Pharmacologic evidence for direct dopaminergic regulation of striatal acetylcholine release

Life Sciences, Vol. 38, pp. 2239-2246 Printed in the U.S.A.

Pergamon Press

PHARMACOLOGIC EVIDENCE FOR DIRECT DOPAMINERGIC REGULATION OF

STRIATAL ~ T [ ' I X ~ O L I l q ~ ~ . R A . ~

Jay M. Gorell I and Bruce Czarneckl Department of Neurology, Henry Ford Hospital Detroit, MI 48202

(Received in final form March 27, 1986)

Summary This study was done to provide pharmacologic evidence for the location of those striatal dopamlne D-1 and D-2 receptors that participate in the regulatlon of local acetylcholine (ACh) release. Strlatal tissue qlices from adult male Sprague-Dawley rats were preloaded with [JH]choline and superfused in separate experiments with buffer containing either: (1) a D-2-specific a g o n i s t (LY141865 o r LY171555), ( 2 ) a D-2 s p e c i f i c a n t a g o n i s t (Lsulpiride), ( 3 ) a D-1 s p e c i f i c a g o n i s t (SKY38393), o r ( 4 ) a D-1 antagonist (SCH23390), i n t h e p r e s e n c e o r a b s e n c e o f t e t r o d o t o x i n (TTX), u s e d t o b l o c k i n t e r n e u r o n a l activity~ With either.D-2 agontst t h e r e was a d o s e - d e p e n d e n d e g t d e c r e a s e i n K - s t i m u l a t e d [SH]ACh release, m a x i m a l l y a t 5 x 1 0 - " - 10-vM [ a g o n i s t ] a n d t o t h e same 3 extent with each drug. B o t h SKF38393 and SCH23390 i n c r e a s e d [ H]ACh release at tested concentrations of these agents. Results ~re u n c h a n g e d whe9 a n y of t h e d r u g s u s e d was s u p e r f u s e d i n t h e p r e s e n c e o f TTX, 5 x l 0 - ' M . These data are consistent with the hypothesis that populations of strfatal D-1 and D-2 r e c e p t o r s e x i s t on l o c a l cholinergtc n e u r o n s , w h e r e t h e y r e g u l a t e ACh r e l e a s e . Alternative interpretations are discussed. Introduction Dopamlnergic suppression of striatal acetylcholine (ACh) release has been shown to occur both in vlvo (I) and in vitro (2). Moreover, this effect is believed to occur via D-2 [non-adenylate cyclase-llnked; (3)] dopamlne receptors. Thus, the D-2 agonlst, LY141865 (4), causes a dose-dependent decrease in ACh release from rat strlatal tissue slices (5,6), whereas the D-I [adenylate cyclase-linked; (3)] agonlst, SKF38393 (7), has appeared to have little i n f l u e n c e on t h i s p r o c e s s i n t h e h a n d s of o t h e r s ( 6 ) . Such D-2 m e d i a t e d s u p p r e s s i o n o f l o c a l ACh r e l e a s e may o c c u r v i a d i r e c t s y n a p t i c contact between nigrostriatal d o p a m i n e r g i c n e r v e e n d i n g s and c h o l t n e r g i c n e u r o n s as one h i s t o c h e m i c a l study has suggested (8), and/or via nonIAddress correspondence to Dr. Gorell, Department of Neurology, Henry Ford Hospital, 2799 W. Grand Blvd., Detroit, MI 48202.

0024-3205/86 $3.00 + .00 Copyright (c) 1986 Pergamon Press Ltd.

