INFLUENCE OF ACUTE AND CHRONIC 1,2,3,4-TETRA-HYDROISOQUINOLINE ADMINISTRATION ON THEEXPRESSION OF PROENKEPHALIN mRNA IN THE RATSTRIATUM

Jadwiga Wardas#, Ma³gorzata Zapa³a, El¿bieta Lorenc-Koci

Department of Neuro-Psychopharmacology, Institute of Pharmacology, Polish Academy of Sciences,Smêtna 12, PL 31-343 Kraków, Poland

Influence of acute and chronic 1,2,3,4-tetrahydroisoquinoline admini-stration on the expression of proenkephalin mRNA in the rat striatum.J. WARDAS, M. ZAPA£A, E. LORENC-KOCI. Pol. J. Pharmacol., 2003,55, 951–956.

Animal studies have shown that a depletion of dopamine or blockade ofdopamine D2 receptors in the striatum produces an increase in striatal proen-kephalin (PENK) mRNA expression and an increase in GABAergic trans-mission in the globus pallidus. Therefore, it has been suggested that an en-hanced striatal PENK mRNA expression may reflect to some extent an in-crease in the activity of the GABAergic striatopallidal pathway whoseoveractivity has been suggested to take place in the course of Parkinson’sdisease. Therefore, the aim of the study was to investigate the role of1,2,3,4-tetrahydroisoquinoline (TIQ), an endogenous substance suspected ofproducing parkinsonism in humans, in the regulation of the activity ofGABAergic striatopallidal pathway in rats. TIQ administered acutely at thedose of 100 mg/kg ip increased the PENK mRNA expression in the dorsalpart of the striatum at two levels I and II (rostral and central striatum, respec-tively). No changes were noticed in the ventral part of the striatum. Moreo-ver, TIQ given chronically to rats for 3 weeks did not modify the level ofPENK mRNA in any examined part of the striatum. The present results showthat the effect of TIQ on the PENK mRNA expression is different from thatdescribed for proparkinsonian model neurotoxins (MPTP, 6-OHDA) as wellas for typical neuroleptics, such as haloperidol.

Key words: 1,2,3,4-tetrahydroisoquinoline, proenkephalin mRNA, in situhybridization, parkinsonism, striatum, rat

Copyright © 2003 by Institute of PharmacologyPolish Academy of Sciences

Polish Journal of Pharmacology

Pol. J. Pharmacol., 2003, 55, 951–956ISSN 1230-6002

# correspondence; e-mail: wardas@if-pan.krakow.pl

INTRODUCTION

According to the current model of basal gangliaconnections, loss of striatal dopamine (DA) inParkinson’s disease (PD), caused by progressivedegeneration of nigrostriatal dopaminergic path-way, results in unbalanced activity of the two mainGABAergic striatal output pathways [10]. The hy-peractivation of the so-called “indirect”, striatopal-lidal pathway seems to result from a disinhibitionof GABAergic neurons, due to the loss of dopamin-ergic inhibition through dopamine D2 receptors.These neurons preferentially co-express GABA,enkephalin and DA D2 receptors [10]. On the otherhand, the so-called “direct” striatonigral pathwayseems to be inhibited due to loss of stimulating ef-fect of DA through DA D1 receptors. These neu-rons, besides DA D1 receptors, co-express GABA,dynorphin and substance P [10]. The observedoveractivation of striatopallidal pathway, reflectedby the increased expression of striatal proenkepha-lin (PENK) mRNA is thought to be one of the mainmechanisms underlying dysfunction in motor be-havior observed in PD and in animal models of thisdisease [10].

Several studies using animal models of PD(6-OHDA in rats, MPTP in mice or monkeys) con-sistently showed the increased expression of stri-atal mRNA for PENK or increased enkephalin im-munoreactivity [1, 12, 27, 29]. Moreover, no chan-ges or a decreased expression of mRNA encodingprodynorphin or substance P was seen in thosemodels [1, 12, 29]. Therefore, it seems, that altera-tions in the level of neuropeptide gene expressioncorrelate with alterations in striatal neuronal activi-ty and that measurement of neuropeptide gene ex-pression in specific cell types can be used to assessthe level of neuronal activity in selected pathways.

