- Email: [email protected]
Receptor-mediated control of neurotransmitter release from brain slices or synaptosomes
83
TIPS - March 1988 [Vol. 9]
Receptor-mediated control of neurotransmitter release from brain slices or synaptosomes With the advent of receptor binding teclmiques during the 1970s, there has beer, an increase in interest in the study of the actions of drugs at CNS receptors. Receptor binding studies play a key role in the search for such agents, as is evidenced by the recent interest displayed in the Nmethyl-v-aspartate (NMDA) antagonist, MK-801 (Ref. 1) and the 5HT1A partial agonist, ipsapirone 2. Their uses are, however, usually limited by their inability to distinguish between agonists and antagonists at receptor sites. The present review highlights a complementary technique, in-vitro radiolabelled neurotransmitter release studies, as a simple and reproducible means of measuring both receptor affinity and efficacy of drugs acting at prejunctional receptors in the CNS. General methodology Slices or synaptosomes derived from specific brain regions of experimental animals are loaded with a radiolabelled precursor for the neurotransmitter of interest by means of the selective, high affinity uptake carriers for these substances which are present in nerve terminals. The tissue is then washed and equilibrated to reduce the background rate of release of the tritiated neurotransmitter. This procedure is usually carded out in a superfusion apparatus, and designs suitable for use with synaptosomes or slices have been published sA. When a low basal rate of release of tritiated neurotransmitter has been established, applying a depolarizing stimulus (15-50mM K + ions or an electrical field oscillating at 0.1 to 10 Hz) increases the overflow of tritium an effect which can be readily quantified by liquid scintillation counting. Agonists which act at presynaptic receptors on the neuroterminal can, when added during the depolarizing stimulus, serve to either reduce or increase the release of neurotransmitter (Table I), and these effects can be blocked by appropriate antagonists. This in-vitro technique has been applied to the study of the receptor-
mediated control of the release of a range of classical transmitters including acetylcholine, dopamine, S-HT and noradrenaline s. A number of receptors have been identified which exert an influence over the release of these neurotransmitters, and the functional effects of agonists, antagonists and partial agonists acting at these receptors can be readily quantified in terms of conventional pharmacological measurements (pA2, pD2 and efficacy). Interestingly, this technique is also amenable to the study of the dependence of receptor activated events on ions such as Ca2+ (ReL 6) or Mg 2+ (Ref. 7). Radiolabelled neurotransmitter release studies can therefore answer questions about the location, function and control of these prejunctional receptors which cannot be addressed using receptor binding techniques. These in-vitro assays complement attempts to study the release of neurotransmitters in vivo, but have advantages in terms of the ease with which the experimental conditions can be modified and the effects of drugs reliably estimated. Examples These techniques have been extensively applied to the study of the release of 5-HT and noradrenaline (Table I). The release of these neu~'otransmitters is controlled by autoreceptors, heteroreceptors or receptors which are acted upon
by co-localized neurotransmitters such as adenosine or neuropeptides. These latter receptors could be te,-'med coreceptors by analogy with auto- and heteroreceptors. All three main types of receptor control the release of both 5-HT and noradrenaline, although each specific receptor type is not always represented on both neuroterminals (Table I). It must, however, be emphasized that direct proof for a prejunctional location of many of these receptors is lacking and indeed in at least one case, that of NMDA-stimulated noradrenaline release from the rat hippocampus, an interneurone may be involved in mediating these excitatory effects7. To date at least 17 different receptor types have been identified as able to control the release of 5-HT and noradrenaline. These effects offer opportunities to study both the brain area and species ~:.electivity of the interactions and their functional relevance in vivo since chronic drug treatment leads to sensitivity changes in both autoand heteroreceptors s'9. []
