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Dopaminergic dorsal raphe neurons in cats and monkeys are sensitive to the toxic effects of MPTP
218
Neuroscience Letters, 94 (1988) 218 22 i Elsevier Scientific Publishers Ireland Lid
NSL 05673
Dopaminergic dorsal raphe neurons in cats and monkeys are sensitive to the toxic effects of MPTP G.A. Unguez and J.S. Schneider Centerfor NeurologicalResearch of the Department of Neurology and Institute of Neuroscience, Hahnernann University School of Medicine, Philadelphia, PA 19102-1192 (U.S.A.) and UCLA School of Medicine, Department of Neurology, Reed Neurological Research Center, Los Angeles, CA 90024-1769 (U.S.A.)
(Received 18 April 1988; Accepted 15 July 1988) Key words: Parkinsonism; Dorsal raphe nucleus; l-Methyl-4-phenyl-l,2,3,6-tetrahydropyridine; Monkey
Cat;
Tyrosine hydroxylase immunohistochemical analysis of the dorsal raphe nucleus (DRN) of severely parkinsonian MPTP-treated cats and cynomolgus monkeys revealed a marked loss of catecholaminergic neurons in this region. Cell loss was more extensive in the ventral portion of the nucleus with a relative sparing of neurons in the dorsal-most portions of the DRN. These results demonstrate that catecholaminergic neurons other than those in the ventral mesencephalon and the locus ceruleus are affected by the toxic effects of MPTP.
Although degeneration of nigrostriatal dopaminergic nerve cells is the primary pathology in Parkinson's disease, the degenerative process also involves other catecholaminergic neurons such as those in the locus ceruleus, dorsal motor vagus nuclei and other regions like the ventral tegmental area (VTA), the hypothalamus, and the raphe nuclei [3, 5]. Neuronal degeneration, Lewy body formation and presence of neurofibrillary tangles are pathological changes seen in neurons in the above-mentioned areas. These changes are characteristic of this multi-system disorder in humans [3].
Recently, the synthetic meperidine analog N-methyl-4-phenyl-l,2,3,6-tetrahydrop y r i d i n e ( M P T P ) has been f o u n d to p r o d u c e s y m p t o m s o f P a r k i n s o n ' s disease in hum a n s [6], n o n - h u m a n p r i m a t e s [1], a n d cats [11] when a d m i n i s t e r e d systemically a n d to result in d a m a g e to n e u r o n s in the s u b s t a n t i a n i g r a p a r s c o m p a c t a a n d o t h e r ventral m e s e n c e p h a l i c cell g r o u p s such as the V T A a n d r e t r o r u b r a l o r A-8 d o p a m i n e cell g r o u p . In a d d i t i o n , p a t h o l o g y has also been o b s e r v e d in the locus ceruleus, p a r ticularly in aged a n i m a l s [4]. These d a t a suggest t h a t M P T P - i n d u c e d p a r k i n s o n i s m m a y be m o r e a n a l o g o u s to i d i o p a t h i c P a r k i n s o n ' s disease t h a n originally t h o u g h t , Correspondence." J.S. Schneider, Hahnemann University School of Medicine, Dept. of Neurology, Broad and Vine Streets, Mail Stop 423, Philadelphia, PA 19102, U.S.A.
0304-3940/88/$ 03.50 © 1988 Elsevier Scientific Publishers Ireland Ltd.
