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Do some tyrosine hydroxylase-immunoreactive neurons in the human ventrolateral arcuate nucleus and globus pallidus produce only l -DOPA?
Neuroscience Letters, 133 (1991) 203-206
203
© 1991 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/91/$ 03.50 NSL 08226
Do some tyrosine hydroxylase-immunoreactive neurons in the human ventrolateral arcuate nucleus and globus pallidus produce only L-DOPA? K. Komori, T. Fujii and I. N a g a t s u Department of Anatomy, School of Medicine, Fujita Health University, Toyoake (Japan) (Received 14 June 1991; Revised version received 4 September 1991; Accepted 4 September 1991)
Key words." L-DOPA; Tyrosine hydroxylase; Aromatic L-amino acid decarboxylase; Immunohistochemistry; Arcuate nucleus; Globus pallidus; Human The presence of 'tyrosine hydroxylase (TH)-only' -immunoreactive (IR) neurons is reported for the first time in the human hypothalamic region and basa ganglia, using immunohistochemical technique. 'TH-only'-IR neurons were demonstrated in the ventrolateral part of the arcuate nucleus and medial segment of globus paUidus. These neurons lacked aromatic L-amino acid decarboxylase (AADC)-, dopamine-/~-hydroxylase (DBH)-, and dopamine (DA)-immunoreactivity. The above results suggest that these 'TH-only'-IR neurons are not dopaminergic, and possibly contain LDOPA as an end-product.
Tyrosine hydroxylase (TH), which catalyzes the ratelimiting step in catecholamine biosynthesis, is able to synthesize L-DOPA. Recently, L-DOPA has been suggested as a new neurotransmitter candidate in 'THonly'-immunoreactive (IR) neurons, namely L-DOPA neurons [9,12]. Similar findings of TH-IR neurons have been reported in some mammalian brains [2, 4, 7, 8, 12, 13, 15]. However, studies concerning the possible existence of L-DOPA neurons in the human brain have not yet been reported. In the present study, we demonstrate that neurons contained TH but lacking aromatic Lamino acid decarboxylase (AADC), dopamine-fl-hydroxylase (DBH) and dopamine (DA) were present in the human ventrolateral arcuate nucleus and medial segment of globus pallidus. The human brain from a lmonth-old male, who died without neurological illness was used in this study. The brain was transported and
Fig. 1. Positive control of the human substantia nigra. A: tyrosine hydroxylase (TH-) immunoreactivity. B: aromatic L-amino acid decarboxylase (AADC)-immunoreactivity. Immunohistochemical results indicate an overlap of TH-IR cells and AADC-IR cells (arrows). x 30. Box: Lower magnification of TH or AADC immunoreactivity, x 10.
Correspondence: I. Nagatsu, Department of Anatomy, School of Medicine, Fujita Health University, Toyoake, Aichi 470-I 1, Japan
204 dissected on crushed ice, and the thin blocks (5 mm thick) were fixed in 4% paraformaldehyde (in 0.1 M phosphate buffer, pH 7.4) or 5% glutaraldehyde (in 0.01 M cacodylate buffer, pH 7.4). The interval from death to fixation was within 10 h. After fixation, the sections (30/lm thickness) were cut on a cryostat. The sections were incubated with anti-TH, anti-AADC, anti-DBH and anti-DA antisera (each diluted 2000-fold). And freefloated sections were prepared for immunohistochemistry by means of peroxidase-antiperoxidase (PAP) method as described previously [12, 13, 15, 16]. As a positive control, we demonstrated that anti-TH and anti-AADC antibodies adequately stained the DA neu-
rons of human substantia nigra (Fig. 1). The specificity and characteristics of these antibodies have been described in detail elsewhere [16]. Ventrolateral arcuate nucleus. Dahlstr6m and Fuxe have already described a presumably DA-containing cell group in the arcuate nucleus, and this cell group has been designated as A12 group [1]. Thereafter, it has been observed that the arcuate nucleus contained at least two subpopulations of T H - I R neurons located respectively in the dorsomedial (A12d) and ventrolateral (A12v) parts of the nucleus with immunostaining discrepancy, a strong TH-like immunoreactivity in the former one and a weaker reaction in the latter one [6]. Thus, their DA nature
A1
A
Fig. 2. A: schematic drawing of the human hypothalamus at frontal sections. TH-IR cells were plotted on each drawing in a one-to-one fashion. Box: area shown in B (ventral part of the arcuate nucleus). B: TH-IR cells. C: AADC-immunoreaction. D: dopamine-fl-hydroxylase(DBH)immunoreaction. E: dopamine (DA)-immunoreaction.TH-IR cells lacked AADC, DBH and DA-immunoreactivity.B,C x 150; D,E x 75.
