Neuroscience Letters, 116 (1990) 347 351 Elsevier Scientific Publishers Ireland Ltd.
347
NSL 07088
Muscarinic receptors mediate direct inhibition of G A B A release from rat striatal nerve terminals M a r i o M a r c h i , Paola Sanguineti a n d M a u r i z i o Raiteri Z~tituto di Farmacologia e Farmacognosia, Universitgt degli Studi di Genova, Genova (Italy) (Received 22 September 1989; Revised version received 17 April 1990; Accepted 2 May 1990)
Key word~. GABA release; Acetylcholine; Muscarinic receptors; Rat corpus striatum; Superfused synaptosomes The effects of acetylcholine (ACh) on the depolarization-evoked release of [3H]?,-aminobutyric acid ([3H]GABA) have been investigated using synaptosomes prepared from rat corpus striatum and depolarized by superfusion with 9 mM KCI. Acetylcholine inhibited the [3H]GABA overflow in a concentrationdependent manner. The maximal effect was about 50%. The IC50 value (concentration producing half-maximal effect) amounted to 1 pM, in the absence of acetylcholinesterase inhibitors. The effect of ACh on the K +-evoked [3H]GABA release was counteracted by the muscarinic receptor antagonist atropine, but not by the nicotinic receptor antagonist mecamylamine or by the selective Mt antagonist pirenzepine. The data show that muscarinic receptors with low affinity for pirenzepine are localized on GABAergic nerve endings in rat corpus striatum where they may directly inhibit the release of GABA,
The corpus striatum contains high concentrations of acetylcholine (ACh) [4], which is essentially present in intrinsic neurons [3, 17]. 7-Aminobutyric acid (GABA), a major striatal transmitter [5, 18, 19], is concentrated both in interneurons [2] and in terminals originating from the recurrent collaterals of efferent neurons [6, 13]. Cholinergic and GABAergic neurons have been reported to interact reciprocally in the corpus striatum. However, no clear evidence has been provided for direct interaction between the two systems. In fact, it was shown that GABA indirectly inhibits the activity of cholinergic interncurons via the nigrostriatal dopaminergic system [12, 15]. Effects of GABA on ACh release [1, 29] or of ACh on GABA release [7, 16] have been observed. However, as discussed by these authors, the complexity of the experimental systems used does not allow to rule out the involvement of indirect intrastriatal interactions. Using an experimental approach different from those employed previously we show here that striatal GABAergic nerve terminals possess cholinergic receptors through which ACh may directly affect the release of GABA. All experiments were carried out using synaptosomes obtained from different brain
Correspondence." M. Raiteri, Ist. Farmacologia e Farmacognosia, Viale Cembrano 4, 16148 Genova, Italy. 0304-3940/90/$ 03.50 ,~'>,1990 Elsevier Scientific Publishers Ireland Ltd.