[]
[]
Although this article has emphasized the receptors controlling the release of 5-HT and noradrenaline, many other examples of the application of in-vitro release techniques to the measurement of receptor affinity and efficacy of drugs have been described s. Some of these studies have monitored the release of endogenous neurotransmitters from brain slices, and compara,~ive studies of radiolabelled and ~mdogenous release
i
TABLE I. Receptors controlling the release of 5-HT and noredreneline Receptor'rype Nerve Terminal Autoreceptor Heterorecoptor Coreceptor RMs 5-HT 5-HT~a ~ GABAa ~, adenoslneA1~ 12, 22,16 muscednlo (MI?) ~ substanceP 1' 18,19 (x2-adroneroic ~ 26 somatostatin 1' 27 dopamine D~ ~ 21 ~,;oredrenal!ne %.edrenergic J, GABAe ~ adenosine A~ ~ 24, 22,17 GABAA 1' neuropeptide Y ~ 14, 23 NMDA t' 7 opiate K ~ 20 opiate I~ ~ 24 somatostatin 1` 28 muecadni¢ J, 29 dopamlne D2 ~ 25 anoiotensin II t' ~ 28,15 prostaglandin E~ ~ 24 Receptors controlling the release of 5-HT and noradrens, ne were identified and cha~o;e~;ze~J using the redlolabelled neurotransmitterrelease technique. & Agonlst at receptor decreases release; 1` agonlst at receptor increases release. ,,,
~) 1908,ElsevierPublications,Csmbddge 0165- 6|,~'/188/$02,00
84
TIPS
-
March 1988 [Vol. 91
15 Bottiglieri, D. F., Sumners, C. and Raizada, M. K. (1987) Brain Res.403,167171 16 Feuerstein, T. J., Hertting, G. and Jackisch, R. (1985) Eur. J. Pha~nacol. 107, 233--242 17 Jackisch, R., Fehr, R. and Hertting, G. (1985) Neuropharmacology 24, 499-507 18 Marchi, M., Paudice, P., Bella, M. and Raiteri, M. (1986) Eur. J. PharmacoL 129, 353--357 19 Solti, M. and Barffai, T. (1987) Brain Res. 401, 377--380 20 Limberger, N., Spath, L., Holting, Th. and Starke, K. (lO86) Naunyn-Schmied. Arch. Pharmacol. 334, 166-171 21 Benkirane, S., Arbilla, S. and Langer, S. Z. (1987) Naunyn-Schmied. Arch. Pharmacol. 335, 502-507 22 Bowery, N. G., Hill, D. R., Hudson, A.L., Doble, A., Middlemiss, D.N., Shaw, J. and Tumbull, M. (1980) Nature 283, 92-94 23 Martire, M., Fuxe, K., Pistritto, G., Preziosi, P. and Agnati, L.F. (1986) I. Neural. Transm. 67, 113-124 24 Taube, H. D., Starke, K. and Borowski, E. (1977) Naunyn-Schmied. Arch. Pharmacol. 299, 123-141 25 Galzin, A. M., Dubocovich, M. L. and Langer, S.Z. (1982) I. PharmacoL Exp. Ther. 221, 461-471 26 Gothert, M., Huth, H. and Schlicker, E. (1981) Naunyn-Schmied. Arch. Pharmacol. 317, 199-203 27 Tanaka, S. and Tsujimoto, A. (1981) Brain Res. 208, 219-222 28 Tsujimoto, A. and Tanaka, S. (1981) Life Sci. 28, 903--910 29 Westfall, T. C. (1974) Life Sci. 14, 16411652 The 'hot' supeffused brain
are n o w b e i n g carried out. A n o t h e r major objective of research i n this area is to verify the s t u d i e s carried o u t on a n i m a l s w i t h those from h u m a n b r a i n tissue: reports o n the effects of d r u g s o n the release of n e u r o t r a n s m i t t e r s from b o t h fresh 1° a n d p o s t - m o r t e m 11 h u m a n tissues are b e g i n n i n g to appear. Such s t u d i e s will b e especially relevant to the t e r m i n a l 5-HT autoreceptor w h i c h , i n a s e m i n a l s t u d y on the rat cortex, h a s b e e n s h o w n to c o r r e s p o n d to the 5-HTzB b i n d i n g site TM, since this recogn i t i o n site is a p p a r e n t l y a b s e n t from the h u m a n cortex 13. The u s e of n e u r o t r a n s m i t t e r release studies, in c o n j u n c t i o n w i t h receptor b i n d i n g t e c h n i q u e s , p r o v i d e s a powerful tool for the identification of d r u g s w i t h effects at CNS receptors. D. N. MIDDLEMISS
Merck Sharp and Dohme Research Laboratories, Neuroscience Research Centre, Terlings Park, Eastwick Road, Harlow, Essex CM20 2QR, UK.
