A D1 dopamine agonist stimulates acetylcholine release from dissociated striatal cholinergic neurons

BRAIN RESEARCH ELSEVIER

B rain Research 727 (1996) 162-168

Research report

A D~ dopamine agonist stimulates acetylcholine release from dissociated striatal cholinergic neurons Ivan S. Login *, Madaline B. Harrison Laboratory for Neurotransmitter Research, Department of Neurology, Box 394, University «~f'Virginia, Health Sciences Center, CharlottesLqlle, VA 22908. USA Accepted 4 Aprit 1996

Abstract

We tested the hypothesis that a D~ dopamine agonist could stimulate acetylcholine release directly from striatal cholinergic neurons. A suspension of viable dissociated striatal cells was made enzymatically and mechanically from normal adult maie rats. The heterogeneous suspension was incubated in [3H]choline to allow synthesis of [3H]acetylcholine selectively by cholinergic neurons. Fractional [3H]acetylcholine release from the cholinergic cells in the suspension was recorded during continuous dynamic perifusion. The D I agonist, 50 ~M ( + ) SKF 38393, increased the basal rate of release from the cholinergic cells by 50% and the action was inhibited by the D 1 antagonist, SKF 83566. Stimulation of [3H]acetylcholine secretion was recorded as low as 500 nM SKF 38393. The ( S , - ) SKF 38393 stereoisomer was significantly less effective than the ( R , + ) isomer in stimulating release. The D~-mediated stimulation of acetylcholine secretion was abolished in a low-calcium environment that also inhibited basal release. The data suggest that striatal cholinergic cells express D 1 receptors functionally coupled to the regulation of acetylcholine release. These D~ actions in the absence of synaptic circuitry imply that such circuitry is not required in situ. In vivo however, indirectly mediated DI actions and those of other transmitters may modify the manifestations of this direct cholinergic stimulation. Keywords: Acetylcholine release; Dopamine D I receptor; Dissociated neuron; Striatum; SKF 38393

1. Introduction

Treatment in vivo with a D 1 dopamine (DA) receptor agonist stimulates striatal cholinergic activity and acetylcholine (ACh) release [6,32]. Several studies bave addressed whether this D~-mediated control is effected outside [7,32] or inside the striatum [2,5,15,23,39] and if inside, directly on the cholinergic cell or indirectly, by a polysynaptic route [3-6]. A small number of large, presumably, cholinergic striatal neurons synthesize D~ receptor m R N A [17,22], suggesting that direct control could occur, as speculated in a recent review [9]. Other opinions hold that D~ actions are probably not mediated directly on cholinergic cells [32,34]. Indirect polysynaptic actions [6], through tachykinins [3] for example, may be more likely. Complex striatal circuitry and the paucity of striatal cholinergic cells [16,21] hamper investigation of the mechanisms regulating their activity. Experiments with striatal slices partially simplify these problems by limiting extras-

* Corresponding author. Fax: (1) (804) 982-1726. 0006-8993/96/$15.00 Published by Elsevier Science B.V. PH S 0 0 0 6 - 8 9 9 3 ( 9 6 ) 0 0 3 6 4 - 2

triatal influence, but reveal discordant results. Under these conditions, the D~ agonist, 1 p~M SKF 38393 enhanced K+-evoked ACh release from rat striatal slices by 15%, but lower concentrations were ineffective [33]. Gorell and Czarnecki observed 30% augmentation of K+-evoked ACh release from rat slices by 10 ~ M SKF 38393 [15]. Fifty ~ M SKF 38393 augmented K+-stimulated release by 56% in slices taken from dopamine-depleted rats but had no effect on evoked release in normal slices, and no effect on basal release under either condition [23]. Dolezal et al. failed to observe an effect of 1 p~M SKF 38393 on electrically stimulated [3H]ACh release from rabbit striatal slices [10]. Thus, a D~ agonist may stimulate ACh release under certain conditions in striatal slices. Residual synaptic connections still exist within slices, however, and may mediate or modulate D~-stimulated ACh release, accounting for some of the conflicting data. The ability to monitor fractional [3H]ACh effiux from dissociated striatal cholinergic neurons [24] offers a new approach to this important issue. Although the cellular suspension in the dissociated striatal preparations is heterogeneous, assaying the secreted product of a single type of