348
areas and prepared essentially according to Gray and Whittaker [8]. Briefly, male Sprague-Dawley rats (200-250 g) were killed by decapitation. The tissues were homogenized (1/40; w/v) in 0.32 M sucrose buffered at pH 7.4 with phosphate. The homogenate was first centrifuged 5 rain at 1000 g and a crude synaptosomal fraction was then isolated from the supernatant by centrifugation at 12,000 g for 20 min. The pellet was resuspended in a physiological solution having the following composition (mM): NaC! 125, KC1 3, MgSO4 1.2, CaCI2 1.2, NaH2PO4 1.0, NaHCO3 22, glucose 10 (aeration with 95% 0 2 and 5% CO2 at 37°C); pH 7.2 to 7.4. The synaptosomes were incubated for 15 min at 37°C in an atmosphere of 95% 02 and 5% CO2 with [3H]GABA (final concentration 0.04/~M). Aminooxyacetic acid (50 ¢tM) was present throughout the experiment to prevent [3H]GABA catabolism. The synaptosomal protein content was determined according to Petersen [20]. After incubation with [3H]GABA, identical aliquots of the synaptosomal suspension were distributed on Millipore filters placed at the bottom of parallel superfusion chambers [22]. Superfusion was then started at a rate of 0.6 ml/min with standard medium aerated with 02 and CO2. After 36 min for equilibration of the system, fractions were collected according to the following scheme: two 3-min samples (basal outflow) before and after one 6-rain sample (evoked overflow). A 90 s period of depolarization (9 mM KC1) was applied at the end of the first fraction collected. Agonists were added concomitantly with high-K + and antagonists 8 rain before depolarization. The fractions collected and the superfused filters were then counted for radioactivity. The amount of radioactivity released into each fraction was expressed as a percentage of the total synaptosomal tritium present on each filter at the start of the respective collection period. The K ÷-evoked overflow was estimated by subtracting from the total the basal outflow. Acetylcholine chloride, atropine sulphate and mecamylamine were purchased from Sigma (St. Louis, MO, U.S.A.). [3H]GABA (spec. act. 51-59 Ci/mmol) was obtained from Amersham Radiochemical Centre (U.K.). As mentioned above, tritium radioactivity was measured during release. However, the presence of aminooxyacetic acid at 50 ¢tM [21], the characteristics of the superfusion technique employed [22] and data obtained previously [16, 26] allow the assumption that at least 90% of the evoked overflow of tritium was accounted for by authentic [3H]GABA. Therefore, in the remainder of the text we refer to the K+-evoked tritium overflow as K +-evoked [3H]GABA release. When added to the superfusion fluid in the presence of 9 mM KCI exogenous ACh inhibited the depolarization-evoked overflow of [3H]GABA in a concentration-dependent manner (Fig. 1). The maximal effect of ACh was about 50%. The ICs0 (agonist concentration producing 50% of the maximal effect) amounted to 1/aM. The inhibitory effect by ACh on [3H]GABA release was much less pronounced in the hippocampus or in the cortex. It should be noted that the experiments were performed in the absence of acetylcholinesterase inhibitors. Fig. 2 illustrates the effects of cholinergic antagonists on the inhibition of [3H]GABA release by ACh. Atropine (0.01 /~M) completely antagonized the effect of 10/tM ACh. In contrast, the effect of ACh was insensitive to 10/tM mecamylamine or to 10 ~zM pirenzepine.
349
lOO
90
80 8
70 60
50
oi~
i
m
~oo
ACh CONCENTRATION [pM]
Fig. I. Effect of A C h on [3H]GABA release from rat hippocampal (A), cortical (11), or striatal (@) synaptosomes. Results are expressed as percentage of inhibition of the K ÷-evoked release. Each point presented is the average + S.E.M. of at least 6 experiments run in triplicate.
=_=
6O
LS
.E
50
T
T
_Y_
"6
40 3O 2O _,_
I0
10
10
10
-
ACETYLCHOUNE
001
-
-
ATROPINE [ p M ]
-
-
10
-
MECAMYLAMINE
-
-
-
1
PIRENZEPINE
-
[pM] [pM]
[pM]
Fig. 2. Antagonism of the effect of A C h on [3H]GABA release from rat corpus striatum synaptosomes. Results are expressed as percentage of inhibition of the K ÷-evoked release. Each point presented is the average ( + S.E.M.) of at least 6 experiments run in triplicate.
