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Calcium-dependent release of radiolabeled catecholamines and serotonin from rat brain synaptosomes in a superfusion system
Brain Research, 99 (1975) 419-424 .© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
419
Calcium-dependent release of radiolabeled catecholamines and serotonin from rat brain synaptosomes in a superfusion system
ARIE H. MULDER, W1M B. VAN DEN BERG AND JOHANNES C. STOOF Department of Pharmacology, Medical Faculty, Free Universit),, Amsterdam (The Netherlands)
(Accepted August 7th, 1975)
Since their discovery some 15 years ago 7,27 isolated presynaptic nerve terminals (synaptosomes) have proven to be a very useful tool for investigations of the presynaptic physiology and pharmacology of CNS neurotransmission. Data from subcellular fractionation studies suggest that neurotransmitters and the enzymes involved in their biosynthesis are mainly localized in the nerve terminals 5-7,16,19,2°,27. Important functional activities of intact neuronal cells are, at least partly, retained in synaptosomes; such as respiration and oxidative phosphorylation 1,z,e7 and biosynthesis of neurotransmitters from their precursors 13,14,19,2s. Furthermore, synaptosomes are able to perform energy-requiring processes like active uptake of (putative) monoamine and amino acid neurotransmitters 23,24 and transmitter release upon membrane depolarization 1,2,17,21. So far, in most studies on central neurotransmitter release in vitro brain or spinal cord slices have been used, which were previously labeled with the radioactive putative transmitters4,S,l°, 11,18,25,e6. However, a brain slice can still be considered as a highly organized structure of both neuronal and non-neuronal cells. It is conceivable that neighboring non-neuronal cells and, in addition, small interneurons, which may also be present in brain slices, have a modulating influence on a particular neuronal release process or the action of drugs thereon. Therefore, synaptosomes would provide a model for investigations on transmitter release phenomena in nerve terminals not influenced by other cells. An important condition in release studies is that reuptake of released transmitter molecules should be prevented, i.e. eft]ux should be the only membrane transport process to occur. For instance, if this is not the case, a reuptake inhibition by a drug may be misinterpreted as a releasing effect. Similar considerations apply to uptake studies, since the effect of drugs on (re)uptake of neurotransmitters may be due to either real reuptake inhibition or, in fact, stimulation of release 1°. Some recent reports have described methods for studying neurotransmitter release with synaptosomal preparations deposited on nylon gauze 1, glass-fiber 17 or Millipore filters 21. In this communication we report on a simple and reproducible technique which
420
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m~ium
cotton plug
G-15 (2001.11)~~~
Sephadex layer
nletd~ m --
iI " ssynaptosome ..~.sion / 150~Jl)
i |
]
cotton plug
Plunger
Fig. 1. Schematic drawing of one of the 4 superfusion chambers of the system. Basically, it consists of a syringe with two movable plungers. Well-oxygenated medium is pumped through the chambers at a rate of 0.25 ml/min and collected in 2 min fractions in vials, which are used for liquid scintillation counting of radioactivity.
we developed for continuous superfusion of synaptosomes. The geometry of the system (Fig. 1) is such that the synaptosomes are present in a very small volume of medium (50 ffl), which is continuously renewed at a comparatively high rate, thereby reducing the possibility of reuptake of released transmitter molecules to a minimum. Twenty per cent (,w/v) homogenates of rat brain tissue (Wistar rats, 140-180 g body wt.) were prepared in 0.32 M sucrose in a Potter-Elvehjem glass homogenizer with a rotating Teflon pestle. The homogenates were centrifuged at 600 × g and the supernatants obtained were used as crude synaptosomal preparations. One ml of the synaptosome suspension (containing the equivalent of 200 mg of fresh tissue) was added to 4 ml of a Krebs-Ringer bicarbonate ( K R B ) medium and preincubated during 5 min at 37 °C under an atmosphere o f O ~ C O ~ (95 ~ : 5 ~ ) in a Dubnoff metabolic shaker. The K R B medium had the following composition ( m M ) : NaC1 (118); KC1 (4.85); CaCI2 (2.5); MgSO4 (1.15); KH2PO4 (1.15); NaHCO3 (25); glucose (11.1); pH 7.2-7.4. In addition the medium normally contained 10 uM iproniazide and 1 mg/ml of ascorbic acid during the uptake incubation. After addition of 5-10/~Ci of the tritiated monoamines (final concentrations in the medium: 2 x 10-7-5 x 10-TM) the incubation was continued for 10 min. At the end of the incubation period the labeled particulate material was collected by centrifugation at 600 x g and the pellet was carefully resuspended in K R B medium (final volume 250 #1). Immediately thereafter 50/~1 of this suspension was applied to each of the 4 chambers of the superfusion system. The superfusion chambers (Fig. 1)basically consist of syrmges surrounded by a
421 3 H - DOPAMINE STRIATUM
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Fig. 2. Calcium-dependence of [3H]dopamine release from rat striatal synaptosomes, induced by 10
l~M veratridine (indicated by the solid bar).
