus striatum seem to be obscured. Thus. these facts make the concept of the ‘extrapyramidal’ sy>iern elusive and untenable. The retrograde fluorescent labeling studies reported here were designed to re-examine whether or not the basal ganglia project fibers directly to the spinal cord in the rat. Our results demonstrate that a distinct group of neurons in the subthalamic nucleus. a constituent of the basal ganglia. unequivocally send axons contralaterally to the upper cervical segments (C,-C,) of the spinal cord. and that a large popula-
University of Toronto. Medical Sciences Building. Toronto. Ont. M5S 1AS.
0006-8993/87/$03.50 @ 1987 Elsevier Science Publishers B .V. (Biomedical Division)
130 tion (60-7"0%) of the subthalamic cells projecting to the contralateral spinal cord have axon collaterals to the ipsilateral globus pallidus. MATERIALS AND METHODS Twenty-two adult male albino rats (Wistar, 250-320 g) were used for this study. Under general anesthesia with sodium pentobarbital (60 mg/kg, i.p.), multiple unilateral injections of each of the following retrograde fluorescent tracers were made into the upper cervical segments (C1-C5) of the spinal cord: True blue (TB, 5% aqueous suspension, 8 rats) and Fluoro-gold (FG, ~uorochrome Inc., 4% aqueous solution, 6 rats). A total volume of 0.6-1.0 /~1of TB or 0.4-0.8/~1 of FG was injected through a 1/~1 Hamilton microsyringe. In an additional series of double-labeling experiments, individual rats received multiple unilateral injections of TB into the C1-C5 levels, coupled with single stereotaxic injections of another fluorescent tracer, Diamidino yellow (DY, 3% aqueous suspension), into the globus pailidus (0.4 btl, 6 rats) contralateral to the spinal injections. The animals were allowed to survive for periods of 4-7 days after TB or FG injections and 1-3 days after DY injections. In the double-labeling experiments, injections of DY were usually made 2-6 days following injections of TB. Then, the rats were deeply reanesthetized and perfused transcardially with 300 ml of 10% formalin in 0.1 M phosphate buffer (pH 7.4). The brains were removed immediately, saturated with 25% sucrose in the same buffer at 4 °C, and cut serially into the coronal plane at 40/~m thickness on a cryostat. The sections were mounted onto clean slides and observed with a Leitz fluorescence microscope. A filter providing a wideband ultraviolet excitation light was used to examine the blue fluorescent TB-containing, gold fluorescent FG-containing and yellow fluorescent DY-containing cells. In 2 rats, a total volume of 0.6-1.0/~! of a 30% solution of horseradish peroxidase (HRP, Sigma, Type VI) in 0.9% saline was injected into the unilateral upper cervical spinal segments. Two days later, the animals were fixed by transcardial perfusion with 10% formalin and 40-/~m thick coronal sections, reacted with tetramethylbenzidine 23, were counterstained with Neutral red. Other technical details were as de-
Fig. 1. The distribution of retrogradely labeled neurons in the subthalamic nucleus followingFG injections into the contralateral upper cervical segments (CrCs) of the spinal cord (upper row). The 5 drawings are from representative coronal sections through the subthalamic nucleus, arranged from rostrai (a) to caudal (e). cp, cerebral peduncle; ot, optic tract.
scribed elsewhere 32. RESULTS Multiple injections of each of 2 different fluorescent tracers (TB and FG) into the upper cervical spinal segments (C1-C5) invariably involved their major portions on one side. However, the tracers did not each infringe on the opposite side (Fig. 1, upper row). From such spinal injections, retrogradely labeled neurons were evident bilaterally in many hypothalamic regions with an ipsilateral predominance and in the red nucleus with a contralateral predominance. Amazingly, a distinct group of labeled cells were consistently detected in the subthalamic nucleus. The perikaryal labeling of the subthalamic nucleus occurred predominantly contralateral to the injection (Figs. 1 and 2), although only an occasional cell was encountered on the ipsilateral side. These labeled subthalamic neurons were located mainly in 1the rostral 2/3 (Fig. la-c), and to a lesser degree in the caudal third of the nucleus (Fig. ld,e). In the rostral two-thirds, they were generally distributed scattered over the nucleus, although at the rostral-most
131
ilii
tia nigra. Similar findings resulted from the HRP study, although fewer cells (one-half to one-third) were usually labeled. The second goal of this experiment was to determine whether the subthalamic cells leading fibers contralaterally to the upper cervical spinal segments give off axon collaterals innervating the ipsilateral globus pailidus (the external segment of the paUidal complex), known as a major recipient of the outflow from the subthalamic nucleus S.9.13,26,35.38. The subthalamic cell labeling with TB from the spinal injections was seen in a fashion similar to that described above; TB-positive subthalamic neurons were observed mostly in the rostral portions of the nucleus contralateral to the injection. From the pallidal injections contralateral to the spinal injections, on the other hand, DY-positive neurons were intensive throughout the entire extent of the subthalamic nucleus ipsilateral to the injection. The dual injections gave rise to dispersed neurons double-labeled with both TB and DY. The double-labeling occurred predominantly in the rostral parts of the nucleus (especially in its ventral, comb-shaped portions) (Fig. 3). A large population (60-70%) of TB-containing cells were double-labeled with DY from the globus pallidus, although the proportion varied, to some extent, from experiment to experiment.