205 has been questioned in the A12v part of the arcuate nucleus. At the midhypothalamic level, we defined T H - I R neurons in the A12d and A12v parts of the arcuate nucleus (Fig. 2A). These neurons were medium in size with variable shapes: fusiform, round and oval. They had short and branched aspiny dendrites but no projecting fibers. When comparing T H and A A D C staining on adjacent sections, there was a discrepancy between the T H - and A A D C - I R cell distribution in ~the ventral part of the arcuate nucleus, where TH-posifive but A A D C -
negative cells were present (Fig. 2B,C). These T H - I R neurons did not react with the antisera for D B H and D A (Fig. 2D,E). On the other hand, T H - and A A D C - I R fibers were observed throughout the sections. Recently, Meister et al. [14], O k a m u r a et al. [17], and K i t a h a m a et al. [11] reported the possible presence of L-DOPA containing neurons in the rat and cat ventrolateral part of the arcuate nucleus using immunohistochemical techniques, but they did not examine primates. Globus pallidus (GP). The G P is one of the main
F Fig. 3. The human basal ganglia at frontal sections. A: TH-IR cells (arrows). B: higher magnification of the TH-IR neuron (arrow). C: AADCimmunoreaction. D: DBH-immunoreaction. E: DA-immunoreaction. TH-IR cells lacked AADC, DBH and DA-immunoreactivity. F: schematic drawing of the human basal ganglia at frontal sections. TH-IR cells were plotted on each drawing in a one-to-one fashion. PU, putamen. A,C x 50; B,D,E x 75.
206 c o m p o n e n t s o f the basal ganglia, a n d m a m m a l i a n G P can be s u b d i v i d e d into two parts, lateral (GP1) a n d medial ( G P m ) segments, by a l a m i n a o f m y e l i n a t e d fibers. The G P receives G A B A e r g i c [3] a n d e n k e p h a l i n e r g i c [5] innervations from neural g r o u p s situated a l o n g the striarum. In this study, we have f o u n d T H - p o s i t i v e b u t A A D C - n e g a t i v e n e u r o n s in the G P m b u t n o t in GPI (Fig. 3A,B,C). F u r t h e r m o r e , these n e u r o n s d i d n o t react with the antisera for D B H a n d D A (Fig. 3D,E). M o s t o f T H - I R n e u r o n s existed t h r o u g h o u t the G P m . Previous reports have d o c u m e n t e d the presence o f interneurons in the G P with s u b s t a n c e - P [5], s o m a t o s t a t i n [10] a n d acetylcholine [18]. G e n e r a l l y , the G P is considered to be d e v o i d o f m o n o a m i n e r g i c p e r i k a r y a . Thus, the possible existence o f T H - I R cells in the h u m a n G P should be noted. O n the o t h e r h a n d , there were no T H - I R perik a r y a in the c a u d o p u t a m e n , b u t T H - I R fibers were seen t h r o u g h o u t the striatum. O u r results clearly showed the existence o f T H - I R n e u r o n s in the h u m a n v e n t r o l a t e r a l p a r t o f the a r c u a t e nucleus a n d G P m , a n d these T H - I R n e u r o n s lacked A A D C , D B H a n d D A . Thus, we speculated that these n e u r o n s m i g h t p r o d u c e L - D O P A as an e n d - p r o d u c t b u t were p r o b a b l y u n a b l e to synthesize c a t e c h o l a m i n e s a n d might p l a y a n y role in the local circuit. Similar findings o f T H - I R n e u r o n s have been r e p o r t e d d u r i n g development, in the rat cortex [13] a n d inferior colliculus [8], m o u s e a n t e r i o r o l f a c t o r y nucleus [15], c a u d o p u t a m e n [12], m o n k e y f o r e b r a i n [2] a n d h u m a n cerebral cortex [4, 7]. H o w e v e r , the existence o f L - D O P A n e u r o n s in the hum a n h y p o t h a l a m u s a n d basal ganglia, has n o t yet been reported. Recently, L - D O P A has been suggested as a new n e u r o t r a n s m i t t e r c a n d i d