thermostatted waterbath. The particle suspension was layered on top of a small column (200 #1) of Sephadex G-15, which was swollen in KRB medium the day before, and superfused with oxygenated medium at a rate of 0.25 ml per minute, maintained by a peristaltic pump (Gilson Minipuls HP-4). The effective volume above the Sephadex layer was adjusted by the movable plunger to about 50 #1. Two-minute fractions were collected and the radioactivity was determined by liquid scintillation counting. A fairly constant rate of efflux of radioactivity was generally obtained within 20 min. Forty minutes after the start of the superfusion, membrane depolarization was effected by changing for a period of 10 min to a medium containing 56 m M potassium (KC1 replacing an equivalent amount of NaCI) or 10 # M veratridine. Thereafter superfusion was continued for another 10 min with normal medium. Finally 200 #1 of a 0.1 N HC1 solution was placed on top of the Sephadex layer and left there for 30 min in order to extract the remaining radioactivity from the synaptosomes. In experiments devised to check the calcium-dependence of the depolarizationinduced release, the uptake incubation of the synaptosomes took place in a KRB medium from which CaCl2 was deleted. Superfusion was also performed with calciumfree medium, except for the controls, where CaCI2 was introduced only during superfusion with the high potassium- or veratridine-containing solution. We studied the release of tritiated dopamine, noradrenaline and serotonin from
422 3H-NORADRENALINE HYPOTHALAMUS
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Figs. 3 and 4. Calcium.dependence of [aH]n0radrenaline release (Fig. 3) and [aH]serotonin release (Fig. 4) from rat hypothalamie synaptosomes, induced by 56 mM potassium chloride (indicated :by the solid bar). both striatal and hypothalamic synaptosomes. Depolarization by high potassium concentrations or by veratridine both effectively released the monoamines (Figs. 2, 3 and 4). Moreover, this depolarization-induced release appeared to be dependent on the presence o f calcium ions, which is thought to be an essential feature o f stimulussecretion couplingg.ZL
423 In some e x p e r i m e n t s we have a n a l y z e d the r a d i o a c t i v i t y in the effluent c h r o m a t o g r a p h i c a l l y on small D o w e x 50 W - X 4 c o l u m n s l L In these e x p e r i m e n t s the m e d i u m , which was used for the u p t a k e i n c u b a t i o n s , did n o t c o n t a i n a M A O inhibitor. U n d e r these c o n d i t i o n s 14.5 ± 1.0 ( 3 ) % o f the basal efflux o f r a d i o a c t i v i t y (last 10 rain before s t i m u l a t i o n ) after labeling with [ 3 H ] d o p a m i n e consisted o f u n c h a n g e d catecholamine. D u r i n g stimulation, however, this percentage increased to 36.8 ± 2.9(3)'),/,. A f t e r labeling with [3H]serotonin these figures were 28.5 -}: 2.3(3) % a n d 52.3 i 2.4 (3) ~o respectively. C a l c u l a t i o n b a s e d on these d a t a showed t h a t at least 75 % o f the radioactivity, which is released u p o n d e p o l a r i z a t i o n (i.e. the r a d i o a c t i v i t y released a b o v e b a s a l level efflux), consists o f the u n m o d i f i e d n e u r o t r a n s m i t t e r s . The presence in the superfusing m e d i u m o f inhibitors o f m o n o a m i n e r e u p t a k e in c o n c e n t r a t i o n s , which are k n o w n to cause an effective i n h i b i t i o n 12,23, h a d no influence on d e p o l a r i z a t i o n - i n d u c e d release in o u r system. Thus, 10-SM d e s i p r a m i n e o r cocaine did n o t affect the release o f t r i t i a t e d n o r a d r e n a l i n e f r o m h y p o t h a l a m i c s y n a p t o s o m e s , while 10 6M b e n z t r o p i n e a n d 10-SM cocaine did n o t change the release o f tritiated d o p a m i n e from striatal s y n a p t o s o m e s . This indicates t h a t r e u p t a k e plays only a m i n o r , if any, role in this superfusion system. In s u m m a r y , the superfusion m e t h o d described in this r e p o r t allows neurot r a n s m i t t e r release from s y n a p t o s o m e s to be studied in a c o n v e n i e n t and r e p r o d u c i b l e m a n n e r , a n d w i t h o u t the interference o f r e u p t a k e processes. U s i n g this m e t h o d we f o u n d t h a t d e p o l a r i z i n g agents induced release o f b o t h r a d i o l a b e l e d c a t e c h o l a m i n e s a n d serotonin from s y n a p t o s o m e s in a c a l c i u m - d e p e n d e n t way. The a u t h o r s t h a n k Sabine T a e k e m a , Bert den Breejen and Franqois H o g e n b o o m for skilful technical assistance.
