Neurochem. Int. Vol. 20, Suppl., pp. 201S-205S, 1992 0197-0186/92 $5.00+0.00 Printed in Great Britain Pergamon Press plc

IN VITRO A N D IN VIVO ACETYLCHOLINE RELEASE FROM RAT STRIATUM AS A FUNCTIONAL PARADIGM OF SIGNAL

TRANSDUCTION VIA A D-2 DOPAMINE RECEPTOR

J. C. STOOF, I B. DRUKARCH, l P. DE BOER 1 and B. H. C. WESTERINK ~

~Department of Neurology, Medical Faculty, Free University, van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands

2Centre for Pharmacy, University of Groningen, Antonius Deusinglaan 2, 9713 AW Groningen, The Netherlands

In the 1970's, the concept of a delicate balance between the dopaminergic and the cholinergic "systems" in the striatum was mainly based on the following major findings:

1. It had been demonstrated that the content of acetylcholine (ACh) in the striatum could be increased by dopamine (DA) receptor agonists and decreased by DA receptor antagonists. Drugs like apomorphine and L-DOPA were able to inhibit the in vivo release of ACh from the striatum; this effect was blocked by DA receptor antagonists (Stadler et al., 1973; Sethy and van Woert, 1974; Guyenet et al., 1975). Data from these in vivo experiments were interpreted in the following way: stimulation of DA receptors, probably located on the cholinergic neurons in the striatum, blocks the release of ACh and thereby increases the content of ACh.

2. The major disturbance in the brains of patients with Parkinson's disease is a degeneration of the nigro- striatal dopaminergic neurons. The loss of striatal DA in these patients disrupts the balance between the striatal dopaminergic and eholinergic systems. This disrupted balance was thought to form an explanation for the therapeutic effects of anticholinergic drugs which were widely used prior to the advent of L- DOPA to alleviate the symptoms of Parkinsonism (Calne, 1978). It was even suggested that some of the therapeutic effects of L-DOPA (after conversion to DA) were achieved by stimulating the DA receptors presumably located on the cholinergic neurons and thereby restoring the balance between the dopa- minergic and cholinergic systems in the striatum.

The aims of the present paper are to give a brief overview of the most important recent data that lend further support to the concept of a functional DA/ ACh interaction in the mammalian striatum and to underline the usefulness of striatal ACb release as a functional paradigm of signal transduction via a D-2 receptor.

DA]ACh INTERACTION

Neuroanatomical aspects o f the DA/ACh interaction.

The mammalian striatum can be divided in the ventral striatum which comprises the nucleus accumbens and the tuberculum olfactorium and the dorsal striatum consisting of the caudate-putamen complex. Likewise, the mesostriatal dopaminergic pathways have a ventral and a dorsal component. The ventral striatum receives most of its doparninergic input from neurons in the ventral tegmentat area (At0), whereas the dorsal striatum is primarily innervated by the nigro-striatal pathway, which originates in the substantia nigra pars compacta (A~) and the retro- rubral (As) cell group (Bjorklund and LindvaU, 1984).

Cholinergic neurons have been identified in the dorsal as well as the ventral striatum (Phelps et al., 1985). The cholinergic elements, only accounting for a small percentage (1-2 %) of the total striatal neuron population, are thought to be intrinsic neurons. On the basis of the effects of DA and dopaminergic drugs on striatal cholinergic neurotransmission it has been assumed that dopaminergic nerve terminals are in synaptic contact with their cholinergic target neurons. However, the cholinergic intrinsic neurons correspond to the morphological category of "large aspiny cells" originally identified by Kemp and Powell (Lehmann and Langer, 1983). "Aspiny" refers to the fact that these cells possess smooth dendrites, which means that dendritic spines are either very rare or entirely absent. Dendritic spines are considered to be specializations that receive the majority of synaptic contacts from axon terminals. Up till now only scarce evidence has been provi~led for the occurrence of synaptic contacts between dgpaminergic terminals and the dendrites or perikarya [of cholinergic intrinsic neurons in the striatum (Kubota et al., 1987; Chang, 1988). This is in contrast to the situation for the other category of neurons occurring in the striatum the so called