a t e in ' T H - o n l y ' - I R neurons, n a m e l y L - D O P A n e u r o n s [9, 12], because they lacked classical amines. The functional significance o f LD O P A in the b r a i n is still u n k n o w n , a n d further studies are needed. A n t i - L - D O P A a n t i s e r u m m a y hopefully indicate m o r e precise answers to the p r o b l e m s . The a u t h o r s are very grateful to m e m b e r s o f the Dep a r t m e n t o f P a t h o l o g y for giving us h u m a n specimens a n d to Dr. K. Y a m a d a for help a n d advice on this study. W e w o u l d like to t h a n k Dr. W.E. W i n t e r ( D e p a r t m e n t o f P a t h o l o g y , U n i v e r s i t y o f F l o r i d a , U . S . A . ) for his v a l u a b l e c o m m e n t s a n d linguistic correction. This w o r k was s u p p o r t e d in p a r t by G r a n t s - i n - A i d for Scientific Research, M i n i s t r y o f E d u c a t i o n , Science a n d Culture, J a p a n , a n d by a G r a n t - i n - A i d from F u j i t a H e a l t h University, J a p a n . 1 Dahlstrrm, A. and Fuxe, K., Evidence for the existence of monoamine-containing neurons in the central nervous system. I. Demonstration of monoamines in the cell bodies of brainstem neurons. Acta. Physiol. Scand, 62 (1964) 1-55. 2 Dubach, M., Schmidt, R., Kunkel, D., Bowden, D.M., Martin, R. and German, D.C., Primate neostriatal neurons containing tyro-
sine hydroxylase: immunohistochemical evidence, Neurosci. Lett., 75 (1987) 205-210. 3 Fonnum, F., Gottesfeld, Z. and Grofova, I., Distribution of glutamate decarboxylase, choline acetyltransferase and aromatic amino acid decarboxylase in the basal ganglia of normal and operated rats. Evidence for striatopallidal, striatoentopeduncular and striatonigral GABAergic fibers. Brain Res., 143 (1978) 125-138. 4 Gaspar, P., Berger, B., Febvret, A., Vigny, A., Krieger-Poulet, M. and Borri-Voltattorni, C., Tyrosine hydroxylase-immunoreactive neurons in the human cerebral cortex: a novel catecholaminergic group?, Neurosci. Lett., 80 (1987) 257-262. 5 Haber, S. and Elde, R., Correlation between met-enkephalin and substance P immuno-reactivity in the primate globus pallidus, Neuroscience, 6 (1981) 1291- 1297. 6 Hrkfelt, T., M~trtensson, R., Bjrrklund, A., Kleinau, S. and Goldstein, M., Distributional maps of tyrosine hydroxylase-immunoreactive neurons in the rat brain. In A. Bjrrklund and T. Hrkfelt (Eds.), Handbook of Chemical Neuroanatomy, Vol. 2, Classical Transmitters in the CNS, Part 1, Elsevier, Amsterdam, 1984, pp. 277 379. 7 Hornung, J.P., Tork, I. and De Tribolet, N., Morphology of tyrosine hydroxylase-immunoreactive neurons in the human cerebral cortex, Exp. Brain Res., 76 (1989) 12-20. 8 Jaeger, C.B. and Joh, T.H., Transient expression of tyrosine hydroxylase in some neurons of the developing inferior colliculus of the rat, Dev. Brain Res., 11 (1983) 128-132. 9 Jaeger, C.B., Ruggiero, D.A., Albert, D.H., Joh, T.H. and Reis, D.J., Aromatic L-amino acid decarboxylase in the rat brain: immunocytochemical localization in neurons of the brain stem, Neuroscience, 11 (1984) 691-713. 10 Johansson, O., Hrkfelt, T. and Elde, R.P., Immunohistochemical distribution of somatostatin-like immunoreactivity in the central nervous system of the adult rat. Neuroscience, 13 (1984) 265 339. 11 Kitahama, K., Geffard, M., Okamura, H., Nagatsu, I., Mons, N. and Jouvet, M., Dopamine- and DOPA-immunoreactive neurons in the cat forebrain with reference to tyrosine hydroxylase-immunohistochemistry, Brain Res., 518 (1990) 83-94. 12 Komori, K., Sakai, M., Karasawa, N., Yamada, K. and Nagatsu, I., Evidence for transient expression of tyrosin hydroxylase immunoreactivity in the mouse striatum and the effect of colchicine, Acta. Histochem. Cytochem., 24 (1991) 223-231. 