Fig. 2. Photomicrographs of retrogradely labeled neurons in the subthalamic nucleus after FG injections into the contralateral Ct-C 5 levels sbown in Fig. 1. a: the rostral level (corresponding with Fig. lb). b: the middle level (corresponding with Fig. lc). x200.
level, its comb-shaped portions embedded within the cerebral peduncle relatively often contained ceP,s labeled with each tracer (Fig. la). The density of labeled cells in the subthalamic nucleus after TB or FG injections varied to some extent; FG injections produced more numerous labeling of subthalamic cells and sometimes up to 80 labeled cells were counted from a single brain with FG injections (Fig. 2). On the other hand, the maximum number of labeled cells counted in the subthalamic nucleus from a single brain with TB injectior:s was 60. From the present spinal injections, no labeled neurons were found in any other components of the basal ganglia, including the striatum, pallidal complex and substan-
DISCUSSION On the basis of a criterion for the basal ganglia, proposed by Nauta and Domesick 28, that requires reciprocal connections with the corpus striatum, the subthalamic nucleus becomes readily included as a part of t~e hasa! ~anglia (Fig. 4). Here, ~e 0rovide the first evidence t aa: a constituent of the basal gangha, the subthalamic nucleus, directly sends long descending axons contralaterally to spinal levels (particularly to the upper cervical spinal segments) in the rat. The total number of subthalamk; ~t~uJoi~s projecting to the spinal cord amounts to maximally 80 cells per brain. Given that the rat subthalamic nucleus contains 7,000-9,500 cells35, this specific cell population can be estimated to make up approximately 1% of subthalamic neurons. Thus, the subthalamospinal projection is sparse but by no means trivial. Moreover, our comparison of subthalamic cell-labeling reveals that FG might be the most sensitive of
132
Fig. 3. Photomicrographs of fluorescent-positive neurons in the subthalamie nucleus. TB was injected into the contralateral upper cervical spinal segments (C1-C5), and DY into the ipsilateral globus pallidus. The two pictures show the ventral, comb-shaped portions of the subthalamic nucleus at its rostral level. Open arrows indicate cells double-labeled with both tracers, and solid arrows cells singlelabeled with TB. Others are all single-labeled with DY. x280.
133 cerebral
cortex
................
t ...............
-,t
striatum pallidum
subthalamus
T [:
~ "
I !
substantia niora ~
s ~inal cord Fig. 4. Schematic summary of the parallelism between the corticospinal (pyramidal) and "extrapyramidal" systems. Major, representative pathways of the basal ganglia (rectangular area surrounded with broken lines) are shown here ":.
the 3 different retrograde tracers (TB, FG and HRP) used, even though a lesser amount was generally injected. These data are greatly consistent with those from our recent experiment with a different system 34. The existence of such projection neurons in the basal ganglia may lead us to the understanding that the 'extrapyramidal' system is an entity at least in the rat brain, thus justifying its parallelism with the co~icospinal (pyramidal) system (Fig. 4). However, the course and termination of the subthalamospina~ pathway remain to be explored definitively. We have not yet examined other mammalian brains from this point of view; if it can be proven that a similar pattern of the projection is common to most mammals (especially to primates), prevalent criticism on the concept of the 'extrapyramidal' motor system will need to be revised. Furthermore, the present study elucidates that single subthalamic neurons simultaneously innervate the ipsilateral globus pallidus as well as the contralateral spinal cord. These bifurcating projection neurons constitute 60-70% of spinal projecting subthalamic cells. In view of the fact that the majority (at least 94%) of rat subthalamic cells give rise to collateral projections to both the globus pallidus and substantia nigra 35, they also probably give off axonal branches to the substantia nigra. With respect to subthalamic afferents, considerable evidence emphasizes the massive projection from the globus pallidus to the subthalamic n u c l e u s 8"9"11"!5"19"24"29"36 Other available evidence suggests the existence of the mo-
tor corticosubthalamic 18"2°'21"3°and striatosubthalamic TM projections. The subthalamic nucleus may integrate the somatic motor information from the cortex, which is conveyed to the nucleus directly through the corticosubthalamic pathway and/or via the corpus striatum, and use it to exert an identical and simultaneous control over both the contralateral spinal cord and ipsilateral globus pallidus (Fig. 4). Both clinically and experimentally, discrete lesions in the subthalamic nucleus or its fiber connections (particularly with the globus pailidus) are regularly followed by a violent dyskinesia, 'hemiballism '4"14" 22.37.39 The subthalamic dyskinesia occurs in the limbs contralateral to the lesion and involves marked hypotonus. This can be abolished by a subsequent lesion of the ipsilateral pallidum 5a4 or a transection of the spinal cord at its cervical level (especially a lesion of the dorsal part of the lateral funiculus) 5"1°. In this light, our results directly provide a clear anatomical substrate for these neuropathological phenomena associated with dysfunction of the subthalamic nucleus. Thus, the specific subthalamic cell population established here, which sends axon collaterals to both the contralateral spinal cord and ipsilateral globus pallidus, might be an essentially fatal cell group in 'hemiballism'. This does not promptly imply that the 'extrapyramidal' system is a functionally complete and independent motor unit, because the fact that ablations of motor cortex and interruption of the corticospinal tract at various levels also abolish the dyskinesia suggests that impulses transmitted to spinal levels via the corticc~-pina! tract must be responsible for the dyskinesia 2"3"~°a2. It is, however, salutary to note that the concept of this so-called system may, in fact, be valid morphologically. In any case, morphological differentiation (of the course and termination) between the corticospinal and subthalamospinal tracts might be an important clue to functional segregation between the pyramidal and 'extrapyramidal" systems. A neo~::otransmitter related to the newly proposed subthalamospinal pathway remains to be determined definitive"1. However, several previous findings respect the notion that y-aminobutyric acid (GABA) may be a neurotransmitter of subthalamopallidal fibers. Two autoradiographic retrograde tracing studies using [3H]GABA suggest the existence of a presumed GABA-containing subthalamopallidai pro-