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~'medium-sized spiny neurons", which clearly has been shown to form synapses with dopaminergic terminals. Moreover, axo-axonic synapses are assumed not to occur in the striatum. Therefore, these findings have led to the hypothesis that signal transduction between dopaminergic terminals and cholinergic intrinsic neu- rons in the striatum is o fa non-synaptic nature (Stool', 1989). Upon depolarization, a dopaminergic nerve terminal releases DA which would then diffuse over a short distance to stimulate DA receptors located on a cholinergic neuron. However, this hypothesis remains to be proven. In addition, it is of importance to note that, up till now, only indirect evidence has been provided for the localization of DA receptors on striatal cholinergic neurons (Dawson et al., 1988).

Biochemica l aspects o f the D A / A Ch interact ion

A. In vitro studies. Results obtained from in vitro superfusion studies strongly support the concept of a functional DA/ACh interaction in the mammalian striatum. In these studies striatal slices are incubated with radiolabeled choline, a precursor of ACh, in order to label the pool of newly synthesized ACh. The tissue is superfused and the calcium- dependent release of radiolabeled ACh can be evoked electrically or with high K*-concentrations. The superfusion technique is a simple procedure for investigating the release of transmitters or hormones and its regulation by com- pounds acting on membrane receptors.

Apomorphine and other DA receptor agonists like bromocriptine inhibit in a concentration-dependent way the evoked release of radiolabeled ACh from striatal tissue. DA receptor antagonists like halo- peridol oppose the inhibitory effects of DA receptor agonists on the evoked release of radiolabeled ACh (Stoofet al., 1979; Hertting et al., 1980). By using this in vitro method it was possible to delineate the pharmacological characteristics of the dopamine re- ceptor involved. IC-50 - and Ki-values of many drugs could easily be calculated. Since the early 1980's it is known that the DA receptor mediating the inhibition of the release of ACh is a D-2 receptor (Helmreich et al., 1982; Stoof and Kebabian, 1982), whereas acti- vation of D- l receptors does not inhibit the release of ACh.

Interestingly, it has also been found that DA is able to inhibit the formation of cyclic AMP in the striatum via D-2 receptors. Thus, selective D-2 receptor ag- onists reduce the cyclic AMP formation induced by selective D-1 receptor agonists or by forskolin. In addition, selective D-2 receptor antagonists potentiate the dopamine (which acts simultaneously on D-I and D-2 receptors) induced cyclic AMP formation (Stoof

and Kebabian, 1984; Onali e t a / . , 1985). A relevant question of course is to find out if the D-2 receptor involved in the regulation of the release of ACh is coupled to adenylate cyclase in an inhibitory way. Surprisingly, we found that cyclic AMP analogs or drugs increasing intracellular cyclic AMP levels are unable to antagonize the inhibition of the release of ACh, brought about by D-2 receptor agonists (Stool" and Kebabian, 1982). These and similar experiments indicated that the D-2 receptor mediated control of striatal ACh release is independent of the activity of the adenylate cyclase system. In recent years much effort has been put in the discovery of other (than adenylate cyclase mediated) signal transduction mechanisms for D-2 receptors. Attention has mainly been focussed on the possibility of a coupling of the D- 2 receptor to phosphoinositide metabolism and/or ion (K ~ and Ca >) channel activity. Reportedly, stim- ulation of the D-2 receptor attenuates phospho- inositide hydrolysis in anterior pituitary gland and striatal tissue (Enjalbert et al., 1986; Pizzi et al., 1987). However, these findings have been refuted by other studies (Kelly et al., 1988; Rubinstein and Hitzemann, 1990). More consistently, the D-2 receptor has been shown to be involved in the regulation of K ÷ channel function. Electrophysiological data indicate that stimulation of the D-2 receptor leads to opening of K " channels resulting in a hyperpolarization of the cell membrane (Israel et al., 1987; Lacey el ai.. 1987). Interestingly, besides in pituitary lactotrophs and substantia nigra zona compacta neurons, this effect of D-2 receptor activation has also been reported to occur in acutely dissociated, but neurochemicalty unidentified, striatal neurons (Freedman and Weight, 1988). Recently, we have collected data indicating that activation of the D-2 receptor inhibits the evoked release of radiolabeled ACh through stimulation of K ÷ efflux i.e. opening o fK ÷ channels (Drukarch et al., 1989).