13 Kosaka, T., Hama, K. and Nagatsu, I., Tyrosine hydroxylaseimmunoreactive intrinsic neurons in the rat cerebral cortex, Exp. Brain Res., 68 (1987) 393~,05. 14 Meister, B., Hrkfelt, T., Steinbusch, H.W.M., Skagerberg, G., Lindvall, O., Geffard, M., Joh, T.H., Cuello, A.C. and Goldstein, M., Do tyrosine hydroxylase-immunoreactive neurons in the ventrolateral arcuate nucleus produce dopamine or only L-DOPA? J. Chem. Neuroanat., 1 (1988) 59~:~4. 15 Nagatsu, I., Komori, K., Takeuchi, T., Sakai, M., Yamada, K. and Karasawa, N., Transient tyrosine hydroxylase-immunoreactive neurons in the region of anterior olfactory nucleus of pre- and postnatal mice do not contain dopamine, Brain Res., 511 (1990) 55-62. 16 Nagatsu, I., Sakai, M., Yoshida, M. and Nagatsu, T., Aromatic Lamino acid decarboxylase immunoreactive neurons in and around the cerebrospinal fluid-contacting neurons of central canal do not contain dopamine of serotonin in the mouse and rat spinal cord, Brain Res., 475 (1988)91-102. 17 Okamura, H., Kitahama, K., Mons, N., Ibata, Y., Jouvet, M. and Geffard, M., L-DOPA-immunoreactive neurons in the rat hypothalamic tuberal region, Neurosci. Lett., 95 (1988) 4246. 18 Zaborszky, L., Eckenstein, F., Leranth, C., Oertel, W., Schmechel, D.E., Alones, V. and Heimer, L., Cholinergic cells of the ventral pallidum: a combined electron microscopic, immunocytochemical, degeneration and HRP study, Soc. Neurosci. Abstr., 10 (1984) 8.
203
© 1991 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/91/$ 03.50 NSL 08226
Do some tyrosine hydroxylase-immunoreactive neurons in the human ventrolateral arcuate nucleus and globus pallidus produce only L-DOPA? K. Komori, T. Fujii and I. N a g a t s u Department of Anatomy, School of Medicine, Fujita Health University, Toyoake (Japan) (Received 14 June 1991; Revised version received 4 September 1991; Accepted 4 September 1991)
Key words." L-DOPA; Tyrosine hydroxylase; Aromatic L-amino acid decarboxylase; Immunohistochemistry; Arcuate nucleus; Globus pallidus; Human The presence of 'tyrosine hydroxylase (TH)-only' -immunoreactive (IR) neurons is reported for the first time in the human hypothalamic region and basa ganglia, using immunohistochemical technique. 'TH-only'-IR neurons were demonstrated in the ventrolateral part of the arcuate nucleus and medial segment of globus paUidus. These neurons lacked aromatic L-amino acid decarboxylase (AADC)-, dopamine-/~-hydroxylase (DBH)-, and dopamine (DA)-immunoreactivity. The above results suggest that these 'TH-only'-IR neurons are not dopaminergic, and possibly contain LDOPA as an end-product.
Tyrosine hydroxylase (TH), which catalyzes the ratelimiting step in catecholamine biosynthesis, is able to synthesize L-DOPA. Recently, L-DOPA has been suggested as a new neurotransmitter candidate in 'THonly'-immunoreactive (IR) neurons, namely L-DOPA neurons [9,12]. Similar findings of TH-IR neurons have been reported in some mammalian brains [2, 4, 7, 8, 12, 13, 15]. However, studies concerning the possible existence of L-DOPA neurons in the human brain have not yet been reported. In the present study, we demonstrate that neurons contained TH but lacking aromatic Lamino acid decarboxylase (AADC), dopamine-fl-hydroxylase (DBH) and dopamine (DA) were present in the human ventrolateral arcuate nucleus and medial segment of globus pallidus. The human brain from a lmonth-old male, who died without neurological illness was used in this study. The brain was transported and
Fig. 1. Positive control of the human substantia nigra. A: tyrosine hydroxylase (TH-) immunoreactivity. B: aromatic L-amino acid decarboxylase (AADC)-immunoreactivity. Immunohistochemical results indicate an overlap of TH-IR cells and AADC-IR cells (arrows). x 30. Box: Lower magnification of TH or AADC immunoreactivity, x 10.