I.S. Login, M.B. Harrison / Brain Resear«h 727(1996) 162 168

neuron (e.g. ACb release from cholinergic cells) in the absence of synaptic control isolates the activity of the cholinergic cell. This is a functional assay that investigates the regulation of cholinergic activity directly at the cell witbout confounding synaptic circuitry. Release of [3H]ACh from the individual cholinergic cells in tbe dissociated striatal suspension is voltage-sensitive, calcium-dependent, and similar to release of native ACh as assayed by HPLC [24]. We tested the hypothesis that one site of Dcstimulated cholinergic activity resides directly at the cholinergic oeil.

2. Methods and materials

Animals were used according to NIH guidelines under experimental protocols approved by tbe University of Virginia Animal Care Committee. Preparation and use of the labeled cells was accomplished as described [24,25]. Briefly, in each experiment striata from two normal adult male rats were dissociated enzymatically and mechanically. Viability was over 90% as measured by trypan blue exclusion. The striatal suspension was incubated in [~H]cboline and tbe labeled cells were perifused to measure fractional [~H]ACh effiux. Data collection began about 5 h after the rats were killed. Krebs Ringer bicarbonate buffer, pli 7.35, bubbled with 95% O~, 5% CO 2 al 37°C was the working solution. The composition was: 118 mM NaCI; 5 mM KCI; 25 mM N a H C Q ; 1.2 mM MgCI~: 1.3 mM CaC12; 10 mM glucose; 1.2 mM KH2PO « A 10 mM K + solution was made by reducing NaCI to rnaintain isotonicity. We prepared a low-calcium buffet by omitting CaCI 2 and adding 60 p,M EGTA, resulting in a free calcium level < 25 I*M (detection limit by atomic absorption spectroscopy), that prevents cellular stimulation by calcium channel activators [26]. Previous investigators have reported that [3H]ACh is the predominant material secreted following striatal slice incubation in [3H]choline [14,15,32]. Suspensions of dissociated striata release [3H]ACh and native ACh in parallel

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3. Results

3. l. The direct effect of SKF 38393 on striatal «holinergi« actit,itv The D~ agonist, SKF 38393. clearly stimulated the release of [~H]ACh from dissociated striata (Fig. 1, solid circles). Cells were exposed to 50 I*M SKF 38393 [br continuity with earlier work [23]. After a short latency SKF 38393 progressively stimulated release over about l0 rein from a resting value of 133 _+ 7 (minute 8) to 196 _+ 23 (minute 28; P < 0.05). Removal of SKF from the perifusate immediately provoked a secondary increase in [~H]ACb release lasting about 14 rein. Compared with the magnitude of the primary stimulation (release at minute 28 less release at minute 8), tbe secondary stimulation increased release by an additional 7 3 ç , to 242 + 24 (minute 31; P < 0.01). Release then decreased toward the basal rate following the secondary stimulation. The spontaneous release from dissociated control preparations (open circles) was stable. The secondary stimulation was apparently related to removal of SKF 38393 from the cells and not to a delayed direct effect of the drug (Fig. 2). When the oeils were continuously exposed to a sustained concentration of SKF 38393 (triangles), the initial stimulation was followed by a slow decline toward the basal rate of release, despite the presence of the agonist. When the concentration of SKF 38393 was abruptly lowered after a 20-rein treatment (solid circles), the initial stimulation was followed by the secondary increased release.

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The racemic mixture of SKF 38393 was used for ail experiments except when the relative effects of the ( + ) and ( - ) stereoisomers were evaluated. The source for ( + ; + ; and - ) SKF 38393, SKF 83566, bicuculline, eticlopride, MK-801 and reserpine was RBI. Atropine, «methyl-para-tyrosine (AMPT) and D-ArgI,D-Trp7'9,Leu IlSubstance P (SPA) were obtained from Sigma. The data in these experiments were derived from 34 independent dispersions. Values in the text and figures represent fractional [3H]ACh efflux (with units, X 104/rein) [24,25] as the mean +_ S.E.M. unless noted otherwise. Statistical analysis was performed using the paired or unpaired t-test, as appropriate, with differences considered significant at P < 0.05.