Somewhat less attention has been paid to the possibility of a direct inhibitory coupling of the D-2 receptor to Ca 2+ influx through closure of Ca 2~ channels and to mobilization of Ca 2" from intra- cellular stores. Nevertheless, studies have been pub- lished suggesting such a role for Ca 2" in D-2 receptor mediated effects (Memo et al., 1986; Fujiwara et al., 1987).

B. In vivo s tudies (rnierodialysis) . Although it is beyond any doubt that DA is able to inhibit the evoked release of ACh in vitro by stimulating D-2 receptors, only the use o f in vivo techniques, e.g. the microdialysis technique, can stress the physiological relevance of the interaction between the dopaminergic projections and

Dopamine 90 203S

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Fig. 1. The selective D-2 receptor agonist ( - ) N0437 inhibits the release of ACh from rat striatum both/n vivo and in vitro. (A) The effect of 0.01/zM (open squares) and 0.1/~M (closed squares) N0437 infused into the striatum of freely moving rats on the release of ACh as a function of time. Data represent mean values ± SEM of 4 observations. (B) N0437 inhibits the release of ACh both/n vivo (closed squares) and in vitro ( o p e n s q u a r e s ) in a concentration-dependent way. Data represent means SEM of 4 (</n vivo) or 8 (< in vitro) observations. In vivo experiments were performed in the presence of neostigmine (0.1 /zM) and 1.2raM Ca 2+ (for further experimental details, see De Boer et al., 1990). In vitro the release of ACh was evoked by 13 mM K ÷ (for further

experimental details, see Drukarch et al., 1989).

the cholinergic intrinsic neurons in the striatum. Initial microdialysis studies of the striatum failed to show a clear inhibition of the release of ACh by DA and D-2 dopamine receptor agonists (Damsma and Westerink, 1989). Apparently, the composition of the perfusion medium has been the main reason for this failure. For instance, a rather high concentration of 3.4 mM Ca 2+ was used in the perfusion medium for the initial experiments and, in addition, the acetylcholinesterase inhibitor neostigmiue had to be present in the medium to generate an outflow of ACh at a detectable level. Under more appropriate experimental conditions,

especially by using a Ca 2+ concentration identical to the concentration used in the/n vitro experiments ( 1.2 mM), we have now been able to demonstrate a clear inhibition of the release of ACh from rat striatum by D-2 receptor agonists added to the perfusion medium (de Boer et al., 1990; Damsma et al., 1990). In Figure 1A, two time response curves of 0.01 and 0.1/zM of the effect of the selective D-2 receptor agonist ( - ) N0437 on the release of A C h / n vivo from the striatum are shown. The concentration-dependency of ( - ) N0437 both on the in vivo as well as on the in vitro release of ACh is shown in Figure lB. As can be seen, N0437 displays an IC-50 value of approximately 0.01/~M on the/n vivo release, which is slightly higher that the IC- 50 value found under in vitro conditions (approx- imately 0.003/z M). These small differences in potency are most likely due to methodological differences between the two kinds of experiments. E.g. in vitro K +- evoked release is used as a parameter, in vitro no acetylcholinesterase inhibitor is present, in vitro no membrane is interposed between the perfusion med- ium and the tissue etc. Other D-2 receptor agonists, like LY 171555 and apomorphine are also able to inhibit the release of ACh in vivo and just like in in vitro studies selective D-2 receptor antagonists such as ( - ) sulpiride antagonize the effects of the D-2 receptor agonists in appropriate concentrations (data not shown). Moreover, also systemic administration of D- 2 receptor agunists induces an inhibition of the/n vivo release of ACh from the striatum (Damsma et al., 1990).