Very few studies have examined the effects of ACh on the release o f G A B A in the corpus striatum. The spontaneous release of [3H]GABA was found to be enhanced by A C h in the rat striatum superfused in vivo with a push-pull cannula. However, attempts to identify the possible receptor involved were not made. N o r was the effect o f ACh on the depolarization-evoked release of G A B A examined [7]. Enhancement
350 by ACh of the basal [3H]GABA outflow was also observed in vitro using slices of rabbit caudate nucleus. The effect was reported to be mediated by nicotinic receptors [16]. According to K u r i y a m a et al. [t4] the K+-evoked release of [3H]GABA from striatal slices was inhibited by pilocarpine, suggesting the involvement of a muscarinic receptor. However, Limberger et al. [16] found muscarinic agonists to be ineffective towards the evoked [3H]GABA overflow. In the present work the possible cholinergic modulation of G A B A release was studied using synaptosomes prepared from rat corpus striatum and depolarized in superfusion with 9 m M KC1. Synaptosomes superfused in a thin layer allow the use of the natural transmitter ACh as an agonist in the absence of acetylcholinesterase inhibitors [23, 25]. Secondly, this experimental set-up eliminates the neuronal loops which may mediate indirect effects in more complex systems. As to the depolarizing stimulus employed, the release of some transmitters evoked by concentrations of K + lower than 15 m M was reported to be tetrodotoxin-sensitive [28]; thus depolarization with 9 m M KCI may be assumed to be a 'quasi-physiological' stimulus. Taken together the above considerations and the results of Fig. 1 allow us to draw a first conclusion: in the rat corpus striatum the natural transmitter ACh may reduce the depolarization-evoked release of G A B A by a direct action on the releasing nerve terminals. The inhibitory effect of ACh on the K ÷-induced [3H]GABA overflow was blocked by the muscarinic receptor antagonist atropine but was insensitive to the nicotinic receptor antagonist mecamylamine or to the selective Mt muscarinic antagonist pirenzepine [9, 11, 24]. Thus the second conclusion of this work is that the inhibitory action of ACh on G A B A release is mediated by muscarinic receptors of the pirenzepine-insensitive subtype. Whether cholinergic nerve endings form axo-axonic synapses with GABAergic terminals or ACh modulates G A B A release through a parasynaptic mechanism implying an action at distance [10, 27] remains to be determined. It is also unknown if all striatai GABAergic terminals, independently of their origin, are endowed with muscarinic receptors. Experiments with striata lesioned with excitotoxins may provide an answer. This work was supported by grants from the Italian Ministry of Education and from the Italian C.N.R. 1 Bianchi, C., Tanganelli, S., Marzola, G. and Beani, L., GABA induced changes in acetylcholinerelease from slices of guinea-pig brain, Naunyn-Schmiedeberg'sArch. Pharmacol., 318 (1982) 253-258. 2 Bolam,J.P., Clarke, D.J., Smith, A.D. and Somogyi,P., A type of aspiny neuron in the rat neostriatum accumulates [3H]gamma-aminobutyricacid: combination of Golgi-staining, autoradiography and electron microscopy,J. Comp. Neurol., 213 (1983) 121 134. 3 Butcher, S.G. and Butcher, L.L., Origin and modulation of acetylcholine activity in the neostriatum, Brain Res., 71 (1974) 167-171. 4 Cheney, D.L., LeFevre, H.F. and Racagni, G., Choline acetyltransferaseactivity and mass fragmentographic measurement of acetylcholine in specificnuclei and tracts of rat brain, Neuropharmacology, 14 (1975) 801-809. 5 Coyle, J.T. and Schwarcz, R., Lesion of striatal neurons with kainic acid provides a model for Huntington's chorea, Nature, 263 (1976) 244-246. 6 Fonnum, F., Gottesfeld, Z. and Grofova, I., Distribution of glutamate decarboxylase,choline acetyl transferase and aromatic decarboxvlase in the basal ,,an21ia of normal and onerated rat~ ~vid~noo
