- Email: [email protected]
Identification of dopamine, noradrenaline and 5-hydroxytryptamine varicosities in a fraction containing nerve ending particles
BRAIN RESEARCH
475
I D E N T I F I C A T I O N OF DOPAMINE, N O R A D R E N A L I N E AND 5-HYDROXY T R Y P T A M I N E VARICOSITIES IN A F R A C T I O N C O N T A I N I N G NERVE E N D I N G PARTICLES
KJELL FUXE, HORST GROBECKER*, TOMAS HOKFELT ANDGOSTA JONSSON Department of Histology, Karolinska Instituter, Stockholm (Sweden)
(Accepted May 19th, 1967)
INTRODUCTION Masuoka 1~ was able to give good support for the view that the catecholamines found in the fraction from the brain stem containing nerve ending particles (NEPs) are localized at least mainly to small rounded structures, which probably represent the varicosities of the central catecholamine nerve terminals. In the present study evidence is given that the dopamine, noradrenaline and 5-hydroxytryptamine in the P2-B-fraction (according to Michaelson and Whittaker 14) are localized at least partly to isolated dopamine, noradrenaline and 5-hydroxytryptamine varicosities respectively. MATERIAL AND METHODS Male Sprague-Dawley rats (150-250 g body weight) were used. The brain stem (without nucleus caudatus-putamen), the spinal cord and the nucleus caudatus-putamen were dissected out from normal rats, or rats pretreated with reserpine (10 mg/kg, i.p., 12 h before sacrifice), nialamide (100-500 mg/kg, i.p., 4-12 h before sacrifice) or nialamide (as before) + L-3,4-dihydroxyphenylalanine (L-DOPA; 100 mg/kg, i.p., 30 min before sacrifice) respectively. The brain stem, the spinal cord and n.caudatusputamen respectively were homogenized in a glass homogenizer with a teflon pestle for 60 sec in l0 volumes of ice-cold 0.3 M sucrose, containing l0 #g/ml of ascorbic acid and 0.5 mg/ml Na2EDTA. The original homogenate was first fractionated into primary fractions by differential centrifugation 9. The first particulate fraction P1 (nuclei and cell debris) was obtained by centrifuging the original homogenate for l0 rain at 1000 × g. Then the supernatant of the P1 preparation was centrifuged for 20 min at 10,000 × g to obtain the P2 fraction. This crude mitochondrial fraction was resuspended in 2 ml of 0.3 M sucrose of which 0.5 ml was carefully layered over a discontinuous density gradient prepared immediately before use, consisting of 1.5 ml 0.8 M * Present address: Department of Pharmacology, University of Frankfurt, Frankfurt/Main (Germany). Brain~.Research, 6~.(1967)475-480
476
K. FUXE et al.
sucrose layered over an equal volume of 1.2 M sucrose and centrifuged for 90 rain at 100,000 x g using a 3 × 5 ml swing-out rotor. All the centrifugations were carried out with an MSE Automatic Superspeed 50'T.C. centrifuge. A distinct separation into these subfractions was obtained : myelin fraction (P2-A), less dense than 0.8 M sucrose; NEPs fraction (P2-B), intermediate in density between 0.8 M and 1.2 M sucrose: and a mitochondrial fraction (P2-C), denser than 1.2 M, as described by Michaelson and Whittaker 14. Smears were prepared on ordinary microscopic slides from all the fractions, dried over night in an exsiccator with phosphorus pentoxide in vacuo. Then the smears were reacted with relatively dry formaldehyde gas (generated from paraformaldehyde containing 0.4% water according to Hamberger 1°) for I-3 h for the histochemical visualization of dopamine, noradrenaline and 5-hydroxytryptamine6, 7. Then the smears were mounted under coverslips in Entellan (Merck) and examined in a Zeiss fluorescence microscope (for technical details on fluorescence microscopy see DahlstrSm and FuxeT). Some of the smears were treated with 0.03 ~ NaBH4 in 90 ~ isopropanol for testing specificity of monoamine fluorescence'L After examining the slides in the fluorescence microscope they were again exposed to formaldehyde gas at +80°C for 1 h as stated above and re-examined. In one experiment the different fractions (P1, P2, P2-A, P2-B and Pz-C) were freeze-dried, treated with formaldehyde gas, and embedded in paraffin, sectioned, mounted