C. Regulatory aspects o f D-2 receptors. Many receptor types desensitize and/or are down-regulated upon long-term stimulation with receptor agonists. For instance, [~-adrenergic receptors desensitize very rapidly (within several minutes) in the presence of the [3-adrenergic receptor isoprenaline (Lefkowitz et al., 1990). Mechanisms underlying this phenomenon are most likely phosphorylation and internalization (plus subsequent sequestration) of the receptor protein.

We have used various experimental approaches to investigate a possible desensitization of the Do2 recep- tor(s). In Table I one of these approaches is visualized.

From the presented data it can be seen that a preincubation of striatal tissue in the presence of 100 g M of D-2 receptor agonist LY 171555 during 1 hour and a subsequent rapid wash-out of this drug does not affect the percentage inhibition of ACh release induced by acute treatment with 1 /JM LY 171555. Interest- ingly, the D-2 receptor mediating the inhibition of ACh release seems hard to desensitize, which could be in line with its normal physiological function. It should be stressed here that most of the physiological

204S Dopamine 90

Table 1. Preincubation of striatal tissue with 100/tM LY 171555 does not affect the acute effect of LY 171555 on electrically evoked ACh

release

[3H]-ACh Release (% of control)

Preincubated tissue Control tissue

No drug 100.0 ± 3.9 100.0 ± 3.0 LY 171555 (0.03/aM) 59.7 ± t.3 53.9 ± 2.1 LY 171555(1.001aM) 16.6 ± 1.9 14.7 ± 1.1

Striatal tissue was preincubated during I hour with the drug. During the last 15 rain of this preincubation period [3H]-choline was added; subsequently, the tissue was washed and superfused. The release of ACh was evoked electrically (l Hz, 4ms, 24 mA during l0 min) 45 rain after washing in the presenc of absence of the indicated concentrations LY 171555; for further experimental details, see Drukarch et al., 1989.

effects mediated by D-2 receptors are (tonic) inhibitory effects, like for instance the inhibition of the release o f prolactin from the anterior pituitary gland, the inhib- ition of the release of a lpha-MSH from the inter- mediate lobe of the pituitary gland, the inhibition of the release and synthesis of DA etc.(for a review see: Stoof and Kebabian, 1984). Taking into consideration that the affinity of D A for the D-2 receptor is in the low nanomolar range, which is similar to the extraceilular concentrations o f DA, one would expect that admin- istration of D-2 receptor antagonists dramatically affects the above mentioned parameters. This is exactly what can be observed after the acute administration of neuroleptics: plasma prolactin and alpha MSH levels rise dramatically just like the release and turnover of DA. Therefore it is tempting to speculate that D-2 receptors in general are not prone to desensitization in case of chronic stimulation (see also Leysen, 1990).

In summary, by using the release of ACh from rat striatum as an experimental paradigm in vitro and in

vivo, we have investigated (a) the pharmacological profile of the D A receptor involved, (b) the way this receptor is coupled to signal transduction pathways and (c) how this receptor reacts upon longterm activation.

F rom in vitro superfusion studies it became ap- parent that dopamine inhibits the depolarization- induced release of ACh by activation of a D-2 receptor. Al though some D-2 receptors in the striatum are coupled to adenylate cyclase in an inhibitory way (via a Gi-protein), the D-2 receptor involved in regulating the release o f ACh is most likely coupled to a K+-channel (via a Go-protein?) and operates inde- pendent on changes in cyclic A M P formation. Int- erestingly, in recent studies, it has been shown that the different subtypes of D-2 receptors, which have been

cloned and sequenced, differ at the level ~t thc cytoplasmic domain involved in the coupling to G- proteins.