References 1 Kemp, J. A., Foster, A. C., Gill, R. and Woodruff, G. N. (1987) Trends Pharmacol. Sci. 8, 414-415 2 Traber, J. and Glaser, T. (1987) Trends Pharmacol. $ci. 8, 432-437 3 Korpi, E. R. and Oja, S. S. (1984) J. Neurochom. 43, 236-242 4 Minnenia, D. and Michaelson, I. A. (1985) J. Neurosci. Methods 14, 193-206 5 Chesselet, M-F. (1984) Neuroscience 12, 347-375 6 Gothert, M. (1980) Naunyn-Schmied. Arch. Pharmacol. 314, 223-230 7 Jones, S. M., Snell, L. D. and Johnson, K. M. (1987) J. Pharmacol. Exp. Thor. 240, 492--497 8 Raiteri, M., Marchi, M. and Maura, G. (1983) Eur. J. Pharmacol. 91, 141-143 9 Maura, G., Bonanno, G. and Raiteri, M. (1985) Eur. J. Pharmacol. 112, 105--110 10 Schlicker, E., Brandt, F., Classen, K. and Gothert, M. (1985) Brain Res. 331, 337341 11 Hardy, J. A. and Dodd, P. R. (1985) in Selected Topics from Neurochemistry (Osborne, N. N., ed.), pp. 25-41, Pergamon Press 12 Engel, G., Gothe~o M~ Hoyer~ D~ 5ch!icker, E. and Hillenbrand, K. (1986) Naunyn-Schmied. Arch. PharmacoL 332, 1-7 13 Hoyer, D., Pazos, A., Probst, A. and Palacios, J. M. (1986) Brain Res. 376, 85-96 14 Bonanno, G. and Raiteri, M. (1987) Synapse 1, 254-257
MK.S01: (+)-5-Methyl-10,11-dihydro-SHdibenzo[a,d]cyclohepten-5,10-imine maleate Ipsapirone: 2-(4-[4-(2-pyrimidinyl)-l-pip-
erazinyl]butyl)l,2-benzisothiazol-3-(2H)one-l,l-dioxidehydrochloride
Pharmacologist to Pharmacologist • I am in urgent need of the following chemicals for my research work: N-methyl-D-aspartate(NIVlDA) c~-amino-3-hydroxy.5-methyl-4isoxazolepropionate (~ P A )
3-3(2-carbo.xypiperazine-4.y|)-
propyl- l-phosphomtto (CPP) phencyclidine (PCP) ~2-amino-4-phosphonobutyrate
(I,-AP4)
I would appreciate any gift: Dr Gang K. Tong, Department of Physiology. Second Mih'tary Medical College, Shanghai 2OO433, Paopl~'~ Republic of t ~ . ~
will print sn~£ actsof ~terest to pbaz~cob~mJ free of e.,~e. Contact: P k m m a ~ o g ~ P/mrmaco/og~ TIPS, 68 Hills Road, Cambridge CB8 ILA, UE.
TIPS - March 1988 [Vol. 9]
Receptor-mediated control of neurotransmitter release from brain slices or synaptosomes With the advent of receptor binding teclmiques during the 1970s, there has beer, an increase in interest in the study of the actions of drugs at CNS receptors. Receptor binding studies play a key role in the search for such agents, as is evidenced by the recent interest displayed in the Nmethyl-v-aspartate (NMDA) antagonist, MK-801 (Ref. 1) and the 5HT1A partial agonist, ipsapirone 2. Their uses are, however, usually limited by their inability to distinguish between agonists and antagonists at receptor sites. The present review highlights a complementary technique, in-vitro radiolabelled neurotransmitter release studies, as a simple and reproducible means of measuring both receptor affinity and efficacy of drugs acting at prejunctional receptors in the CNS. General methodology Slices or synaptosomes derived from specific brain regions of experimental animals are loaded with a radiolabelled precursor for the neurotransmitter of interest by means of the selective, high affinity uptake carriers for these substances which are present in nerve terminals. The tissue is then washed and equilibrated to reduce the background rate of release of the tritiated neurotransmitter. This procedure is usually carded out in a superfusion apparatus, and designs suitable for use with synaptosomes or slices have been published sA. When a low basal rate of release of tritiated neurotransmitter has been established, applying a depolarizing stimulus (15-50mM K + ions or an electrical field oscillating at 0.1 to 10 Hz) increases the overflow of tritium an effect which can be readily quantified by liquid scintillation counting. Agonists which act at presynaptic receptors on the neuroterminal can, when added during the depolarizing stimulus, serve to either reduce or increase the release of neurotransmitter (Table I), and these effects can be blocked by appropriate antagonists. This in-vitro technique has been applied to the study of the receptor-