219
since as in the idiopathic disorder, neurons other than the substantia nigra pars compacta dopaminergic cells are affected. The present study further examines the extent of MPTP-induced neuronal pathology by studying the dorsal raphe nucleus (DRN). Previous histochemical studies have demonstrated the existence of dopaminergic cell bodies in the D R N [2, 8], and cell loss in the human D R N as a result of Parkinson's disease has been reported [! 9], Since M P T P is considered primarily a dopaminergic toxin, we chose to examine whether dopaminergic neurons in the D R N might be affected by this toxin. A semiquantitative analysis of the D R N in non-human primates (M. fascicularis) and felines was performed. Brains from 5 normal adult male monkeys and 4 adult male MPTP-treated monkeys of similar age with severe parkinsonian m o t o r deficits were used along with brains from 2 normal adult male cats and 4 adult male, MPTPtreated cats of similar age. Cell counts were obtained from camera lucida drawings of 50-/~m-thick sections processed for tyrosine hydroxylase (TH) immunoreactivity. T H like immunoreactivity was detected by the avidin biotin immunoperoxidase technique described elsewhere [11]. Only TH-positive cells with distinct nuclei were counted within the area corresponding to the D R N (see below). The presence or absence of cells was confirmed with cresyl violet counterstaining of adjacent sections. Care was taken to match sections as closely as possible using gross landmarks so that similar anatomical levels of the D R N could be compared between the normal and MPTP-treated animals. Neuroanatomical landmarks such as the cerebral aqueduct and medial longitudinal fasciculus were used to help define the borders of the D R N . Since we did not obtain total cell counts from serial sections through the D R N , we selected sections for quantitation that best represented each of 4 distinct anatomical levels in the D R N defined empirically. For the monkeys, Level 1, the most rostral, included D R N cells at approximately the level of the oculomotor nucleus (pars dorsolateralis) and the decussation of the superior cerebellar peduncle. This corresponded to an approximate stereotaxic coordinate A:2.5 [15]. Extending caudally, Level 2 was the D R N cell group sampled at approximately A: 1.0. Further caudally, Level 3 corresponded to the D R N cell group located at the level of the trochlear nerve which is seen at the base of the periaqueductal grey matter (AP:0). Level 4, the most caudal, included the D R N cell population sampled at the level corresponding approximately to P:0.5. All coordinates are taken from the Snider and Lee atlas [15]. A total of 33 sections were sampled from control animals and 46 sections were sampled from MPTP-treated monkeys. Similarly, 4 anatomical levels of the D R N were analyzed in the cat. Level 1 corresponded to the D R N at approximately A:I.0. Level 2 included the D R N at the rostral extent of the trochlear nucleus (A:0.0). Extending caudally, our Level 3 was approximately at P:l.0 and Level 4, our most caudal level, corresponded to the raphe cell group located at approximately P:2.0. All coordinates were taken from the Snider and Nieman atlas [14]. Thirty-three sections were sampled from control animals while 58 sections were sampled from MPTP-treated cats. F o r both cats and monkeys, mean cell counts (and S.D.) were obtained for each defined anatomical level and the Student's t-test was used to statistically compare counts obtained from normal and
220
MPTP-treated animals. In normal control animals, TH-positive neuronal cell bodies were readily observed in the D R N of both cats and monkeys. In cats, numerous round and oval cells, ranging in size from 12 to 1 6 / t m in diameter, were distributed throughout the D R N , spanning from the cerebral aqueduct (dorsal D R N ) to the medial longitudinal fascicull (ventral D R N ) , as previously described by Leger and Wiklund [7]. Other THpositive neurons were fusiform in shape and ranged in size from 16 to 35/zm in diameter. The smaller of these fusiform neurons tended to be located in the mid-region of the D R N along the midline. The larger of these fusiform-shape neurons