351 for striato-pallidal, striato-entopeduncular and striato-nigral GABAergic fiber, Brain Res., 143 (1974) 125 138. 7 Girault, J.A., Spampinato, U., Savaki, H.E., Glowinski, J. and Besson, M.J., In vivo release of [3H]7aminobutyric acid in the rat neostriatum. I. Characterization and topographical heterogeneity of the effects of dopaminergic and cholinergic agents, Neuroscience, 19 (1986) 1101-1108. 8 Gray, E.G. and Whittaker, V.P., The isolation of nerve endings from brain: an electron microscopic study of cell fragments derived by homogenization and centrifugation, J. Anat., 96 (1962) 79 87. 9 Hammer, R., Berrie, C.P,, Birdsall, N.J.M., Burgen, A.S.V. and Hulme, E.C., Pirenzepine distinguishes between different subclasses of muscarinic receptors, Nature (Lond.), 283 (1980) 90~ 92. 10 Herkenham, M., Mismatches between neurotransmitter and receptor localizations in brain: observations and implications, Neuroscience, 23 (1987) 1-38. 11 Hirschowitz, B.I., Hammer, R., Giachetti, A., Keirns, J.J. and Levine, R.R., Subtypes of Muscarinic Receptors, Elsevier, Amsterdam, 1984, 103 pp. 12 Javoy, F., Euvrard, C., Herbet, A. and Glowinski, J., Involvement of the dopamine nigrostriatal system in the picrotoxin effect on striatal acetylcholine levels, Brain Res., 126 (1977) 382 386. 13 Jessell, T.M., Emson, P.C., Paxinos, G. and Cuello, A.C., Topographic projections of substance P and GABA pathways in the striato- and pallido-nigral system: a biochemical and immunohistochemical study, Brain Res., 152 (1978) 487-498. 14 Kuriyama, K., Kanmori, K., Taguchi, J. and Yoneda, Y., Stress-induced enhancement of suppression of [3H]GABA release from striatal slices by presynaptic autoreceptor, J. Neurochem., 42 (1984) 943 950. 15 Ladinsky, K., Consolo, S., Bianchi, S. and Jori, A., Increase in striatal acetylcholine by picrotoxin in the rat: evidence for a GABAergic-dopaminergic-cholinergic link, Brain Res., 108 (1976) 351 361. 16 Limberger, N., Sp~ith, L. and Starke, K., A search for receptors modulating the release of 7-[3H]amino butyric acid in rabbit caudate nucleus slices, J. Neurochem., 46 (1986) 1109 1117. 17 McGeer, P.L., McGeer, E.G., Fibiger, H.C. and Wickson, V., Neostriatal choline acetylase and cholinesterase following selective brain lesions, Brain Res., 35 ( 1971 ) 308 314. 18 McGeer, E.G. and McGeer, P.L., Duplication of biochemical changes of Huntington's chorea by intrastriatal injections of glutamic and kainic acid, Nature, 263 (1976) 517 519. 19 Ottersen, O.P. and Storm-Mathisen, J., Glutamate- and GABA-containing neurons in the mouse and rat brain, as demonstrated with a new immunocytochemical technique, J. Comp. Neurol., 229 (1984) 374 392. 20 Petersen, G.L., A simplification of the protein assay method of Lowry et al. which is more generally applicable, Anal. Biochem., 83 (1977) 346 356. 21 Pittaluga, A., Asaro, D., Pellegrini, G. and Raiteri, M., Studies on [3H]GABA and endogenous GABA release in rat cerebral cortex suggest the presence of autoreceptors of the GABAB type, Eur. J. Pharmacol., 144(1987)45 52. 22 Raiteri, M., Angelini, F. and Levi, G., A simple apparatus for studying the release of neurotransmitters from synaptosomes, Eur. J. Pharmacol., 25 (1974) 411-414. 23 Raiteri, M., Marchi, M. and Maura, G., Release ofcatecholamines, serotonin, and acetylcholine from isolated brain tissues. In A. Lajtha (Ed.), Handbook of Neurochemistry, Plenum, New York, 1984, pp. 431-462. 24 Raiteri, M., Leardi, R. and Marchi, M., Heterogeneity of presynaptic muscarinic receptors regulating neurotransmitter release in the rat brain, J. Pharmacol. Exp. Ther., 228 (1984) 209 214. 25 Raiteri, M., Maura, G., Bonanno, G. and Pittaluga, A., Differential pharmacology and function of two 5-HT~ receptors modulating transmitter release in rat cerebellum, J. Pharmacol. Exp. Ther., 237 (1986) 644-649. 26 Raiteri, M., Pellegrini, G., Cantoni, C. and Bonanno, G., A novel type of GABA receptor in rat spinal cord?, Naunyn-Schmiedeberg's Arch. Pharmacol., 340 (1989) 6664570. 27 Schmitt, F.O., Molecular regulators of brain function: a new view, Neuroscience, 13 (1984) 991 1001. 28 Schoffelmeer, A.N.M., Wemer, J. and Mulder, A.H., Comparison between electrically evoked and potassium induced 3H-noradrenaline release from rat neocortex slices: role of calcium ions and transmitter pools, Neurochem. Int., 3 (1981) 129 136. 29 Stool J.C., Den Breejen, E.J.S. and Mulder, A.H., GABA modulates the release of dopamine and acetylcholine from rat caudate nucleus slices, Eur. J. Pharmacol., 57 (1979) 35--42.