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Direct DA Regulation of Strlatal ACh

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cholinergtc intrastriatal interneurons. D-1 d o p a m i n e r g i c r e c e p t o r s have so f a r not been a s s o c i a t e d w i t h p a r t i c u l a r s t r i a t a l neurons r e l e a s i n g i d e n t i f i e d n e u r o t r a n s m i t t e r s , but d a t a d e r i v e d from e x p e r i m e n t a l k a i n i c a c i d l e s i o n s of r a t s t r i a t u m (9) s t r o n g l y s u g g e s t t h a t neurons b e a r i n g D-1 r e c e p t o r s a r e either nearly all entirely intrastrtatal or p r o j e c t from t h i s CNS r e g i o n (10). The work r e p o r t e d h e r e p r o v i d e s p h a r m a c o l o g i c e v i d e n c e t h a t p o p u l a t i o n s of s t r i a t a l D-1 and D-2 r e c e p t o r s r e g u l a t i n g ACh r e l e a s e may be l o c a t e d on l o c a l ACh r e l e a s e s i t e s ( i . e . , n e r v e t e r m i n a l s or d e n d r i t e s ) . Materials and Methods Preparation of Strlatal Tissue Slices. Adult male Sprague-Dawley rats (Charles River, Wilmington, MA) were decapitated by guillotine, and strlata from each rat were dissected and sliced twice with a Mcllwaln chopper at 0.3 mm t h i c k n e s s , r o t a t i n g t h e t i s s u e 90 ° b e f o r e the second c u t t i n g . Tissue s l i c e s were i m m e d i a t e l y p l a c e d i n i c e c o l d K r e b s - b i c a r b o n a t e b u f f e r , pH 7 . 3 7 . 4 , c o n s i s t i n g of (mM) 116.4 NaC1, 5.4 KC1, 26.2 NaHCO~, 1.0 NaH2PO4, 0 .6 MgS04, 5.6 g l u c o s e , and 1.3 CaC12 under 95% 02/5Z CO2 aEmosphere. S l i c e s were p o o l e d , washed once w i t h 5 ml of f r e s h b u f f e r , and p r e i n c u b a t e d ( a p p r o x i m a t e l y 25 mg t i s e u e / m l b u f f e r ) under c o n s t a n t 95% 02/5% CO2 atmosphere f o r 10 min at 37°C. The medium was a ~ p i r a t e d and r e p l a c e d w i t h f r e s h b u f f e r c o n t a i n i n g ( f i n a l c o n c e n t r a t i o n ) [ = H ] c h o l i n e , 1.2 vEi/ml (New England N u c l e a r , Boston, MA), f o l l o w e d by i n c u b a t i o n f o r a f u r t h e r 15 min. The medium was then a s p i r a t e d and s l i c e s were washed t w i c e with n o n - r a d i o a c t i v e b u f f e r b e f o r e b ei n g p l a c e d i n t o each of 24 s u p e r f u s t o n chambers ( a p p r o x i m a t e l y 10 mg tissue/chamber). Superfuslon Apparatus and Experimental Protocols. The fluid path of the superfuslon apparatus began in capped glass reservolrs, constantly gassed with 95% O2:5% C O 2 a n d placed in a water bath at 37°C. Reservoirs con~alned Krebsbicarbonate buffer with 5.4,~ KCI to measure resting release of [=H]ACh or 17 mM KCI (with NaCI decreased to 104.8 mM to maintain osmolallty) to measure neurotransmttter released during periods of K+-depolarlzatlon. Reservoirs were connected by polyethylene tubing to plastic superfuslon chambers containing strlatal slices between two layers of cotton. The total volume of each chamber was adjusted to 0.2 ml, and a rack with all sealed chambers was immersed in a 37°C water bath. Exit llnes from each chamber were connected to Indlvidual flow-callbrated (200~I/mln) Technlcon ® plastlc tubing in a Technlcon ® (Pump III, slngle speed) perlstaltlc roller pump. Pump exit lines were led to separate sclntillatlon vials to collect superfusate. Sclntillant (ACS; Aqueous Counting Sc~ntillant; Amersham Corp., Arlington Hts.,IL) was added to all samples and H was counted (Beckman LS7800) at 38% efficiency. Superfuston protocols and data analysis of simultaneously run control and test conditions exactly followed the methods detailed by Stool and Kebablan (5). Percent control drug effects with agents used at each test concentration were analyzed for significant differences by an unpaired, two-tailed Student's t-test with significance taken as p < 0.05. No l~ss than 3 concurrent controls were compared ~rlth drugs at levels tested in any one experimental run. [3H]ACh Specificity of 3H R~leased. Separate experiments were done to determine what fractlon o f ~ H released during brain tissue superfuslon with bicarbonate buffer containing 5.4 or 17 ~q KCI resided within ACh