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Fig. 1. Direct stimulation of [3H]ACh releasc from striatal cholinergic neurons by SKF 38393. Dissociated. labeled striatal cells fl'om normal adult maie rats were used to record the rate of fractional [ !H]ACh efflux during continuous perifusion. Cells in six independen[ studies exposed to 50 tzM SKF 38393 for 20 rein (solid circles; horizontal time bar) showed direct stimulation of release and a secondary stîmulation (starting at the arrow) provoked by withdrawing SKE 38393. The open circtes represent spontaneous release from nine independcntly dissociated control preparations.

I.S. Loçit. M.B. Harrison / Brain Resear«h 727 ( 19961 162-168

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Fig. 2. The secondary stimulation of release depends on terminating the SKF 38393 treatment. In three columns of labeled cells studied concurrently, one was a control (open circles) and two were treated with SKF 38393: one exposed continuously (triangles) and one for only 20 min (solid circles). In this example of three similar studies, the secondary stimulation was evoked by drug withdrawal but hOt by sustained treatment.

Fig. 4. Acetylcholine release from normal and DA-depleted dissociated striata is acutely and reversibly inhibited by 20 IxM S K F 83566. In four independent experiments dissociated striata were prepared from n o r m a l rats (two studies; solid symbols) or rats depleted of catecholamines ( t w o studies; open symbols). The D I antagonist, 20 IxM S K F 83566, was infused in each study during the time indicated by the horizontal bar. Rats treated with reserpine (5.0 m g / k g , i.p., at 0 h) and AMPT (100 m g / k g , i.p., at 18 h) had ptosis and akinesia and were killed 1 h after the second injection [35].

3.2. Pharmacological specificity of Dl-mediated stimulation of ACh release The D~ antagonist SKF 83566 inhibited the spontaneous release of [BH]ACh by 57% and reduced both the primary and secondary stimulation of ACh release by the D~ agonist (Fig. 3). The net primary stimulation (maximum rate during administration of SKF 38393 less the basal rate when SKF 38393 was added) was calculated for each column in each experiment in the absence (control group) or presence of the antagonist. The D j antagonist reduced tbe primary stimulation by SKF 38393 to 2 6 _ 17 com-

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Fig. 3. A D I antagonist inhibits the direct primary and secondary stimulatory effects o f a D I agonist. Two columns of labeled cells were perifused simultaneously. The control stimulatory effect o f 50 IxM SKF 38393 was evaluated in the absence (open circles; uppermost time bar) and presence of the D I antagonist, 20 txM S K F 8 3 5 6 6 (solid circles; lower two time bars). This graph shows a single experiment representing four similar independent studies.

pared with the control value of 54 +_ 22 (n = 4; P < 0.05). The net secondary stimulation (peak rate «(ter D~ treatment less the maximum value during D~ treatment) was 23 +_ 7 in the presence of the antagonist compared with 47 _ 3 for the control D l effect ( P < 0.05). We questioned whether SKF 83566-mediated inhibition of basal ACh release was due to blockade of residual stimulatory dopaminergic tone in the dissociated perifused cells. The SKF 83566 rapidly, continuously and reversibly inhibited release in both normal striatal cells and those prepared from dopamine-depleted rats (Fig. 4). Concentrations as low as 500 nM SKF 38393 (Fig. 5) induced a small amplitude sustained increase of [~H]ACh release that terminated when the ligand was withdrawn, without a secondary augmentation. One IxM SKF 38393 stimulated release in a pattern similar to 500 nM SKF 38393 (data not shown). Basal release was hOt inhibited by 1 txM SKF 83566 which did however, block the stimulatory effect of 500 nM SKF 38393. We compared the effects on ACh release of the active (R, + ) and less active ( S , - ) stereoisomers of SKF 38393 [39]. Like the racemate, ( + ) 50 IxM SKF 38393 directly stimulated ACh release over 8 - l 0 rein to reach a sustained plateau (Fig. 6). The net primary stimulation by the ( + ) isomer was 34_+ 6 compared with 17 + 7 for the ( - ) isomer ( P < 0.05). The net secondary effects however. were 22_+ 1 for the ( + ) isomer and 33 _+ 1 for the ( - ) isomer ( P < 0.05) resulting in a net total response that was similar for the two agents: 56 +_+_7 for ( + ) isomer and 50 ± 7 lbr ( - ) isomer. The duration of the secondary stimulation provoked by removal of the ( - ) isomer was almost as long as the direct primary effect.

