Apparent lack of a dopaminergic-cholinergic link in the rat nucleus accumbens septi-tuberculum olfactorium

Braia Research, 135 (1977) 255-263 Elsevier/North-Holland Biomedical Press

255

APPARENT LACK OF A DOPAMINERGIC-CHOLINERGIC LINK IN THE RAT NUCLEUS ACCUMBENS SEPT1-TUBERCULUM OLFACTORIUM

S. CONSOLO, H. LADINSKY, S. BIANCHI and D. GHEZZI Istitato ~" Ric rche Farmacologiche "Mario Negri', Via Eritrea, 62-20157 Milan (Italy)

(Accepted February 10th, 1977)

SUMMARY

The nucleus accumbens septi and tuberculum olfactorium (NAS-TO), which from part of the mesolimbic dopaminergic system, and the striatum, which is part of the nigrostriatal dopaminergic system, contain high levels of both dopamine (DA) and acetylcholine and resemble each other in some other biochemical properties. We determined whether blockade or stimulation of DA receptors by agonists or antagonists affects the cholinergic neurons in this brain structure. The DA receptor antagonists haloperidol, pimozide, chlorpromazine and clozapir,,e had no effect on the acetylcholine level in the NAS-TO even at 2-8 times the minimum dose required to maximally decrease striatal acetylcholine. Similarly, Damphetamine and bromocriptine (CB 154), DA receptor stimulating drugs, had nc effect on the acetylcholine level in this brain area at doses up to 3 times higher than those that produced a maximum increase in the striatum. Piribedil (15-120 mg/kg) and apomorphine (4 mg]kg) did increase acetylcholine in the NAS-TO but the action was not blocked by pimozide and is therefore not attributable to DA receptor action. The data thus indicate an apparent lack of a dopaminergic-cholinergic link in the NAS-TO.

INTRODUCTION

The mesolimbic dopaminergic pathway has been mapped out ~,3~ using the histochemical fluorescence technique combined with the biochemical determination of dopamine. This dopaminergic system, anatomically distinguishable from the nigroneostriatal pathway, has its origin in the At0 group of neurons 13 lying medial to the A9 group in the midbrain and has terminals in the nucleus (n.) accumbens septi (NAS), tuberculum olfactorium (TO), dorsolateral part of the nucleus interstitialis striae terminalis and the central nucleus of the amygdala.

256 The mesolimbic and nigro-neostriatal dopaminergic systems resemble each other in some biochemical pro gmties. Horn et al. x6 have shown that two neurochemical phenomena present in the striatum also occur in the HAS-TO: a benztropinesensitive dopamine uptake and a dopamine-sensitive adenylate cyclase activity. This finding suggests that the dopamine receptors act equally in both dopaminergic tracts since these receptors appear to be functionally linked to adenylat: cyclase. Similarly, antipsychotic drugs are approximately as effective in increasing dopamine metabofism in the limbic forebrain structures as they are in the striatum89. 46. Finally, it was shown by several workersT,x0 that the dopamine-rich HAS and TO possess a high concentration of acetylcholine as well as elevated choline acetyltransferase and cholinesterase activitiesg,Xg,ga,27. A flurry of reports in the past few years showed that dopaminergic agonists and antagonists affected the release, level and turnover of acetylcholine in the striatum and therefore that a link existed between these two neuronal systemsx°.xx.xS.9+x,aa,aS. No data is presently available on the relationship between the dopaminergic and cholinergic pathways in the rat HAS-TO. It was of interest to establish whether a link exists by determining if the level of acetylcholine in the HAS-TO is altered by dopaminergic agonists and antagonists. MATERIALS AND METHODS Female CDx rats of 210-220 g body wt. were killed by decapitation and the head was immediately immersed in liquid H2 for 6-7 sex. The nucleus accumbens septi (HAS) together with the tuberculum ~lfactorium (TO) were isolated as described by Koob et al. TMunder n-pentane at --5 °Cs and then frozen in liquid H2. The striatum was also dissected out from other animals under n-pentane. Each tissue was weighed in the frozen state and pulverized e2. Acetylcholine and choline were determined by the radiochemical method of Saelen~ et al. 2a with some modifications~0 and the tissue concentration of these amines is expressed as nmoles]g fresh wt. Pretreatment with 6-hydroxydopamine (6-OHDA) was performed as follows: the rats were given desmethylimipramine, 25 mg/kg, i.p., 30 min prior to the intracerebral administration of 6-OHDA in order to prevent noradrenergic fiber degeneration~. 6-OHDA was perfused unilaterally under light ether anesthesia into the dopamine axon bundle in the area ventralis tegmenti (AVT) of the right side through a stainless steel cannula ~tereotaxicaUy implanted x7 according to the coordinates A 2.6; L 1; H --2.3. 6-OHDA hydrochloride (8/~g calculated as the free base) was dissolved in saline (4/41) containing ascorbic acid (1 mg/ml) to prevent oxidation. The solution was delivered at a constant rate of I/d/min witn a Harvard pump. Vehicle-treated groups received desmethylimipramine, i.p. and saline--ascorbic acid solution intracerebraily. The experim~.r,ts were performed 9 days after 6-OHDA perfusion, This treatment reduced dopamine in the ipsilateral striatum by about 95 ~ and in the ipsilateral HAS-TO by about 85-90~ as measured by the method of Palkovits et al. ae without affecting noradrenaline content.

