Evidence of an interaction between serotoninergic and cholinergic neurons in the corpus striatum and hippocampus of the rat brain

Brain Research, 151 (1978) 73-82 © Elsevier/North-Holland Biomedical Press

73

EVIDENCE OF AN I N T E R A C T I O N BETWEEN S E R O T O N I N E R G I C A N D C H O L I N E R G I C N E U R O N S IN T H E CORPUS S T R I A T U M A N D HIPPOCAMPUS OF T H E RAT BRAIN*

R. SAMANIN, A. QUATTRONE**, G. PERI, H. LADINSKY and S. CONSOLO Istituto di Ricerche Farmacologiche 'Mario Negri', Via Eritrea 62, 20157 Milan (Italy)

(Accepted December 1st, 1977)

SUMMARY The existence of an interaction between serotoninergic and cholinergic neurons in the brain has been investigated by studying the effects of quipazine and D-fenfluramine on regional brain acetylcholine in various experimental conditions. Quipazine, at a dose of 10 mg/kg, i.p., significantly increased the levels of acetylcholine in the striatum and hippocampus but not in the telencephalon and brain stem. The striatal increase was not significantly modified by electrolytic lesions placed in the midbrain raphe nuclei, an important site of origin of serotonin-containing neurons in the brain. On the other hand, pretreatment with serotonin antagonists such as methergoline and cinanserin or with parachlorophenylalanine, a serotonin synthesis blocker, prevented the increase of striatal acetylcholine induced by quipazine. Impairment of nigrostriatal dopaminergic mechanisms by local application of 6hydroxydopamine or by pretreatment with alpha-methylparatyrosine did not modify the effect of quipazine on acetylcholine. The quipazine-induced increase in hippocampal acetylcholine was instead completely blocked by an electrolytic lesion of the nucleus medianus raphe. D-Fenfluramine also significantly increased striatal acetylcholine, this effect being completely prevented by parachlorophenylalanine pretreatment. These findings are compatible with the hypothesis that serotoninergic neurons originating in the raphe nuclei may normally serve to inhibit cholinergic neurons in two areas of the rat brain, i.e. the corpus striatum and the hippocampus.

INTRODUCTION Changes of striatal acetylcholine (ACh) levels and release have been found by * A preliminary report on these studies was presented at the Symposium on Interactions between Putative Neurotransmitters in the Brain, Milan, October 26-28, 1976. ** Present Address: Clinica Neurologica, Universitb.di Messina, Messina, Italy.

74 various authors following administraUon of drugs believed to exert a relatively specific effect on dopaminergic mechanisms in the brainl,10,z4, 36. These findings have led to the suggestion that cholinergic neurons in the striatum might be under an inhibitory influence of dopaminergic nerve terminals whose cell bodies are localized mainly in the pars compacta of the substantia nigra 2. The striatum contains relatively high concentrations of serotonin (5-HT) 2,5 and an interaction between 5-HT and ACh in this area has been suggested in relation to Parkinson's disease 4, and by some recent studies in the rat 7,12. In the present experiments we investigated the effects of quipazine, a central 5-HT receptor stimulant 31,3~,34, on the ACh concentrations in the striatum and other brain areas of the rat, and whether changes in ACh induced by quipazine are mediated through a serotoninergic mechanism. The effect on striatal ACh concentration of Dfenfluramine, a 5-HT releaser 1~,16, was also studied. MATERIALS AND METHODS Female CD1 rats (Charles River, Italy), weighing 200 ~ 20 g, were used. The animals were housed in groups of 6 in plastic (Makrolon) cages at constant room temperature (22 °C) and relative humidity (60 ~o). They were fed ad libitum and maintained under a light-dark cycle (12 h dark, 7.00 p.m.-7.00 a.m., and 12 h light). All the experiments were performed at the same time of the day to avoid interferences due to circadian variations. In all the experiments quipazine maleate was administered at a dose of 10 mg/kg, i.p., and the animals were killed 60 min later for biochemical assay. The following doses and time schedules were used in the pretreatments with various compounds. Methergoline maleate (6 mg/kg, i.p.) and cinanserin hydrochloride (10 mg/kg, i.p.) were dissolved in saline and injected respectively 3 and I h before quipazine. Parachlorophenylalanine (PCPA) was suspended in 0.5 ~ carboxymethylcellulose and given in 3 consecutive daily oral doses of 100 mg/kg. Quipazine was administered 24 h after the last PCPA administration; alpha-methylparatyrosine (a-MPT) methyl ester HC1 was administered as two consecutive injections of 300 mg/ kg, i.p., and 200 mg/kg, i.v., respectively 20 and 4 h before quipazine. I)-Fenfluramine hydrochloride was dissolved in saline and injected intraperitoneally at a dose of 7.5 mg/kg 60 min before the animals were killed.