mediated control of the release of a range of classical transmitters including acetylcholine, dopamine, S-HT and noradrenaline s. A number of receptors have been identified which exert an influence over the release of these neurotransmitters, and the functional effects of agonists, antagonists and partial agonists acting at these receptors can be readily quantified in terms of conventional pharmacological measurements (pA2, pD2 and efficacy). Interestingly, this technique is also amenable to the study of the dependence of receptor activated events on ions such as Ca2+ (ReL 6) or Mg 2+ (Ref. 7). Radiolabelled neurotransmitter release studies can therefore answer questions about the location, function and control of these prejunctional receptors which cannot be addressed using receptor binding techniques. These in-vitro assays complement attempts to study the release of neurotransmitters in vivo, but have advantages in terms of the ease with which the experimental conditions can be modified and the effects of drugs reliably estimated. Examples These techniques have been extensively applied to the study of the release of 5-HT and noradrenaline (Table I). The release of these neu~'otransmitters is controlled by autoreceptors, heteroreceptors or receptors which are acted upon
by co-localized neurotransmitters such as adenosine or neuropeptides. These latter receptors could be te,-'med coreceptors by analogy with auto- and heteroreceptors. All three main types of receptor control the release of both 5-HT and noradrenaline, although each specific receptor type is not always represented on both neuroterminals (Table I). It must, however, be emphasized that direct proof for a prejunctional location of many of these receptors is lacking and indeed in at least one case, that of NMDA-stimulated noradrenaline release from the rat hippocampus, an interneurone may be involved in mediating these excitatory effects7. To date at least 17 different receptor types have been identified as able to control the release of 5-HT and noradrenaline. These effects offer opportunities to study both the brain area and species ~:.electivity of the interactions and their functional relevance in vivo since chronic drug treatment leads to sensitivity changes in both autoand heteroreceptors s'9. []
[]
[]
Although this article has emphasized the receptors controlling the release of 5-HT and noradrenaline, many other examples of the application of in-vitro release techniques to the measurement of receptor affinity and efficacy of drugs have been described s. Some of these studies have monitored the release of endogenous neurotransmitters from brain slices, and compara,~ive studies of radiolabelled and ~mdogenous release
i
TABLE I. Receptors controlling the release of 5-HT and noredreneline Receptor'rype Nerve Terminal Autoreceptor Heterorecoptor Coreceptor RMs 5-HT 5-HT~a ~ GABAa ~, adenoslneA1~ 12, 22,16 muscednlo (MI?) ~ substanceP 1' 18,19 (x2-adroneroic ~ 26 somatostatin 1' 27 dopamine D~ ~ 21 ~,;oredrenal!ne %.edrenergic J, GABAe ~ adenosine A~ ~ 24, 22,17 GABAA 1' neuropeptide Y ~ 14, 23 NMDA t' 7 opiate K ~ 20 opiate I~ ~ 24 somatostatin 1` 28 muecadni¢ J, 29 dopamlne D2 ~ 25 anoiotensin II t' ~ 28,15 prostaglandin E~ ~ 24 Receptors controlling the release of 5-HT and noradrens, ne were identified and cha~o;e~;ze~J using the redlolabelled neurotransmitterrelease technique. & Agonlst at receptor decreases release; 1` agonlst at receptor increases release. ,,,
~) 1908,ElsevierPublications,Csmbddge 0165- 6|,~'/188/$02,00
84
TIPS
-
March 1988 [Vol. 91