It has been postulated that 1,2,3,4-tetrahydro-isoquinoline (TIQ) and its derivatives which arestructurally similar to MPTP are suspected of pro-ducing parkinsonism in humans [2, 20, 22]. Thesecompounds are present both endo- and exoge-nously (in plants and some food) and readily crossthe blood-brain barrier [16]. It has already beenshown that TIQ given at high doses (50–100 mg/kg)to animals evoked some behavioral and biochemi-cal symptoms similar to those found in parkinso-nian patients [4, 19, 23]. Therefore, the aim of thepresent study was to examine the influence of acute

and chronic TIQ administration on the expressionof PENK mRNA measured in the rat striatum.

MATERIALS and METHODS

The experiment was carried out in compliancewith the Animal Protection Bill [Dziennik Ustawno. 111/1997, item 724] and according to the NIHGuide for the Care and Use of Laboratory Animals.

Male Wistar rats weighing 230–280 g were usedfor all experiments. Rats were kept in the well-ven-tilated room on an artificial light/dark cycle (12/12 h,light on from 7 am to 7 pm), under standard condi-tions (21°C) with a free access to food and water.

TIQ was obtained from Aldrich Chemical Com-pany (Milwaukee, WI, USA), dissolved in redis-tilled water and given acutely (single dose) or chro-nically once daily for three weeks (100 mg/kg ip).The rats were killed either 4 h after the single doseof TIQ or 4 h and 72 h after the last dose of chronicadministration. Control rats were administered withthe redistilled water. The number of animals ineach treatment group was N = 7–8.

Rat brains were rapidly removed, frozen in coldheptane (–70°C), cut into frontal sections (10 �mthick) which were thaw-mounted on gelatin-coatedmicroscopic slides, and processed for in situ hy-bridization [8]. Briefly, the sections were postfixedin 4% paraformaldehyde, dehydrated, delipidated,rehydrated and air-dried. A 48-mer synthetic deoxy-oligonucleotide, complementary to bases 388–435of rat PENK mRNA (New England Nuclear, LifeSci. Products, Inc.), was labelled using [35S]dATP(1,200 Ci/mmol, New England Nuclear) to obtainspecific activity of about 4 × 105 cpm/�l. The sec-tions were hybridized with the labeled oligonucleo-tide for 20 h at 37°C. After washing (4 × 15 minin 2 × SSC at 40°C, 1 × SSC for 15 min at a roomtemperature), the sections were air-dried and ex-posed to a [3H]Hyperfilms (Amersham) for 4 weeksat 4°C.

The specificity of the probe was assessed bypretreatment of some tissue sections with RNAaseA (20 �g/ml) for 40 min at 30°C, which completelyeliminated the hybridization signal with the cDNAprobe. Moreover, when hybridization was carriedout in the presence of a 100-fold excess of the unla-belled probe, the signal also disappeared.

After exposure, the films were developed witha Dektol developer (Kodak), fixed with a GBXFixer and Replenisher (Kodak) and dried. Autora-

952 Pol. J. Pharmacol., 2003, 55, 951–956

J. Wardas, M. Zapa³a, E. Lorenc-Koci

diograms were analyzed by a computer-assisteddensitometry using an image-analyzing system(MCID, St. Catharines, Ontario, Canada). The av-eraged optical density (OD) values were calculatedafter subtraction of the film background density.Means of the OD values were obtained by averag-ing measurements of both sides of the brain in2 sections per each brain structure. The level ofPENK mRNA was estimated in the dorsal (CPd)and the ventral (CPv) striatum at three levels: levelI (A = 1.7 to 1.2 mm from the bregma), level II(A = 0.48 to –0.26 mm from the bregma, and levelIII (A = –0.40 to –0.92 mm from the bregma) [26](Fig. 1). The data are presented in OD values(means ± SEM).

The results were statistically assessed by theone-way analysis of variance (ANOVA) followedby an LSD (Least Significance Difference) test forpost hoc comparisons.