257 The following drugs were used: apomorphine hydrochloride and pimozide (gifts from Janssen Pharmaceutica, Beerse) dissolved in distilled water and 0.2 M D-tartaric acid, respectively; piribedil monomethylsulfonate (gi~'t from Servier Laboratories, Paris) dissolved in distilled water; D-amphetamine sulfate (gift from Recordati, Milan) dissolved in distilled water; CB 154 methanesulfonate (2 Br-a-ergocryptine) and clozapine (gifts from Sandoz, Basel) dissolved in I part ethanol plus 19 parts propylene glycol and distilled water, respectively; haloperidol (gift from Lusofarmaco, Milan) dissolved in distilled water with the dropwise addition of I N HCI; chlorpromazine hydrochloride (gift from Farmitalia, Milan) dissolved in distilled water; trihexyphenidyl (gift from Lederle Laboratories) suspended in 0.5 % carboxymethylcellulose; diazepam (gift from Roche, Italy) dissolved in 1 part diethylacetamide plus 10 parts Tween 80 plus 39 parts distilled water. Picrotoxin (purchased i'rom Sigma) was dissolved in distilled water. All drugs, except diazepam, were administered intraperitoneally. The doses refer to the salts. Control animals received the appropriate volume of saline or vehicle. RESULTS Pimozide (0.5-2 mg/kg), haloperidol (0.5-4 mg/kg), c h l o r p r o m a z m e (15 and 25 m g / k g ) a n d clozapine (15 a n d 60 mg/kg), did n o t alter the acetylcholine level in the N A S - T O (Table I) at times at which these drugs are known to produce a d o p a m i n e receptor blocking activity in the n.accumbens septi. These doses were 2-8 times higher t h a n the m i n i m u m doses producing m a x i m u m decreases in the acetylcholine level o f the striatum. Table 11 shows t h a t a m o n g the dopaminergic agonists used in this TABLE I Comparison of the action of some central dopam e receptor antagonists on the acetylcholine level in the NAS-TO and striatum of the rat Drug

Dose Time ( min) (mglkgi.p.)

Solvent Pimozide

0.5 1.0 2.0

240 240 240

0.5 1.0 4.0

60

Solvent

Haloperidol Saline Chlorpromazine Saline Clozapine

--Ii5 25 15 6O

60 60 -60 60 -60 60

* P < 0.01, Dunnett's test or Student's t-test.

NAS-TO Striatum (nmole/g wet wt. + S.E.) (nmole/g wet wt. ± S.E.)