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Direct DA Regulation of Strlatal ACh

2241

specifically. Striatal slices prepared and preloaded with [3H]chollne as described, were superfused with Krebs-blca~bonate buffer containing paraoxon, 40 ~M, to prevent hydrolysis of released [OH]ACh. Superfusates were collected during periods 1 (5.4 ~M KCI buffer; time (t) ffi 30-50 min. of superfusion), 2 (17~M KCI, t ffi 50-55 min; 5.4 mM KCI, t ffi 55-70 mln.), and 3 (5.4 mM KCI, t = 70-90 min.), and samples from each period were divided into two 2 ml portions. Radioactivity in the first portion was determined directly by liquid scintillation counting as described above. The second 2 ml portion from each collection period received 4 ml of chloroform, was briefly vortexed and centrifuged, and was extracted using the following specific modification of the method of Freeman et al (II): back-extraction from the organic phase after the second wash with 0.01 M TAPS (trls-Hydroxymethyl-methylaminopropane sulfonic acid) buffer was done with 0.2 ml of 0.4N HCI. Two 90 ~I allquots of the final aqueous phase from each sample were dried completely (Bio-Dryer; VirTis, Gardiner, NY). Dried residue from the first aliquot of each extracted sample was resuspended in 10 ~i of an incubation medium consisting of (final concentration) 50 mM sodium phosphate buffer, pH 8.0, 0.001 U/~I of choline kinase (ChK; E.C. 2.7.1.32; Sigma Chemical Co., St. Louis, MO), 0.4 ~M ATP and 5 mM MgCI 2. Dried residue from the second, matched aliquot of each sample was resuspended in the same volume of the same incubation medium, which also contained acetylchollnesterase (ACHE, type V from eel; Sigma Chemical Co, St. Louis, MO), 0.001U/ul. All samples were incubated at 37°C for 30 mln and extracted with 50 ~I of sodium tetraphenylboron in 3-heptanone (5 mg/ml). Measured aliquots of the organic phase wer~ added to I0 ml of ACS (Amersham Corp., Arlington Hts.,IL) and counted for H (Beckman LS7800). With this procedure [(see (12)], when corrected for matched blank values [i.e., (ChK) (ChK+AChE)~, aliquot volume factors and3recovery of simultaneously run external [~H]ACh standard (60%), those ~ c.p.m, in the TPB/heptanone phase represent °H specifically within ACh. [°H]choline external standards carried through this procedure were at blank level in the final TPB/heptanone phase. Results Virtually all 3H released from superfused rat strlatal slices under these conditions appeared to reside in ACh specifically (Table I). These results are hlgh~r than those found by others, namely~ 85% of choline acet~lated during K -depolarization (13,14) and 48% as [=H]ACh unde~ normal K conditions (13). Possibly this is because we used just 0.0125 ~M [ H]choline (final concentration), about .01 x Km for hlgh3affinity choline transport (15), whereas other studies employed 0.5 ~M [ H]choline in incubation media. As shown in Fig. I, there was a dose-dependent decrease in [3H]ACh release from strlatal slices during superfuslon with progressively increasing concentrations of the D-2 agonlst, LY141865, reaching a maximum at 5 x 10-'M drug (54 ± 4%; x% control ± SEM, n=3). 7Durlng superfusion of LY141865 in the presence of tetrodotoxln (TTX), 5 x I0- M, there was no difference (p > .05) in the ability of the levels of LY141865 tested to depress release of stri~tal ACh when compared with LY141865 in the absence of TTX (e.g., LY141865, 10-vM, plus TTX; 50 ± 2%, n=9). As shown in Fig. I, ~Y171555, the L-isomer of LY141865 (16), depressed the release of striatal [ H]ACh in a dose-dependent manner6as well, and to the same extent, as the racemate compound (e.g., LY171555, 10- M, 52 ± 3%, n=9). Further studies with LY171555 demonstrated that its maximally depressant

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Direct DA Regulation of Striatal ACh

Vol. 38, No. 24~ 1986

TABLE I. Specificity of 3H Released During Striatal Slice Superfuslon Period (Superfusio n Time) [K+],mM 1 (30-50 mln) 2 (50-70 min) 3 (70-90 min)

5.4

17

5.4

Fraction-I (C.P~M.)

