Brain Research, 159 (1978) 223-227 O Elsevier/North-Holland Biomedical Press
223
Increased dopamine metabolism in rat striatum after infusions of substance P into the substantia nigra
P. C. WALDMEIER, R. KAM and K. STt)CKLIN Research Department, Pharmaceuticals Divisions, CIBA-G EIG Y Ltd., Basle (Switzerland)
(Accepted August 24th, 1978)
The substantia nigra (SN) of rat and human brain contains high amounts of substance P (SP) 1,8,15, which seems to reside in axon terminals of neurons projecting from the striatum6, 9A4. The peptide was shown to be released from nigral tissue 7 and to excite nigral neurons4,16, ~9. Intraventricular injection of SP enhances tyrosine hydroxylation in rat brainL Perfusion of the cat SN with SP increases the release of newly synthesized dopamine (DA) in the caudate nucleus 3. Repeated treatment of rats with haioperidol reduces SP levels in the SN 5. These and other results support the idea that SP containing neurons increase firing in dopaminergic neurons in the SN 2. As changes in the endogeneous levels of the deaminated DA metabolites are thought to reflect changes in the activity of dopaminergic neuronsl2, ~7, we decided to investigate the effect of local application of SP to the SN on the levels of homovanillic (HVA) and 3,4-dihydroxyphenylacetic acid (DOPAC) in the corpus striatum. Female Tif:RAIf (SPF) rats (Tierfarm Sisseln, Switzerland) weighing 145-160 g were used throughout. Two or 3 days before the experiments, rats were anesthetized with Nembutal (50 mg/kg i.p.). A cannula guide (i.d. 0.50 mm, length 9 mm, stainless steel) embedded in a precast socket made of dental cement was mounted in a stereotaxic apparatus (type K 2C; La Pr6cision cin6matographique, Asni6res s/Seine, France). The lower end of the cannula guide was not more than 0.50 mm below the inner surface of the skull. The position of the guide was such that the tip of the cannula to be used (see below) was in the right SN at the coordinates A = 2.4; L = 2.4; H -- --2.01°. The guides were closed with stylets. Six 5/~l Hamilton syringes (type 85 RN, Micromeasure AG, Bonaduz, Switzerland) were mounted in an aluminum rack on an infusion pump (type 1830, B. Braun, Melsungen, G.F.R.) and connected by polyethylene tubing (length 50 cm) to stainless steel cannulas (o.d. 0.45 mm, i.d. 0.20 mm, overall length 40 mm). The tubing was filled with H20. Then, 2 #l toluene (to prevent diffusion and control flow) followed by 3/~l saline or solutions of SP in saline (1-90/~g//~l; Protein Research Foundation, Osaka, Japan; purity ~ 97%, checked before use by thin layer and high pressure liquid chromatography) were aspirated to fill the cannula and the end of the polyethylene tubing. The animals were held and, (to avoid air bubbles), the cannulas inserted while the pump was already running. Thin
224
layer chromatography of the SP solutions remaining in the cannulas after the infusions occasionally revealed the presence of the methionine-sulfoxide derivative of SP. However, in no case was more than about 30 ~ of the original SP content found to be oxidized after the infusion. In each experiment, 3 cannulas were filled with saline and 3 others with solutions of SP. Infusion rates varied between 0.88 and 1.76/~l/h. The animals were placed in separate cages and were fully conscious during the infusions. No turning of the rats was observed after any of the doses of SP used in this study. Immediately after the infusions the cannulas were removed, the animals decapitated and the brains dissected. Infusion sites were first examined histologically. In the actual experiments, a cut was made along the cannula trace in each brain after the removal of the striata. A small spot of blood had formed at the place where the cannula tip had