were predominantly located lateral to the midline and were more numerous near the ventral portion of the nucleus. In normal monkeys, the morphology and distribution pattern of TH-positive neurons were basically similar to those observed in normal cats. Round and oval cells, ranging in size from 8 to 18 j~m in diameter were scattered throughout the D R N . Larger angular and fusiform-shaped neurons, ranging from approximately 16 to 39 /tm in diameter were found mostly lateral to the midline and toward the ventral portion of the nucleus. In normal cats, we found our levels 2 and 3 to contain the most TH-positive neurons. MPTP-treated cats showed a significant loss of TH-positive cells in our levels 1, 3 and 4 (Table IB). Following M P T P exposure, definite cell loss was observed throughout the D R N in the cat, with perhaps more pronounced cell loss in the more ventral portions of the nucleus in some animals. Fig. 1B shows the extent of D R N
TABLE 1 A V E R A G E C O U N T S O F T H - I M M U N O R E A C T I V E N E U R O N S IN T H E D O R S A L R A P H E OF N O R M A L A N D M P T P - T R E A T E D M O N K E Y S A N D CATS Anatomical levels extend from the most rostral D R N (level 1) to the most caudal D R N (level 4). See text for further description. Animals
Monkeys Control (n=5) MPTP-treated (n=4) Cats Control (n=2) MPTP-treated (n=4)
N u m b e r of TH-immunoreactive D R N cells (average) Level 1
Level 2
Level 3
Level 4
155.43___59.11
66.53+31.66
18.29-+ 6.45
10.00_+3.91
44.47+24.47 P > 0.002
26.91+ 8.64 P > 0.002
13.25_+ 6.90
3.9-t-3.87 P > 0.05
25.54_+25.88
74.17 +43.81
68.27_+66.30
17.33+4.72
10.31 + 17.58 P>0.05
63.86_+48.83
16.94_+ 7.60 P>0.005
5.0-+3.74 P>0.01
221
Fig. 1. A: tyrosine hydroxylase (TH) immunoreactivity in the normal cat dorsal raphe nucleus (DRN). B: TH immunoreactivity in the DRN of a cat with signs of severe parkinsonism following 7 days administration of MPTP. C: TH immunoreactivity in DRN of normal monkey. D: TH immunoreactivity in the DRN of a severely parkinsonian monkey. Bar= 100 ~m.
cell loss observed in one of the more severely affected cats. Although we observed a decrease in the number of all TH-positive cell types in the cat, the larger fusiform cells in the ventral D R N appeared to be most affected by the MPTP. In monkeys, TH-positive neurons in both control and MPTP-treated animals were found to be most abundant in the more rostral portions of the D R N (our levels 1 and 2). In MPTP-treated monkeys, we observed a significant loss of TH-positive neurons at our levels 1, 2 and 4 (Table 1A). The pattern of D R N cell loss in monkeys was similar to that observed in the cats with cell loss evident throughout the nucleus but with perhaps more pronounced cell loss in the ventral and lateral portions of the nucleus. Again, predominantly large fusiform and angular cells appeared to be most susceptible to toxic effects of MPTP. Fig. 1D shows the extent of cell loss in a severely affected monkey (compared to the normal monkey, Fig. IC). The pathology of idiopathic Parkinson's disease suggests that the degenerative changes affect a variety of systems in addition to the nigrostriatal dopamine system
222 [3, 5]. Similarly, cell groups other than the substantia nigra pars compacta are found to sustain damage in MPTP-induced parkinsonism [12]. The present results further demonstrate that the toxic effects o f M P T P are not confined to the substantia nigra pars compacta. Since the D R N in both monkeys and cats is not considered to be a neuromelanin-containing structure, it is unlikely that the observed cell loss was due to n e u r o m e l a n i n - M P T P interactions. One possible alternative explanation for the D R N cell loss is that serotonergic D R N cells, which contain M A O - B [10, 18], took up MPTP, converted it to and released M P P ÷ [13], which in turn was toxic to the adjacent dopaminergic neurons. In the past, we have shown that at least in the cat, M P T P accumulates to a great extent in the D R N [10]. Dopaminergic neurons in the D R N have been previously described by others using TH-immunohistochemical techniques [2] and by a combination of microspectrofluorometry with fluorescence histochemistry [8]. In