347
NSL 07088
Muscarinic receptors mediate direct inhibition of G A B A release from rat striatal nerve terminals M a r i o M a r c h i , Paola Sanguineti a n d M a u r i z i o Raiteri Z~tituto di Farmacologia e Farmacognosia, Universitgt degli Studi di Genova, Genova (Italy) (Received 22 September 1989; Revised version received 17 April 1990; Accepted 2 May 1990)
Key word~. GABA release; Acetylcholine; Muscarinic receptors; Rat corpus striatum; Superfused synaptosomes The effects of acetylcholine (ACh) on the depolarization-evoked release of [3H]?,-aminobutyric acid ([3H]GABA) have been investigated using synaptosomes prepared from rat corpus striatum and depolarized by superfusion with 9 mM KCI. Acetylcholine inhibited the [3H]GABA overflow in a concentrationdependent manner. The maximal effect was about 50%. The IC50 value (concentration producing half-maximal effect) amounted to 1 pM, in the absence of acetylcholinesterase inhibitors. The effect of ACh on the K +-evoked [3H]GABA release was counteracted by the muscarinic receptor antagonist atropine, but not by the nicotinic receptor antagonist mecamylamine or by the selective Mt antagonist pirenzepine. The data show that muscarinic receptors with low affinity for pirenzepine are localized on GABAergic nerve endings in rat corpus striatum where they may directly inhibit the release of GABA,
The corpus striatum contains high concentrations of acetylcholine (ACh) [4], which is essentially present in intrinsic neurons [3, 17]. 7-Aminobutyric acid (GABA), a major striatal transmitter [5, 18, 19], is concentrated both in interneurons [2] and in terminals originating from the recurrent collaterals of efferent neurons [6, 13]. Cholinergic and GABAergic neurons have been reported to interact reciprocally in the corpus striatum. However, no clear evidence has been provided for direct interaction between the two systems. In fact, it was shown that GABA indirectly inhibits the activity of cholinergic interncurons via the nigrostriatal dopaminergic system [12, 15]. Effects of GABA on ACh release [1, 29] or of ACh on GABA release [7, 16] have been observed. However, as discussed by these authors, the complexity of the experimental systems used does not allow to rule out the involvement of indirect intrastriatal interactions. Using an experimental approach different from those employed previously we show here that striatal GABAergic nerve terminals possess cholinergic receptors through which ACh may directly affect the release of GABA. All experiments were carried out using synaptosomes obtained from different brain
Correspondence." M. Raiteri, Ist. Farmacologia e Farmacognosia, Viale Cembrano 4, 16148 Genova, Italy. 0304-3940/90/$ 03.50 ,~'>,1990 Elsevier Scientific Publishers Ireland Ltd.