been, which could be taken as an indication of the position of the latter. These sections were photographed under the microscope as a record. HVA, DOPAC and DA were isolated on Sephadex columns 20ml and quantitated fluorometricallylS,2°m 1. In preliminary experiments, intranigral injections of 5/2g SP in I /,l saline 25 or 50 min before the decapitation did not cause any changes in HVA, DOPAC and DA levels in the ipsilateral striata. In view of the rapid enzymatic degradation of SP, it seemed more promising to infuse the peptide at a constant rate to ensure its presence at the receptor sites during a reasonable length of time. Subsequent experiments with infusions of SP (30 #gila in saline at a rate of 2.34 #l/h, corresponding to a cumulative dose of 70.4 #g/h) for 15, 30 and 60 min were performed. Significant increases of HVA and DOPAC in the ipsilateral striata were only found after 60 min infusions. Therefore, we investigated the effects of 60 min infusions of different concentrations of SP at different rates on striatal DA metabolism. The means ± S.E.M. of the absolute values of HVA (or DOPAC, in brackets) in the striata contralateral to the SN infused with saline varied between 462 ± 45 ng/g, n = 3 ( 1 1 1 6 ± 45, n = 3) and 6 3 0 ± 9 2 n g / g , n - - 3 ( 1 4 8 4 ~ 195, n = 3 ) i n t h e 10 experiments performed. The average was 529 ± 20 rig/g, n -= 29 (1309 ~ 58, n ---- 30). In the striata ipsilateral to the saline infusion they varied between 536 + 12 ng/g, n = 3 (1284 ~ 73, n = 3) and 876 ± 102 rig/g, n = 3 (1782 ± 279, n = 3), the average being 709 ± 40 ng/g, n = 30 (1542 ± 59, n = 30). Therefore, HVA levels were 34 + 8 ~ (P < 0.001) and those of DOPAC 18 + 5 ~ (P < 0.01) higher in the striata of the same side, indicating some degree of impairment of the nigrostriatal DA neurons by the infusion of saline alone. Consequently, the results obtained with SP infusions were expressed in per cent of the values measured in the striata ipsilateral to saline infusion. SP infusions into the right SN for l h had no effect on striatal D A concentrations and these results are therefore not shown. However, the levels of HVA, and to a somewhat lesser degree of DOPAC, were increased significantly in the ipsilateral striata at cumulative doses of 52.8 #g/h (both HVA and DOPAC ~ +409/o) and 70.4/tg/h (HVA ~-~ +1009/o; DOPAC ~ + 6 0 ~ ) (Fig. 1). At the higher cumulative doses of 105.6 and 158.4 #g/h, the effect of SP was smaller and more variable. Occasional spontaneous gel formation from such concentrated solutions indicated that SP was
225 %of NaCIcontrols
HVA p,¢0.01 6
@"--~ SP
~
3
200,
=
:NaO
/l~
3
100
~
3
"-t T'q
3
~I
37--1
~
1;6
~2 52'~~
lo~ I~A ~ / .
SPinfused
1~ ; /
e~
i
%o f NaCIcontrols
200
I
DOPAC
p,¢0.01
= NaCI
-
6
p,o.o~ T 5
•
100,
3
~"
3
3
3
3
3
!
-
-
3
SPinfused
H
I.~BI / '
I~
17.6
35.2 52",B~
I0~
I~IA ju~/h
Fig. 1. Effect of a 1 h infusion of graded doses of substance P into the substantia nigra on striatal HVA and D O P A C concentrations. Saline or solutions of substance P in saline (1-90/~g//zl) were infused into the right substantia nigra at rates of 0.88, 1.32 or 1.76/~l/h for I h. Immediately after the end of the infusion, the animals were decapitated and the left and right striata dissected and analyzed separately. The HVA (upper panel) and D O P A C values (lower panel) obtained in the right striata after substance P infusion are given as the percentage of those found in the right striata after saline infusion. Points and bars represent means 4- S.E.M. The number of animals used per dose is indicated above or below each point in the graph. Significances were calculated by means of Student's t-test. The doses given on the abscissa are the cumulative doses the animals received during the 1 h infusion.