and examined in the fluorescence microscope. Generally, the same results were obtained with this technique compared with the 'smear' technique. To confirm the presence of NEPs, the Pe-B fraction from the spinal cord, the brain stem and the n. caudatus-putamen was examined in the electron microscope. An aliquot of each fraction was carefully resuspended in isotonic sucrose and centrifuged down on flat discs consisting of polymerized Epon 18, after which the pellets were fixed in glutaraldehyde 17 and osmium tetroxide. After dehydration in alcohol the specimen were embedded in Epon 12. The sections (500-800 A thick) were contrasted with uranyl acetate followed by lead citrate 16. An RCA-EM U-3A electron microscope was used. RESULTS
Brain s t e m
In the normal rat there were observed in the P2-B fraction large numbers of weak to strongly green-fluorescent dots of varying sizes (0.3-2/D. The colour of the emitted light from these dots was typical for fluorescence due to presence of primary catecholamines (see Fig. 1). Sometimes dots in the same size range having a weak yellow fluorescence very sensitive to irradiation with ultraviolet light, typical for the presence of 5-hydroxytryptamine, were observed. These fluorescent dots were somewhat difficult to differentiate direct visually from the autofluorescent granules usually present in the mentioned fraction. The fluorescence of these autofluorescent granules was, however, not formaldehyde-induced, fairly stable against irradiation to ultraviolet light, not abolished by NaBH4 treatment and did not disappear after reserpine Brain Research, 6 (1967) 475-480
MONOAMINE-CONTAINING NERVE ENDING PARTICLES
477
Fig. 1. Section of a freeze-dried, paraffin-embedded P2-B fraction of brain stem of rat treated with nialamide (500 mg/kg i.p., 4 h before sacrifice). A large number of densely packed weakly to strongly green- (the vast majority) and yellow-fluorescent dots of various diameters (0.3-2/~) are observed. These in all probability represent noradrenaline and 5-hydroxytryptamine varicosities respectively. 300 ×. pretreatment. Furthermore, they did not show any signs of diffusion of the fluorescent compounds after reaction with humid formaldehyde gas, which was the case with the dots emitting typical monoamine fluorescence. The other fractions investigated (P1, P2-A and P2-C) showed a relatively much smaller number of fluorescent dots. In the nialamide-pretreated rat there were observed distinct increases in number and intensity of the dots emitting a yellow light, due to the presence of 5-hydroxytryptamine. The green-fluorescent dots did not show any certain changes. After nialamide-L-DOPA, however, the green-fluorescent dots were markedly increased in number and intensity. In the reserpinized rats the number of green- and yellowfluorescent dots emitting a specific monoamine fluorescence had practically disappeared. Those remaining after this treatment usually had an irregular appearance and more distinctloutlines than the vast majority of those observed in normal rats, part of which probably represent autofluorescent granules. The specificity of monoamine fluorescence was tested by NaBH4 treatment, which completely abolished the fluorescence. After renewed formaldehyde gas treatment, the fluorescence could partly be regenerated. Spinal cord About the same fluorescence microscopical picture was obtained in the Pz-B fraction as from that in the smears from the brain stem. However, the smears from the normal and especially from the nialamide-treated rats showed much higher amounts of weak to medium yellow-fluorescent dots. Furthermore, the yellow-fluorescent dots usually appeared to be larger in size (1-2 #) as seen in the fluorescence microscope than in the brain stem. Brain Research, 6 (1967) 475-480
478
K. VUXE et al.
Fig. 2. Electron micrograph of a P~-B fraction of brain stem of rat. In spite of deficiency in morphology a large number ofNEPs (n) can be identified. 23,000 ×. Fig. 3. P2-B fraction of brain stem of rat. A higher magnification of a single NEP. 41,000 x.