By using microdialysis, we were able to demonstrate also in vivo that D-2 receptor agonists inhibit the release of ACh from rat striatum to a significant extent.

Longterm preincubation of rat striatal tissue m the presence of a D-2 receptor agonist did not affect the extent to which acute administration e r a D-2 receptor agonist inhibits the release of ACh in vitro. This could imply that desensitization of D-2 receptors does not occur, a finding that might be in line with their physiological function(s), since many D-2 receptors are involved in tonic inhibitory processes, such as the inhibition of the release of several hormones and neurotransmitters.

REFERENCES

Bjorklund A, Lindvall O. Dopamine-containing systems in the CNS. In: Bjorklund A, Hokfelt T, eds. Handbook of Chemical Neuroanatomy. Amsterdam: Elsevier, 1984: 55-122.

de Boer P, Damsma G, Fibiger HC, Timmerman W, De Vries JB, Westerink BHC. Dopaminergic-cholinergic interactions in the striatum: the critical significance of calcium concentrations in brain microdialysis. N-S Arch Pharmacol, in press.

Calne DB. Parkinsonism, clinical and neuropharmacological aspects. Postgrad Med 1978; 64: 82-88.

Chang HT. Dopamine-acetylcholine interaction in the rat striatum: a dual-labeling immunoeytoehemieal study. Brain Res Bull I988; 21: 295-304.

Damsma G, De Boer P, Westerink BHC, Fibiger HC. Dopaminergic regulation of striatal cholinergic inter- neurons: an in rive microdialysis study. N-S Arch Pharmacol, in press.

Dawson VL, Dawson TM, Fitloux FM, Wamsley JK. Evidence for dopamine D-2 receptors on cholinergic interneurons in the rat caudate-putamen. Life Sci 1988; 42: 1933-1939.

Drukarch B, Sehepens E, Schoffelmeer ANM, Stoof JC. Stimulation of D-2 dopamine receptors decreases the evoked in vitro release of [3H]- acetylcholine from rat neostriatum: role of K + - and Ca+ %ions. J Neurochem 1989; 52: 1680-1685.

Enjalbert A, Sladeczek A, Guillon G, Bertrand P, Shu C, Epelbaum J, Garcia-Sainz A, Ja.rd S, Lombard C, Kordon C, Bockaert J. Angiotensin II and dopamine modulate both cAMP and inositol phosphate produc- tions in anterior pituitary cells. J Biol Chem 1986; 261: 4071-4075.

Freedman JE, Weight FF. Single K + channels activated by D-2 dopamine receptors in acutely dissociated neurons from rat corpus striatum. Proc Natl Acad Sci (USA) 1988; 85: 36t8-3622.

Fujiwara H, Kate N, Shuntoh H, Tanaka C. D-2 dopamine receptor-mediated inhibition of intracellular Ca ~ ~ mob- ilization and release of acetylcholine from guinea-pig

Dopamine 90 205S

neostriatal slices. Br J Pharmacol 1987; 91: 28%297. Guyenet PG, Agid Y, Javoy F, Beaujouan JC, Rossicr JC,

Glowinski J. Effects of dopaminergic receptor agonists and antagonists on the activity of the neostriatal cholinergic system. Brain Res 1975; 84:227-244

Helmreich I, Reimann W, Hertting G, Starke K. Are presynaptic dopamine autoreceptors and postsynaptic dopamine receptors in the rabbit caudate nucleus pharmacologically different. Neuroscience 1982; 7:1557- 1566.