no longer truly in solution (the concentrations were 80 and 90 #g//~l in these particular experiments). This could have influenced the actual amount of peptide available at the site of action. On the other hand, it should be noted that Krnjevic and Morris n also found a reversal of the excitatory effect of SP on cat cuneate neurons at high doses. The high amounts of SP needed to produce an effect on DA metabolism are surprising, especially in view of the fact that Ch6ramy et al.a found an increase in the
226 release o f newly synthesized D A in the cat s t r i a t u m after perfusion of the S N for 1 h with 10 -s M SP. However, the species, e x p e r i m e n t a l a r r a n g e m e n t and m e a s u r e d p a r a m e t e r s were entirely different. M e a s u r a b l e increases o f H V A a n d D O P A C might only occur if substantial a m o u n t s o f the totally available D A are released. This would n o t imply changes in the levels o f D A , since increased utilization is in general a c c o m p a n i e d by increased synthesis. T h e r e are several possible explanations for the fact t h a t SP infusions of 1 h d u r a t i o n are necessary to p r o d u c e a significant effect. It m a y take a long time to build up effective c o n c e n t r a t i o n s o f SP in the tissue because o f r a p i d d e g r a d a t i o n o f the peptide. F u r t h e r m o r e , as SP is strongly b o u n d to b r a i n lipid m a t e r i a l at physiological p H 1~, the p e p t i d e m a y become available only at the r e c e p t o r sites when the s u r r o u n d i n g lipid m a t e r i a l is saturated. Finally, p r o l o n g e d presence o f SP at the receptor sites could be necessary to p r o d u c e a n effect on D A m e t a b o l i s m . Interestingly, H V A a n d D O P A C increases i n d u c e d by SP were only m o d e r a t e . This m a y relate to the observation, that the absolute increase in the rate o f n e u r o n a l discharge o f nigral cells was low (20-50 ~ ) , even if high ejection currents were used 4. In conclusion, our results are c o m p a t i b l e with the hypothesis o f an excitatory SP i n p u t on d o p a m i n e r g i c cells in the SN. It remains to be shown w h e t h e r this excitatory effect o f SP is n o t due to an unspecific m o d u l a t o r y influence o f the peptide, possibly in the sense o f the a b o v e - m e n t i o n e d binding to lipid m e m b r a n e c o m p o n e n t s . The a u t h o r s wish to t h a n k Prof. K o e l l a for valuable suggestions for the preparation o f the manuscript.
1 Brownstein, M. J., Mroz, E. A., Kizer, K. S., Palkovits, M. and Leeman, S. E., Regional distribution of substance P in the brain of the rat, Brain Research, 116 (1976) 299-305. 2 Carlsson, A., Magnusson, T., Fisher, G. H., Chang, D. and Folkers, K., Effect of substance P on central monoaminergic mechanisms, In U. S. von Euler and B. Pernow (Eds.), Substance P, Raven Press, New York, 1977, pp. 201-205. 3 Ch6ramy, A., Nieoullon, A., Michelot, R. and Glowinski, J., Effects of intranigral application of dopamine and substance P on the in vivo release of newly synthesized [3H]dopamine in the i psilateral caudate nucleus of the cat, Neurosci. Lett., 4 (1977) 105-109. 4 Davies, J. and Dray, A., Substance P in the substantia nigra, Brain Research, 107 (1976) 623-627. 5 Hong, J. S., Yang, H.-Y. T. and Costa, E., Substance P content of substantia nigra after chronic treatment with antischizophrenic drugs, Neuropharmacology, 17 (1978) 83-85. 6 Hong, J. S., Yang, H.-Y. T., Racagni, G. and Costa, E., Projections of substance P containing neurons from neostriatum to substantia nigra, Brain Research, 122 (1977) 541-544. 7 Jessell, T. M., Inhibition of substance P release from the isolated rat substantia nigra by GABA, Brit. J. Pharm~coL, 59 (1977) 486P. 8 Kanazawa, I. and Jessell, T., Post mortem changes and regional distribution of substance P in the rat and mouse nervous system, Braia Research, 117 (1976) 362-367. 9 Kanazawa, I., Emson, P. C. and Cuello, A. C., Evidence for the existence of substance P containing fibres in striato-nigral and pallido-nigral pathways in rat brain, Brain Research, 119 (1977) 447-453. 10 K6nig, J. F. R. and Klippel, R., The Rat Brain. A Stereotaxic Atlas, Krieger, Huntington, N.Y., 1967. 11 Krnjevic, K. and Morris, M. E., An excitatory action of substance P on cuneate neurones, Canad. J. Physiol. Pharmacol., 52 (1974) 736-744. 12 Korf, J., Grasdijk, L. and Westerink, B. H. C., Effects of electrical stimulation of the nigrostriatal pathway of the rat on dopamine metabolism, J. Neurochem., 26 (1976) 579-584. 13 Lembeck, F., Mayer, N. and Schindler, G., Lipid bound substance P in central nervous system, Naunyn-Schrniedeberg's Arch. exp. Path. Pharmak., Suppl. 297 (1977) R 55.