Correspondence: I. Nagatsu, Department of Anatomy, School of Medicine, Fujita Health University, Toyoake, Aichi 470-I 1, Japan
204 dissected on crushed ice, and the thin blocks (5 mm thick) were fixed in 4% paraformaldehyde (in 0.1 M phosphate buffer, pH 7.4) or 5% glutaraldehyde (in 0.01 M cacodylate buffer, pH 7.4). The interval from death to fixation was within 10 h. After fixation, the sections (30/lm thickness) were cut on a cryostat. The sections were incubated with anti-TH, anti-AADC, anti-DBH and anti-DA antisera (each diluted 2000-fold). And freefloated sections were prepared for immunohistochemistry by means of peroxidase-antiperoxidase (PAP) method as described previously [12, 13, 15, 16]. As a positive control, we demonstrated that anti-TH and anti-AADC antibodies adequately stained the DA neu-
rons of human substantia nigra (Fig. 1). The specificity and characteristics of these antibodies have been described in detail elsewhere [16]. Ventrolateral arcuate nucleus. Dahlstr6m and Fuxe have already described a presumably DA-containing cell group in the arcuate nucleus, and this cell group has been designated as A12 group [1]. Thereafter, it has been observed that the arcuate nucleus contained at least two subpopulations of T H - I R neurons located respectively in the dorsomedial (A12d) and ventrolateral (A12v) parts of the nucleus with immunostaining discrepancy, a strong TH-like immunoreactivity in the former one and a weaker reaction in the latter one [6]. Thus, their DA nature
A1
A
Fig. 2. A: schematic drawing of the human hypothalamus at frontal sections. TH-IR cells were plotted on each drawing in a one-to-one fashion. Box: area shown in B (ventral part of the arcuate nucleus). B: TH-IR cells. C: AADC-immunoreaction. D: dopamine-fl-hydroxylase(DBH)immunoreaction. E: dopamine (DA)-immunoreaction.TH-IR cells lacked AADC, DBH and DA-immunoreactivity.B,C x 150; D,E x 75.
205 has been questioned in the A12v part of the arcuate nucleus. At the midhypothalamic level, we defined T H - I R neurons in the A12d and A12v parts of the arcuate nucleus (Fig. 2A). These neurons were medium in size with variable shapes: fusiform, round and oval. They had short and branched aspiny dendrites but no projecting fibers. When comparing T H and A A D C staining on adjacent sections, there was a discrepancy between the T H - and A A D C - I R cell distribution in ~the ventral part of the arcuate nucleus, where TH-posifive but A A D C -
negative cells were present (Fig. 2B,C). These T H - I R neurons did not react with the antisera for D B H and D A (Fig. 2D,E). On the other hand, T H - and A A D C - I R fibers were observed throughout the sections. Recently, Meister et al. [14], O k a m u r a et al. [17], and K i t a h a m a et al. [11] reported the possible presence of L-DOPA containing neurons in the rat and cat ventrolateral part of the arcuate nucleus using immunohistochemical techniques, but they did not examine primates. Globus pallidus (GP). The G P is one of the main
F Fig. 3. The human basal ganglia at frontal sections. A: TH-IR cells (arrows). B: higher magnification of the TH-IR neuron (arrow). C: AADCimmunoreaction. D: DBH-immunoreaction. E: DA-immunoreaction. TH-IR cells lacked AADC, DBH and DA-immunoreactivity. F: schematic drawing of the human basal ganglia at frontal sections. TH-IR cells were plotted on each drawing in a one-to-one fashion. PU, putamen. A,C x 50; B,D,E x 75.
