P-11-2 Serotonin4 receptors in the rat brain are not located on dopaminergic nerve endings

404

P-l l Basic neuroscience and neuropsychopharrnacology

iS, at least partially, cor'trolled by somato-denorltlc serotonerglc 5 m-1~. a u toreceptors in bralnstem rapne nuclei Thus, stimulation of such receptors by the systemic or local administration of the 5-HT1A autoreceptor ago nist 8-hydroxy-2-(di-n-prepylamino)tetrahn (8-Or4-DPAT) results in decreased forebrain 5-HT synthesis and release (see H,Ilegaart 1991) These oPser rations prompted the present study where we investigated *,he effects ot 8-OH-DPAT on treadmill locomotion in the rat Adult male Wistar rats were trained for two consecutive days to walk on the treadmill (~ 166 m m 16 "pm), corresponding to a speed o ~ approximately8 m rain 1 On thetqird gay, separate groups of ammals(n -- 6-17) were gwen one of 5 d~fferent doses of 8-OH-DPAT Hi~r (0 05, O 1 0.2, 0 4 or 0.8 mg kg 1 SC) and were placed on thetreadm~lt 10 mln after injection Controls(n 17)were given thesaline vehicle(2 mL kg 1) The time the anlmais remained on the treadmill was recorded up to a maximum of 40 rain 8-OH-DPAT (rag kg' SC) 000

005

010

Time(rnin) 8 2 ± 4 9 133±170 ns 3 4 1 : 1 5 6 "

020

040

080

!02:~-18 ~ns 12±198 ns 0 7 1 0 8 " "

*'P<002 The results are presented as medqaqs ± seml-lnterguartlle ra'lge As shown in the Table, there was a ma'ked, and statistically significant in crease in duration of treadmill locomotion at the 0.1 mg dose of 8-OH [}PA~whereas the performance gradually deteriorated with higher doses The re suits shown, in all probabihty, reflect preferential pre- and post-synaDtlc effects of 8 OH DPAT at dram 5-H-1A receptors. The results also erowde strong support for the notion that increased availability of bra~n 5 HT cos tributes to fatigue from physical activity (see references above). it is concluded that 8-OH-DPA T produces biphasic effects on treadrrm s e r f o r m a n c e l n t h e r a t Enhanced duration at low doses (0.05 0 2 " ~ g k g !, and a decreased performance at bighe- doses ( 0 4 - 0 8 mg kg 1, These two effects in all Drobab,lity, reflects preferential stlmuiation ot ~re arc post-synaptlc serotonm receptors respectively by 8-OH DPAT

(6 OH DAJ This might suggest that, ~n the caudate putamen, the receptors are located on inteneurons, probably GABAergic. However. receptor binding studies in animals with lesions of neurochemical pathways are not conclusbve, since the density of presynaptic sites located on degenerating nerve endings could be low and masked by post-synaptic localization. A sensitive approach to evaluate the presynaptic localization of these heteroreceptors can be the functional assay of the effect of agonists and antagonists on [3H 1dopamine release from isolated and superfused striatal syqaptosomes. The present study, still in progress, was therefore designed to directly verify the presence of 5-HT 4 receptors on dopaminergic nerve endings ~n rat striatum. Our experiments were all conducted in the presence of 1 # M nomifensine, a monoamine uptake blocker, to prevent a possible action of the agonist via uptake carrier. We have found that BIMU8, a specific 5-HT 4 agonist, induced a concentration-dependent increase of [3H]dopamine release with doses ranging from 3 # M up to 30 # M . For further experiments we used the concentration giving an intermediate effect, that is 20/xM, which led to an overflow of 9.47 4- 0.83. This effect has been shown to de additive with that of K+ 15 raM, being the overflow produced by K+ alone 8.32 ± 0 19 and 15.83 ± 1.78 the one given by the combination of the two releasing stimuli We were not able to antagonize this releasing effect ustng a selective 5-HT 4 antagonist such as GR 113808 at the concentrat,on of 3 /zM, which is the highest possible since this drug has a releasing effect at higher concentrations. Other studies are in progress with @fferent agonists and antagonists. At present, the results obtained suggest that the ,eported modulation of 5-HT 4 receptors on dopamine release is not directly mediated, since these receptors do not seem to be located on eooamlnerglc nerve endings in the rat striatum.

References Benlouc I S r Keegan M J and Galloway M P (1992). Serotonin-facilitated dopamine release ,n rive pharmachological characterization J Pharmacol Exp Ther. 265, 373 377 Patel S , ReaviHC, RobertsJ and Moorman J (1994) Localizationof 5-HT4 receptors m the stnato-nigral pathway in rat brain Br J Pharmacol 113 Suppl.. 135R

References

I

Chaouloff, F. Laude. D and Elghozl J L (1989)Pr'vslcal exercise evidence lot d fferent a consequences of tryptophan oq 5 r~P synthesis and metabohsm in centra, serotoqer glc cell bodies and terminals J Neu,al Transm IGenSect 78 121 130 Bailey, S P, Davis JM and Ahlborn E N (~g92} Effect of increased braiP serctorerg, activity on endurance performance in the rat Acta Physiol Scand !45 75 77 Hillegaart, V {1991) Functional topography o~era q serotonerglc pathways in 1he ral Act: Phyaiol Scand 142 (Supal 598)