Nucleus caudatus-putamen In the P2-B fraction f r o m n o r m a l and n i a l a m i d e - p r e t r e a t e d rat there a p p e a r e d a large n u m b e r o f green-fluorescent dots ranging mainly between 0.2 and I ,u in d i a m eter. N o yellow fluorescent dots exhibiting a specific fluorescence were observed, however. In a c c o r d a n c e with the results o f m a n y earlier electron m i c r o s c o p i c studies 9 a large n u m b e r o f N E P s could be identified in all three P2-B fractions studied (Figs. 2 a n d 3). A l t h o u g h no statistical e v a l u a t i o n was m a d e a clear difference in size could be observed between the N E P s in the P2-B fraction o f the b r a i n stem and the n. caud a t u s - p u t a m e n . The m a j o r i t y o f the N E P s o f the f o r m e r fraction had a d i a m e t e r between 0.5 a n d 1.5/z, while m o s t o f the N E P s o f the latter one h a d a d i a m e t e r between Brain Research, 6 (1967) 475-480
MONOAMINE-CONTAINING NERVE ENDING PARTICLES
479
0.3 and 0.6 # as measured in the ultrathin sections. In addition also isolated mitochondria were seen. DISCUSSION
The present results give support for the view that the green- and yellow-fluorescent dots observed in the P2-B fraction represent dopamine, noradrenaline and 5hydroxytryptamine varicosities respectively la. The evidence is as follows: (1) Several of the histochemical criteria for the specificity of the fluorescence are satisfied (diffusion of the fluorescent compounds after reaction with humid formaldehyde gas, colour of fluorescence, NaBH4-reduction test, ultraviolet sensitivity, observed especially in the yellow-fluorescent dots). Furthermore, in the non-reacted smears these fluorescent dots could not be observed 6. (2) Many of the pharmacological criteria are satisfied for the specificity of the fluorescence observed. The green- and yellow-fluorescence disappeared after reserpine treatment which is known to deplete the endogenous monoamines a and is markedly increased after a nialamide injection which produces increases in the noradrenaline and especially the 5-hydroxytryptamine levels in the brain and the spinal cord 15. (3) The green- and yellow-fluorescent dots with specific monoamine fluorescence in all probability represent varicosities of disrupted dopamine, noradrenaline and 5hydroxytryptamine nerve terminals, since they lie (a) in the same size range as the varicosities (0.3-2 #) (ref. 8) and (b) electron microscopy of the same fraction revealed the presence of large numbers of NEPs in about the same size range as the fluorescent dots. Furthermore, in the Pe-B fraction of n. caudatus-putamen which contains a large number of very fine dopamine nerve terminals1, 8, the NEPs as observed in the electron microscope were much finer than in the other areas studied. (4) 5-Hydroxytryptamine and noradrenaline assays on brain subfractions are in agreement with the presented resultsZ,4,11,13. Thus, it seems possible to identify catecholamine- and 5-hydroxytryptaminecontaining varicosities by the fluorescence histochemical technique of Falck and Hillarp in a pinched-off nerve ending fraction (P2-B). The use of fluorescence histochemistry may be a useful complement for studies on monoaminergic mechanisms by subfractionation techniques, especially for checking the homogenization procedure. SUMMARY
Using the histochemical fluorescence technique of Falck and Hillarp for the demonstration of dopamine, noradrenaline and 5-hydroxytryptamine in combination with histochemical and pharmacological specificity tests, it has been possible to show that there is in all probability a high accumulation of dopamine, noradrenaline and 5-hydroxytryptamine varicosities in the nerve ending particle fraction P2-B (according to Whittaker et al.19). The subfractionation technique used in the present investigation was checked with electron microscopy. Brain Research, 6 (1967) 475-480
480
K. FUXE et al.
ACKNOWLEDGEMENTS T h e i n v e s t i g a t i o n has b e e n s u p p o r t e d Medical
Research
Council
by r e s e a r c h g r a n t s f r o m t h e S w e d i s h
(B 67-12X-715-03
and
B 68-12X-2295-01),
'Magnus
Bergvalls Stiftelse' a n d the T h e r e s e and J o h a n A n d e r s s o n M e m o r i a l F o u n d a t i o n . F o r g e n e r o u s supplies o f drugs, we a r e i n d e b t e d to t h e f o l l o w i n g c o m p a n i e s : S w e d i s h C i b a (Serpasil ® ) a n d S w e d i s h Pfizer, S t o c k h o l m ( N i a m i d ®).