Electrolytic and chemical lesions Separate electrolytic lesions of the raphe nuclei medianus (MR) and dorsalis (DR) or a concomitant lesion of both nuclei were made using a 2.5 mA direct current for 15 sec with a stainless steel electrode (tip 0.5 mm, diameter 0.3 mm) implanted stereotaxically according to the following co-ordinates: MR, A 0.4, L 0, H - 2 . 6 and DR, A 0.4, L 0, H --0.62L Control animals were similarly operated on but not lesioned. Lesion placement was verified in each animal after removal of the experimental tissue samples. The brain stem (diencephalon + mesencephalon q- pons q- medulla oblongata)

75 was placed in 5 ~ formalin. After adequate fixation, sections were cut at 20 #m and stained with cresyl violet. 6-Hydroxydopamine (6-OHDA) hydrobromide was dissolved in saline containing ascorbic acid (1 mg/ml) and injected through an infusion pump (Harvard mod. 975) at the level of the right nigrostriatal pathway according to co-ordinates A 2.6, L 1, H --2.32~. Eight #g of 6-OHDA (calculated as free base) in 4 #1 were administered at a rate of 1 #1 per min. The animals received desipramine (DMI) 25 mg/kg, i.p., 30 min before the 6-OHDA in order to protect noradrenergic terminals from the action of the latter% Control animals received DMI and were injected intracerebrally with an equal volume of the vehicle. Biochemical assay

For the acetylcholine assay, the rats were killed by decapitation and the head was immediately immersed in liquid N2 for 6-7 sec. The brain stem, striatum and hippocampus of the right side and remaining telencephalon were isolated under n-pentane at - - 5 °C and frozen in liquid N2. Acetylcholine was determined by the radiochemical method of Saelens et al. 3z with some modifications z3 and the tissue concentration of the amine was expressed as nmoles/g wet wt. Choline-O-acetyltransferase activity was determined by the radiochemical method of McCaman and Hunt zs with slight modification 9, and cholinesterase activity by the method by McCaman et al. 29. Separate groups of animals, selected at random, were used for dopamine (DA) and serotonin (5-HT) assays. The brains were removed immediately after decapitation and the right striata and hippocampus dissected on a plate placed on dry ice. Striatal DA was determined fluorimetrically according to Chang s. 5-HT was assayed in the striatum and hippocampus by the method of Curzon and Green 11. The data were statistically analyzed by Anova (2 × 2) factorial analysis and Tukey's test for unconfounded means. RESULTS Quipazine, at a dose of 10 mg/kg, i.p., significantly increased the level of ACh in the striatum and hippocampus but not in the telencephalon and brain stem (Table I). The drug did not affect choline-O-acetyltransferase or cholinesterase activities of striatal and hippocampal homogenate in vitro at a concentration of 20 #g/ml. Table II gives the data on the effect of quipazine in animals lesioned in the midbrain raphe nuclei. Electrolytic lesions placed in the nucleus medianus raphe or the nucleus dorsalis raphe or in both nuclei produced marked decreases of 5-HT concentrations. Representative lesions of both raphe nuclei are shown in Fig. 1. A slight but significant increase of basal levels of ACh was found in animals lesioned in the nucleus medianus raphe. The effect of quipazine on striatal ACh was not significantly modified by the lesions. On the other hand, quipazine was completely blocked in animals pretreated with methergoline or cinanserin (Table IID. Pretreatment with PCPA also significantly