Fractlon-2 No AChE

(C.P.M.) + AChE

3H in ACh (%)

12,469 ¢ 235

1,264 ± 114

40 ± 4

96 ± 7

19,352 ± 585

2,194 ± 124

48 ± 18

108 ± 8

5,512 ± 129

588 ± 65

45 ± 7

I01 ± 9

Rat striatal slices, prepared and preloaded with [3H]choline (80 Ci/mmol; 0.0125~M final concentration) as described in the text, were superfused wlth Krebs bicarbonat~ buffer containing paraoxon (40pM) to prevent hydrolysis of released [ H]ACh. Superfusate effluents were divided into two 2 ml volumes, the first (fraction I) being counted directly, and the second (fraction 2) being extracted to remove paraoxon and to concentrate quaternary a~aonlum compounds (see text). Faired, extracted final aqueous aliquots were dried and resuspended in buffer substrates containing either ch~llne klnase (ChK) alone or ChK plus true acetylchollnesterase (ACHE), [~H]ACh was calculated as (net c.p.m.) in the equation: [(ChK) - (ChK + ACHE)] fraction 1 Data are presented as the x ± SEM of three determinations for each condition tested.

effect on striatal [3H]ACh release (10-6M LY171555) was fully inhibited during cosuperfusion with the substituted benzamlde D-2 dopamine receptor blocker (3), L-sulplrlde. That is, there was 113 + 4% (X + SEM; n=6) of drug-free control [3H]ACh output during K+-~epolariz~tion wi~h L-sulplrlde alone at IO-~M versu~ll9.+ 1% (n~6) of control with both LY171555 and L-sulplrlde, each at IO-~M (t=l.40, p > .05). The D-I dopamln~rgic agog±st SKF38393 (7) increased striatal ACh output significantly at I0-~ and 10-~M (Table 2). The~e responses were unchanged when superfused in the presence of TTX, 5 x 10- M. Finally, the apparent D-I dopam/nergic antagonist SCH23~90 (17) also increased ACh release from strlatal tissue slices at I0-v and I0- M in a dose-dependent manne~7(Table 3). These effects were also unchanged in the presence of TTX 5 x I0 M. Discussion The data presented confirm the observations of Stoof and Kebab±an (5) and Scatton (6) demonstrating a dose-dependent suppression of ACh release from superfused rat strlatal slices by the I)-2 agonlst, L¥141865, to about 50% of control values. Our results indicate that compound's L-Isomer, LY171555, is as effective in this process, and is fully inhibited by the I>-2 antagonist, Lsulpiride. Moreover, the demonstration here that LY141865 and LY171555 are as effective in the presence of a concentration of TTX known to block axonal

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Direct DA Regulation of Striatal ACh

2243

RELEASE OF ACH FROM RAT STRIATAL SLICES

\

100

\\

~o 90 0

-\ I

8O n"

70

tO

'< 60

I

k___J

50

-£

I

~

i

I

I

10 -s

5 X 1 0 -8

10 -7

5 X 1 0 -7

10 -6

M

FIG.