REFERENCES l ANDEN, N.-E., FUXE, K., HAMBERGER,B., AND H()KFELT, T., A quantitative study on the nigro-
neostriatal dopamine neuron system in the rat, Actaphysiol. scand., 67 (1966) 306-312. 2 CARLINI, E. A., AND GREEN, J. P.,thThe subcellular distribution of histamine, slow-reacting substance and 5-hydroxytryptamine in the brain of the rat, Brit. J. Pharmaeol., 20 (1963) 264-277. 3 CARLSSON, A., Drugs which block the storage of 5-hydroxytryptamine and related amines. In O. EICHLERAND A. FARAH(Eds.), Handbuch der experimentellen Pharmakologie, Vol. X1X, Springer, Heidelberg, 1966, pp. 529-592. 4 CHRUSCIEL,T., Adrenergic Mechanisms. Churchill, London, 1960, p. 539. 5 CORRODI, H., HILLARP, N.-fl~., AND JONSSON, G., Fluorescence methods for the histochemical demonstration of monoamines. 3. Sodium borohydride reduction of the fluorescent compounds as a specificity test, J. Histochem. Cytochem., 12 (1964) 582-586. 6 CORRODI, H., AND JONSSON, G., The formaldehyde fluorescence method for the histochemical demonstration of biogenic monoamines. A review on the methodology, J. Histochem. Cytochem., 15 (1967) 65-78. 7 DAHLSTROM,A., AND FUXE, K., Evidence for the existence of monoamine neurons in the central nervous system. 1. Demonstration of monoamines in the cell-bodies of brain stem neurons, Acta physioL scand., 62 (1964) Suppl. 232. 8 FUXE, K., Evidence for the existence of monoamine neurons in the central nervous system. III. The monoamine nerve terminal, Z. Zellforsch., 65 (1965) 573-596. 9 GRAY, E. G., AND WHITTAKER, V. P., The isolation of nerve-endings from brain: an electronmicroscopic study of cell fragments derived by homogenization and centrifugation, J. Anat. (Lond.), 96 (1962) 79-88. l0 HAMBERGER,B., Reserpine-resistant uptake of catecholamines in isolated tissues of the rat, Acta physiol, scand., (1967) Suppl. 295. I I LEVI, R., AND MAYNERT, E. W., The subcellular localization of brain stem norepinephrine and 5-hydroxytryptamine in stressed rats, Biochem. Pharmacol., 13 (1964) 615-62 I. 12 LUFT, J. H., Improvements in epoxy resin embedding methods, J. biophys, biochem. Cytol., 9 (1961) 409--414. 13 MASUOKA,O., Monoamines in isolated nerve-ending particles, Biochem. Pharmacol., 14 (1965) 1688-1689. 14 MICHAELSON,I. A., AND WHITTAKER,V. P., The subcellular localization of 5-hydroxytryptamine in guinea-pig brain, Biochem. Pharmacol., 12 (1963) 203-21 I. 15 PLETSCHER,A., GEY, K. F., AND BURKARD, W. P., Inhibitors of monoamine oxidase and decarboxylase of aromatic amino acids. In O. EICHLER AND A. FARAH (Eds.), Handbuch der experimentellen Pharmakologie, Vol. X1X, Springer, Heidelberg, 1966, pp. 593-735. 16 REYNOLDS,E. S., The use of lead citrate at high pH as an electron-opaque stain in electron microscopy, J. Cell Biol., 17 (1963) 208-212. 17 SABATINI,D. D., BENSCH, K., AND BARRNETT,R.. J., The preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation, J. Cell Biol., 17 (1963) 19-58. 18 SJOSTRAND,F., Electron microscopy of cellular constituents. In S. P. COLOWICKAND N. O. KAPLAN (Eds.), Methods in Enzymology, VoL 4, Academic Press, New York, 1957, p. 391. 19 WHITTAKER,V. P., MICHAELSON,|. A., AND KIRKLAND, R. JEANETTEA., The separation of synaptic vesicles from nerve-ending particles ('synaptosomes'), Biochem. J., 90 (1964) 293-303.