76 TABLE I Effect of quipazine on acetylcholine levels in rat brain

Quipazine was administered at a dose of 10 mg/kg, i.p., and the animals were killed 60 min after treatment. The number of animals for each experiment is indicated in brackets. Brain area

Acetylcholine (nmole/g ± S.E.) Controls

Striatum Hippocampus Telencephalon Brain stem

30.5 ~ 2.0 15.5 4- 0.6 13.3 4- 1.8 20.5 4- 0.7

Treated

(4) (9) (4) (4)

40.7 4- 1.5' (4) 20.2 4- 0.6* (9) 11.6 4- 0.3 (4) 19.7 ± 0.6 (4)

* P < 0.01 (Student's t-test). TABLE lI Effect of quipazine on striatal acetylcholine levels of rats lesioned in the nucleus medianus ( M R ) or dorsalis (DR) raphe or both ( M R + DR)

Quipazine was administered at a dose of 10 mg/kg, i.p., and the animals were killed 60 min after treatment. Statistics: unweighted means analysis for completely randomized factorial (2 × 3) or (2 × 2) design. F interaction not significant. !!: Treatment

Striatal levels Acetylcholine (nmoles/g 4- S.E.)

Serotonin (ng/g 4- S.E.)

Controls

Quipazine

Sham-operated MR-lesioned DR-lesioned

30.2 ± 1.1 (5) 36.4 ± 0.6*** (5) 32.4 :~ 1.5 (9)

38 ! 1.7'* (15) 44.4 i 2.3** (8) 37.6 4- 1.8"* (8)

321 ± 14 206 ± 10" 146 i 11"

Sham-operated DR + MR-lesioned

30.7 4- 1.4 33.6 i 1.8

41.1 £ 2.8** (7) 45.6 4- 3.2** (7)

300 4- 10 35 ± 1*

(7) (7)

* P < 0.01 compared with sham-operated animals (Duncan's test). ** P < 0.01 vs. control group. *** P < 0.05 vs. the sham-operated group.

reduced the quipazine-induced increase in striatal A C h (Table III). F u r t h e r m o r e , neither injection of 6 - O H D A at the level of the nigrostriatal dopaminergic pathway, which markedly decreased striatal D A , n o r pretreatment with a - M P T significantly modified the effect of quipazine on striatal A C h (Table IV). D-Fenfluramine significantly increased the level of A C h in the striatum, a n effect which was completely prevented by P C P A p r e t r e a t m e n t (Table V). The increase in striatal A C h appears to be specific for the D-isomer of fenfluramine. The L-isomer, by contrast, tended to produce a decrease in striatal A C h (data n o t shown). The increase in h i p p o c a m p a l A C h induced by quipazine was completely blocked in animals lesioned in M R (Table VI).

77

Fig. 1. Photograph of representative combined lesions of nucleus medianus and dorsal raphe. The lesioning electrodes were directed to the target areas according to the following co-ordinates given in the K6nig and Klippel rat brain atlas (1963): nucleus medianus, A 0.4, L 0, H --2.6 and nucleus dorsalis, A 0.4, L 0, H ---0.623.

DISCUSSION Quipazine increases the levels o f acetylcholine in the striatum and hippocampus but not in the telencephalon and brain stem. The regional specificity o f its effects, together with the fact that quipazine does not affect in vitro choline acetyltransferase or cholinesterase, argue against it acting directly on cholinergic neurons. Quipazine is, however, k n o w n to activate serotoninergic transmission in the brain 17,19,

78 TABLE III Effect of methergoline, einanserin and parachlorophenylalanine increase in acetylcholine in the rat striatum

(PCPA) on the quipazine-induced

Methergoline was suspended in 0.5 ~ carboxymethylcellulose and administered at a dose of 6 mg/kg, i.p. Cinanserin was administered at a dose of l0 mg/kg, i.p. Quipazine was administered at a dose of 10 mg/kg, i.p. The animals were killed 60 min after quipazine, 180 rain after methergoline, 90 min after cinanserin and 24 h after the last administration of PCPA (see Methods). Treatment

Acetylcholine (nmole/g ± S.E.) Saline

Quipazine

Control Methergoline

31.8 ± 1.1 (6) 27.6 ± 0.6 (6)