134 jection 27'33. Small localized injections of G A B A an-
cending contralaterally to the spinal cord, in the sub-
tagonists (i.e. picrotoxin or bicuculline) into the pallidum/subthalamic nucleus produce contralateral violent choreoid dyskinesia 16a7, which is identical to that resulting from small discrete electrolytic lesions in the subthalamic nucleus TM. Taken together, the data from our double-labeling experiment strongly favor G A B A e r g i c neurons, with long axons des-
thalamic nucleus,
REFERENCES
and pharmacological analysis of hyperkinesia ~'esulting from lesions of the subthalamic nucleus of Luys, J. Comp. Neurol., 92 (1950) 293-332. 15 Carter, D.A. and Fibiger, H.C., The projections of the entopeduncular nucleus and globus pallidus in rat as demonstrated by autoradiography and horseradish peroxidase histochemistry, J. Comp. Neurol., 177 (1978) 113-124. 16 Crossman, A.R., Sambrook, M.A. and Jackson, A., Experimental hemiballismus in the baboon produced by injection of a gamma-aminobutyric acid antagonist into the basal ganglia, Neurosci. Lett., 20 (1980) 369-372. 17 Crossman, A.R., Sambrook, M.A. and Jackson, A., Experimental hemichorea/hemiballismus in the monkey: studies on the intracerebral site of action in a drug-induced dyskinesia, Brain, 107 (1984) 579-596. 18 Hartmann-von Monakow, K., Akert, K.and Kiinzle, H., Projections of the precentral motor cortex and other cortical areas of the frontal lobe to the subthalamic nucleus in the monkey, Exp. Brain Res., 33 (1978) 395-403. 19 Kim, R., Nakano, K., Jayaraman, A. and Carpenter, M.B., Projections of the globus pallidus and adjacent structures: an autoradiographic study in the monkey, J. Comp. Neurol., 169 (1976) 263-290. 20 Kitai, S.T. and Deniau, J.M., Cortical inputs to the subthalamus: intracellular analysis, Brain Research, 214 (1981) 411-415. 21 Kiinzle, H. and Akert, K., Efferent connections of cortical area 8 (frontal eye field) in Macacafascicularis. A reinvestigation using the autoradiographic technique, J. Comp. Neurol., 173 (1977) 147-164. 22 Martin, J.P., Hemichorea resulting from a local lesion of the brain (syndrome of body of Luys), Brain, 50 (1927) 637-651. 23 Mesulam, M.-M., Tetramethyl benzidine for horseradish peroxidase neurohistochemistry: a non-carcinogenic blue reaction-product with superior sensitivity for visualizing afferents and efferents, J. Histochem. Cytochem., 26 (1978) 106-117. 24 Nauta, H.J.W., Projections of the pallidal complex: an autoradiographic study in the cat, Neuroscience, 4 (1979) 1853-1873. 25 Nauta, H.J.W., A proposed conceptual reorganization of the basal ganglia and telencephalon, Neuroscience, 4 (1979) 1875-1881. 26 Nauta, H.J.W. and Cole, M., Efferent projections of the subthalamic nucleus: an autoradiographic study in the monkey and cat, J. Comp. Neurol., 180 (1978) 1-16. 27 Nauta, H.J.W. and Cu6nod, M., Perikaryai cell labeling in the subthalamic nucleus following the injection of [3H]),-
1 Beckstead, R.M., A reciprocal axonal connection between the subthalamic nucleus and the neostriatum in the cat, Brain Research, 275 (1983) 137-142. 2 Bucy, P.C., The cortico-spinal tract and tremor. In W.S. Fields (Ed.), Pathoge.,.esis and Treatment of Parkinsonism, Charles C. Thomas, Springfield, 1958, pp. 271-293. 3 Bucy, P.C., The surgical treatment of abnormal involuntary movements, Neurologia, 1 (1959) 1-15. 4 Carpenter, M.B., Ballism associated with partial destruction of the subthalamic nucleus of Luys, Neurology, 5 (1955) 479-489. 5 Carpenter, M.B., Brainstem and infratentorial neuraxis in experimental dyskinesia, Arch. Neurol. (Chic.), 5 (1961) 504-524. 6 Carpenter, M.B., Anatomical organization of the corpus striatum and related nuclei. In M.D. Yahr (Ed.), The Basal Ganglia, Raven, New York, 1976, pp. 1-36. 7 Carpenter, M.B., Interconnections between the corpus striatum and brainstem nuclei. In J.S. McKenzie, R.E. Kemm and L.N. Wilcock (Eds.), The Basal Ganglia: Structure and Function, Advances in Behavioral Biology, Vol. 27, Plenum, New York, 1984, pp. 1-68. 8 Carpenter, M.B., Batton III, R.R., Carleton, S.C. and Keller, J.T., Interconnections and organization of pallidal and subthalamic nucleus neurons in the monkey, J. Comp. Neurol., 197 (1981) 579-603. 9 Carpenter, M.B., Carleton, S.C., Keller, J.T. and Conte, P., Connections of the subthalamic nucleus in the monkey, Brain Research, 224 (198!) 1-29. l0 Carpenter, M.B., Correll, J.W. and Hinman, A., Spinal tracts mediating subthalamic hyperkinesia. Physiological effects of selective partial cordotomies upon dyskinesia in rhesus monkey, J. Neurophysiol., 23 (1960) 288-304. 11 Carpenter, M.B., Fraser, R.A.R. and Shriver, J.E., The organization of the pallidosubthalamic fibers in the monkey, Brain Research, 11 (1968) 522-559. 12 Carpenter, M.B. and Mettler, F.A., Analysis of subthalamic hyperkinesia in the monkey, with special reference to ablations of agranular cortex, J. Comp. Neurol., 95 (1951) 125-158. 13 Carpenter, M.B. and Strominger, N.L., Efferent fibers of the subthalamic nucleus in the monkey. A comparison of the efferent projections of the subthalamic nucleus, substantia nigra and globus pailidus, Am. J. Anat., 121 (1967) 41-72. 14 Carpenter, M.B., Whittier, J.R. and Mettler, F.A., Analysis of choreoid hyperkinesia in the rhesus monkey. Surgical
ACKNOWLEDGEMENTS This work was supported by the Medical Research Council of Canada. M.T. is a recipient of an M R C Postprofessional Fellowship.