Brain Research, 6 (1967) 475-480
475
I D E N T I F I C A T I O N OF DOPAMINE, N O R A D R E N A L I N E AND 5-HYDROXY T R Y P T A M I N E VARICOSITIES IN A F R A C T I O N C O N T A I N I N G NERVE E N D I N G PARTICLES
KJELL FUXE, HORST GROBECKER*, TOMAS HOKFELT ANDGOSTA JONSSON Department of Histology, Karolinska Instituter, Stockholm (Sweden)
(Accepted May 19th, 1967)
INTRODUCTION Masuoka 1~ was able to give good support for the view that the catecholamines found in the fraction from the brain stem containing nerve ending particles (NEPs) are localized at least mainly to small rounded structures, which probably represent the varicosities of the central catecholamine nerve terminals. In the present study evidence is given that the dopamine, noradrenaline and 5-hydroxytryptamine in the P2-B-fraction (according to Michaelson and Whittaker 14) are localized at least partly to isolated dopamine, noradrenaline and 5-hydroxytryptamine varicosities respectively. MATERIAL AND METHODS Male Sprague-Dawley rats (150-250 g body weight) were used. The brain stem (without nucleus caudatus-putamen), the spinal cord and the nucleus caudatus-putamen were dissected out from normal rats, or rats pretreated with reserpine (10 mg/kg, i.p., 12 h before sacrifice), nialamide (100-500 mg/kg, i.p., 4-12 h before sacrifice) or nialamide (as before) + L-3,4-dihydroxyphenylalanine (L-DOPA; 100 mg/kg, i.p., 30 min before sacrifice) respectively. The brain stem, the spinal cord and n.caudatusputamen respectively were homogenized in a glass homogenizer with a teflon pestle for 60 sec in l0 volumes of ice-cold 0.3 M sucrose, containing l0 #g/ml of ascorbic acid and 0.5 mg/ml Na2EDTA. The original homogenate was first fractionated into primary fractions by differential centrifugation 9. The first particulate fraction P1 (nuclei and cell debris) was obtained by centrifuging the original homogenate for l0 rain at 1000 × g. Then the supernatant of the P1 preparation was centrifuged for 20 min at 10,000 × g to obtain the P2 fraction. This crude mitochondrial fraction was resuspended in 2 ml of 0.3 M sucrose of which 0.5 ml was carefully layered over a discontinuous density gradient prepared immediately before use, consisting of 1.5 ml 0.8 M * Present address: Department of Pharmacology, University of Frankfurt, Frankfurt/Main (Germany). Brain~.Research, 6~.(1967)475-480
476
K. FUXE et al.
sucrose layered over an equal volume of 1.2 M sucrose and centrifuged for 90 rain at 100,000 x g using a 3 × 5 ml swing-out rotor. All the centrifugations were carried out with an MSE Automatic Superspeed 50'T.C. centrifuge. A distinct separation into these subfractions was obtained : myelin fraction (P2-A), less dense than 0.8 M sucrose; NEPs fraction (P2-B), intermediate in density between 0.8 M and 1.2 M sucrose: and a mitochondrial fraction (P2-C), denser than 1.2 M, as described by Michaelson and Whittaker 14. Smears were prepared on ordinary microscopic slides from all the fractions, dried over night in an exsiccator with phosphorus pentoxide in vacuo. Then the smears were reacted with relatively dry formaldehyde gas (generated from paraformaldehyde containing 0.4% water according to Hamberger 1°) for I-3 h for the histochemical visualization of dopamine, noradrenaline and 5-hydroxytryptamine6, 7. Then the smears were mounted under coverslips in Entellan (Merck) and examined in a Zeiss fluorescence microscope (for technical details on fluorescence microscopy see DahlstrSm and FuxeT). Some of the smears were treated with 0.03 ~ NaBH4 in 90 ~ isopropanol for testing specificity of monoamine fluorescence'L After examining the slides in the fluorescence microscope they were again exposed to formaldehyde gas at +80°C for 1 h as stated above and re-examined. In one experiment the different fractions (P1, P2, P2-A, P2-B and Pz-C) were freeze-dried, treated with formaldehyde gas, and embedded in paraffin, sectioned, mounted and examined in the fluorescence microscope. Generally, the same