15 Bottiglieri, D. F., Sumners, C. and Raizada, M. K. (1987) Brain Res.403,167171 16 Feuerstein, T. J., Hertting, G. and Jackisch, R. (1985) Eur. J. Pha~nacol. 107, 233--242 17 Jackisch, R., Fehr, R. and Hertting, G. (1985) Neuropharmacology 24, 499-507 18 Marchi, M., Paudice, P., Bella, M. and Raiteri, M. (1986) Eur. J. PharmacoL 129, 353--357 19 Solti, M. and Barffai, T. (1987) Brain Res. 401, 377--380 20 Limberger, N., Spath, L., Holting, Th. and Starke, K. (lO86) Naunyn-Schmied. Arch. Pharmacol. 334, 166-171 21 Benkirane, S., Arbilla, S. and Langer, S. Z. (1987) Naunyn-Schmied. Arch. Pharmacol. 335, 502-507 22 Bowery, N. G., Hill, D. R., Hudson, A.L., Doble, A., Middlemiss, D.N., Shaw, J. and Tumbull, M. (1980) Nature 283, 92-94 23 Martire, M., Fuxe, K., Pistritto, G., Preziosi, P. and Agnati, L.F. (1986) I. Neural. Transm. 67, 113-124 24 Taube, H. D., Starke, K. and Borowski, E. (1977) Naunyn-Schmied. Arch. Pharmacol. 299, 123-141 25 Galzin, A. M., Dubocovich, M. L. and Langer, S.Z. (1982) I. PharmacoL Exp. Ther. 221, 461-471 26 Gothert, M., Huth, H. and Schlicker, E. (1981) Naunyn-Schmied. Arch. Pharmacol. 317, 199-203 27 Tanaka, S. and Tsujimoto, A. (1981) Brain Res. 208, 219-222 28 Tsujimoto, A. and Tanaka, S. (1981) Life Sci. 28, 903--910 29 Westfall, T. C. (1974) Life Sci. 14, 16411652 The 'hot' supeffused brain
are n o w b e i n g carried out. A n o t h e r major objective of research i n this area is to verify the s t u d i e s carried o u t on a n i m a l s w i t h those from h u m a n b r a i n tissue: reports o n the effects of d r u g s o n the release of n e u r o t r a n s m i t t e r s from b o t h fresh 1° a n d p o s t - m o r t e m 11 h u m a n tissues are b e g i n n i n g to appear. Such s t u d i e s will b e especially relevant to the t e r m i n a l 5-HT autoreceptor w h i c h , i n a s e m i n a l s t u d y on the rat cortex, h a s b e e n s h o w n to c o r r e s p o n d to the 5-HTzB b i n d i n g site TM, since this recogn i t i o n site is a p p a r e n t l y a b s e n t from the h u m a n cortex 13. The u s e of n e u r o t r a n s m i t t e r release studies, in c o n j u n c t i o n w i t h receptor b i n d i n g t e c h n i q u e s , p r o v i d e s a powerful tool for the identification of d r u g s w i t h effects at CNS receptors. D. N. MIDDLEMISS
Merck Sharp and Dohme Research Laboratories, Neuroscience Research Centre, Terlings Park, Eastwick Road, Harlow, Essex CM20 2QR, UK.
References 1 Kemp, J. A., Foster, A. C., Gill, R. and Woodruff, G. N. (1987) Trends Pharmacol. Sci. 8, 414-415 2 Traber, J. and Glaser, T. (1987) Trends Pharmacol. $ci. 8, 432-437 3 Korpi, E. R. and Oja, S. S. (1984) J. Neurochom. 43, 236-242 4 Minnenia, D. and Michaelson, I. A. (1985) J. Neurosci. Methods 14, 193-206 5 Chesselet, M-F. (1984) Neuroscience 12, 347-375 6 Gothert, M. (1980) Naunyn-Schmied. Arch. Pharmacol. 314, 223-230 7 Jones, S. M., Snell, L. D. and Johnson, K. M. (1987) J. Pharmacol. Exp. Thor. 240, 492--497 8 Raiteri, M., Marchi, M. and Maura, G. (1983) Eur. J. Pharmacol. 91, 141-143 9 Maura, G., Bonanno, G. and Raiteri, M. (1985) Eur. J. Pharmacol. 112, 105--110 10 Schlicker, E., Brandt, F., Classen, K. and Gothert, M. (1985) Brain Res. 331, 337341 11 Hardy, J. A. and Dodd, P. R. (1985) in Selected Topics from Neurochemistry (Osborne, N. N., ed.), pp. 25-41, Pergamon Press 12 Engel, G., Gothe~o M~ Hoyer~ D~ 5ch!icker, E. and Hillenbrand, K. (1986) Naunyn-Schmied. Arch. PharmacoL 332, 1-7 13 Hoyer, D., Pazos, A., Probst, A. and Palacios, J. M. (1986) Brain Res. 376, 85-96 14 Bonanno, G. and Raiteri, M. (1987) Synapse 1, 254-257
MK.S01: (+)-5-Methyl-10,11-dihydro-SHdibenzo[a,d]cyclohepten-5,10-imine maleate Ipsapirone: 2-(4-[4-(2-pyrimidinyl)-l-pip-
erazinyl]butyl)l,2-benzisothiazol-3-(2H)one-l,l-dioxidehydrochloride
Pharmacologist to Pharmacologist • I am in urgent need of the following chemicals for my research work: N-methyl-D-aspartate(NIVlDA) c~-amino-3-hydroxy.5-methyl-4isoxazolepropionate (~ P A )
3-3(2-carbo.xypiperazine-4.y|)-
propyl- l-phosphomtto (CPP) phencyclidine (PCP) ~2-amino-4-phosphonobutyrate
(I,-AP4)
I would appreciate any gift: Dr Gang K. Tong, Department of Physiology. Second Mih'tary Medical College, Shanghai 2OO433, Paopl~'~ Republic of t ~ . ~
will print sn~£ actsof ~terest to pbaz~cob~mJ free of e.,~e. Contact: P k m m a ~ o g ~ P/mrmaco/og~ TIPS, 68 Hills Road, Cambridge CB8 ILA, UE.