addition, dopaminergic D R N afferent projections to the striatum have been demonstrated by combined TH-immunohistochemistry and radioautography [2] as well as with H R P retrograde labeling [9]. Both of these studies found dopaminergic neurons predominantly located close to the midline and within the ventral portion of the D R N . Striatally projecting D R N neurons have also been reported to be restricted to the more rostral parts of the D R N [9]. In the present study, TH-positive cells located in the ventral and rostral portions of the D R N were destroyed to a larger extent than TH-positive cells located in the dorsal and caudal portions of the nucleus. This pattern of dopaminergic cell destruction in the D R N following administration of M P T P closely parallels the localization of dopaminergic D R N neurons which are found to project to both the substantia nigra and striatum [2]. Therefore, the possibility exists that MPTP-induced damage to D R N dopaminergic neurons may further impair striatal functioning and further contribute to the severe striatal dopamine depletion seen following M P T P exposure. The functional significance of D R N dopaminergic neurons has not been clearly defined. Dopamine-containing neurons in the D R N are much fewer in number than serotonin (5-HT)-containing neurons [I 6]. The D R N 5-HT pathways innervating the striatum [2, 17] are thus also more extensive than the corresponding dopaminergic projections [9]. Yet, it is possible that D R N dopaminergic neurons may play an important role in modulating serotonergic neuronal activity within the D R N , thus affecting the interactions between the D R N and the striatum with a resultant influence on m o t o r output. This research was supported in part by U S P H S Grants DE7282 and MH41645. 1 Burns, R.S., Chiueh, C.C., Markey, S., Ebert, M.H., Jacobowitz, D. and Kopin, l.J., A primate model of Parkinson's disease: selectivedestruction of substantia nigra pars compacta dopaminergic neurons by N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine,Proc. Natl. Aead. Sci. U.S.A., 80 (1983) 4546-4550. 2 Descarries, L., Berthelet, F., Garcia, S. and Beaudet, A., Dopaminergic projection from nucleus raphe dorsalis to neostriatum in the rat, J. Comp. Neurol., 249 (1986) 511 520. 3 Forno, L.S., Pathology of Parkinson's disease. In C.D. Marsden and S. Fahn (Eds.), Neurology 2, Movement Disorders, Butterworth, London, 1982, pp. 25-40.
223 4 Forno, L.S., Langston, J.W., Delanney, L.E., Irwin, I. and Ricaurte, G.A., Locus ceruleus lesions and eosinophilic inclusions in MPTP-treated monkeys, Ann. Neurol., 20 (1986) 449~455. 5 Jellinger, K., Overview of morphological changes in Parkinson's disease. In M.D. Yahr and K.J. Bergmann (Eds.), Advances in Neurology, Vol. 45, Raven, New York, 1986, pp. 1 18. 6 Langston, J.W., Ballard, P., Tetrud, J.W. and Irwin, I., Chronic parkinsonism in humans due to a product of meperidine-analog synthesis, Science, 219 (I 983) 979 980. 7 Leger, L. and Wiklund, L., Distribution and numbers of indoleamine cell bodies in the cat brainstem determined with Falck-Hillarp fluorescence histochemistry, Brain Res. Bull., 9 (1982) 245 251. 8 Ochi, J. and Shimizu, K., Occurrence of dopamine-containing neurons in the midbrain raphe nuclei of the rat, Neurosci. Lett., 8 (1978) 317 320. 9 Pasquier, D.A., Kemper, T.L., Forbes, W.B. and Morgane, P.J., Dorsal raphe, substantia nigra and locus ceruleus: interconnections with each other and the neostriatum, Brain Res. Bull., 2 (1977) 323 339. 10 Schneider, J.S., MPTP Parkinsonism in the cat: Pattern of neuronal loss may partially be explained by the distribution of MAO-B in the brain. In M.B. Carpenter and A. Jayaraman (Eds.), The Basal Ganglia I1." Structure and Function." Current Concepts, Plenum, New York, 1987, pp. 405 413. 11 Schneider, J.S., Yuwiler, A. and Markham, C.H., Neurotoxic effects of N-methyl-4-phenyl-l,2,3,6tetrahydropyridine (MPTP) in the cat. Tyrosine hydroxylase immunohistochemistry, Brain Res., 373 (1986) 258 267. 12 Schneider, J.S., Yuwiler, A. and Markham, C.H., Selective loss of subpopulations of ventral mesencephalic dopaminergic neurons in the monkey following exposure to MPTP, Brain Res., 411 (1987) 144 150.