33.3:1:1.4 -34.3 4- 1.4 32.4 -t- 1.7 31.2 ± 1.3

(6)

30.4 4- 0.6 12.9 -t- 0.4 11.1 ± 0.5 -29.0 4- 0.8

(6) (6)* (6)*

--

11.4 -t- 1.1

(8)*

-31.6 4- 1.3 (5) 33.8 -4- 1.7 (6)

11.6 ± 0.6 -28.6 ± 0.6 20.3 ± 2.5 -30.3 :t: 0.8 23.3 :[: 0.6 22.8 -t- 1.4

(6)*

(6) (6) (5)

--

37.0 -4- 1.8 34.3 :[: 1.0 36.7 4- 1.6 38.0 4- 1.5

(6) (12) (6) (11)

(7)

(6) (6)* (6) (6)* (6)*

258 TABLE I!

Co~n o f the action o f some central dopamine receptor agonists on the acetylcholine ievel in the NAS-TO and striatum o f the rat Drug

Dose Time (rain) (mglkgi.p.)

NAS-TO Striatum (nmolelg wet wt. + &E.) (nmolelg wet wt. 4-&£.)

Saline Apomorphine

-9.5 1.0 4.0

-30 30 30

32.0 4- 0.9 (17) m 34.4 4- 1.$ (13) 38.8 4- 1.2 (14)*

-15 60 120

-30 30 30

31.8 42.2 40.5 47.3

-$ 15 15 15

-30 30 60 120

-5 15

-120 120

Saline

Piribedil

Saline D-Amphetamine

Solven: Bromocriptine

--

(6) (6)* (6)* (6)*

28.2 -4- 1.2 (6) 43.7 -I- 4.2 (6)* 53.1 4- 3.3 (6)*

32.6 -I- 0.6 0 6 ) 32.9 -4- 1.6 (9) 32.5 -4- 1.0 (6) 31.7 4- 1.9 (6)

28.0 4- 0.5 (9) 41.1 4- 2.4 (9)* ----

30.7 4- 0.9 (5) 33.2 4- 2.0 (5) 34.0 4- 1.1 (5)

30.2 4- 0.9 (11) 58.7 4- 2.1 (11)* --

-

4- 1.2 -4- 1.7 4- 1.5 4- 1.2

30.5 4- 1.1 (9) 48.6 4- 1.7 (9)* 50.5 -4- 2.3 (6)*

-

* P < 0.01, Dunnett's test or Student's t-test.

study (D-amphetamine, apomorphine, piribedil and bromocriptine) only piribedil and apomorphine increased the acetylcholine level in the NAS-TO. However, the dose of apomorphine which yielded a 21% increase was 8 times higher than the minimum dose required to produce a maximal increase of about 60 % in the striatum. None of the dopaminergic antagonists or agonists altered the level of choline in the NAS-TO (data not shown). The normal level of choline was 71.3 -4- 1.6 nmole/g wet wt. (mean 4- S.E.). The dose-response effects of piribedil on the acetylcholine level in the striatum TABLE I!I

Lack o f effect of pimozide or 6-OHDA pretreatment on the increase in the acet¥1choline level produced by piribedil in the NAS-TO o f the rat The animals were killed 240 rain after pimozide and 30 rain after piribedil (120 mg/kg, i.p.). See Methods for 6-OHDA pretreatment. Acetylcholine was determined in the right NAS-TO ipsilateral to the 6-OHDA injection.

Drug in C and D

Pimozide (2 mg/kg, i.p.) 6-OHDA

Acetylcholine in NAS-TO (nmole/g wet wt. ~=S.E.) A, saline

B, piribedil

C, drug

D, drug + pirtbedi!

30.0 -4- 2.1 (6) 32.8 4- 0.7 (5)

46.7 -4- 2.0 (6)* 49.3 -4- 3.8 (5)*

33.9 -4- 2.7 (6) 38.2 -4- 1.2 (5)

43.9 -4- 1.8 (6)* 48.0 4- 0.6 (5)*

* P < 0.01 vs. control. There was no interaction between pimozide and piribedil or 6-OHDA and piribedil. Statistics: Factorial Experiment (2 × 2) Anova. Tuhey's t e s t - Tuhey's test for unconfounded means.

259 TABLE IV Lack o f e.Orectof pimozide on the apomorphine-induced increase in acetylcholine in the NAS-TO of the rat

Statistics: Factorial Experiment (2 x 2) Anova. Tuke/s test - - Tukey's test for unconfounded means. There was no interaction between pimozide and apomorphine. Drug

Dose (mg/kg i.p.)