227 14 Mroz, E. A., Brownstein, M. J. and Leeman, S. E., Evidence for substance P in the striato-nigral tract, Brain Research, 125 (1977) 305-311. 15 Niisson, G., H/~kfelt, T. and Pernow, B., Distribution of substance P-like immuno-reactivityin the rat central nervous system as revealed by immunohistochemistry, ivied. Biol., 52 (1974) 424-427. 16 Olpe, H.-R. and Koella, W. P., Rotatory behavior in rats by intranigral application of substance P and an eledoisin fragment, Brain Research, 126 (1977) 576-579. 17 Roth, R. H., Murrin, L. C. and Waiters, J. R., Central dopaminergic neurons: effects of alterations in impulse flow on the accumulation of dihydroxyphenylacetic acid, Europ. J. Pharmacol., 36 (1976) 163-171. 18 Waldmeier, P., de Herdt, P. and Maitre, L., Simultaneous automated estimation of noradrenalin and dopamine in brain tissue, Clin. Chem., 20 (1974) 81-83. 19 Walker, R. J., Kemp, J. A., Yajima, H., Kitagawa, K. and Woodruff, G. N., The action of substance P on mesencephalic reticular and substantia nigral neurones of the rat, Experientia (Basel), 32 (1976) 214-215. 20 Westerink, B. H. C., Regional Dopamine Metabolism in the Rat Brain, Ph.D. thesis, Groningen, Netherlands, 1977. 21 Westednk, B. H. C. and Korf, J., Regional rat brain levels of 3,4-dihydroxyphenylacetic acid and homovanillicacid: concurrent fluorometric measurement and influence of drugs, Europ. J. Pharmacol., 38 (1976) 281-291.
223
Increased dopamine metabolism in rat striatum after infusions of substance P into the substantia nigra
P. C. WALDMEIER, R. KAM and K. STt)CKLIN Research Department, Pharmaceuticals Divisions, CIBA-G EIG Y Ltd., Basle (Switzerland)
(Accepted August 24th, 1978)
The substantia nigra (SN) of rat and human brain contains high amounts of substance P (SP) 1,8,15, which seems to reside in axon terminals of neurons projecting from the striatum6, 9A4. The peptide was shown to be released from nigral tissue 7 and to excite nigral neurons4,16, ~9. Intraventricular injection of SP enhances tyrosine hydroxylation in rat brainL Perfusion of the cat SN with SP increases the release of newly synthesized dopamine (DA) in the caudate nucleus 3. Repeated treatment of rats with haioperidol reduces SP levels in the SN 5. These and other results support the idea that SP containing neurons increase firing in dopaminergic neurons in the SN 2. As changes in the endogeneous levels of the deaminated DA metabolites are thought to reflect changes in the activity of dopaminergic neuronsl2, ~7, we decided to investigate the effect of local application of SP to the SN on the levels of homovanillic (HVA) and 3,4-dihydroxyphenylacetic acid (DOPAC) in the corpus striatum. Female Tif:RAIf (SPF) rats (Tierfarm Sisseln, Switzerland) weighing 145-160 g were used throughout. Two or 3 days before the experiments, rats were anesthetized with Nembutal (50 mg/kg i.p.). A cannula guide (i.d. 0.50 mm, length 9 mm, stainless steel) embedded in a precast socket made of dental cement was mounted in a stereotaxic apparatus (type K 2C; La Pr6cision cin6matographique, Asni6res s/Seine, France). The lower end of the cannula guide was not more than 0.50 mm below the inner surface of the skull. The position of the guide was such that the tip of the cannula to be used (see below) was in the right SN at the coordinates A = 2.4; L = 2.4; H -- --2.01°. The guides were closed with stylets. Six 5/~l Hamilton syringes (type 85 RN, Micromeasure AG, Bonaduz, Switzerland) were mounted in an aluminum rack on an infusion pump (type 1830, B. Braun, Melsungen, G.F.R.) and connected by polyethylene tubing (length 50 cm) to stainless steel cannulas (o.d. 0.45 mm, i.d. 0.20 mm, overall length 40 mm). The tubing was filled with H20. Then, 2 #l toluene (to prevent diffusion and control flow) followed by 3/~l saline or solutions of SP in saline (1-90/~g//~l; Protein Research Foundation, Osaka, Japan; purity ~ 97%, checked before use by thin layer and high pressure liquid chromatography) were aspirated to fill the cannula and the end of the polyethylene tubing. The animals were held and, (to avoid air bubbles), the cannulas inserted while the pump was already running. Thin