135 aminobutyric acid into the pallidal complex: an autoradiographic study in cat, Neuroscience, 7 (1982) 2725-2734. 28 Nauta, W.J.H. and Domesick, V.B., The anatomy of the extrapyramidal system. In K. Fuxe and D.B. Calne (Eds.), Dopaminergic Ergot Derivatives and Motor Function, Wenner-Gren Certer lnternatioaal Symposium Series, Vol. 31, Pergamon, Oxford, 1979, pp. 3-22. 29 Nauta, W.J.H. and Mehler, W.R., Projections of the lentiform nucleus in the monkey, Brain Research, 1 (1966) 3-42. 30 Romansky, K.V., Usunoff, K G . , Ivanov, D.P. and Galaboy, G.P., Corticosubthalamic projection in the cat: an electron microscopic study, Brain Research, 163 (1979) 319-322. 31 Royce, G.J. and Laine, E.J., Efferent connections of the c-audate nucleus, including cortical projections of the striaturn and other basal ganglia: an autoradiographic and horseradish peroxidase investigation in the cat, J. Comp. NeuroL, 226 (1984) 28-49. 32 Takada, M. and Hattori, T., The rat striatum: a target nucleus for ascending axon collaterals of the entopedunculohabenular pathway, Brain Research, in press. 33 Takada, M. and Hattori, T., Glyclne: an alternative transmitter candidate of the paUidosubthalamic projection neurons in the rat, J. Comp. Neurol., in press. 34 Takada, M., Li, Z.K. and Hattori, T., A direct projection
from the tuberomammillary nucleus to the spinal cord in the rat, Neurosci. Len., in press. 35 Van der Kooy, D. and Hattori, T., Single subthalamic nucleus neurons project to b~th the globus pallidus and substantia nigra in rat, J. Comp. Neurol., 192 (1980) 751-768. 36 Van der Kooy, D., Hattori, T., Shannak, K. and Hornvkiewicz, O., The paUido-subthalamic projection in the rat: anatomical and biochemical studies, Brain Research, 204 (1981) 253-268. 37 Whittier, J.R., Ballism and the subthalamic nucleus (nucleus hypothalamicus, corpus Luysi). Review of the literature and study of thirty cases, Arch. Neurol. Psychiat. (Chic.), 58 (1947) 672-692. 38 Whittier, J.R. and Mettler, F.A., Studies on the subthalamus of the rhesus monkey. I. Anatomy and fiber connections of the subthalamic nucleus of Luys, J. Comp. Neurol., 90 (1949) 281-317. 39 Whittier, J.R. and Merrier, F.A., Studies on the subthalamus of the rhesus monkey. II. Hyperkinesia and other physiologic effects of subthalamic lesions, with special reference to the subthalamic nucleus of Luys, J. Comp. Neurol., 90 (1949) 319-372. 40 Wilson, S.A.K., Progressive lenticular degeneration: a familial nervous disease associated with cirrhosis of the liver, Brain, 34 (1912) 295-509.
436 ( 1987) 129- I.35
BRE 13116
M. Takada, 2X. Li and T. Department of Anatomy, Umiversity of Toronto, Toronto, Ont. (Canada) (Accepted 26 May 1987) Key words: Subthalamic nucleus; Spinal cord; Globus pallidus; Extrapyramidal system: Basal ganglia; Retrograde fluorescent labeling; Rat
Our retrograde fluorescent labeling study shows that a distinct cell group of the subthalamic nucleus. posited in the basal ganglia. directly sends long descending axons contralaterally to the upper cervical segments (C,-C,) of the spinal cord in the rat. A large population (60-70%) of these subthalamic cells projecting to contralateral spinal levels give off axonal branches innervating the ipsilateral globus pallidus. Now, the classical concept of the ‘extrapyramidal’ motor system needs to be reconsidered. Furthermore. our results may provide a morphological substrate for the onset of a violent form of dyskinesia. ‘hemiballism’. which occurs in the contratateral limbs both clinically and experimentally following discrete lesions in the subthalamic nucleus or its fiber connections with the globus pallidus.