RESULTS

The patterns of hybridization found in brain re-gions of control rats fully agreed with the well-known distribution of PENK mRNA (Fig. 1). Thehighest level of PENK mRNA expression wasfound in the ventrolateral part of the striatum at allthree levels examined (Fig. 1, 2).

ISSN 1230-6002 953

TIQ AND PROENKEPHALIN mRNA

Fig. 1. Typical examples of proenkephalin mRNA expression incontrol rats are shown in three frontal sections of the striatum:rostral (level I, A = 1.7 to 1.2 mm from the bregma); central(level II, A = 0.48 to –0.26 mm from the bregma) and the caudal(level III, A = –0.40 to –1.3 mm from the bregma), according to[26]. Black outlines show the regions studied. CP – striatum;dl – dorsolateral part of the striatum; vl – ventrolateral part ofthe striatum; NA – nucleus accumbens; GP – globus pallidus

dorsal ventral dorsal ventral dorsal ventral0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

**

Control

TIQ 100 mg/kg acute (4 h)

TIQ 100 mg/kg chronic (72 h)

TIQ 100 mg/kg chronic (4 h)

OD

un

its

level I level II level III

Fig. 2. The influence of single or chronic TIQ administration onthe PENK mRNA expression in the striatum. The data are pre-sented as a mean ± SEM in optical density (OD) values. Thenumber of animals per group: control n = 7, TIQ 100 mg/kgacute n = 8, TIQ 100 mg/kg chronic (72 h) n = 6; TIQ 100 mg/kgchronic (4 h) n = 8. Statistics: a one-way ANOVA, followed byan LSD (Least Significance Difference) test for post hoc com-parisons. * p < 0.05 vs. control. For further explanations seeFigure 1

TIQ injected at a single dose of 100 mg/kg ipsignificantly increased the PENK mRNA expres-sion only in the dorsal part of the CP at two levels:level I (15.5% of increase vs. control) and II (10.7%of increase vs. control) (Fig. 2). No changes in thePENK mRNA expression in the dorsal part of thestriatum at the level III and in the ventrolateral partof this structure at all three levels (I-II-III) werefound (Fig. 2). Moreover, TIQ given at the dose of100 mg/kg ip chronically for 3 weeks did notchange the level of PENK mRNA expression inany examined part of the striatum (Fig. 2).

DISCUSSION

Numerous studies have shown that DA exertsa potent influence on striatal neuropeptide expres-sion since depletion of this neurotransmitter byproparkinsonian neurotoxins or neuroleptic-inducedblockade of DA D2 receptors in the striatum resultsin increased expression of PENK mRNA in thestriatopallidal neurons and an increase in GABA-ergic transmission in the globus pallidus [1, 6, 11,12, 29]. Therefore, it has been suggested that an in-crease in the striatal PENK expression may reflect,at least to some extent, an increase in the activity ofthe striatopallidal neurons and may be related tomanifestation of symptoms of PD or the neurolep-tic-induced parkinsonism. Although studies withanimal models of PD produced rather consistent re-sults in regard to increases in PENK mRNA ex-pression, studies of enkephalin expression in par-kinsonian patients have produced conflicting results(decrease, increase or no changes in met-enkepha-lin level in caudate-putamen) [9, 17, 24]. In the lat-ter case, however, expression of neuropeptides inPD patients may be affected by e.g. the long-termuse of various anti-parkinsonian drug therapiesand/or variability in the degree of striatal DA de-pletion.

The results of the present study show that effectof chronic TIQ administration on the PENKmRNA expression in the striatum is different fromthat observed after a single dose of this compound.It has been previously suggested that TIQ in somerespect resembled the action of typical neurolep-tics, but several actions of this compound are in-compatible with the idea that it may have neuro-leptic-like properties. Firstly, in contrast to classicneuroleptics, TIQ did not produce sedation norcatalepsy even at doses that produce muscle rigid-

ity [4, 19]. Secondly, it produces very small bio-chemical effects by itself and does not show syner-gism with the neuroleptics in the action on DA me-tabolism [4]. Moreover, Antkiewicz-Michaluk etal. [3] have recently described that acutely givenTIQ did not bind to the antagonistic site of DA D1or D2 receptors, however, it displaced [3H]-apomor-phine from its binding sites with effectiveness com-parable to that of DA [3]. The fact that TIQ, simi-larly to DA, binds to the agonistic binding sites ofthe DA receptors, suggests that this compound maysuppress dopaminergic transmission at site differ-ent from the neuroleptic binding site. This antido-paminergic effect of TIQ may be responsible forthe observed increase in the expression of mRNAencoding PENK in the striatum after acute treat-ment.