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3.3. DFstimulated ACh release is calcium dependent We tested the hypothesis that DE-mediated cholinergic stimulation required calcium influx (Fig. 7). Fractional effiux was stimulated by 10 IxM SKF 38393 in a pattern that resembled 50 IxM SKF 38393 with a slowly increasing primary effect followed by a small post-treatment secondary augmentation. The low-calcium conditions immediately, continuously and reversibly suppressed basal ACh release and prevented D t-mediated stimulation. The 225 ,

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Fig. 5. The interactions of I txM SKF 83566 and 500 nM SKF 38393 on release of [3H]ACh. Dissociated striatal cells from rive independent preparations of normal rats were exposed to 1 IxM SKF 83566 (open circles, lowermost horizontal line). The antagonist had no inhibitory effect at this concentration. Cells were also treated with a 28 min infusion of 500 nM SKF 38393 that included a 10 min puise of 1 t~M SKF 83566 (ligand delivery represented by the upper two horizontal lines). This experiment was independently repeated twice and the values for the mean response are shown (solid circles). The agonist induced a small stimulation of [3H]ACh release and the effect was reversibly inhibited by the antagonist. There was no post-treatment secondary stimulation when lhe agonist was withdrawn.

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Fig. 6. Comparison of the effects of the SKF 38393 stereoisomers on [~H]ACh release from dissociated, labeled, perifused, striatal cholinergic neurons. Parallel columns of cells in three independent studies were exposed to either tlle (R, + ) isomer (solid circles) or the ( S , - ) isomer (open circles) of 50 p.M SKF 38393, as indicated by the horizontal rime line.

Fig. 7. A low-calcium buffer inhibits the spontaneous release of [~H]ACh and the primary and secondary stimulatory effects of 10 ixM SKF 38393. Three columns of cells were studied simuhaneously. One was an untreated control (open circles). Two were treated with 10 IxM SKF 38393 (short time bar) in the absence (solid circles) or presence (triangles) of a low-calcium buffet. The low-calcium buffer inhibited basal release (by 67-L-_3% in lbur studies) and abolished D~-mediated stimulalion.

primary and secondary stimulatory effects of 50 IxM SKF 38393 were also abolished by the low-calcium buffer in two additional independent experiments (data hOt shown).

3.4. Absence of paracrine effects during perifusion We postulated that ACh release from dissociated cholinergic neurons was hOt influenced either by paracrine effects of transmitters released into the perifusion column from other neurons or by synaptic effects of retained boutons. Potassium depolarization releases endogenous transmitter stores in the cell suspension. If indirect stimulatory or inhibitory effects of other transmitters modulated K+-stimulated [3H]ACh release, then antagonists to such other transmitters should alter the K + action. Depolarization with 10 mM K + stimulated calcium-dependent ACh release (Fig. 8). In each column of cells we determined the net effect of 10 mM K * on ACh release (peak minus basal) in the absence (Control) or presence of antagonists to striatal transmitters. Each test column received a continuous infusion of a robust concentration of a single antagonist starting 10 rein before K+: 20 IxM SKF 83566 as a D~ antagonist [27]; 20 IxM eticlopride as a D~ antagonist [18]; 10 IxM bicuculline as a GABA A antagonist[8]; 1 ixM atropine as a cholinergic antagonist [12]; 100 ixM MK-801 as an NMDA antagonist [1]; or 10 IxM SPA as an antagonist of the tachykinin family [4]. Three independent replicates of each experimental group were collected. For all Control columns, the net response to K ~ was 369 _+ 38 (mean +_ S.E.M.: n = I I). The values in the experimental groups for K+-stimulated ACh release were (% of control; mean _+ S.E.M.; /7 = 3): SKF 83566:98 + 4%; eticlopride: 93 _+_4%; bicuculline: 105 + 5%" atropine: 104 + 6%; M K - 8 0 1 : 1 1 4 + 8%: and for SPA: 107 _+ 9%.