INTRODUCTION
Since the basal ganglia represent large subcortical nuclear masses comprising the corpus striatum and its related brainstem nuclei6*‘*“, neurologists have found it expedient to use the term ‘extrapyramidal’ motor system, which groups together these classically telencephalic derivatives subserving somatic motor functions. This term, coined but not defined by Wilso#, has been utilized widely to parallel the corticospinal (pyramidzl) motor system and emphasize functional relationships between the basal ganglia and specific brainstem nuclei. However, there are many objections to the use of the term. The components of this so-called system. in addition to the corpus striatum (the striatum and the pallidal complex), include the subthalamic nucleus, substantia nigra. red nucleus, and brainstem reticular formation. Explicit neuroanatomical relationships wi‘th the corpus striatum are established only for the subthalamic nucleus and substantia nigra6.‘. Although the corpus Correspondence: M. Takada. Department Canada.
of Anatomy.
striatum and these two nuclei are considered to play a critical role in somatic motor functions, none of them have so far been shown to send their axons directly to spinal levels. Indeed. attempts by many neurologists to establish direct neuronal connections between the basal ganglia and spinal cord have been made in vain. On the other hand, both the red nucleus and braInstem reticular formation project to spinal levels. giving rise to part of a non-pyramidal motor pathway. However, relationships between these two regions and the corpus striatum seem to be obscured. Thus. these facts make the concept of the ‘extrapyramidal’ sy>iern elusive and untenable. The retrograde fluorescent labeling studies reported here were designed to re-examine whether or not the basal ganglia project fibers directly to the spinal cord in the rat. Our results demonstrate that a distinct group of neurons in the subthalamic nucleus. a constituent of the basal ganglia. unequivocally send axons contralaterally to the upper cervical segments (C,-C,) of the spinal cord. and that a large popula-
University of Toronto. Medical Sciences Building. Toronto. Ont. M5S 1AS.
0006-8993/87/$03.50 @ 1987 Elsevier Science Publishers B .V. (Biomedical Division)
130 tion (60-7"0%) of the subthalamic cells projecting to the contralateral spinal cord have axon collaterals to the ipsilateral globus pallidus. MATERIALS AND METHODS Twenty-two adult male albino rats (Wistar, 250-320 g) were used for this study. Under general anesthesia with sodium pentobarbital (60 mg/kg, i.p.), multiple unilateral injections of each of the following retrograde fluorescent tracers were made into the upper cervical segments (C1-C5) of the spinal cord: True blue (TB, 5% aqueous suspension, 8 rats) and Fluoro-gold (FG, ~uorochrome Inc., 4% aqueous solution, 6 rats). A total volume of 0.6-1.0 /~1of TB or 0.4-0.8/~1 of FG was injected through a 1/~1 Hamilton microsyringe. In an additional series of double-labeling experiments, individual rats received multiple unilateral injections of TB into the C1-C5 levels, coupled with single stereotaxic injections of another fluorescent tracer, Diamidino yellow (DY, 3% aqueous suspension), into the globus pailidus (0.4 btl, 6 rats) contralateral to the spinal injections. The animals were allowed to survive for periods of 4-7 days after TB or FG injections and 1-3 days after DY injections. In the double-labeling experiments, injections of DY were usually made 2-6 days following injections of TB. Then, the rats were deeply reanesthetized and perfused transcardially with 300 ml of 10% formalin in 0.1 M phosphate buffer (pH 7.4). The brains were removed immediately, saturated with 25% sucrose in the same buffer at 4 °C, and cut serially into the coronal plane at 40/~m thickness on a cryostat. The sections were mounted onto clean slides and observed with a Leitz fluorescence microscope. A filter providing a wideband ultraviolet excitation light was used to examine the blue fluorescent TB-containing, gold fluorescent FG-containing and yellow fluorescent DY-containing cells. In 2 rats, a total volume of 0.6-1.0/~! of a 30% solution of horseradish peroxidase (HRP, Sigma, Type VI) in 0.9% saline was injected into the unilateral upper cervical spinal segments. Two days later, the animals were fixed by transcardial perfusion with 10% formalin and 40-/~m thick coronal sections, reacted with tetramethylbenzidine 23, were counterstained with Neutral red. Other technical details were as de-
Fig. 1. The distribution of retrogradely labeled neurons in the subthalamic nucleus followingFG injections into the contralateral upper cervical segments (CrCs) of the spinal cord (upper row). The 5 drawings are from representative coronal sections through the subthalamic nucleus, arranged from rostrai (a) to caudal (e). cp, cerebral peduncle; ot, optic tract.