1 BELLEROCHE,J. S. DE, AND BRADFORD,H. F., Metabolism of beds of mammalian cortical synaptosomes: responses to depolarizing influences, J. Neurochem., 19 (1972) 585-602. 2 BLAUSTEIN, M. P., JOHNSON, E. M., AND NEEDLEMAN, P., Calcium-dependent norepinephrine release from presynaptic nerve endings in vitro, Proc. nat. Acad. Sci. (Wash.), 69 (1972) 2237-2240. 3 BRADFORD, H. F., Respiration in vitro of synaptosomes from mammalian cerebral cortex, J. Neurochem., 16 (1969) 675 684. 4 CHASE,T. N., KATZ, R. I., AND KOPIN, |. J., Release of 3H-serotonin from brain slices, J. Neurochem., 16 (1969) 607-615.
5 COYLE,J. T., Tyrosine hydroxylase in rat brain; cofactor requirements, regional and subcellular distribution, Biochem. Pharmacol., 21 (1972) 1935-1944. 6 COYLE,J. T., AND KUHAR,M. J., Subcellular localization ofdopamine-fl-hydroxylaseand endogenous norepinephrine in the rat hypothalamus, Brain Research, 65 (1974) 475-487. 7 DE ROBERTIS, E., DE IRALDI, A. P., DE LOREZARNAIZ, G. R., AND SALGANICOFF,L., Cholinergic and non-cholinergic nerve endings in rat brain. 1. Isolation and subcellular distribution of acetylcholine and acetylcholine esterase, J. Neurochem., 9 (1962) 23-35. 8 FARNEBO,L. O., HAMBERGER,B., ANDJONSSON,G., Release of 3H-noradrenaline and 3H-dopamine from field stimulated cerebral cortex slices. Effect of tyrosine hydroxylase and dopamine-/4hydroxylase inhibition, J. Neurochem., 18 (1971) 2491-2500. 9 HALL, Z. W., Release of neurotransmitters and their interaction with receptors, Ann. Rev. Biochem., 41 (1972) 925-952. 10 HEIKKILA,R. E., ORLANSKY,H., ANDCOHEN, G., Studies on the distinction between uptake inhibition and release of 3H-dopamine in rat brain tissue slices, Biochem. Pharmacol., 24 (1975) 847 852. 11 HOPKIN,J., ANDNEAL,M. J., Effect of electrical stimulation and high potassium concentrations on the efflux of 14C-glycine from slices of spinal cord, Brit. J. Pharmacol., 42 (1971) 215-223.