39.3 zk 2.2* (6) 27.9 + 2.0 (6)

Control Cinanserin

31.0 4- 0.8 (6) 35.1 ± 2.3 (6)

42.2 4- 1.0" (6) 35.4 :k 2.7 (6)

Control PCPA

27.8 ± 1.6 (6) 31.2 ± 2.0 (6)

46.7 ± 2.0** (6) 37.8 + 2.0**(6)

* P < 0.01 vs. all the other groups. P < 0.01 vs. saline group. Methergoline-quipazine: F interaction 4.9 (1,20 df) P < 0.05. Cinanserin-quipazine: F interaction 8.4 (1,20 df) P < 0.05. PCPA-quipazine: F interaction 11.2 (1,20 d f ) P < 0.01. **

TABLE IV Effect of lesioning of the nigrostriatal dopaminergic path way with 6-OHDA or blockade of catecholamine synthesis with alpha-methylparatyrosone (a-MPT) on the quipazine-induced increase in ACh in the rat striatum The experiment was performed 10 days after 6-OHDA administration (see Methods). The animals were killed 60 min after quipazine (10 mg/kg, i.p.) and 4 h after the last dose of a-MPT (see Methods). Treatment

Acetylcholine (nmole/g -k S.E.) Vehicle

Quipazine

Control 6-OHDA

30.5 ± 0.8 (8) 34.9 + 0.9 (8)

39.1 ± 0.4* (8) 50.9 ± 3.9** (8)

Control a-MPT

30.9 ± 1.0 (6) 36.5 ± 2.4 (6)

43.3 -k 2.6* (6) 49.6 ± 2.9* (6)

* P < 0.01 vs. vehicle. ** P < 0.01 vs. all other groups. F interaction not significant. 20,31,3a,34. T h e r e f o r e , t h e p o s s i b i l i t y was e x a m i n e d in t h e p r e s e n t s t u d y t h a t t h e d r u g m i g h t influence c h o l i n e r g i c n e u r o n s t h r o u g h its a c t i o n o n s e r o t o n i n . S e r o t o n i n e r g i c n e u r o n s o r i g i n a t i n g in the n u c l e u s m e d i a n u s a n d dorsalis r a p h e h a v e b e e n r e p o r t e d to h a v e different d i s t r i b u t i o n s in v a r i o u s b r a i n areas, i n c l u d i n g t h e s t r i a t u m 26. T h e r e f o r e , in this s t u d y s e p a r a t e a n d c o m b i n e d e l e c t r o l y t i c lesions

79 TABLE V The effect of parachlorophenylalanine (PCP.4) on the increase of striatal ACh induced by o-fenfluramine

The animals were killed 60 min after D-fenfluramine, 7.5 mg/kg, i.p. Treatment

Vehicle D-Fenfluramine

Acetylcholine (nmole/g 4- S.E.) Controls

PCPA

33.2 4- 1.1 (11) 50.1 ± 2.2* (11)

33.6 4- 1.2 35.7 -4- 3.3

(11) (11)

* P < 0.01 vs. all other groups. Finteraction = 12.1 (1, 40 df) P < 0.01. TABLE VI The effect of quipazine on hippocampal acetylcholine in rats with lesions of the nucleus medianus raphe (MR)

The animals were killed 60 min after quipazine (10 mg/kg, i.p.). Hippocampal levels of serotonin in controls and MR-lesioned rats were respectively 298 4- 13 and 36 q- 5 (ng/g 4- S.E.). Treatment

Saline Quipazine

Aeetylcholine nmole/g 4- S.E. Sham

MR-lesioned

17.6 ± 0.8 (14) 20.3 -4- 0.6* (14)

17.3 i 0.4 (14) 16.9 4- 0.4 (14)

* P < 0.01 vs. all other groups. Finteraction = 7.29 (1,52 df), P < 0.01.