13 Shen, R.-S., Abell, C.W., Gessner, W. and Brossi, A., Serotonergic conversion of MPTP and dopaminergic accumulation of MPP ÷, FEBS Lett., 189 (1985) 22.%230. 14 Snider, R.S. and Niemer, W.T., Stereotaxic Atlas of the Cat Brain, The University of Chicago Press, Chicago, 1961. 15 Snider, R.S. and Lee, J.C., Stereotaxic Atlas of the Monkey Brain, The University of Chicago Press, Chicago, 1961. 16 Steinbusch, H.W.M. and Nieuwenhuys, R., The raphe nuclei of the rat brainstem: a cytoarchitectonic and immunohistochemical study. In P.C. Emson (Ed.), Chemical Neuroanatomy, Raven, New York, 1983, pp. 131 207. 17 Van Der Kooy, D. and Hattori, T., Dorsal raphe cells with collateral projections to the caudate-putamen and substantia nigra: a fluorescent retrograde double labeling study in the rat, Brain Res., 186 (1980) 1 7. 18 Westlund, K.N., Denny, R.M., Kochersperger, UM., Rose, R.M. and Abell, C.W., Distinct monoamine oxidase A and B populations in primate brain, Science, 230 (1985) 181 183. 19 Yamamoto, T. and Hirano, A., Nucleus raphe dorsalis in parkinsonism-dementia complex of Guam, Acta Neuropathol., 67 (1985) 29(~299.
Neuroscience Letters, 94 (1988) 218 22 i Elsevier Scientific Publishers Ireland Lid
NSL 05673
Dopaminergic dorsal raphe neurons in cats and monkeys are sensitive to the toxic effects of MPTP G.A. Unguez and J.S. Schneider Centerfor NeurologicalResearch of the Department of Neurology and Institute of Neuroscience, Hahnernann University School of Medicine, Philadelphia, PA 19102-1192 (U.S.A.) and UCLA School of Medicine, Department of Neurology, Reed Neurological Research Center, Los Angeles, CA 90024-1769 (U.S.A.)
(Received 18 April 1988; Accepted 15 July 1988) Key words: Parkinsonism; Dorsal raphe nucleus; l-Methyl-4-phenyl-l,2,3,6-tetrahydropyridine; Monkey
Cat;
Tyrosine hydroxylase immunohistochemical analysis of the dorsal raphe nucleus (DRN) of severely parkinsonian MPTP-treated cats and cynomolgus monkeys revealed a marked loss of catecholaminergic neurons in this region. Cell loss was more extensive in the ventral portion of the nucleus with a relative sparing of neurons in the dorsal-most portions of the DRN. These results demonstrate that catecholaminergic neurons other than those in the ventral mesencephalon and the locus ceruleus are affected by the toxic effects of MPTP.
Although degeneration of nigrostriatal dopaminergic nerve cells is the primary pathology in Parkinson's disease, the degenerative process also involves other catecholaminergic neurons such as those in the locus ceruleus, dorsal motor vagus nuclei and other regions like the ventral tegmental area (VTA), the hypothalamus, and the raphe nuclei [3, 5]. Neuronal degeneration, Lewy body formation and presence of neurofibrillary tangles are pathological changes seen in neurons in the above-mentioned areas. These changes are characteristic of this multi-system disorder in humans [3].