I

Rat strlatal tissue slices, preloaded wlth [3H] choline to label [3HI ACh speclflcally (see text), were superfused w l~h LY141865 alone [(0) n=3-15 at each level], LY141865 plus TrX, 5x10 [(D), n=9-11], or LY171555 alone [(*), n=8-15] at the concentrations indicated. Results are expressed as the ~ean ± SEM % of concurrently run control fractional rates of K+-Induced [°H] ACh release. conduction (18) (and thus interneuronal activity) suggests that a populatlon of D-2 receptors regulating strlatal ACh release exists on chollnerglc neurons (i.e., nerve terminals, dendrites) directly. The same interpretation would seem to apply to the data presented here showing no alteration of effect in the action of SKF38393 and SCH23390 in the presence of TrX, namely, that a population of striatal D-I receptors may exist on local chollnerglc neurons. The argument for the location of some I>-I receptors on strlatal cholinerglc neurons seems clearer with SKF38393 data than with SCH23390 observations because the former drug is known to be an exclusive, if partial, 1>-I agonlst (19), whereas SCH23390 at the levels used may be blocking D-2 sites as well (17) or even acting at non-dopaminergic receptors (20). Neuropharmacologic mechanisms responsible for the rise in striatal ACh output by SKF38393 and

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Direct DA Regulation of Striatal ACh

Vol. 38, No. 24, 1986

SCH23390 are discussed in an accompanying paper (21). TABLE 2. Effect of SKF38393 ± TTX on [3R]ACh Release from Rat Strlatal Slices

Dru~

Concentration ~ ~M

[3H]ACh Release, % Control

None

--

100 ± 7 (12)

TTX

0.5

108 ± 4 ( 6 )

SKF38393

10

SKF38393 + TTX SKF38393

10 + 0.5 I00

SKF38393 + TTX

100 + 0.5

130 ± 4 ( 6)** 132 ± 1 ( 6)*** 138 ± 3 (

3)***

138 ± 2 ( 5)***

TABLE 3. Effect of SCH23390 ± TTX on [3H]ACh Release from Rat Strlatal Slices

Drug

Concentration, ~M

[3H]ACh Release % Control

None

--

I00 ± 4

(12)

TTX

0.5

108 ± 9

(9)

SCH23390

1.0

128 ± 6

( 6)**

SCH23390 + TTX SCH23390 SCH23390 + TTX

1.0 + 0.5 I0 I0 + 0.5

125 ± I0 ( 6)* 160 ± 9

( 6)***

166 ± 7

( 6)***

Rat strlatal tissue slices were superfused in separate experlments w ~ t h either ~KF38393 or SCH23390 alone or in the presence of TTX (5 x 10-'M) while [ H]ACh output was measured. Results are presented as the x% of control values ± SEM with the numbers of observations given in parentheses. (*), p<.05; (**), p<.01; (***), p<.O01 vs no drug control. Future work will be needed to conclusively prove the hypothesis that both D-2 and D-I receptors exist on strlatal cholinerglc neurons, however, since one can imagine an anatomical arrangement in which DA terminals synapse with a striatal interneuronal terminal, itself synapslng directly with a cholinergic release site. In such a model, directly apposed synapses would be unaffected by TTX, but would continue to release neurotransmltter in response to K +depolarization. With current pharmacologic tools, such a possibility can be tested if brain slices are cosuperfused with neurotransmltter-speclfic

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Direct DA Regulation of Striatal ACh

2245

antagonists in the presence of TTX. Using thls rationale, we cosuperfused plcrotoxln, a selective blocker of chlorlde c_~nnels associated wlt~ the GABA receptor (22), in the presence of TTX (5 x I0- M) and LY141865 (10- M) in separate experiments (data not shown). Plcrotoxln was unable to inhibit LYI41865's action on strlatal ACh release, providing evidence that strlatal GABAerglc Interneurons are not Involved in thls effect. Other strfatal fnterneuronal te:mfnals hypothetically having direct contact with chollnergfc release sites may be involved, however, and It would require testing wlth other speclflc neurotransmitter antagonists to be sure that each was not involved In D-I or D-2 dopamlnergfc regulation of local ACh release. Ultimately, the issue of whether, or to what extent, nlgrostrlatal nerve endings synapse directly with strlatal chollnergfc neurons may have to be settled using future Immunohlstochemlcal methods, perhaps involving a demonstration of specific dopamlne receptor staining on choline acetyltransferase-posltlve strlatal neurons. At the present time, the closest investlgators have come to thls goal is to show a hlgh correlation between the topography of D-2 receptor binding, chollne acetyltransferase activity and high-afffnlty chollne uptake in rat caudate-putamen (23). Acknowledgments