I.S. Login, M.B. Harrison / Brain Research 727 (1996) 162-168

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In a matched-group, two-tailed t-test, there were no significant differences between any of the experimental groups and their paired controls.

4. Discussion With experience studying hormone release and fractional 45Ca2+ effiux from dissociated anterior pituitary cells [25], we combined dissociation, labeling and dynamic perifusion to study [3H]ACh secretion from isolated striatal cholinergic neurons. Brain tissue dissociation is an established means to harvest viable responsive individual neurons for electrophysiological investigation. Acetylcholine release in this system could theoretically be modulated by stimulatory or inhibitory paracrine actions. Perifusion continuously limits such paracrine effects, however, by diluting and removing released transmitter. Effects on ACh release might also occur through the activity of synaptic boutons retained on cholinergic cells after dissociation. This indirect effect is also minimized because the calcium and magnesium levels in the Krebs Ringer Bicarbonate buffer deplete synaptic vesicles from residual terminal boutons [11,19]. Antagonists against a wide range of striatal transmitters failed to alter K+-stimulated ACh release, indicating that paracrine actions and bouton activity are hOt likely to have an impact on ACh release during perifusion of dissociated striata. As the dissociated cholinergic cells receive no synaptic modulation, the spontaneous release of ACh represents the intrinsic secretory rate at which the cells are programmed. Agents added to the perifusate regulate the secretory rate through actions mediated directly at the cholinergic cell. Despite the loss of distal dendrites, spines, shafts or heads during dispersion, a secretory response to a

specific ligand in the perifusate demonstrates that the required receptive elements are present and functional [16,21,31], similar to the physiological response of isolated neurons in patch clamp analysis. The SKF 38393 stimulated ACh release in a concentration-dependent manner. Five hundred nM SKF 38393 generated a small amplitude stimulation that began and ended abruptly as the ligand was delivered and removed. The stimulatory etTects of l0 and 50 ~M SKF 38393 were more complex. A large primary stimulation increased over about 10 rein. The slowly increasing response to higher levels of SKF 38393 differs from the abrupt K +- or glutamate-evoked cholinergic stimulation [24]. The change in the stimulatory profile at the higher concentrations of SKF 38393 could indicate D~ receptor activation of additional intracellular signaling systems [36]. Withdrawal of the D~ agonist provoked a secondary increase in ACh secretion that averaged 73% of the primary stimulation and lasted almost as long. Low-calcium conditions abolished the primary and secondary effects of SKF 38393 on ACh release which implicates a role for calcium influx in both these stimulatory actions. The D~ receptor antagonist, SKF 83566, inhibited ail stimulatory effects of 500 nM and 50 >M SKF 38393. The ( + and - ) stereoisomers of SKF 38393 reproduced the primary and the secondary effects of the racemate and the ( - ) stereoisomer was less active than the ( + ) isomer. These observations support the conclusion that SKF 38393 can regulate ACh release through D) DA receptors [30] expressed directly on cholinergic neurons [17,22]. These directly mediated actions are likely to be modulated in vivo by indirectly mediated D~ effects as well as those of other transmitters, emphasizing the complexity of striatal circuitry. Our approach adds another dimension to electrical, immunological and anatomical studies. The secondary augmentation of ACh release in the same direction as the primar3' stimulation is an atypical response during in vitro perifusion experiments, seen only at higher concentrations of SKF 38393. Itis not a technical artefact in our system because it does not occur upon withdrawal of either low D l levels (Fig. 5), D I antagonists (Fig. 4) or 10 mM K + (Fig. 8). Further, the secondary increased release is not simply a delayed stimulatory effect of SKF 38393. The secondary response does nol occur when the concentration of SKF 38393 is sustained by prolonged infusion (Fig. 2), and the continuous perifusion design rapidly clears drug from the cells wben the programmed exposure is completed. Similar primary and secondary cholinergic responses to treatment with SKF 38393 also occur in vivo. Ten, 25 [3] and 100 DM SKF 38393 [39] each stimulate ACh release about 15-20% dnring microdialysis infusion. After terminating the infusion however, ACh release increases an additional 34-58% over the next 20-40 min. Given the enhanced temporal resolution in out system, the rapid drug washout from the dissociated cells and the direct effects of