Recently, the synthetic meperidine analog N-methyl-4-phenyl-l,2,3,6-tetrahydrop y r i d i n e ( M P T P ) has been f o u n d to p r o d u c e s y m p t o m s o f P a r k i n s o n ' s disease in hum a n s [6], n o n - h u m a n p r i m a t e s [1], a n d cats [11] when a d m i n i s t e r e d systemically a n d to result in d a m a g e to n e u r o n s in the s u b s t a n t i a n i g r a p a r s c o m p a c t a a n d o t h e r ventral m e s e n c e p h a l i c cell g r o u p s such as the V T A a n d r e t r o r u b r a l o r A-8 d o p a m i n e cell g r o u p . In a d d i t i o n , p a t h o l o g y has also been o b s e r v e d in the locus ceruleus, p a r ticularly in aged a n i m a l s [4]. These d a t a suggest t h a t M P T P - i n d u c e d p a r k i n s o n i s m m a y be m o r e a n a l o g o u s to i d i o p a t h i c P a r k i n s o n ' s disease t h a n originally t h o u g h t , Correspondence." J.S. Schneider, Hahnemann University School of Medicine, Dept. of Neurology, Broad and Vine Streets, Mail Stop 423, Philadelphia, PA 19102, U.S.A.
0304-3940/88/$ 03.50 © 1988 Elsevier Scientific Publishers Ireland Ltd.
219
since as in the idiopathic disorder, neurons other than the substantia nigra pars compacta dopaminergic cells are affected. The present study further examines the extent of MPTP-induced neuronal pathology by studying the dorsal raphe nucleus (DRN). Previous histochemical studies have demonstrated the existence of dopaminergic cell bodies in the D R N [2, 8], and cell loss in the human D R N as a result of Parkinson's disease has been reported [! 9], Since M P T P is considered primarily a dopaminergic toxin, we chose to examine whether dopaminergic neurons in the D R N might be affected by this toxin. A semiquantitative analysis of the D R N in non-human primates (M. fascicularis) and felines was performed. Brains from 5 normal adult male monkeys and 4 adult male MPTP-treated monkeys of similar age with severe parkinsonian m o t o r deficits were used along with brains from 2 normal adult male cats and 4 adult male, MPTPtreated cats of similar age. Cell counts were obtained from camera lucida drawings of 50-/~m-thick sections processed for tyrosine hydroxylase (TH) immunoreactivity. T H like immunoreactivity was detected by the avidin biotin immunoperoxidase technique described elsewhere [11]. Only TH-positive cells with distinct nuclei were counted within the area corresponding to the D R N (see below). The presence or absence of cells was confirmed with cresyl violet counterstaining of adjacent sections. Care was taken to match sections as closely as possible using gross landmarks so that similar anatomical levels of the D R N could be compared between the normal and MPTP-treated animals. Neuroanatomical landmarks such as the cerebral aqueduct and medial longitudinal fasciculus were used to help define the borders of the D R N . Since we did not obtain total cell counts from serial sections through the D R N , we selected sections for quantitation that best represented each of 4 distinct anatomical levels in the D R N defined empirically. For the monkeys, Level 1, the most rostral, included D R N cells at approximately the level of the oculomotor nucleus (pars dorsolateralis) and the decussation of the superior cerebellar peduncle. This corresponded to an approximate stereotaxic coordinate A:2.5 [15]. Extending caudally, Level 2 was the D R N cell group sampled at approximately A: 1.0. Further caudally, Level 3 corresponded to the D R N cell group located at the level of the trochlear nerve which is seen at the base of the periaqueductal grey matter (AP:0). Level 4, the most caudal, included the D R N cell population