Time ( r a i n )

Acetyicholine (nmole/g wet wt. ± S.E.)

Controls Apomorphine Pimozide Pimozide -I- apomorphine

-4 1 1 4

30 240 210 3O

31.4 + 38.9 i 34.3 + 39.9 +

1.7 2.3 1.4 1.6

(6) (6)* (6) (6)*

* P < 0.05 vs. controls. and in the NAS-TO are different. Wherea~ piribedil in the striatum produces a dosedependent response between 15 and 60 mg/kg (Table II), in the N A S - f O it increased the acetylcholine content maximally to about the same extent (40 %) at doses of 15-120 mg/kg. The increase in acetylcholine in the NAS-TO by piribedil was not prevented by prior treatment with either pimozide or 6 - O H D A (Table III). Thus its action is probably not mediated directiy or indirectly through the dopaminergic system. Similarly, pimozide pretreatment did not block the action of apomorphine (Table IV). Table V shows the effect of some other classes of drugs on the acetylcholine level in the NAS-TO. Picrotoxin at a dose of 2 mg/kg increased the acetylcholine concentration in the rat striatum by about 60% at 30 min after its administration but had no affect on the acetylcholine level in the NAS-TO. The inability of this dose of picrotoxin to affect acetylcholine in other areas investigated, i.e. the cerebellum, diencephalon, mesencephalon, cerebral hemispheres and hippocampus, w~ts shown previously 9°. At this dose, picrotoxin produced convulsions in about 90 % of the animals within 30 min. Doses above 2 mg/kg were not used because they produced high mortality within 30 min. The antimuscarinic drug, trihexyphenidyl 10 mg/kg, proTABLE V Comparison of the action of some other drugs on the acetylcholine level in the NAS-TO and striatum

Drug

Dose Time (rain) NAS-TO Striatum (mglkg, Lp.) (nmolelg wet wt. + S.E.) (nmole/g wet wt. ~ S.E.)

Saline Picrotoxin

Vehicle Trihexyphentdyl Solvent Diazepam

--

2 10

30 -60 --

5 (i.v.)

* P < 0.01, Student's t-test.

15

33.8 + 1.6 33.5 + 1.'. 33.6 -I- 1.7 19.9 -I- 0.8 31.7 ± 1.2 40.4 u_ 2.4

(6) (6) (6) (6)* (11) (11)*

30.5 + 0.9 49.9 + 2.9 29.7 ± 1.4 14.9 :t: 0.6 28.4 + 0.5 42.7 + 1.8

(9) (8)* (6) (6)* (9) (9)*

260 TABLE VI

EITect o f pimozide on the diazepam.induced increase in acetylcholine in the NA$-TO o f the rat Statistics: Factorial Exlgriment (2 × 2) Anova. Tukey's test - - Tukey's test for unconfonnded means. There was no interaction between pimozide and diazepam.

Drug

Dose (mg/kg i.p.)

Time (rain)

Acetylcholine (nmole/g wet wt 4- S.E.)

Controls Dia~.~am Pimozide Pimozide ÷ diazepam

-5 0.v.) 1 1

-15 240 225

32.0 4- 1.2 41.2 4- 1.7 33.0 4- 1.5 38.4 4- 1.5

5 (i.v.)

15

~ (6)* (6) (6)*

* P < 0.01 vs. controls.

duced a marked decrease in the level of this quaternary amine while diazepam 5 mg/kg, a benzodiazepine, increased this level. Both drugs produced approximately equivalent changes in the striatum as well. Pimozide pretreatment did not block the effect of diazepam on acetylcholine (Table VI). DISCUSSION