l.S. Login, M.B. Harrison / Brain Research 72711996) 162-168

S K F 38393 at the cholinergic neuron (without paracrine or circuit-dependent input), we conclude that the secondary augmentation of ACh release is associated with, and induced by, falling levets of SKF38393. The greater secondary response to the ( - ) isomer seen here and in vivo [39] might suggest the S K F effect was not mediated through a D~ DA receptor, although the D i antagonist inhibited both the primary and secondary stimulatory actions of S K F 38393. Two mechanisms might explain the secondary augmentation. As S K F 38393 stimulates ACh release, another stimulatory intracellular signal may be generated concurrently, but its action may be blocked by the D~ ligand. When the S K F 38393 level falls below a critical value, the effect of the secondary stimulator becomes apparent. Alternatively, S K F 38393 might activate another signaling system within cholinergic cells that inhibits cholinergic tone but whose effect is buried within the 'net' stimulation by S K F 38393. Acetylcholine release might respond to cessation of this inhibitory input with a p o s t - S K F 38393 rebound or, secondary stimulation when the ligand is removed. If mediated by a specific D t receptor, the secondarily increased ACh release from falling S K F 38393 levels could potentially contribute to dyskinesias seen in Parkinson's disease during f a l l i n g levodopa levels ('end of dose dyskinesia') [28]. The basal fractional [3H]ACh efflux from s l i c e s of normal or DA-dep[eted rats is unaffected by S K F 38393 [23], yet this ligand stimulates release from dissociated normal striata. Transmitters other than D A therefore, may tonically inhibit D l-mediated cholinergic stimulation. The slices might not respond to S K F 38393 because there was no D z ligand in the buffer [20], but there was none in the buffer of the dissociated cells either. Basal release from slices could represent a non-activated state [29] preventing modulation by D~. Systemic [6,7,13] or intra striatal [6] delivery of a D~ antagonist lowers striatal ACh release [6,7] and raises tissue ACh content [13]. These results bave been attributed to the combined effects of inhibiting tonic DI stimulation and unmasking tonic D: inhibition [6]. However, the D~ antagonist, S K F 83566, directly inhibited [3H]ACh release from dissociated cells in the absence of D l or D= receptor activation (Fig. 4). The S K F 83566 [37] and another commonly used D t antagonist, SCH 23390 [15,34], bave actions besides D I receptor blockade [27] that warrant caution in ascribing results specifically to effects mediated by blockade of D I receptors. Our studies however, with Iower concentrations of S K F 83566 that lack direct actions and with enantiomers of S K F 38393 support the position that D j receptor-mediated mechanisms control striatal ACh secretion. This new approach provides functional neurophysiological evidence for D~ DA receptor-mediated stimulation of ACh release directly on striatal cholinergic interneurons. Cholinergic cells are few in number but are critically poised to modulate striatal activity and ultimately behavior

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with their tonic firing and vast axonal and dendritic fields [38]. Demonstration of D l D A control of cholinergic function at the cellular level at once not only simplifies out understanding of striatal interactions, but also ãdds greater complexity to studies with intact circuitry.

Acknowledgements W e recognize the expert technical assistance of Kate Borland. The work was supported, in part, by NIH research grants CA 38228 and NS 01454 and the American Parkinson Disease Association.

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[16]

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[18]

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