scribed elsewhere 32. RESULTS Multiple injections of each of 2 different fluorescent tracers (TB and FG) into the upper cervical spinal segments (C1-C5) invariably involved their major portions on one side. However, the tracers did not each infringe on the opposite side (Fig. 1, upper row). From such spinal injections, retrogradely labeled neurons were evident bilaterally in many hypothalamic regions with an ipsilateral predominance and in the red nucleus with a contralateral predominance. Amazingly, a distinct group of labeled cells were consistently detected in the subthalamic nucleus. The perikaryal labeling of the subthalamic nucleus occurred predominantly contralateral to the injection (Figs. 1 and 2), although only an occasional cell was encountered on the ipsilateral side. These labeled subthalamic neurons were located mainly in 1the rostral 2/3 (Fig. la-c), and to a lesser degree in the caudal third of the nucleus (Fig. ld,e). In the rostral two-thirds, they were generally distributed scattered over the nucleus, although at the rostral-most
131
ilii
tia nigra. Similar findings resulted from the HRP study, although fewer cells (one-half to one-third) were usually labeled. The second goal of this experiment was to determine whether the subthalamic cells leading fibers contralaterally to the upper cervical spinal segments give off axon collaterals innervating the ipsilateral globus pailidus (the external segment of the paUidal complex), known as a major recipient of the outflow from the subthalamic nucleus S.9.13,26,35.38. The subthalamic cell labeling with TB from the spinal injections was seen in a fashion similar to that described above; TB-positive subthalamic neurons were observed mostly in the rostral portions of the nucleus contralateral to the injection. From the pallidal injections contralateral to the spinal injections, on the other hand, DY-positive neurons were intensive throughout the entire extent of the subthalamic nucleus ipsilateral to the injection. The dual injections gave rise to dispersed neurons double-labeled with both TB and DY. The double-labeling occurred predominantly in the rostral parts of the nucleus (especially in its ventral, comb-shaped portions) (Fig. 3). A large population (60-70%) of TB-containing cells were double-labeled with DY from the globus pallidus, although the proportion varied, to some extent, from experiment to experiment.
Fig. 2. Photomicrographs of retrogradely labeled neurons in the subthalamic nucleus after FG injections into the contralateral Ct-C 5 levels sbown in Fig. 1. a: the rostral level (corresponding with Fig. lb). b: the middle level (corresponding with Fig. lc). x200.
level, its comb-shaped portions embedded within the cerebral peduncle relatively often contained ceP,s labeled with each tracer (Fig. la). The density of labeled cells in the subthalamic nucleus after TB or FG injections varied to some extent; FG injections produced more numerous labeling of subthalamic cells and sometimes up to 80 labeled cells were counted from a single brain with FG injections (Fig. 2). On the other hand, the maximum number of labeled cells counted in the subthalamic nucleus from a single brain with TB injectior:s was 60. From the present spinal injections, no labeled neurons were found in any other components of the basal ganglia, including the striatum, pallidal complex and substan-
DISCUSSION On the basis of a criterion for the basal ganglia, proposed by Nauta and Domesick 28, that requires reciprocal connections with the corpus striatum, the subthalamic nucleus becomes readily included as a part of t~e hasa! ~anglia (Fig. 4). Here, ~e 0rovide the first evidence t aa: a constituent of the basal gangha, the subthalamic nucleus, directly sends long descending axons contralaterally to spinal levels (particularly to the upper cervical spinal segments) in the rat. The total number of subthalamk; ~t~uJoi~s projecting to the spinal cord amounts to maximally 80 cells per brain. Given that the rat subthalamic nucleus contains 7,000-9,500 cells35, this specific cell population can be estimated to make up approximately 1% of subthalamic neurons. Thus, the subthalamospinal projection is sparse but by no means trivial. Moreover, our comparison of subthalamic cell-labeling reveals that FG might be the most sensitive of
132
Fig. 3. Photomicrographs of fluorescent-positive neurons in the subthalamie nucleus. TB was injected into the contralateral upper cervical spinal segments (C1-C5), and DY into the ipsilateral globus pallidus. The two pictures show the ventral, comb-shaped portions of the subthalamic nucleus at its rostral level. Open arrows indicate cells double-labeled with both tracers, and solid arrows cells singlelabeled with TB. Others are all single-labeled with DY. x280.
133 cerebral
cortex
................
t ...............
-,t
striatum pallidum
subthalamus
T [:
~ "
I !
substantia niora ~
s ~inal cord Fig. 4. Schematic summary of the parallelism between the corticospinal (pyramidal) and "extrapyramidal" systems. Major, representative pathways of the basal ganglia (rectangular area surrounded with broken lines) are shown here ":.