348
areas and prepared essentially according to Gray and Whittaker [8]. Briefly, male Sprague-Dawley rats (200-250 g) were killed by decapitation. The tissues were homogenized (1/40; w/v) in 0.32 M sucrose buffered at pH 7.4 with phosphate. The homogenate was first centrifuged 5 rain at 1000 g and a crude synaptosomal fraction was then isolated from the supernatant by centrifugation at 12,000 g for 20 min. The pellet was resuspended in a physiological solution having the following composition (mM): NaC! 125, KC1 3, MgSO4 1.2, CaCI2 1.2, NaH2PO4 1.0, NaHCO3 22, glucose 10 (aeration with 95% 0 2 and 5% CO2 at 37°C); pH 7.2 to 7.4. The synaptosomes were incubated for 15 min at 37°C in an atmosphere of 95% 02 and 5% CO2 with [3H]GABA (final concentration 0.04/~M). Aminooxyacetic acid (50 ¢tM) was present throughout the experiment to prevent [3H]GABA catabolism. The synaptosomal protein content was determined according to Petersen [20]. After incubation with [3H]GABA, identical aliquots of the synaptosomal suspension were distributed on Millipore filters placed at the bottom of parallel superfusion chambers [22]. Superfusion was then started at a rate of 0.6 ml/min with standard medium aerated with 02 and CO2. After 36 min for equilibration of the system, fractions were collected according to the following scheme: two 3-min samples (basal outflow) before and after one 6-rain sample (evoked overflow). A 90 s period of depolarization (9 mM KC1) was applied at the end of the first fraction collected. Agonists were added concomitantly with high-K + and antagonists 8 rain before depolarization. The fractions collected and the superfused filters were then counted for radioactivity. The amount of radioactivity released into each fraction was expressed as a percentage of the total synaptosomal tritium present on each filter at the start of the respective collection period. The K ÷-evoked overflow was estimated by subtracting from the total the basal outflow. Acetylcholine chloride, atropine sulphate and mecamylamine were purchased from Sigma (St. Louis, MO, U.S.A.). [3H]GABA (spec. act. 51-59 Ci/mmol) was obtained from Amersham Radiochemical Centre (U.K.). As mentioned above, tritium radioactivity was measured during release. However, the presence of aminooxyacetic acid at 50 ¢tM [21], the characteristics of the superfusion technique employed [22] and data obtained previously [16, 26] allow the assumption that at least 90% of the evoked overflow of tritium was accounted for by authentic [3H]GABA. Therefore, in the remainder of the text we refer to the K+-evoked tritium overflow as K +-evoked [3H]GABA release. When added to the superfusion fluid in the presence of 9 mM KCI exogenous ACh inhibited the depolarization-evoked overflow of [3H]GABA in a concentration-dependent manner (Fig. 1). The maximal effect of ACh was about 50%. The ICs0 (agonist concentration producing 50% of the maximal effect) amounted to 1/aM. The inhibitory effect by ACh on [3H]GABA release was much less pronounced in the hippocampus or in the cortex. It should be noted that the experiments were performed in the absence of acetylcholinesterase inhibitors. Fig. 2 illustrates the effects of cholinergic antagonists on the inhibition of [3H]GABA release by ACh. Atropine (0.01 /~M) completely antagonized the effect of 10/tM ACh. In contrast, the effect of ACh was insensitive to 10/tM mecamylamine or to 10 ~zM pirenzepine.
349
lOO
90
80 8
70 60
50
oi~
i
m
~oo
ACh CONCENTRATION [pM]
Fig. I. Effect of A C h on [3H]GABA release from rat hippocampal (A), cortical (11), or striatal (@) synaptosomes. Results are expressed as percentage of inhibition of the K ÷-evoked release. Each point presented is the average + S.E.M. of at least 6 experiments run in triplicate.
=_=
6O
LS
.E
50
T
T
_Y_
"6
40 3O 2O _,_
I0
10
10
10
-
ACETYLCHOUNE
001
-
-
ATROPINE [ p M ]
-
-
10
-
MECAMYLAMINE
-
-
-
1
PIRENZEPINE
-
[pM] [pM]
[pM]
Fig. 2. Antagonism of the effect of A C h on [3H]GABA release from rat corpus striatum synaptosomes. Results are expressed as percentage of inhibition of the K ÷-evoked release. Each point presented is the average ( + S.E.M.) of at least 6 experiments run in triplicate.