results were obtained with this technique compared with the 'smear' technique. To confirm the presence of NEPs, the Pe-B fraction from the spinal cord, the brain stem and the n. caudatus-putamen was examined in the electron microscope. An aliquot of each fraction was carefully resuspended in isotonic sucrose and centrifuged down on flat discs consisting of polymerized Epon 18, after which the pellets were fixed in glutaraldehyde 17 and osmium tetroxide. After dehydration in alcohol the specimen were embedded in Epon 12. The sections (500-800 A thick) were contrasted with uranyl acetate followed by lead citrate 16. An RCA-EM U-3A electron microscope was used. RESULTS
Brain s t e m
In the normal rat there were observed in the P2-B fraction large numbers of weak to strongly green-fluorescent dots of varying sizes (0.3-2/D. The colour of the emitted light from these dots was typical for fluorescence due to presence of primary catecholamines (see Fig. 1). Sometimes dots in the same size range having a weak yellow fluorescence very sensitive to irradiation with ultraviolet light, typical for the presence of 5-hydroxytryptamine, were observed. These fluorescent dots were somewhat difficult to differentiate direct visually from the autofluorescent granules usually present in the mentioned fraction. The fluorescence of these autofluorescent granules was, however, not formaldehyde-induced, fairly stable against irradiation to ultraviolet light, not abolished by NaBH4 treatment and did not disappear after reserpine Brain Research, 6 (1967) 475-480
MONOAMINE-CONTAINING NERVE ENDING PARTICLES
477
Fig. 1. Section of a freeze-dried, paraffin-embedded P2-B fraction of brain stem of rat treated with nialamide (500 mg/kg i.p., 4 h before sacrifice). A large number of densely packed weakly to strongly green- (the vast majority) and yellow-fluorescent dots of various diameters (0.3-2/~) are observed. These in all probability represent noradrenaline and 5-hydroxytryptamine varicosities respectively. 300 ×. pretreatment. Furthermore, they did not show any signs of diffusion of the fluorescent compounds after reaction with humid formaldehyde gas, which was the case with the dots emitting typical monoamine fluorescence. The other fractions investigated (P1, P2-A and P2-C) showed a relatively much smaller number of fluorescent dots. In the nialamide-pretreated rat there were observed distinct increases in number and intensity of the dots emitting a yellow light, due to the presence of 5-hydroxytryptamine. The green-fluorescent dots did not show any certain changes. After nialamide-L-DOPA, however, the green-fluorescent dots were markedly increased in number and intensity. In the reserpinized rats the number of green- and yellowfluorescent dots emitting a specific monoamine fluorescence had practically disappeared. Those remaining after this treatment usually had an irregular appearance and more distinctloutlines than the vast majority of those observed in normal rats, part of which probably represent autofluorescent granules. The specificity of monoamine fluorescence was tested by NaBH4 treatment, which completely abolished the fluorescence. After renewed formaldehyde gas treatment, the fluorescence could partly be regenerated. Spinal cord About the same fluorescence microscopical picture was obtained in the Pz-B fraction as from that in the smears from the brain stem. However, the smears from the normal and especially from the nialamide-treated rats showed much higher amounts of weak to medium yellow-fluorescent dots. Furthermore, the yellow-fluorescent dots usually appeared to be larger in size (1-2 #) as seen in the fluorescence microscope than in the brain stem. Brain Research, 6 (1967) 475-480
478
K. VUXE et al.
Fig. 2. Electron micrograph of a P~-B fraction of brain stem of rat. In spite of deficiency in morphology a large number ofNEPs (n) can be identified. 23,000 ×. Fig. 3. P2-B fraction of brain stem of rat. A higher magnification of a single NEP. 41,000 x.