224
layer chromatography of the SP solutions remaining in the cannulas after the infusions occasionally revealed the presence of the methionine-sulfoxide derivative of SP. However, in no case was more than about 30 ~ of the original SP content found to be oxidized after the infusion. In each experiment, 3 cannulas were filled with saline and 3 others with solutions of SP. Infusion rates varied between 0.88 and 1.76/~l/h. The animals were placed in separate cages and were fully conscious during the infusions. No turning of the rats was observed after any of the doses of SP used in this study. Immediately after the infusions the cannulas were removed, the animals decapitated and the brains dissected. Infusion sites were first examined histologically. In the actual experiments, a cut was made along the cannula trace in each brain after the removal of the striata. A small spot of blood had formed at the place where the cannula tip had been, which could be taken as an indication of the position of the latter. These sections were photographed under the microscope as a record. HVA, DOPAC and DA were isolated on Sephadex columns 20ml and quantitated fluorometricallylS,2°m 1. In preliminary experiments, intranigral injections of 5/2g SP in I /,l saline 25 or 50 min before the decapitation did not cause any changes in HVA, DOPAC and DA levels in the ipsilateral striata. In view of the rapid enzymatic degradation of SP, it seemed more promising to infuse the peptide at a constant rate to ensure its presence at the receptor sites during a reasonable length of time. Subsequent experiments with infusions of SP (30 #gila in saline at a rate of 2.34 #l/h, corresponding to a cumulative dose of 70.4 #g/h) for 15, 30 and 60 min were performed. Significant increases of HVA and DOPAC in the ipsilateral striata were only found after 60 min infusions. Therefore, we investigated the effects of 60 min infusions of different concentrations of SP at different rates on striatal DA metabolism. The means ± S.E.M. of the absolute values of HVA (or DOPAC, in brackets) in the striata contralateral to the SN infused with saline varied between 462 ± 45 ng/g, n = 3 ( 1 1 1 6 ± 45, n = 3) and 6 3 0 ± 9 2 n g / g , n - - 3 ( 1 4 8 4 ~ 195, n = 3 ) i n t h e 10 experiments performed. The average was 529 ± 20 rig/g, n -= 29 (1309 ~ 58, n ---- 30). In the striata ipsilateral to the saline infusion they varied between 536 + 12 ng/g, n = 3 (1284 ~ 73, n = 3) and 876 ± 102 rig/g, n = 3 (1782 ± 279, n = 3), the average being 709 ± 40 ng/g, n = 30 (1542 ± 59, n = 30). Therefore, HVA levels were 34 + 8 ~ (P < 0.001) and those of DOPAC 18 + 5 ~ (P < 0.01) higher in the striata of the same side, indicating some degree of impairment of the nigrostriatal DA neurons by the infusion of saline alone. Consequently, the results obtained with SP infusions were expressed in per cent of the values measured in the striata ipsilateral to saline infusion. SP infusions into the right SN for l h had no effect on striatal D A concentrations and these results are therefore not shown. However, the levels of HVA, and to a somewhat lesser degree of DOPAC, were increased significantly in the ipsilateral striata at cumulative doses of 52.8 #g/h (both HVA and DOPAC ~ +409/o) and 70.4/tg/h (HVA ~-~ +1009/o; DOPAC ~ + 6 0 ~ ) (Fig. 1). At the higher cumulative doses of 105.6 and 158.4 #g/h, the effect of SP was smaller and more variable. Occasional spontaneous gel formation from such concentrated solutions indicated that SP was
225 %of NaCIcontrols
HVA p,¢0.01 6
@"--~ SP
~
3
200,
=
:NaO
/l~
3
100
~
3
"-t T'q
3
~I
37--1
~
1;6
~2 52'~~
lo~ I~A ~ / .