the 3 different retrograde tracers (TB, FG and HRP) used, even though a lesser amount was generally injected. These data are greatly consistent with those from our recent experiment with a different system 34. The existence of such projection neurons in the basal ganglia may lead us to the understanding that the 'extrapyramidal' system is an entity at least in the rat brain, thus justifying its parallelism with the co~icospinal (pyramidal) system (Fig. 4). However, the course and termination of the subthalamospina~ pathway remain to be explored definitively. We have not yet examined other mammalian brains from this point of view; if it can be proven that a similar pattern of the projection is common to most mammals (especially to primates), prevalent criticism on the concept of the 'extrapyramidal' motor system will need to be revised. Furthermore, the present study elucidates that single subthalamic neurons simultaneously innervate the ipsilateral globus pallidus as well as the contralateral spinal cord. These bifurcating projection neurons constitute 60-70% of spinal projecting subthalamic cells. In view of the fact that the majority (at least 94%) of rat subthalamic cells give rise to collateral projections to both the globus pallidus and substantia nigra 35, they also probably give off axonal branches to the substantia nigra. With respect to subthalamic afferents, considerable evidence emphasizes the massive projection from the globus pallidus to the subthalamic n u c l e u s 8"9"11"!5"19"24"29"36 Other available evidence suggests the existence of the mo-
tor corticosubthalamic 18"2°'21"3°and striatosubthalamic TM projections. The subthalamic nucleus may integrate the somatic motor information from the cortex, which is conveyed to the nucleus directly through the corticosubthalamic pathway and/or via the corpus striatum, and use it to exert an identical and simultaneous control over both the contralateral spinal cord and ipsilateral globus pallidus (Fig. 4). Both clinically and experimentally, discrete lesions in the subthalamic nucleus or its fiber connections (particularly with the globus pailidus) are regularly followed by a violent dyskinesia, 'hemiballism '4"14" 22.37.39 The subthalamic dyskinesia occurs in the limbs contralateral to the lesion and involves marked hypotonus. This can be abolished by a subsequent lesion of the ipsilateral pallidum 5a4 or a transection of the spinal cord at its cervical level (especially a lesion of the dorsal part of the lateral funiculus) 5"1°. In this light, our results directly provide a clear anatomical substrate for these neuropathological phenomena associated with dysfunction of the subthalamic nucleus. Thus, the specific subthalamic cell population established here, which sends axon collaterals to both the contralateral spinal cord and ipsilateral globus pallidus, might be an essentially fatal cell group in 'hemiballism'. This does not promptly imply that the 'extrapyramidal' system is a functionally complete and independent motor unit, because the fact that ablations of motor cortex and interruption of the corticospinal tract at various levels also abolish the dyskinesia suggests that impulses transmitted to spinal levels via the corticc~-pina! tract must be responsible for the dyskinesia 2"3"~°a2. It is, however, salutary to note that the concept of this so-called system may, in fact, be valid morphologically. In any case, morphological differentiation (of the course and termination) between the corticospinal and subthalamospinal tracts might be an important clue to functional segregation between the pyramidal and 'extrapyramidal" systems. A neo~::otransmitter related to the newly proposed subthalamospinal pathway remains to be determined definitive"1. However, several previous findings respect the notion that y-aminobutyric acid (GABA) may be a neurotransmitter of subthalamopallidal fibers. Two autoradiographic retrograde tracing studies using [3H]GABA suggest the existence of a presumed GABA-containing subthalamopallidai pro-
134 jection 27'33. Small localized injections of G A B A an-
cending contralaterally to the spinal cord, in the sub-
tagonists (i.e. picrotoxin or bicuculline) into the pallidum/subthalamic nucleus produce contralateral violent choreoid dyskinesia 16a7, which is identical to that resulting from small discrete electrolytic lesions in the subthalamic nucleus TM. Taken together, the data from our double-labeling experiment strongly favor G A B A e r g i c neurons, with long axons des-
thalamic nucleus,
REFERENCES
and pharmacological analysis of hyperkinesia ~'esulting from lesions of the subthalamic nucleus of Luys, J. Comp. Neurol., 92 (1950) 293-332. 15 Carter, D.A. and Fibiger, H.C., The projections of the entopeduncular nucleus and globus pallidus in rat as demonstrated by autoradiography and horseradish peroxidase histochemistry, J. Comp. Neurol., 177 (1978) 113-124. 16 Crossman, A.R., Sambrook, M.A. and Jackson, A., Experimental hemiballismus in the baboon produced by injection of a gamma-aminobutyric acid antagonist into the basal ganglia, Neurosci. Lett., 20 (1980) 369-372. 17 Crossman, A.R., Sambrook, M.A. and Jackson, A., Experimental hemichorea/hemiballismus in the monkey: studies on the intracerebral site of action in a drug-induced dyskinesia, Brain, 107 (1984) 579-596. 18 Hartmann-von Monakow, K., Akert, K.and Kiinzle, H., Projections of the precentral motor cortex and other cortical areas of the frontal lobe to the subthalamic nucleus in the monkey, Exp. Brain Res., 33 (1978) 395-403. 19 Kim, R., Nakano, K., Jayaraman, A. and Carpenter, M.B., Projections of the globus pallidus and adjacent structures: an autoradiographic study in the monkey, J. Comp. Neurol., 169 (1976) 263-290. 20 Kitai, S.T. and Deniau, J.M., Cortical inputs to the subthalamus: intracellular analysis, Brain Research, 214 (1981) 411-415. 21 Kiinzle, H. and Akert, K., Efferent connections of cortical area 8 (frontal eye field) in Macacafascicularis. A reinvestigation using the autoradiographic technique, J. Comp. Neurol., 173 (1977) 147-164. 22 Martin, J.P., Hemichorea resulting from a local lesion of the brain (syndrome of body of Luys), Brain, 50 (1927) 637-651. 23 Mesulam, M.-M., Tetramethyl benzidine for horseradish peroxidase neurohistochemistry: a non-carcinogenic blue reaction-product with superior sensitivity for visualizing afferents and efferents, J. Histochem. Cytochem., 26 (1978) 106-117. 24 Nauta, H.J.W., Projections of the pallidal complex: an autoradiographic study in the cat, Neuroscience, 4 (1979) 1853-1873. 25 Nauta, H.J.W., A proposed conceptual reorganization of the basal ganglia and telencephalon, Neuroscience, 4 (1979) 1875-1881. 26 Nauta, H.J.W. and Cole, M., Efferent projections of the subthalamic nucleus: an autoradiographic study in the monkey and cat, J. Comp. Neurol., 180 (1978) 1-16. 27 Nauta, H.J.W. and Cu6nod, M., Perikaryai cell labeling in the subthalamic nucleus following the injection of [3H]),-