424 12 HORN, A. S., COYLE, J. T., AND SNYDER, S. H., Catecholamine uptake by synaptosomes from rat brain. Structure-activity relationships of drugs with differential effects on dopamine and norepinephrine neurons, Molec. Pharmacol., 7 (1971) 66-80. 13 KAROBATH,M., Catecholamines and the hydroxylation of tyrosine in synaptosomes isolated from rat brain, Proc. nat. Acad. Sci. (Wash.), 68 (1971) 2370-2373. 14 KAROBATH, M., Serotonin synthesis with rat brain synaptosomes, Biochem. Pharmacol,, 21 (1972) 1253-1263. 15 KEHR, W., A method for the isolation and determination of 3-methoxytyramine in brain tissue, Arch. Pharmacol., 284 (1974) 149-158. 16 KUHAR, M. J., SHASKAN,E. G., AND SNYDER, S. H., The subcellular distribution of endogenous serotonin in brain tissue: comparison of synaptosomes storing serotonin, norepinephrine and ~,,aminobutyric acid, J. Neurochem., 18 (1971) 333-343. 17 LEVY, W. B., REDaURN, D. A., AND COTMAN, C. W., Stimulus-coupled secretion of GABA from rat brain synaptosomes, Science, 181 (1973) 676-678. 18 MULDER, A. H., AND SNYDER, S. H., Potassium-induced release of amino acids from cerebral cortex and spinal cord slices of the rat, Brain Research, 76 (1974) 297-308. 19 PATRICK, R. L., AND BARCHAS,J. D., Regulation of catecholamine synthesis in rat brain synaptosomes, J. Neurochem., 23 (1974) 7-15. 20 POTTER, L. T., GLOVER, V. A. S., AND SAELENS,J. K., Choline acetyltransferase from rat brain, J. biol. Chem., 243 (1968) 3864-3870. 21 RAITERI,M., LEW, G., AND FEDERICO,R., Stimulus-coupled release of unmetabolized 3H-norepinephrine from rat brain synaptosomes, Pharmaeol. Res. Commun., 7 (1975) 181-187. 22 RUB1N, R. P., The role of calcium in the release of neurotransmitter substances and hormones, Pharmacol. Rev., 22 (1970) 389-428. 23 SNYDER,S. H., KUHAR, M. J., GREEN, A. 1., COYLE, J. T., AND SHASKAN,E. G., Uptake and subcellular localization of neurotransmitters in the brain, Int. Rev. Neurobiol., 13 (1970) 127-158. 24 SNVDER, S. H., YOUN(;, A. B., BENNETT, J. P., AND MOLDER, A. H., Synaptic biochemistry of amino acids, Fed. Proc., 32 (1973) 2039-2047. 25 SOMOGYI,G. T., AND SZERa, J. C., Demonstration of acetylcholine release by measuring efflux of labeled choline from cerebral cortical slices, J. Neurochem., 19 (1972) 2667-2677. 26 STARKE, K., AND MONTEL, H., Alpha-receptor mediated modulation of transmitter release from central noradrenergic neurons, Arch. Pharmacol., 279 (1973) 53-60. 27 WHITTAKER, V. P., The synaptosome. In A. LAJTHA(Ed.), Handbook of Neurochemistry, Vol. II, Plenum Press, New York, 1969, pp. 327-364. 28 YAMAMURA,H. l., AND SNYDER, S. H., High affinity transport of choline into synaptosomes of rat brain, J. Neurochem., 21 (1973) 1355-t374.
419
Calcium-dependent release of radiolabeled catecholamines and serotonin from rat brain synaptosomes in a superfusion system
ARIE H. MULDER, W1M B. VAN DEN BERG AND JOHANNES C. STOOF Department of Pharmacology, Medical Faculty, Free Universit),, Amsterdam (The Netherlands)
(Accepted August 7th, 1975)
Since their discovery some 15 years ago 7,27 isolated presynaptic nerve terminals (synaptosomes) have proven to be a very useful tool for investigations of the presynaptic physiology and pharmacology of CNS neurotransmission. Data from subcellular fractionation studies suggest that neurotransmitters and the enzymes involved in their biosynthesis are mainly localized in the nerve terminals 5-7,16,19,2°,27. Important functional activities of intact neuronal cells are, at least partly, retained in synaptosomes; such as respiration and oxidative phosphorylation 1,z,e7 and biosynthesis of neurotransmitters from their precursors 13,14,19,2s. Furthermore, synaptosomes are able to perform energy-requiring processes like active uptake of (putative) monoamine and amino acid neurotransmitters 23,24 and transmitter release upon membrane depolarization 1,2,17,21. So far, in most studies on central neurotransmitter release in vitro brain or spinal cord slices have been used, which were previously labeled with the radioactive putative transmitters4,S,l°, 11,18,25,e6. However, a brain slice can still be considered as a highly organized structure of both neuronal and non-neuronal cells. It is conceivable that neighboring non-neuronal cells and, in addition, small interneurons, which may also be present in brain slices, have a modulating influence on a particular neuronal release process or the action of drugs thereon. Therefore, synaptosomes would provide a model for investigations on transmitter release phenomena in nerve terminals not influenced by other cells. An important condition in release studies is that reuptake of released transmitter molecules should be prevented, i.e. eft]ux should be the only membrane transport process to occur. For instance, if this is not the case, a reuptake inhibition by a drug may be misinterpreted as a releasing effect. Similar considerations apply to uptake studies, since the effect of drugs on (re)uptake of neurotransmitters may be due to either real reuptake inhibition or, in fact, stimulation of release 1°. Some recent reports have described methods for studying neurotransmitter release with synaptosomal preparations deposited on nylon gauze 1, glass-fiber 17 or Millipore filters 21. In this communication we report on a simple and reproducible technique which
420
...... ~. . . . .