o f these nuclei were made in an a t t e m p t to distinguish their possible roles in the mediation o f the effects of quipazine. The basal levels o f striatal A C h were not significantly modified by lesions o f the nucleus dorsalis or b o t h nuclei, while a small increase was observed in the animals lesioned in the nucleus medianus. This effect has been also f o u n d by Butcher et al. 7, who interpreted the increase as due to destruction of excitatory 5-HT neurons projecting u p o n nigral neurons which, in turn, exert a facilitatory influence on striatal cholinergic interneurons. The end result is inhibition o f the cholinergic neurons. However, the m a n y reports indicating an inhibitory influence of nigroneostriatal D A neurons u p o n striatal cholinergic interneurons 1,1°,24,38 ought to be taken into account. Obviously, it c a n n o t be excluded that other mechanisms are involved in this effect. Raphe lesions did n o t significantly affect the quipazine-induced increase o f striatal ACh. This does n o t necessarily exclude the involvement o f 5-HT in the effect o f quipazine since the drug, in addition to acting on presynaptic 5-HT nerve terminals, can directly stimulate postsynaptic receptorslg,31,aa, 34. On the other hand, reduction o f quipazine effects by P C P A at a dose and schedule reported to be fairly specific for blocking 5-HT synthesis ~1 suggests that there also m a y be a presynaptic serotoninergic

80 component in the mechanism by which this drug increases striatal levels of ACh in the rat. These differences in results could be due to the presence of a small but functionally important 5-HT neural population left intact in raphe-lesioned animals and/or to the development of denervation supersensitivity which unmasks the direct action of quipazine on postsynaptic receptors. Although some controversy exists as to the specificity and efficacy of peripheral 5-HT antagonists in blocking 5-HT receptors TM, the hypothesis that the effects of quipazine on ACh are mediated through the serotoninergic system is further supported by the fact that methergoline and cinanserin, two 5-HT antagonists a,27, completely prevented its effects. It has been reported that quipazine inhibits dopamine uptake into nerve terminals, although at concentrations well above those required to inhibit 5-HT uptake13, a0. In view of the dopaminergic-cholinergic interaction in the striatum suggested by various authors1,1°,24, 36, the effect on dopamine might also be involved in the mechanism by which quipazine increases striatal ACh. However, this possibility appears to be ruled out by the fact that pretreatment with alpha-methylparatyrosine, a potent, specific blocker of catecholamine synthesisa~, or injection of 6-OHDA directly into the nigrostriatal dopaminergic pathway, which causes almost complete disappearance of striatal dopamine, did not significantly affect the quipazine-induced increase of striatal ACh. While these studies were in progress, Euvrard et al. 12 reported a similar effect of quipazine alone and in combination with 6-OHDA and PCPA on striatal ACh. The data obtained in the present study with D-fenfluramine also suggest that cholinergic neurons in the striatum are regulated in some manner by serotoninergic neurons. In fact, D-fenfluramine, which releases 5-HT from presynaptic terminals and inhibits its reuptake15, ~6, markedly increased striatal ACh concentrations, this effect being significantly reduced by pretreatment with PCPA. The increase of hippocampal ACh induced by quipazine appears to depend on a serotoninergic mechanism as well, since it was completely prevented by electrolytic lesions of the nucleus medianus raphe, which is known to provide almost all the serotoninergic innervation in the hippocampus 26. This finding suggests that in the hippocampus quipazine must depend mainly on 5-HT release, while in the striatum there is both pre- and postsynaptic action. Euvrard et al. 12 found no significant changes of hippocampal ACh concentrations following quipazine administration. Since the only apparent difference between these two studies is that Euvrard et al. used a higher dose of quipazine (30 mg/kg, i.p.), it is possible that additional mechanisms present at higher doses may have contributed to masking the serotonin-dependent effect of quipazine on hippocampal acetylcholine. Although information is needed on the anatomical and physiological connections between serotoninergic and cholinergic neurons in the brain, these findings, in analogy with other observations 1,25, are compatible with the hypothesis that 5-HT may normally serve to reduce acetylcholine release at least in two brain areas of the rat, i.e. the corpus striatum and the hippocampus.

81 ACKNOWLEDGEMENTS The a u t h o r s t h a n k Mr. M. Recchia for the statistical analysis o f the data. Quipazine maleate was kindly d o n a t e d by Miles Lab., I n d i a n a , U.S.A.

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