SPinfused
1~ ; /
e~
i
%o f NaCIcontrols
200
I
DOPAC
p,¢0.01
= NaCI
-
6
p,o.o~ T 5
•
100,
3
~"
3
3
3
3
3
!
-
-
3
SPinfused
H
I.~BI / '
I~
17.6
35.2 52",B~
I0~
I~IA ju~/h
Fig. 1. Effect of a 1 h infusion of graded doses of substance P into the substantia nigra on striatal HVA and D O P A C concentrations. Saline or solutions of substance P in saline (1-90/~g//zl) were infused into the right substantia nigra at rates of 0.88, 1.32 or 1.76/~l/h for I h. Immediately after the end of the infusion, the animals were decapitated and the left and right striata dissected and analyzed separately. The HVA (upper panel) and D O P A C values (lower panel) obtained in the right striata after substance P infusion are given as the percentage of those found in the right striata after saline infusion. Points and bars represent means 4- S.E.M. The number of animals used per dose is indicated above or below each point in the graph. Significances were calculated by means of Student's t-test. The doses given on the abscissa are the cumulative doses the animals received during the 1 h infusion.
no longer truly in solution (the concentrations were 80 and 90 #g//~l in these particular experiments). This could have influenced the actual amount of peptide available at the site of action. On the other hand, it should be noted that Krnjevic and Morris n also found a reversal of the excitatory effect of SP on cat cuneate neurons at high doses. The high amounts of SP needed to produce an effect on DA metabolism are surprising, especially in view of the fact that Ch6ramy et al.a found an increase in the
226 release o f newly synthesized D A in the cat s t r i a t u m after perfusion of the S N for 1 h with 10 -s M SP. However, the species, e x p e r i m e n t a l a r r a n g e m e n t and m e a s u r e d p a r a m e t e r s were entirely different. M e a s u r a b l e increases o f H V A a n d D O P A C might only occur if substantial a m o u n t s o f the totally available D A are released. This would n o t imply changes in the levels o f D A , since increased utilization is in general a c c o m p a n i e d by increased synthesis. T h e r e are several possible explanations for the fact t h a t SP infusions of 1 h d u r a t i o n are necessary to p r o d u c e a significant effect. It m a y take a long time to build up effective c o n c e n t r a t i o n s o f SP in the tissue because o f r a p i d d e g r a d a t i o n o f the peptide. F u r t h e r m o r e , as SP is strongly b o u n d to b r a i n lipid m a t e r i a l at physiological p H 1~, the p e p t i d e m a y become available only at the r e c e p t o r sites when the s u r r o u n d i n g lipid m a t e r i a l is saturated. Finally, p r o l o n g e d presence o f SP at the receptor sites could be necessary to p r o d u c e a n effect on D A m e t a b o l i s m . Interestingly, H V A a n d D O P A C increases i n d u c e d by SP were only m o d e r a t e . This m a y relate to the observation, that the absolute increase in the rate o f n e u r o n a l discharge o f nigral cells was low (20-50 ~ ) , even if high ejection currents were used 4. In conclusion, our results are c o m p a t i b l e with the hypothesis o f an excitatory SP i n p u t on d o p a m i n e r g i c cells in the SN. It remains to be shown w h e t h e r this excitatory effect o f SP is n o t due to an unspecific m o d u l a t o r y influence o f the peptide, possibly in the sense o f the a b o v e - m e n t i o n e d binding to lipid m e m b r a n e c o m p o n e n t s . The a u t h o r s wish to t h a n k Prof. K o e l l a for valuable suggestions for the preparation o f the manuscript.