sampled at the level corresponding approximately to P:0.5. All coordinates are taken from the Snider and Lee atlas [15]. A total of 33 sections were sampled from control animals and 46 sections were sampled from MPTP-treated monkeys. Similarly, 4 anatomical levels of the D R N were analyzed in the cat. Level 1 corresponded to the D R N at approximately A:I.0. Level 2 included the D R N at the rostral extent of the trochlear nucleus (A:0.0). Extending caudally, our Level 3 was approximately at P:l.0 and Level 4, our most caudal level, corresponded to the raphe cell group located at approximately P:2.0. All coordinates were taken from the Snider and Nieman atlas [14]. Thirty-three sections were sampled from control animals while 58 sections were sampled from MPTP-treated cats. F o r both cats and monkeys, mean cell counts (and S.D.) were obtained for each defined anatomical level and the Student's t-test was used to statistically compare counts obtained from normal and
220
MPTP-treated animals. In normal control animals, TH-positive neuronal cell bodies were readily observed in the D R N of both cats and monkeys. In cats, numerous round and oval cells, ranging in size from 12 to 1 6 / t m in diameter, were distributed throughout the D R N , spanning from the cerebral aqueduct (dorsal D R N ) to the medial longitudinal fascicull (ventral D R N ) , as previously described by Leger and Wiklund [7]. Other THpositive neurons were fusiform in shape and ranged in size from 16 to 35/zm in diameter. The smaller of these fusiform neurons tended to be located in the mid-region of the D R N along the midline. The larger of these fusiform-shape neurons were predominantly located lateral to the midline and were more numerous near the ventral portion of the nucleus. In normal monkeys, the morphology and distribution pattern of TH-positive neurons were basically similar to those observed in normal cats. Round and oval cells, ranging in size from 8 to 18 j~m in diameter were scattered throughout the D R N . Larger angular and fusiform-shaped neurons, ranging from approximately 16 to 39 /tm in diameter were found mostly lateral to the midline and toward the ventral portion of the nucleus. In normal cats, we found our levels 2 and 3 to contain the most TH-positive neurons. MPTP-treated cats showed a significant loss of TH-positive cells in our levels 1, 3 and 4 (Table IB). Following M P T P exposure, definite cell loss was observed throughout the D R N in the cat, with perhaps more pronounced cell loss in the more ventral portions of the nucleus in some animals. Fig. 1B shows the extent of D R N
TABLE 1 A V E R A G E C O U N T S O F T H - I M M U N O R E A C T I V E N E U R O N S IN T H E D O R S A L R A P H E OF N O R M A L A N D M P T P - T R E A T E D M O N K E Y S A N D CATS Anatomical levels extend from the most rostral D R N (level 1) to the most caudal D R N (level 4). See text for further description. Animals
Monkeys Control (n=5) MPTP-treated (n=4) Cats Control (n=2) MPTP-treated (n=4)
N u m b e r of TH-immunoreactive D R N cells (average) Level 1
Level 2
Level 3
Level 4
155.43___59.11
66.53+31.66
18.29-+ 6.45
10.00_+3.91
44.47+24.47 P > 0.002
26.91+ 8.64 P > 0.002
13.25_+ 6.90
3.9-t-3.87 P > 0.05
25.54_+25.88
74.17 +43.81
68.27_+66.30
17.33+4.72
10.31 + 17.58 P>0.05
63.86_+48.83
16.94_+ 7.60 P>0.005
5.0-+3.74 P>0.01
221
Fig. 1. A: tyrosine hydroxylase (TH) immunoreactivity in the normal cat dorsal raphe nucleus (DRN). B: TH immunoreactivity in the DRN of a cat with signs of severe parkinsonism following 7 days administration of MPTP. C: TH immunoreactivity in DRN of normal monkey. D: TH immunoreactivity in the DRN of a severely parkinsonian monkey. Bar= 100 ~m.