Thls work was supported by the Fund for Henry Ford Hospital and The Gossett Fund. The authors wish to thank Messrs. James Ewlng and Stephen McGee for their development of a computer program used in the analysis of data derived from 11quld sclnt111atlon counting. Dopamlnerglc drugs were generously supplled by Ell Lllly and Co. (Indlanapolls, IN; LY141865, LY171555), Ravlzza Pharmaceuticals (Milan, Italy; L-sulplrlde), Smlth, Kllne and French Laboratories (Phlladelphla, PA; SKF38393) and Scherlng Corporation (Bloomfleld, NJ; SCH23390). References I. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

G. BARTHOLINI, H. STADLER, M. GADEA CIRIA and K.G. LLOYD. Neuropharmacol. 15:515-19 (1976). J. DEBELLEROCHE, J. COUTINHO-NETTO and H.F. BRADFORD. J. Neurochem. 39:217-22 (1982). J.W. KEBABIAN and D.B. CALNE. Nature. 227:93-6 (1979). K. TSURUTAp E.A. FREY, C.W. GREW, T.E. COTE, R.L. ESKAY and J.W. KEBABIAN. Nature. ( L o n d . ) 292:463-5 (1981). J.C. STOOF and J.W. KEBABIAN. B r a i n Res. 250:263-70 (1982). B. SCATTON. L i f e Sct. 31:2883-90 (1982). P.E. SETLER, H.M. SARAU, C.L. ZIRKLE and H.L. SAUNDERS. European J. Pharmacol. 50:419-30 (1978). T. HATTORI, V.K. SINGH, E.G. HC GEER, and P.L. HCGEER. Brain Res. 102:164-173 (1976). E.G. MC GEER, V.T. INNANEN, and P.L. MCGEER. B r a i n Res. 118:356-358 (1976). J . T . COYLE, M.E. HOLLIVER and M . J . KUHAR. J , Comp. N e u r . 180:301-324 (1970). J . J . FREEMAN, R.L. CHOI and D . J . JENDEN. J . Neurochem. 24:729-34 (1975). A.M. GOLDBERG and R.E. MC CAHAN. I n : H a n t n I , e d . C h o l i n e and Acetylcholine: Handbook of C h e m i c a l A s s a y M e t h o d s . New Y o r k : Raven P r e s s , 1974; 4 7 - 6 1 . A.H. MULDER, H . I , YAMAMURA, M.H. KUHAR, and S.H. SNYDER. B r a i n Res.

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70:372-6 (1974). 14.

EoJ. BURGESS, C.K. ATTERWILL, and A.K. PRINCE.

J. Neurochem.

31:1027-33

(1978). 15. 16. 17. 18. 19. 20. 21, 22, 23.

R.S. JOPE. Brain Res. Rev. 1:313-44 (I~79). R.D. TITUS, E.C. KORNFELD, E.D. JONES, et al. J. Med. Chem. 26:1112-1116 (1983). L.C. IORIO, A. BARNETT, F.H. LEITZ, V,P. HOUSER, and C.A. KORDUBA. J. Pharmacol. Exp. Ther. 226:462-468 (1983). C.V. KAO. Pharmacol. Rev. 18:997-1049 (1966). K.J. NATLING and J.E. DONLING. J. Neurochem. 36:559-568 (1981). P.E. HICKS, H. SCHOEIdAKER and S.Z. LANGER. European J. Pharmacol. 105:339-342 (1984). J.M. GORELL, B. CZARNECKI and S. HUBBELL. Life Sci. 38:2247-2254 (1986). S.J. ENNA. Bioehem. Pharmacol. 30:907-13 (1981). J.N. JOYCE and J.F. MARSHALL. Neuroseienee Letters 53:127-131 (1985).