The changes in PENK mRNA expression afterTIQ administration were found only in the dorsalbut not in other parts of the striatum. At the mo-ment we have no explanation for the lack of TIQeffect in the ventral part of the striatum. However,a similar increase in PENK gene expression, par-ticularly in the dorsolateral sensorimotor territorieshas been previously observed [12]. Moreover, a di-stinction between the dorsal and ventral parts of thestriatum has already been suggested by others [5,14]. The dorsal part of the striatum (associative,motor striatum) seems to be the most important forthe control of DA-mediated motor behavior [14].On the other hand, in the ventral striatum, whichcomprises the nucleus accumbens and the ventro-medial part of the caudate-putamen [14], DA seemsto play a primary role in the control of affective andpsychomotor behaviors [5, 14].

The results obtained in animal models of PDsupport the proposal that DA exerts a tonic inhibi-tory control over enkephalin-containing GABA-ergic striatopallidal output neurons [10]. However,most of these studies have been conducted on acutemodels of parkinsonism, i.e. on animals with rapidonset and short duration of dopaminergic neurondegeneration. Recent studies conducted on mon-keys treated with MPTP showed that the most criti-cal factor in modulating striatal PENK expressionis the duration, but not the extent of striatal dener-vation, as prolonged denervation appeared to haveeven the opposite effect on enkephalin biosynthe-sis, causing a down-regulation in many striatal re-gions [27]. Moreover, a variety of neurochemicaland morphological changes occur in the striatum as

954 Pol. J. Pharmacol., 2003, 55, 951–956

J. Wardas, M. Zapa³a, E. Lorenc-Koci

a consequence of lesion of the mesostriatal DA sys-tem [7, 27, 28, 30]. These changes reflect both theacute loss of DA innervation (e.g. loss of DA con-tent) and compensatory responses which includee.g. increased DA release and turnover [27, 28, 30].Since the PENK mRNA expression is tightly linkedto DA transmission, striatal change or lack ofchange in PENK mRNA expression may reflect theextent of extracellular DA normalization. There-fore, the lack of TIQ effect on PENK mRNA ex-pression after chronic treatment, as seen in thepresent study, may reflect the DA normalizationdue to compensatory mechanisms.

The prolonged treatment with TIQ evokes onlyslight decreases in DA concentration in the stria-tum and tyrosine hydroxylase immunoreactivity inthe substantia nigra [19], in contrast to MPTP,which drastically lowered these parameters [7].Moreover, TIQ does not affect the level of DAtransporter in the striatum [18], which is also acharacteristic parameter of PD [15, 21, 25], whileMPTP changes it distinctly [7, 13]. In addition, ourcurrent study shows that chronically administeredTIQ does not affect PENK mRNA expression inthe striatum and this effect is different from that de-scribed by other authors for proparkinsonian modelneurotoxins as well as for typical neuroleptics, suchas haloperidol.

All the abovementioned studies show that neu-rochemical profile of TIQ action on the nigrostri-atal dopaminergic system is different from that oftypical parkinsonism-inducing neurotoxins. There-fore, it is assumed that TIQ cannot be longer con-sidered as potentially neurotoxic compound butrather as neuromodulator of dopaminergic neuro-transmission.

Acknowledgment. This study was supported by a statu-tory fund from the Institute of Pharmacology, Polish Acad-emy of Sciences, Kraków, Poland.

REFERENCES

1. Angulo J.A.: Involvement of dopamine D1 and D2 re-ceptors in the regulation of proenkephalin mRNAabundance in the striatum and accumbens of the ratbrain. J. Neurochem., 1992, 58, 1104–1109.