206 c o m p o n e n t s o f the basal ganglia, a n d m a m m a l i a n G P can be s u b d i v i d e d into two parts, lateral (GP1) a n d medial ( G P m ) segments, by a l a m i n a o f m y e l i n a t e d fibers. The G P receives G A B A e r g i c [3] a n d e n k e p h a l i n e r g i c [5] innervations from neural g r o u p s situated a l o n g the striarum. In this study, we have f o u n d T H - p o s i t i v e b u t A A D C - n e g a t i v e n e u r o n s in the G P m b u t n o t in GPI (Fig. 3A,B,C). F u r t h e r m o r e , these n e u r o n s d i d n o t react with the antisera for D B H a n d D A (Fig. 3D,E). M o s t o f T H - I R n e u r o n s existed t h r o u g h o u t the G P m . Previous reports have d o c u m e n t e d the presence o f interneurons in the G P with s u b s t a n c e - P [5], s o m a t o s t a t i n [10] a n d acetylcholine [18]. G e n e r a l l y , the G P is considered to be d e v o i d o f m o n o a m i n e r g i c p e r i k a r y a . Thus, the possible existence o f T H - I R cells in the h u m a n G P should be noted. O n the o t h e r h a n d , there were no T H - I R perik a r y a in the c a u d o p u t a m e n , b u t T H - I R fibers were seen t h r o u g h o u t the striatum. O u r results clearly showed the existence o f T H - I R n e u r o n s in the h u m a n v e n t r o l a t e r a l p a r t o f the a r c u a t e nucleus a n d G P m , a n d these T H - I R n e u r o n s lacked A A D C , D B H a n d D A . Thus, we speculated that these n e u r o n s m i g h t p r o d u c e L - D O P A as an e n d - p r o d u c t b u t were p r o b a b l y u n a b l e to synthesize c a t e c h o l a m i n e s a n d might p l a y a n y role in the local circuit. Similar findings o f T H - I R n e u r o n s have been r e p o r t e d d u r i n g development, in the rat cortex [13] a n d inferior colliculus [8], m o u s e a n t e r i o r o l f a c t o r y nucleus [15], c a u d o p u t a m e n [12], m o n k e y f o r e b r a i n [2] a n d h u m a n cerebral cortex [4, 7]. H o w e v e r , the existence o f L - D O P A n e u r o n s in the hum a n h y p o t h a l a m u s a n d basal ganglia, has n o t yet been reported. Recently, L - D O P A has been suggested as a new n e u r o t r a n s m i t t e r c a n d i d a t e in ' T H - o n l y ' - I R neurons, n a m e l y L - D O P A n e u r o n s [9, 12], because they lacked classical amines. The functional significance o f LD O P A in the b r a i n is still u n k n o w n , a n d further studies are needed. A n t i - L - D O P A a n t i s e r u m m a y hopefully indicate m o r e precise answers to the p r o b l e m s . The a u t h o r s are very grateful to m e m b e r s o f the Dep a r t m e n t o f P a t h o l o g y for giving us h u m a n specimens a n d to Dr. K. Y a m a d a for help a n d advice on this study. W e w o u l d like to t h a n k Dr. W.E. W i n t e r ( D e p a r t m e n t o f P a t h o l o g y , U n i v e r s i t y o f F l o r i d a , U . S . A . ) for his v a l u a b l e c o m m e n t s a n d linguistic correction. This w o r k was s u p p o r t e d in p a r t by G r a n t s - i n - A i d for Scientific Research, M i n i s t r y o f E d u c a t i o n , Science a n d Culture, J a p a n , a n d by a G r a n t - i n - A i d from F u j i t a H e a l t h University, J a p a n . 1 Dahlstrrm, A. and Fuxe, K., Evidence for the existence of monoamine-containing neurons in the central nervous system. I. Demonstration of monoamines in the cell bodies of brainstem neurons. Acta. Physiol. Scand, 62 (1964) 1-55. 2 Dubach, M., Schmidt, R., Kunkel, D., Bowden, D.M., Martin, R. and German, D.C., Primate neostriatal neurons containing tyro-