Hertting G, Zumstein A, Jackisch R, Hoffmann I, Starke K. Modulation by endogenous dopamine of the release of acetylcholine in the caudate nucleus of the rabbit. Naunyn- Schmiedeberg's Arch Pharmacol 1980; 315:111-117.

Israel JM, Kirk C, Vincent JD. Electrophysiological re- sponses to dopamine of rat hypophysial cells in lacto- troph-enriched primary cultures. J Physio11987; 390: 1-22.

Kelly E, Batty I, Nahorski SR. Dopamine receptor stimu- lation does not affect phosphoinositide hydrolysis in slices of rat striatum. J Neurochem 1988; 51; 918-924.

Kubota Y, Inagaki S, Shimada S, Kito S, Eckenstein F, Tohyama. Neotsriatal cholinergic neurons receive direct synaptic inputs from dopaminergic axons. Brain Res 1987; 413: 179-184.

Lacey MG, Mercuri NB, North RA. Dopamine acts on D-2 receptors to increase potassium conductance in neurones of the rat substantia nigra zona compacta. J Physio11987; 392: 397-416.

Lefkowitz R J, Hausdorff WP, Caron MG. Role of phos- phorylation in desensitization of the -adrenoceptor. TIPS 1990, 11: 190-194.

Lehmann J, Langer SZ. The striatai cholinergic interneuron: synaptic target of dopaminergic terminals? Neuroscience 1983; 10:1105-1120

Leysen JE. Receptor modulation by repeated psychotropic drug administration: an integrative view. TIPS, in press.

Memo M, Castelletti L, Missale C, Valerio A, Carruba M, Spano PF. Dopaminergic inhibition of prolactin release and calcium influx induced by neurotensin in anterior pituitary is independent of cyclic AMP system. J Neuro-

chem 1986; 47: 1689-1695. Onali P, Ofianas MC, Gessa GL. Characterization of

dopamine receptors mediating inhibition of adenylate cyclase activity in rat striatum. Mol Pharmacol 1985; 28: 138-145.

Phelps PE, Houser CR, Vanghn JE. Immunocytochemical localization of choline acetyltransferase within the rat neostriatum: A correlated light and electron microscopic study of cholinergic neurons and synapses. J Comp Neurol 1985; 238: 286-307.

Pizzi M, D'Agostini F, Da Prada M, Spano PF, Haefely WE. Dopamine D-2 receptor stimulation decreases the ino- sitol triphosphate level of rat striatal slices. Eur J Pharmacol 1987; 136: 263-264.

Rubinstein JE, Hitzemann RJ. Further evidence against the coupling of dopamine receptors to phosphoinositide hydrolysis in rat striatum. Biochem Pharmacol 1990; 39: 1965-1970.

Sethy VH, Van Woert MH. Regulation of striatal ace- tylcholine concentration by dopamine receptors. Nature 1974; 251: 528-530.

Stadler H, Lloyd KG, Gadea-Ciria, Bartholini G. Enhanced striatal acetylcholine release by chlorpromazine and its reversal by apomorphine. Brain Res 1973; 55: 476-480.

Stoof JC. Localization and pharmacology of some dopamine receptor complexes in the striatum and the pituitary gland: synaptic and non-synaptic communication. Acta Morphol Neerl-Scand 1989; 26:115-130.

Stoof JC, Kebabian JW. Independent in vitro regulation by the D-2 dopamine receptor of dopamine stimulated cyclic AMP efflux and K ÷ -stimulated release of acetylcholine from rat neostriatum. Brain Res 1982; 250: 263-270.

Stoof JC, Kebabian JW. Two dopamine receptors: bio- chemistry, physiology and pharmacology. Life Sci 1984; 35:2281-2296.

Stoof JC, Thieme RE, Vrijmoed-de Vries MC, Mulder AH. In vitro acetylcholine release from rat caudate nucleus as a new model for testing drugs with dopamine receptor activity. Naunyn-Schmiedebcrg's Arch Pharmacol 1979; 309:119-124.