2. Antkiewicz-Michaluk L.: Endogenous risk factors inParkinson’s disease: dopamine and tetrahydroisoqui-nolines. Pol. J. Pharmacol., 2002, 54, 567–572.

3. Antkiewicz-Michaluk L., Michaluk J., Romañska I.,Papla I., Vetulani J.: Antidopaminergic effects of 1,2,3,4-

tetrahydroisoquinoline and salsolinol. J. Neural Transm.,2000, 107, 1009–1019.

4. Antkiewicz-Michaluk L., Romañska I., Papla I., Mi-chaluk J., Bakalarz M., Vetulani J., Krygowska-WajsA., Szczudlik A.: Neurochemical changes induced byacute and chronic administration of 1,2,3,4-tetrahy-droisoquinoline and salsolinol in dopaminergic struc-ture of rat brain. Neuroscience, 2000, 96, 59–64.

5. Brown L.L., Sharp F.R.: Metabolic mapping of ratstriatum: somatotopic organization of sensorimotoractivity. Brain Res., 1995, 686, 207–222.

6. Chapman M.A., See R.E.: Differential effects of uni-que profile antipsychotic drugs on extracellular aminoacids in the ventral pallidum and globus pallidus ofrats. J. Pharmacol. Exp. Ther., 1996, 277, 1586–1594.

7. Dauer W., Przedborski S.: Parkinson’s disease: me-chanism and models. Neuron, 2003, 39, 889–909.

8. Dziedzicka-Wasylewska M., Rogo¿ R.: The effect ofprolonged treatment with imipramine and electrocon-vulsive shock on the levels of endogenous enkepha-lins in the nucleus accumbens and the ventral tegmen-tum of the rat. J. Neural Transm.-Gen. Sect., 1995,102, 221–228.

9. Fernandez A., de Ceballos M.L., Rose S., Jenner P.,Marsden C.D.: Alterations in peptide levels in Parkin-son’s disease and incidental Lewy bodies disease.Brain, 1996, 119. 823–830.

10. Gerfen C.R.: Molecular effects of dopamine on stria-tal-projection pathways, Trends Neurosci., 2000, 23,Suppl., S64–S70.

11. Grimm J.W., See R.E.: Chronic haloperidol-inducedalterations in pallidal GABA and striatal D(1)-me-diated dopamine turnover as measured by dual probemicrodialysis in rats. Neuroscience, 2000, 100, 507–514.

12. Herrero M.-T., Augood S.J., Hirsch E.C., Javoy-AgidF., Luqin M.R., Agid Y., Obeso A., Emson P.C.: Ef-fects of L-DOPA on preproenkephalin and prepro-tachykinin gene expression in the MPTP-treated mon-key striatum. Neuroscience, 1995, 68, 1189–1198.

13. Ishiwata K., Koyanagi Y., Abe K., Kawamura K., Ta-guchi K., Saitoh T., Toda J., Senda M., Sano T.: Eva-luation of neurotoxicity of TIQ and MPTP and of par-kinsonism preventing effect of 1-MeTIQ by in vivomeasurement of pre-synaptic dopamine transporterand postsynaptic dopamine D2 receptors in the mousestriatum. J. Neurochem., 2001, 79, 868–876.

14. Joel D., Weiner I.: The connections of the dopaminer-gic system with the striatum in rats and primates: ananalysis with respect to the functional and compart-mental organization of the striatum. Neuroscience,2000, 96, 451–474.

15. Kaufman M.J., Madras B.K.: Severe depletion of co-caine recognition sites associated with the dopaminetransporter in Parkinson’s-diseased striatum. Synapse,1991, 9, 43–49.

16. Kikuchi K., Nagatsu Y., Makino Y., Mashino T., OhtaS., Hirobe M.: Metabolism and penetration throughblood-brain barrier of parkinsonism-related compounds1,2,3,4-tetrahydroisoquinoline and 1-methyl-tetrahy-

ISSN 1230-6002 955

TIQ AND PROENKEPHALIN mRNA

droisoquinoline. Drug Metab. Disposition, 1990, 19,257–262.