Very few studies have examined the effects of ACh on the release o f G A B A in the corpus striatum. The spontaneous release of [3H]GABA was found to be enhanced by A C h in the rat striatum superfused in vivo with a push-pull cannula. However, attempts to identify the possible receptor involved were not made. N o r was the effect o f ACh on the depolarization-evoked release of G A B A examined [7]. Enhancement
350 by ACh of the basal [3H]GABA outflow was also observed in vitro using slices of rabbit caudate nucleus. The effect was reported to be mediated by nicotinic receptors [16]. According to K u r i y a m a et al. [t4] the K+-evoked release of [3H]GABA from striatal slices was inhibited by pilocarpine, suggesting the involvement of a muscarinic receptor. However, Limberger et al. [16] found muscarinic agonists to be ineffective towards the evoked [3H]GABA overflow. In the present work the possible cholinergic modulation of G A B A release was studied using synaptosomes prepared from rat corpus striatum and depolarized in superfusion with 9 m M KC1. Synaptosomes superfused in a thin layer allow the use of the natural transmitter ACh as an agonist in the absence of acetylcholinesterase inhibitors [23, 25]. Secondly, this experimental set-up eliminates the neuronal loops which may mediate indirect effects in more complex systems. As to the depolarizing stimulus employed, the release of some transmitters evoked by concentrations of K + lower than 15 m M was reported to be tetrodotoxin-sensitive [28]; thus depolarization with 9 m M KCI may be assumed to be a 'quasi-physiological' stimulus. Taken together the above considerations and the results of Fig. 1 allow us to draw a first conclusion: in the rat corpus striatum the natural transmitter ACh may reduce the depolarization-evoked release of G A B A by a direct action on the releasing nerve terminals. The inhibitory effect of ACh on the K ÷-induced [3H]GABA overflow was blocked by the muscarinic receptor antagonist atropine but was insensitive to the nicotinic receptor antagonist mecamylamine or to the selective Mt muscarinic antagonist pirenzepine [9, 11, 24]. Thus the second conclusion of this work is that the inhibitory action of ACh on G A B A release is mediated by muscarinic receptors of the pirenzepine-insensitive subtype. Whether cholinergic nerve endings form axo-axonic synapses with GABAergic terminals or ACh modulates G A B A release through a parasynaptic mechanism implying an action at distance [10, 27] remains to be determined. It is also unknown if all striatai GABAergic terminals, independently of their origin, are endowed with muscarinic receptors. Experiments with striata lesioned with excitotoxins may provide an answer. This work was supported by grants from the Italian Ministry of Education and from the Italian C.N.R. 1 Bianchi, C., Tanganelli, S., Marzola, G. and Beani, L., GABA induced changes in acetylcholinerelease from slices of guinea-pig brain, Naunyn-Schmiedeberg'sArch. Pharmacol., 318 (1982) 253-258. 2 Bolam,J.P., Clarke, D.J., Smith, A.D. and Somogyi,P., A type of aspiny neuron in the rat neostriatum accumulates [3H]gamma-aminobutyricacid: combination of Golgi-staining, autoradiography and electron microscopy,J. Comp. Neurol., 213 (1983) 121 134. 3 Butcher, S.G. and Butcher, L.L., Origin and modulation of acetylcholine activity in the neostriatum, Brain Res., 71 (1974) 167-171. 4 Cheney, D.L., LeFevre, H.F. and Racagni, G., Choline acetyltransferaseactivity and mass fragmentographic measurement of acetylcholine in specificnuclei and tracts of rat brain, Neuropharmacology, 14 (1975) 801-809. 5 Coyle, J.T. and Schwarcz, R., Lesion of striatal neurons with kainic acid provides a model for Huntington's chorea, Nature, 263 (1976) 244-246. 6 Fonnum, F., Gottesfeld, Z. and Grofova, I., Distribution of glutamate decarboxylase,choline acetyl transferase and aromatic decarboxvlase in the basal ,,an21ia of normal and onerated rat~ ~vid~noo
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