1 Beckstead, R.M., A reciprocal axonal connection between the subthalamic nucleus and the neostriatum in the cat, Brain Research, 275 (1983) 137-142. 2 Bucy, P.C., The cortico-spinal tract and tremor. In W.S. Fields (Ed.), Pathoge.,.esis and Treatment of Parkinsonism, Charles C. Thomas, Springfield, 1958, pp. 271-293. 3 Bucy, P.C., The surgical treatment of abnormal involuntary movements, Neurologia, 1 (1959) 1-15. 4 Carpenter, M.B., Ballism associated with partial destruction of the subthalamic nucleus of Luys, Neurology, 5 (1955) 479-489. 5 Carpenter, M.B., Brainstem and infratentorial neuraxis in experimental dyskinesia, Arch. Neurol. (Chic.), 5 (1961) 504-524. 6 Carpenter, M.B., Anatomical organization of the corpus striatum and related nuclei. In M.D. Yahr (Ed.), The Basal Ganglia, Raven, New York, 1976, pp. 1-36. 7 Carpenter, M.B., Interconnections between the corpus striatum and brainstem nuclei. In J.S. McKenzie, R.E. Kemm and L.N. Wilcock (Eds.), The Basal Ganglia: Structure and Function, Advances in Behavioral Biology, Vol. 27, Plenum, New York, 1984, pp. 1-68. 8 Carpenter, M.B., Batton III, R.R., Carleton, S.C. and Keller, J.T., Interconnections and organization of pallidal and subthalamic nucleus neurons in the monkey, J. Comp. Neurol., 197 (1981) 579-603. 9 Carpenter, M.B., Carleton, S.C., Keller, J.T. and Conte, P., Connections of the subthalamic nucleus in the monkey, Brain Research, 224 (198!) 1-29. l0 Carpenter, M.B., Correll, J.W. and Hinman, A., Spinal tracts mediating subthalamic hyperkinesia. Physiological effects of selective partial cordotomies upon dyskinesia in rhesus monkey, J. Neurophysiol., 23 (1960) 288-304. 11 Carpenter, M.B., Fraser, R.A.R. and Shriver, J.E., The organization of the pallidosubthalamic fibers in the monkey, Brain Research, 11 (1968) 522-559. 12 Carpenter, M.B. and Mettler, F.A., Analysis of subthalamic hyperkinesia in the monkey, with special reference to ablations of agranular cortex, J. Comp. Neurol., 95 (1951) 125-158. 13 Carpenter, M.B. and Strominger, N.L., Efferent fibers of the subthalamic nucleus in the monkey. A comparison of the efferent projections of the subthalamic nucleus, substantia nigra and globus pailidus, Am. J. Anat., 121 (1967) 41-72. 14 Carpenter, M.B., Whittier, J.R. and Mettler, F.A., Analysis of choreoid hyperkinesia in the rhesus monkey. Surgical
ACKNOWLEDGEMENTS This work was supported by the Medical Research Council of Canada. M.T. is a recipient of an M R C Postprofessional Fellowship.
135 aminobutyric acid into the pallidal complex: an autoradiographic study in cat, Neuroscience, 7 (1982) 2725-2734. 28 Nauta, W.J.H. and Domesick, V.B., The anatomy of the extrapyramidal system. In K. Fuxe and D.B. Calne (Eds.), Dopaminergic Ergot Derivatives and Motor Function, Wenner-Gren Certer lnternatioaal Symposium Series, Vol. 31, Pergamon, Oxford, 1979, pp. 3-22. 29 Nauta, W.J.H. and Mehler, W.R., Projections of the lentiform nucleus in the monkey, Brain Research, 1 (1966) 3-42. 30 Romansky, K.V., Usunoff, K G . , Ivanov, D.P. and Galaboy, G.P., Corticosubthalamic projection in the cat: an electron microscopic study, Brain Research, 163 (1979) 319-322. 31 Royce, G.J. and Laine, E.J., Efferent connections of the c-audate nucleus, including cortical projections of the striaturn and other basal ganglia: an autoradiographic and horseradish peroxidase investigation in the cat, J. Comp. NeuroL, 226 (1984) 28-49. 32 Takada, M. and Hattori, T., The rat striatum: a target nucleus for ascending axon collaterals of the entopedunculohabenular pathway, Brain Research, in press. 33 Takada, M. and Hattori, T., Glyclne: an alternative transmitter candidate of the paUidosubthalamic projection neurons in the rat, J. Comp. Neurol., in press. 34 Takada, M., Li, Z.K. and Hattori, T., A direct projection
from the tuberomammillary nucleus to the spinal cord in the rat, Neurosci. Len., in press. 35 Van der Kooy, D. and Hattori, T., Single subthalamic nucleus neurons project to b~th the globus pallidus and substantia nigra in rat, J. Comp. Neurol., 192 (1980) 751-768. 36 Van der Kooy, D., Hattori, T., Shannak, K. and Hornvkiewicz, O., The paUido-subthalamic projection in the rat: anatomical and biochemical studies, Brain Research, 204 (1981) 253-268. 37 Whittier, J.R., Ballism and the subthalamic nucleus (nucleus hypothalamicus, corpus Luysi). Review of the literature and study of thirty cases, Arch. Neurol. Psychiat. (Chic.), 58 (1947) 672-692. 38 Whittier, J.R. and Mettler, F.A., Studies on the subthalamus of the rhesus monkey. I. Anatomy and fiber connections of the subthalamic nucleus of Luys, J. Comp. Neurol., 90 (1949) 281-317. 39 Whittier, J.R. and Merrier, F.A., Studies on the subthalamus of the rhesus monkey. II. Hyperkinesia and other physiologic effects of subthalamic lesions, with special reference to the subthalamic nucleus of Luys, J. Comp. Neurol., 90 (1949) 319-372. 40 Wilson, S.A.K., Progressive lenticular degeneration: a familial nervous disease associated with cirrhosis of the liver, Brain, 34 (1912) 295-509.