cell loss observed in one of the more severely affected cats. Although we observed a decrease in the number of all TH-positive cell types in the cat, the larger fusiform cells in the ventral D R N appeared to be most affected by the MPTP. In monkeys, TH-positive neurons in both control and MPTP-treated animals were found to be most abundant in the more rostral portions of the D R N (our levels 1 and 2). In MPTP-treated monkeys, we observed a significant loss of TH-positive neurons at our levels 1, 2 and 4 (Table 1A). The pattern of D R N cell loss in monkeys was similar to that observed in the cats with cell loss evident throughout the nucleus but with perhaps more pronounced cell loss in the ventral and lateral portions of the nucleus. Again, predominantly large fusiform and angular cells appeared to be most susceptible to toxic effects of MPTP. Fig. 1D shows the extent of cell loss in a severely affected monkey (compared to the normal monkey, Fig. IC). The pathology of idiopathic Parkinson's disease suggests that the degenerative changes affect a variety of systems in addition to the nigrostriatal dopamine system
222 [3, 5]. Similarly, cell groups other than the substantia nigra pars compacta are found to sustain damage in MPTP-induced parkinsonism [12]. The present results further demonstrate that the toxic effects o f M P T P are not confined to the substantia nigra pars compacta. Since the D R N in both monkeys and cats is not considered to be a neuromelanin-containing structure, it is unlikely that the observed cell loss was due to n e u r o m e l a n i n - M P T P interactions. One possible alternative explanation for the D R N cell loss is that serotonergic D R N cells, which contain M A O - B [10, 18], took up MPTP, converted it to and released M P P ÷ [13], which in turn was toxic to the adjacent dopaminergic neurons. In the past, we have shown that at least in the cat, M P T P accumulates to a great extent in the D R N [10]. Dopaminergic neurons in the D R N have been previously described by others using TH-immunohistochemical techniques [2] and by a combination of microspectrofluorometry with fluorescence histochemistry [8]. In addition, dopaminergic D R N afferent projections to the striatum have been demonstrated by combined TH-immunohistochemistry and radioautography [2] as well as with H R P retrograde labeling [9]. Both of these studies found dopaminergic neurons predominantly located close to the midline and within the ventral portion of the D R N . Striatally projecting D R N neurons have also been reported to be restricted to the more rostral parts of the D R N [9]. In the present study, TH-positive cells located in the ventral and rostral portions of the D R N were destroyed to a larger extent than TH-positive cells located in the dorsal and caudal portions of the nucleus. This pattern of dopaminergic cell destruction in the D R N following administration of M P T P closely parallels the localization of dopaminergic D R N neurons which are found to project to both the substantia nigra and striatum [2]. Therefore, the possibility exists that MPTP-induced damage to D R N dopaminergic neurons may further impair striatal functioning and further contribute to the severe striatal dopamine depletion seen following M P T P exposure. The functional significance of D R N dopaminergic neurons has not been clearly defined. Dopamine-containing neurons in the D R N are much fewer in number than serotonin (5-HT)-containing neurons [I 6]. The D R N 5-HT pathways innervating the striatum [2, 17] are thus also more extensive than the corresponding dopaminergic projections [9]. Yet, it is possible that D R N dopaminergic neurons may play an important role in modulating serotonergic neuronal activity within the D R N , thus affecting the interactions between the D R N and the striatum with a resultant influence on m o t o r output. This research was supported in part by U S P H S Grants DE7282 and MH41645. 1 Burns, R.S., Chiueh, C.C., Markey, S., Ebert, M.H., Jacobowitz, D. and Kopin, l.J., A primate model of Parkinson's disease: selectivedestruction of substantia nigra pars compacta dopaminergic neurons by N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine,Proc. Natl. Aead. Sci. U.S.A., 80 (1983) 4546-4550. 2 Descarries, L., Berthelet, F., Garcia, S. and Beaudet, A., Dopaminergic projection from nucleus raphe dorsalis to neostriatum in the rat, J. Comp. Neurol., 249 (1986) 511 520. 3 Forno, L.S., Pathology of Parkinson's disease. In C.D. Marsden and S. Fahn (Eds.), Neurology 2, Movement Disorders, Butterworth, London, 1982, pp. 25-40.
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