sine hydroxylase: immunohistochemical evidence, Neurosci. Lett., 75 (1987) 205-210. 3 Fonnum, F., Gottesfeld, Z. and Grofova, I., Distribution of glutamate decarboxylase, choline acetyltransferase and aromatic amino acid decarboxylase in the basal ganglia of normal and operated rats. Evidence for striatopallidal, striatoentopeduncular and striatonigral GABAergic fibers. Brain Res., 143 (1978) 125-138. 4 Gaspar, P., Berger, B., Febvret, A., Vigny, A., Krieger-Poulet, M. and Borri-Voltattorni, C., Tyrosine hydroxylase-immunoreactive neurons in the human cerebral cortex: a novel catecholaminergic group?, Neurosci. Lett., 80 (1987) 257-262. 5 Haber, S. and Elde, R., Correlation between met-enkephalin and substance P immuno-reactivity in the primate globus pallidus, Neuroscience, 6 (1981) 1291- 1297. 6 Hrkfelt, T., M~trtensson, R., Bjrrklund, A., Kleinau, S. and Goldstein, M., Distributional maps of tyrosine hydroxylase-immunoreactive neurons in the rat brain. In A. Bjrrklund and T. Hrkfelt (Eds.), Handbook of Chemical Neuroanatomy, Vol. 2, Classical Transmitters in the CNS, Part 1, Elsevier, Amsterdam, 1984, pp. 277 379. 7 Hornung, J.P., Tork, I. and De Tribolet, N., Morphology of tyrosine hydroxylase-immunoreactive neurons in the human cerebral cortex, Exp. Brain Res., 76 (1989) 12-20. 8 Jaeger, C.B. and Joh, T.H., Transient expression of tyrosine hydroxylase in some neurons of the developing inferior colliculus of the rat, Dev. Brain Res., 11 (1983) 128-132. 9 Jaeger, C.B., Ruggiero, D.A., Albert, D.H., Joh, T.H. and Reis, D.J., Aromatic L-amino acid decarboxylase in the rat brain: immunocytochemical localization in neurons of the brain stem, Neuroscience, 11 (1984) 691-713. 10 Johansson, O., Hrkfelt, T. and Elde, R.P., Immunohistochemical distribution of somatostatin-like immunoreactivity in the central nervous system of the adult rat. Neuroscience, 13 (1984) 265 339. 11 Kitahama, K., Geffard, M., Okamura, H., Nagatsu, I., Mons, N. and Jouvet, M., Dopamine- and DOPA-immunoreactive neurons in the cat forebrain with reference to tyrosine hydroxylase-immunohistochemistry, Brain Res., 518 (1990) 83-94. 12 Komori, K., Sakai, M., Karasawa, N., Yamada, K. and Nagatsu, I., Evidence for transient expression of tyrosin hydroxylase immunoreactivity in the mouse striatum and the effect of colchicine, Acta. Histochem. Cytochem., 24 (1991) 223-231. 13 Kosaka, T., Hama, K. and Nagatsu, I., Tyrosine hydroxylaseimmunoreactive intrinsic neurons in the rat cerebral cortex, Exp. Brain Res., 68 (1987) 393~,05. 14 Meister, B., Hrkfelt, T., Steinbusch, H.W.M., Skagerberg, G., Lindvall, O., Geffard, M., Joh, T.H., Cuello, A.C. and Goldstein, M., Do tyrosine hydroxylase-immunoreactive neurons in the ventrolateral arcuate nucleus produce dopamine or only L-DOPA? J. Chem. Neuroanat., 1 (1988) 59~:~4. 15 Nagatsu, I., Komori, K., Takeuchi, T., Sakai, M., Yamada, K. and Karasawa, N., Transient tyrosine hydroxylase-immunoreactive neurons in the region of anterior olfactory nucleus of pre- and postnatal mice do not contain dopamine, Brain Res., 511 (1990) 55-62. 16 Nagatsu, I., Sakai, M., Yoshida, M. and Nagatsu, T., Aromatic Lamino acid decarboxylase immunoreactive neurons in and around the cerebrospinal fluid-contacting neurons of central canal do not contain dopamine of serotonin in the mouse and rat spinal cord, Brain Res., 475 (1988)91-102. 17 Okamura, H., Kitahama, K., Mons, N., Ibata, Y., Jouvet, M. and Geffard, M., L-DOPA-immunoreactive neurons in the rat hypothalamic tuberal region, Neurosci. Lett., 95 (1988) 4246. 18 Zaborszky, L., Eckenstein, F., Leranth, C., Oertel, W., Schmechel, D.E., Alones, V. and Heimer, L., Cholinergic cells of the ventral pallidum: a combined electron microscopic, immunocytochemical, degeneration and HRP study, Soc. Neurosci. Abstr., 10 (1984) 8.