All of the powerful dopaminergic agonists and antagonists employed in this study were shown by several workers 8s-40 to act equally on dopamine metabolism in different rat brain structures. However, they failed to alter the acetylcholine level in the NAS-TO at doses of up to one order of magnitude higher than those which produced their effects in the striatum with the exception of apomorphine and piribedil. The increase in acetylcholin~ produced by these drugs was not blocked by pimozide or 6-OHDA pretreatment. These data suggest that a dopaminergic--cholinergic link similar to that in the striatum is not present in the NAS-TO. These results are in accordance with release experiments 5.34 showing that chlorpromazine and haloperidol, contrary to that found in the caudate, did not modify acetylcholine liberation from the perfused lqAS of the cat. Fig. 1 depicts our concept of the difference in the relationship between the dopaminerglc and cholinergic neurons in the NAS-TO and in the striatum. In the latter structure, the dopaminergic neurons are linked in series to cholinergic interneurons whereas in the NAS-TO system the two types of neurons appear to have separate, independent pathways. The possibility c,f a ~opaminergic-cholinergic link arising in the mesolimbic dopaminergic system and terminating elsewhere cannot be excluded. However, the lack of effect of piribedil 91, D-amphetamine 81, chlorpromazines~ and haloperidol x0 on the acetylcholine level in all brain areas with the exception of the striatum speaks against this possibility. Thus, these two areas (NAS-TO vs. striatum), which contain biochemically similar dopaminergic and cholinergic components, appear to be organized differently.

261

NAS-TO

Striatum

Fig. 1. Concept of the difference in the relationship between dopaminergicand cholinergicneurons in the n.accumbens septi-tuberculum olfactorium and in the striatum of the rat. The dopaminergic neurons appear to be linked in series to cholinergicinterneurons in the striatum while in the NAS-TO the two types of neurons appear to form part of separate, independentpathways. The location of the cell bodies of the cholinergicneurons terminating in these limbicareas is unknown. A hypothesis for the inhibitory gabergic regulation of nerve impulse flow to the striatal dopaminergic nerve terminals was set forth by And6n et al. 1,3, and Stock et al. 36 and supported by the experiments of Bartholini and Stadler 4 showing that GABA decreased dopamine output from the cat caudate while both picrotoxin and bicuculline, GABA receptor blockers, markedly increased the dopamine outp~,t. Furthermore, we suggested that picrotoxin increased the acetylcholine level in ~ striatum, not directly, but through a ~equential gabergic (inhibitory)-dopaminerg~ (inhibitory)-cho~inergic reaction z°. It follows that the negative effect of picrotoxia on the acetylcholine level in the NAS-TO, where an inhibitory modulation of DA neurons by GABA also appears to exist 14, is a consequence of a lack of a dopaminergic-cholinergic link in this brain area. It has been proposed that clozapine has both central antimuscarinic and antidopaminergic actions 25.~9,83, either of which effect could account for its decreasing rat striatal acetylcholine10. If trihexyphenidyl decreases the acetylcholine content in the NAS-TO specifically through its antimuscarinic action, then it is reasonable to suppose that clozapine lacks substantial antimuscarinic action in vivo in this region where it failed to alter the acetyicholine level. Diazepam increased the acetylcholine level in the NAS-TO in the same proportion as it did in the hippocampus and striatum 12 and fits in with electrophy~iological studies indicating an action of this drug on limbic areas ao. Based mainly upon biochemical studies in the cerebellum, it has been suggested that diazepam has GABA-Iike activity24. If one assumes that GABA neurons inhibit cholinergic cells directly in the NAS-TO, then diazepam might increase the acetylcholine level in this region by mimicking GABA. However, the lack of effect of picrotoxin in the NAS-TO would speak against such a mechanism in this limbic region. Furthermore, there is no evidence at present to indicate that this benzodiazepine acts on the cholinergic system directly or indirectly through a G:~,BAergic mechanism in any brain area. In conclusion, from these data it appears evident that no link exists between dopaminergic and cholinergic neurons within the n. accumbens septi-tuberculum

262 olfactorium. A t this point it is interesting to speculate that the p h e n o m e n o n o f inhibitory dopamhlergic neurons modulating cholinergic activity, and perhaps function, is unique to the striatum a n d may be one o f the features that characterizes this brain region. However, we cannot exclude the possibility t h a t there exist in the striatum links o f the cholinergic system with neurotransmitters other than dopamine. ACKNOWLEDGEMENT We thank Mr. M. Recchia for advice on statistical handling of the data.

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