Nucleus caudatus-putamen In the P2-B fraction f r o m n o r m a l and n i a l a m i d e - p r e t r e a t e d rat there a p p e a r e d a large n u m b e r o f green-fluorescent dots ranging mainly between 0.2 and I ,u in d i a m eter. N o yellow fluorescent dots exhibiting a specific fluorescence were observed, however. In a c c o r d a n c e with the results o f m a n y earlier electron m i c r o s c o p i c studies 9 a large n u m b e r o f N E P s could be identified in all three P2-B fractions studied (Figs. 2 a n d 3). A l t h o u g h no statistical e v a l u a t i o n was m a d e a clear difference in size could be observed between the N E P s in the P2-B fraction o f the b r a i n stem and the n. caud a t u s - p u t a m e n . The m a j o r i t y o f the N E P s o f the f o r m e r fraction had a d i a m e t e r between 0.5 a n d 1.5/z, while m o s t o f the N E P s o f the latter one h a d a d i a m e t e r between Brain Research, 6 (1967) 475-480
MONOAMINE-CONTAINING NERVE ENDING PARTICLES
479
0.3 and 0.6 # as measured in the ultrathin sections. In addition also isolated mitochondria were seen. DISCUSSION
The present results give support for the view that the green- and yellow-fluorescent dots observed in the P2-B fraction represent dopamine, noradrenaline and 5hydroxytryptamine varicosities respectively la. The evidence is as follows: (1) Several of the histochemical criteria for the specificity of the fluorescence are satisfied (diffusion of the fluorescent compounds after reaction with humid formaldehyde gas, colour of fluorescence, NaBH4-reduction test, ultraviolet sensitivity, observed especially in the yellow-fluorescent dots). Furthermore, in the non-reacted smears these fluorescent dots could not be observed 6. (2) Many of the pharmacological criteria are satisfied for the specificity of the fluorescence observed. The green- and yellow-fluorescence disappeared after reserpine treatment which is known to deplete the endogenous monoamines a and is markedly increased after a nialamide injection which produces increases in the noradrenaline and especially the 5-hydroxytryptamine levels in the brain and the spinal cord 15. (3) The green- and yellow-fluorescent dots with specific monoamine fluorescence in all probability represent varicosities of disrupted dopamine, noradrenaline and 5hydroxytryptamine nerve terminals, since they lie (a) in the same size range as the varicosities (0.3-2 #) (ref. 8) and (b) electron microscopy of the same fraction revealed the presence of large numbers of NEPs in about the same size range as the fluorescent dots. Furthermore, in the Pe-B fraction of n. caudatus-putamen which contains a large number of very fine dopamine nerve terminals1, 8, the NEPs as observed in the electron microscope were much finer than in the other areas studied. (4) 5-Hydroxytryptamine and noradrenaline assays on brain subfractions are in agreement with the presented resultsZ,4,11,13. Thus, it seems possible to identify catecholamine- and 5-hydroxytryptaminecontaining varicosities by the fluorescence histochemical technique of Falck and Hillarp in a pinched-off nerve ending fraction (P2-B). The use of fluorescence histochemistry may be a useful complement for studies on monoaminergic mechanisms by subfractionation techniques, especially for checking the homogenization procedure. SUMMARY
Using the histochemical fluorescence technique of Falck and Hillarp for the demonstration of dopamine, noradrenaline and 5-hydroxytryptamine in combination with histochemical and pharmacological specificity tests, it has been possible to show that there is in all probability a high accumulation of dopamine, noradrenaline and 5-hydroxytryptamine varicosities in the nerve ending particle fraction P2-B (according to Whittaker et al.19). The subfractionation technique used in the present investigation was checked with electron microscopy. Brain Research, 6 (1967) 475-480
480
K. FUXE et al.
ACKNOWLEDGEMENTS T h e i n v e s t i g a t i o n has b e e n s u p p o r t e d Medical
Research
Council
by r e s e a r c h g r a n t s f r o m t h e S w e d i s h
(B 67-12X-715-03
and
B 68-12X-2295-01),
'Magnus
Bergvalls Stiftelse' a n d the T h e r e s e and J o h a n A n d e r s s o n M e m o r i a l F o u n d a t i o n . F o r g e n e r o u s supplies o f drugs, we a r e i n d e b t e d to t h e f o l l o w i n g c o m p a n i e s : S w e d i s h C i b a (Serpasil ® ) a n d S w e d i s h Pfizer, S t o c k h o l m ( N i a m i d ®).