17. Levy R., Vila M., Herrero M.T., Faucheux B., AgidY., Hirsch E.C.: Striatal expression of substance P andmethionin-enkephalin in genes in patients with Par-kinson’s disease. Neurosci. Lett., 1995, 199, 220–224.

18. Lorenc-Koci E., Antkiewicz-Michaluk L., Wardas J.,Zapa³a M., Wieroñska J.: Effect of 1,2,3,4-tetrahydro-isoquinoline (TIQ) administration under conditionsof CYP2D inhibition on dopamine metabolism, levelof tyrosine hydroxylase protein and the binding of[3H]GBR 12,935 to dopamine transporter in the rat ni-grostriatal, dopaminergic system. Brain Res., 2003,submitted.

19. Lorenc-Koci E., Œmia³owska M., Antkiewicz-Micha-luk L., Go³embiowska K., Bajkowska M., Wolfarth S.:Effect of acute and chronic administration of 1,2,3,4-tetrahydroisoquinoline on muscle tone, metabolism ofdopamine in the striatum and tyrosine hydroxylaseimmunohistochemistry in the substantia nigra in rats.Neuroscience, 2000, 95, 1049–1059.

20. McNaught K.S., Carrupt P.A., Altomare C., CellamareS., Carotti A., Testa B., Jenner P., Marsden C.D.: Iso-quinoline derivatives as endogenous neurotoxins inthe aetiology of Parkinson’s disease. Biochem. Phar-macol., 1998, 56, 921–933.

21. Mizukawa K., McGeer E.G., McGeer P.L.: Autoradio-graphic study on dopamine uptake sites and their cor-relation with dopamine levels and their striata frompatients with Parkinsons disease, Alzheimer diseaseand neurologically normal controls. Mol. Chem. Neu-ropathol., 1993, 18, 133–144.

22. Nagatsu T.: Isoquinoline neurotoxins in the brain andParkinson’s disease. Neurosci. Res., 1987, 29, 99–111.

23. Nagatsu T., Yoshida M.: An endogenous substance ofthe brain, tetrahydroisoquinoline, produces parkinson-

ism in primates with decreased dopamine, tyrosine hy-droxylase and biopterin in the nigrostriatal regions.Neurosci. Lett., 1988, 87, 178–182.

24. Nisbet A.P., Foster O.J., Kingsbury A., Eve D.J., Dan-iel S.E., Marsden C.D., Lees A.J.: Preproenkephalinand preprotachykinin messenger RNA expression innormal human basal ganglia and in Parkinson’s dis-ease. Neuroscience, 1995, 66, 361–176.

25. Niznik H.B., Fogel E.F., Fassos F.F., Seeman P.: Thedopamine transporter is absent in parkinsonian puta-men and reduced in the caudate nucleus. J. Neuro-chem., 1991, 56, 192–198.

26. Paxinos G., Watson C.: The Rat Brain in StereotaxicCoordinates. Academic Press, San Diego, 1986.

27. Schneider J.S., Decamp E., Wade T.: Striatal prepro-enkephalin gene expression is upregulated in acute butnot chronic parkinsonian monkeys: implications forthe contribution of the indirect striatopallidal circuit toparkinsonian symptomatology. J. Neurosci., 1999, 19,6643–6649.

28. Song D.D., Hauber S.N.: Striatal responses to partialdopaminergic lesion: evidence for compensatory sprout-ing. J. Neurosci., 2000, 20, 5102–5114.

29. Voorn P., Roest G., Groenewegen H.J.: Increase of en-kephalin and decrease of substance P immunoreactiv-ity in the dorsal and ventral striatum of the rat aftermidbrain 6-hydroxydopamine lesions. Brain Res.,1987, 412, 391–396.

30. Zigmond M.J., Abercrombie E.D., Berger T.W., GraceA.A., Stricker E.M.: Compensations after lesions ofcentral dopaminergic neurons: some clinical and basicimplications. Trends Neurosci., 1990, 13, 290–296.

Received: July 29, 2003; in revised form: October 5, 2003.

956 Pol. J. Pharmacol., 2003, 55, 951–956

J. Wardas, M. Zapa³a, E. Lorenc-Koci