Serotonin4 receptors in the rat brain are not located on dopaminergic nerve endings D Crespi, M Gchbi, T Menn~n~ /st/tuto dlRtcerche Farrnaco/oglche

"'Mane Negn" Vm Entrea 62, 20757 Mi/ano, /~aly in human, guinea pig aqd rat bra qs, the nighest densities of seroto',~rq4 {5-HT4) receptors occur in the caudate #Jtamen, substantia nig~a, nucleus accumbens and globus palildus wRn moderate levels in the h~ppc:am pus and cortex Theserecepto~s, wh,ch arepositv@ycoupled t o a d e p v a t e cyc:ase, have been found to modulate the release of dopamlne in the n~ gro striatal s y s t e m In f a c t it has been shown that the administratlop o* 10,uM renzapride or 10/zM (S)-zacopride both 5-HT 4 agon~sts, err*,arcee the release of dopamme in vitro in ra'~ stnata; shoes (Steward and 3ames unpublished) In another functlor'a~ study, done (n rive using the mlcrodlal ysls technique, it has beer- found that tqe perfuslon of antenor s t n a t , / t with 5-methoxytryptamlne(5-MT) a qonoselective 5-HT agomst, fac, lt~:ee dopamlne release, and the coperfuslon with ICS 205 9 3 0 a weak ,~)t~Ta antagonist, resulted in an overall reduction of the 5 MTinduced faclhta tlon of dopamlne release (Benloucif et al 1992} D,rect proof that *!'es~release-regulating 5 HT4 receptors are present on axonterminals s n o t r e : avalaDle In f a c t from t h e a b o v e ' e l e a s e s t u c l e s d o n e / r l v / y o or u s m o l v a r slices, it is not possible to understand ,f the effect of 5-HT 4 agonlsts o:cur directly, tbrough 5-HT 4 receptors located on dopamme nerve endings ,:)' indirectly, tqrough 5-HT 4 recepto-s located oP Interneuroqs wh~cq ,!h ' , q modulate doparmne release :n a binding study attempting tc c arlfv the cellJla~ localization of s ; s t 5 HT4 r e c e p t o r s ( a a t e l e t a l , 1994) lesions w i t h s e e c t w e neurotoxns were made both in the substantla nigra arid r~ the caudate putamen The result~ showed a decrease of the bindir'g of a soeof,c 5 H[ 4 antagornst only wrlen the cellular bodies ,n the caudate puta'qe'~ were destroyed w th ka.s~c as~c but not whep tqe nlgra~ projections were les o" ed w~th 6 hydroxyoo~)am ,1~

I

P-11-3 J Dopamine

receptor blockade decreases nocturnal pineal activity in rats

R M o n t e l e o n e l . L S t e a r d o 2 I. Serino3, A T o r t o r e l l a l , M . d ' l s t r i a 3 , M. Mal 1 1 Institute of Psychlatzy, 3 Department of Human Physiology and

Biology "F Bottazz/', Second UniversW of Naples, 80138 Naples, Italy, 2 Institute of Pharmacology, University of Ban: Ban} Italy The mammalian pineal gland synthesizes melatonin in a characteristic circadian pattern with the highest levels occurring at night. It is commonly accepted that, at least in rats, norepinephrine (NE) released from sympathetic fibres arising from superior cervical ganglia is the main regulator of pineal activity (Reiter, 1991) However, evidence has been provided that, n] addition to NE, also dopamine (DA) stimulates the production of meiatonin through the interaction with D 1 and D 2 receptors located on the pn~ealocytes (Simonneaux et al.. 1990) At present, however, the modulatory role of DA on pineal actiwty has not been exhaustively investigated In particular, no data have been prowded on the role of DA on nocturnal melatonin production. Therefore, in the present study, we investigated the effects of both acute and chronic DA receptor blockade by haloperidol on the rat pineal actiwty m v w o To this purpose, in a first experiment, male Sdrague Dawley rats were acutely injected with halopebdol (l mg.kg I s.c.) or volume matched saline at 22.00 h and killed 1 or 2 hours later, in dark cond,tion In a second experiment, male rats were chronically treated with haloperidol (1 m g k g -1 s.c., at midday) or volume-matched saline for 10 days: on the day after the last injection, rats were killed at 23.00, 24.00 a n d 0 ! 00h, rndarkcond,tion The activity of the pineaIN acetyltransferase ',NAT). the key enzyme involved in the melatonin biosynthesis, was detertuned by a radioenzymat~c m e t h o d The levels of melatonin in the pineal gand were determined by radloimmunoassay. As compared to saline, both acute and chronic treatment with haloperidol induced a significant decrease in nocturnal pineal NAT activity (p < 0.001) and melatonln content (p < 0.001 ). Since haloperidol is provided of blocking activity mainly, although not exclusively, on DA receptors, these results support the hypothesis that DA may have a tonic stimulatory role on the pnysioiog~cal production of pineal melatonin in rats.

References Relter. RJ i1991) Pineal melatonln: cell biology of its synthesis and its physiological interactions Endocr Ray 12, 151 179 S,monneaux,V Mumn, L C , and Ebadi, M 0990) Characterization of D1 dopamine re-