REFERENCES l ANDEN, N.-E., FUXE, K., HAMBERGER,B., AND H()KFELT, T., A quantitative study on the nigro-
neostriatal dopamine neuron system in the rat, Actaphysiol. scand., 67 (1966) 306-312. 2 CARLINI, E. A., AND GREEN, J. P.,thThe subcellular distribution of histamine, slow-reacting substance and 5-hydroxytryptamine in the brain of the rat, Brit. J. Pharmaeol., 20 (1963) 264-277. 3 CARLSSON, A., Drugs which block the storage of 5-hydroxytryptamine and related amines. In O. EICHLERAND A. FARAH(Eds.), Handbuch der experimentellen Pharmakologie, Vol. X1X, Springer, Heidelberg, 1966, pp. 529-592. 4 CHRUSCIEL,T., Adrenergic Mechanisms. Churchill, London, 1960, p. 539. 5 CORRODI, H., HILLARP, N.-fl~., AND JONSSON, G., Fluorescence methods for the histochemical demonstration of monoamines. 3. Sodium borohydride reduction of the fluorescent compounds as a specificity test, J. Histochem. Cytochem., 12 (1964) 582-586. 6 CORRODI, H., AND JONSSON, G., The formaldehyde fluorescence method for the histochemical demonstration of biogenic monoamines. A review on the methodology, J. Histochem. Cytochem., 15 (1967) 65-78. 7 DAHLSTROM,A., AND FUXE, K., Evidence for the existence of monoamine neurons in the central nervous system. 1. Demonstration of monoamines in the cell-bodies of brain stem neurons, Acta physioL scand., 62 (1964) Suppl. 232. 8 FUXE, K., Evidence for the existence of monoamine neurons in the central nervous system. III. The monoamine nerve terminal, Z. Zellforsch., 65 (1965) 573-596. 9 GRAY, E. G., AND WHITTAKER, V. P., The isolation of nerve-endings from brain: an electronmicroscopic study of cell fragments derived by homogenization and centrifugation, J. Anat. (Lond.), 96 (1962) 79-88. l0 HAMBERGER,B., Reserpine-resistant uptake of catecholamines in isolated tissues of the rat, Acta physiol, scand., (1967) Suppl. 295. I I LEVI, R., AND MAYNERT, E. W., The subcellular localization of brain stem norepinephrine and 5-hydroxytryptamine in stressed rats, Biochem. Pharmacol., 13 (1964) 615-62 I. 12 LUFT, J. H., Improvements in epoxy resin embedding methods, J. biophys, biochem. Cytol., 9 (1961) 409--414. 13 MASUOKA,O., Monoamines in isolated nerve-ending particles, Biochem. Pharmacol., 14 (1965) 1688-1689. 14 MICHAELSON,I. A., AND WHITTAKER,V. P., The subcellular localization of 5-hydroxytryptamine in guinea-pig brain, Biochem. Pharmacol., 12 (1963) 203-21 I. 15 PLETSCHER,A., GEY, K. F., AND BURKARD, W. P., Inhibitors of monoamine oxidase and decarboxylase of aromatic amino acids. In O. EICHLER AND A. FARAH (Eds.), Handbuch der experimentellen Pharmakologie, Vol. X1X, Springer, Heidelberg, 1966, pp. 593-735. 16 REYNOLDS,E. S., The use of lead citrate at high pH as an electron-opaque stain in electron microscopy, J. Cell Biol., 17 (1963) 208-212. 17 SABATINI,D. D., BENSCH, K., AND BARRNETT,R.. J., The preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation, J. Cell Biol., 17 (1963) 19-58. 18 SJOSTRAND,F., Electron microscopy of cellular constituents. In S. P. COLOWICKAND N. O. KAPLAN (Eds.), Methods in Enzymology, VoL 4, Academic Press, New York, 1957, p. 391. 19 WHITTAKER,V. P., MICHAELSON,|. A., AND KIRKLAND, R. JEANETTEA., The separation of synaptic vesicles from nerve-ending particles ('synaptosomes'), Biochem. J., 90 (1964) 293-303.
Brain Research, 6 (1967) 475-480