m~ium
cotton plug
G-15 (2001.11)~~~
Sephadex layer
nletd~ m --
iI " ssynaptosome ..~.sion / 150~Jl)
i |
]
cotton plug
Plunger
Fig. 1. Schematic drawing of one of the 4 superfusion chambers of the system. Basically, it consists of a syringe with two movable plungers. Well-oxygenated medium is pumped through the chambers at a rate of 0.25 ml/min and collected in 2 min fractions in vials, which are used for liquid scintillation counting of radioactivity.
we developed for continuous superfusion of synaptosomes. The geometry of the system (Fig. 1) is such that the synaptosomes are present in a very small volume of medium (50 ffl), which is continuously renewed at a comparatively high rate, thereby reducing the possibility of reuptake of released transmitter molecules to a minimum. Twenty per cent (,w/v) homogenates of rat brain tissue (Wistar rats, 140-180 g body wt.) were prepared in 0.32 M sucrose in a Potter-Elvehjem glass homogenizer with a rotating Teflon pestle. The homogenates were centrifuged at 600 × g and the supernatants obtained were used as crude synaptosomal preparations. One ml of the synaptosome suspension (containing the equivalent of 200 mg of fresh tissue) was added to 4 ml of a Krebs-Ringer bicarbonate ( K R B ) medium and preincubated during 5 min at 37 °C under an atmosphere o f O ~ C O ~ (95 ~ : 5 ~ ) in a Dubnoff metabolic shaker. The K R B medium had the following composition ( m M ) : NaC1 (118); KC1 (4.85); CaCI2 (2.5); MgSO4 (1.15); KH2PO4 (1.15); NaHCO3 (25); glucose (11.1); pH 7.2-7.4. In addition the medium normally contained 10 uM iproniazide and 1 mg/ml of ascorbic acid during the uptake incubation. After addition of 5-10/~Ci of the tritiated monoamines (final concentrations in the medium: 2 x 10-7-5 x 10-TM) the incubation was continued for 10 min. At the end of the incubation period the labeled particulate material was collected by centrifugation at 600 x g and the pellet was carefully resuspended in K R B medium (final volume 250 #1). Immediately thereafter 50/~1 of this suspension was applied to each of the 4 chambers of the superfusion system. The superfusion chambers (Fig. 1)basically consist of syrmges surrounded by a
421 3 H - DOPAMINE STRIATUM
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40OO
NORMAL
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1'
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'
i
,
~
I
L_
..J ~ w
2000
i
"1-
1000
STIM
30
4o
s'o
60 TIME (MIN)
Fig. 2. Calcium-dependence of [3H]dopamine release from rat striatal synaptosomes, induced by 10
l~M veratridine (indicated by the solid bar).
thermostatted waterbath. The particle suspension was layered on top of a small column (200 #1) of Sephadex G-15, which was swollen in KRB medium the day before, and superfused with oxygenated medium at a rate of 0.25 ml per minute, maintained by a peristaltic pump (Gilson Minipuls HP-4). The effective volume above the Sephadex layer was adjusted by the movable plunger to about 50 #1. Two-minute fractions were collected and the radioactivity was determined by liquid scintillation counting. A fairly constant rate of efflux of radioactivity was generally obtained within 20 min. Forty minutes after the start of the superfusion, membrane depolarization was effected by changing for a period of 10 min to a medium containing 56 m M potassium (KC1 replacing an equivalent amount of NaCI) or 10 # M veratridine. Thereafter superfusion was continued for another 10 min with normal medium. Finally 200 #1 of a 0.1 N HC1 solution was placed on top of the Sephadex layer and left there for 30 min in order to extract the remaining radioactivity from the synaptosomes. In experiments devised to check the calcium-dependence of the depolarizationinduced release, the uptake incubation of the synaptosomes took place in a KRB medium from which CaCl2 was deleted. Superfusion was also performed with calciumfree medium, except for the controls, where CaCI2 was introduced only during superfusion with the high potassium- or veratridine-containing solution. We studied the release of tritiated dopamine, noradrenaline and serotonin from
422 3H-NORADRENALINE HYPOTHALAMUS
1000
A
800
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NORMAL
0 u.