1 Brownstein, M. J., Mroz, E. A., Kizer, K. S., Palkovits, M. and Leeman, S. E., Regional distribution of substance P in the brain of the rat, Brain Research, 116 (1976) 299-305. 2 Carlsson, A., Magnusson, T., Fisher, G. H., Chang, D. and Folkers, K., Effect of substance P on central monoaminergic mechanisms, In U. S. von Euler and B. Pernow (Eds.), Substance P, Raven Press, New York, 1977, pp. 201-205. 3 Ch6ramy, A., Nieoullon, A., Michelot, R. and Glowinski, J., Effects of intranigral application of dopamine and substance P on the in vivo release of newly synthesized [3H]dopamine in the i psilateral caudate nucleus of the cat, Neurosci. Lett., 4 (1977) 105-109. 4 Davies, J. and Dray, A., Substance P in the substantia nigra, Brain Research, 107 (1976) 623-627. 5 Hong, J. S., Yang, H.-Y. T. and Costa, E., Substance P content of substantia nigra after chronic treatment with antischizophrenic drugs, Neuropharmacology, 17 (1978) 83-85. 6 Hong, J. S., Yang, H.-Y. T., Racagni, G. and Costa, E., Projections of substance P containing neurons from neostriatum to substantia nigra, Brain Research, 122 (1977) 541-544. 7 Jessell, T. M., Inhibition of substance P release from the isolated rat substantia nigra by GABA, Brit. J. Pharm~coL, 59 (1977) 486P. 8 Kanazawa, I. and Jessell, T., Post mortem changes and regional distribution of substance P in the rat and mouse nervous system, Braia Research, 117 (1976) 362-367. 9 Kanazawa, I., Emson, P. C. and Cuello, A. C., Evidence for the existence of substance P containing fibres in striato-nigral and pallido-nigral pathways in rat brain, Brain Research, 119 (1977) 447-453. 10 K6nig, J. F. R. and Klippel, R., The Rat Brain. A Stereotaxic Atlas, Krieger, Huntington, N.Y., 1967. 11 Krnjevic, K. and Morris, M. E., An excitatory action of substance P on cuneate neurones, Canad. J. Physiol. Pharmacol., 52 (1974) 736-744. 12 Korf, J., Grasdijk, L. and Westerink, B. H. C., Effects of electrical stimulation of the nigrostriatal pathway of the rat on dopamine metabolism, J. Neurochem., 26 (1976) 579-584. 13 Lembeck, F., Mayer, N. and Schindler, G., Lipid bound substance P in central nervous system, Naunyn-Schrniedeberg's Arch. exp. Path. Pharmak., Suppl. 297 (1977) R 55.
227 14 Mroz, E. A., Brownstein, M. J. and Leeman, S. E., Evidence for substance P in the striato-nigral tract, Brain Research, 125 (1977) 305-311. 15 Niisson, G., H/~kfelt, T. and Pernow, B., Distribution of substance P-like immuno-reactivityin the rat central nervous system as revealed by immunohistochemistry, ivied. Biol., 52 (1974) 424-427. 16 Olpe, H.-R. and Koella, W. P., Rotatory behavior in rats by intranigral application of substance P and an eledoisin fragment, Brain Research, 126 (1977) 576-579. 17 Roth, R. H., Murrin, L. C. and Waiters, J. R., Central dopaminergic neurons: effects of alterations in impulse flow on the accumulation of dihydroxyphenylacetic acid, Europ. J. Pharmacol., 36 (1976) 163-171. 18 Waldmeier, P., de Herdt, P. and Maitre, L., Simultaneous automated estimation of noradrenalin and dopamine in brain tissue, Clin. Chem., 20 (1974) 81-83. 19 Walker, R. J., Kemp, J. A., Yajima, H., Kitagawa, K. and Woodruff, G. N., The action of substance P on mesencephalic reticular and substantia nigral neurones of the rat, Experientia (Basel), 32 (1976) 214-215. 20 Westerink, B. H. C., Regional Dopamine Metabolism in the Rat Brain, Ph.D. thesis, Groningen, Netherlands, 1977. 21 Westednk, B. H. C. and Korf, J., Regional rat brain levels of 3,4-dihydroxyphenylacetic acid and homovanillicacid: concurrent fluorometric measurement and influence of drugs, Europ. J. Pharmacol., 38 (1976) 281-291.