60O i
0 X ..I i1
E
F- ' 4O0
~
.
|
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/ e,)
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60 TIME (MIN)
3H-SEROTONtN HYPOTHALAMUS
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Figs. 3 and 4. Calcium.dependence of [aH]n0radrenaline release (Fig. 3) and [aH]serotonin release (Fig. 4) from rat hypothalamie synaptosomes, induced by 56 mM potassium chloride (indicated :by the solid bar). both striatal and hypothalamic synaptosomes. Depolarization by high potassium concentrations or by veratridine both effectively released the monoamines (Figs. 2, 3 and 4). Moreover, this depolarization-induced release appeared to be dependent on the presence o f calcium ions, which is thought to be an essential feature o f stimulussecretion couplingg.ZL
423 In some e x p e r i m e n t s we have a n a l y z e d the r a d i o a c t i v i t y in the effluent c h r o m a t o g r a p h i c a l l y on small D o w e x 50 W - X 4 c o l u m n s l L In these e x p e r i m e n t s the m e d i u m , which was used for the u p t a k e i n c u b a t i o n s , did n o t c o n t a i n a M A O inhibitor. U n d e r these c o n d i t i o n s 14.5 ± 1.0 ( 3 ) % o f the basal efflux o f r a d i o a c t i v i t y (last 10 rain before s t i m u l a t i o n ) after labeling with [ 3 H ] d o p a m i n e consisted o f u n c h a n g e d catecholamine. D u r i n g stimulation, however, this percentage increased to 36.8 ± 2.9(3)'),/,. A f t e r labeling with [3H]serotonin these figures were 28.5 -}: 2.3(3) % a n d 52.3 i 2.4 (3) ~o respectively. C a l c u l a t i o n b a s e d on these d a t a showed t h a t at least 75 % o f the radioactivity, which is released u p o n d e p o l a r i z a t i o n (i.e. the r a d i o a c t i v i t y released a b o v e b a s a l level efflux), consists o f the u n m o d i f i e d n e u r o t r a n s m i t t e r s . The presence in the superfusing m e d i u m o f inhibitors o f m o n o a m i n e r e u p t a k e in c o n c e n t r a t i o n s , which are k n o w n to cause an effective i n h i b i t i o n 12,23, h a d no influence on d e p o l a r i z a t i o n - i n d u c e d release in o u r system. Thus, 10-SM d e s i p r a m i n e o r cocaine did n o t affect the release o f t r i t i a t e d n o r a d r e n a l i n e f r o m h y p o t h a l a m i c s y n a p t o s o m e s , while 10 6M b e n z t r o p i n e a n d 10-SM cocaine did n o t change the release o f tritiated d o p a m i n e from striatal s y n a p t o s o m e s . This indicates t h a t r e u p t a k e plays only a m i n o r , if any, role in this superfusion system. In s u m m a r y , the superfusion m e t h o d described in this r e p o r t allows neurot r a n s m i t t e r release from s y n a p t o s o m e s to be studied in a c o n v e n i e n t and r e p r o d u c i b l e m a n n e r , a n d w i t h o u t the interference o f r e u p t a k e processes. U s i n g this m e t h o d we f o u n d t h a t d e p o l a r i z i n g agents induced release o f b o t h r a d i o l a b e l e d c a t e c h o l a m i n e s a n d serotonin from s y n a p t o s o m e s in a c a l c i u m - d e p e n d e n t way. The a u t h o r s t h a n k Sabine T a e k e m a , Bert den Breejen and Franqois H o g e n b o o m for skilful technical assistance.
1 BELLEROCHE,J. S. DE, AND BRADFORD,H. F., Metabolism of beds of mammalian cortical synaptosomes: responses to depolarizing influences, J. Neurochem., 19 (1972) 585-602. 2 BLAUSTEIN, M. P., JOHNSON, E. M., AND NEEDLEMAN, P., Calcium-dependent norepinephrine release from presynaptic nerve endings in vitro, Proc. nat. Acad. Sci. (Wash.), 69 (1972) 2237-2240. 3 BRADFORD, H. F., Respiration in vitro of synaptosomes from mammalian cerebral cortex, J. Neurochem., 16 (1969) 675 684. 4 CHASE,T. N., KATZ, R. I., AND KOPIN, |. J., Release of 3H-serotonin from brain slices, J. Neurochem., 16 (1969) 607-615.
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