The effect of prolonged administration of lithium on the level of D2 mRNA in the rat and

Marta Dziedzicka-Wasylewska and Krzysztof Wqdzony

Department of Pharmacology, Institute of Pharmacology, Polish Academy of Sciences, 12 Smetna St., 31-343 Cracow, Poland

Abstract. In the present study the alterations in the levels of mRNA coding for D2, were determined in the rat striatum (STR) and the nucleus accumbens septi (NAS), in dependence on duration of lithium administration. The levels of mRNA coding for D2 receptor (determined using an in situ hybridization technique) were determined after 1,7 and 14 days of lithium administration (LiC1,6 mEq/kg, p.o.), at 3 and 24 h after the last dose of the drug. This treatment resulted in the increase in the levels of mRNA coding for dopamine receptor D2 in all brain regions examined and the effect depended on the time after lithium administration. However the effect was most pronounced in the shell region of NAS ,24 h after the 14-day treatment.

Key words: lithium, mRNA D2, striatum, nucleus accumbens, rat 30 M. Dziedzicka-Wasylewska and K. Wedzony INTRODUCTION drug treatment, although long-term changes in neuronal synaptic function are known to be depend- Lithium salts are among the most effective drugs ent upon selective regulation of expression. in the prevention and treatment of manic-depressive Therefore the present study was designed in order episodes (Post 1986). The substantial number of to obtain information whether repeated administra- clinical and pharmacological investigations suggest tion of LiCl modifies the bisynthesis of postsynaptic that overactivity of pathway may, at dopamine D2 receptors in the rat striatum (STR) and least in part, mediate human mania (Schildkraut the nucleus accumbens septi (NAS) core and shell. 1965, Bunney et al. 197 1, Gerner et al. 1976, Garver and Davis 1979). Behavioural experiments indeed METHODS suggest that lithium renders less active dopaminer- gic system. For example it has been reported that Lithium administration rats maintained on a lithium diet for three weeks ex- hibited less spontaneous activity during habituation Male Wistar rats (weight 180-200 g) were ad- to a new environment that their controls did (Bloom ministered LiCl(6 rnEq/kg) by means of intragas- et al. 1983). In addition, acute and chronic lithium tric intubation, once daily, for the period of 1,7 or treatment have been repeatedly reported to reduce 14 days. Animals were sacrificed 3 or 24 h after the the locomotor stimulation induced by indicated dose of lithium. The trunk blood was col- (Bergren 1985) as well as the responses to apomor- lected when the animals were sacrificed and the phine in animals rendered supersensitive by chronic serum was separated. The lithium content of the treatment (Bloom et al. 1983, Bunney serum was determined by atomic absorption spec- and Garland 1983). trometry (Pybus and Bowers 1970). When the issue of the effects of prolonged li- On the repeated administration of lithium the le- thium treatment on the activity of dopaminergic vels in serum varied as a function of time after each system was addressed via the parameters charac- number of doses. At 3 h following the single as well teristic for DA receptors, the outcome of numerous as repeated administration of lithium the mean serum studies neither had indicated any clear answer. level rose to 0.75L0.02 mmol/l. This level dim- Original finding that chronic lithium administration inished to 0.35L0.02 mmol/l by 24 h after each dose. interferes with the development of DA postsynaptic supersensitivity was reported by Pert et al. (1987) In situ hybridization but it could not be confirmed by subsequent studies (Reches et al. 1982, Staunton et al. 1982, Bloom et For the in situ hybridization study the brains of al. 1983, Pittman et al. 1984). Also in our laboratory the rats were rapidly removed at 3 or 24 h after the we were not able to see any changes in the binding indicated dose of LiCl and frozen on dry ice. Coro- parameters of ['HISCH~~~~Oor ['~ls~i~erone to nal sections (12 pm thick) were made on cryostat the membranes of striatum or limbic forebrain of through the NAS and STR. The sections were thaw- rats treated with LiCl for 21 days (Dziedzicka- mounted onto chrome alum pretreated slides, post- -Wasylewska and Klimek, unpublished results). fixed in 4% paraformaldehyde for 10 min and Since clinical effect of lithium is generally ob- processed for in situ hybridization as described pre- served only after the prolonged treatment, apparent- viously (Dziedzicka-Wasylewska and Rogoz ly the biochemical changes requiring such 1995). Briefly, a 48-mer synthetic oligonucleotide prolonged administration of the drug suggest alter- mixture (New England Nuclear) complementary to ations at the genomic level. Until recently, however, the rat dopamine D2 receptor was labelled using little has been known about transcriptional and [35~]d~~~(1200 Cilmmol, New England Nuclear) posttranscriptional factors regulated by chronic to obtain a specific activity of about 4 x lo5cpmlpl. Lithium and dopamine receptor D2 mRNA 31

The sections were hybridized with the labelled oli- group differences were analysed by Duncan's gonucleotide for 20 h at 37OC in a humidified incu- multiple-range test. bator. After washing at 40°C, the sections were dried in a cool-air stream and exposed to a film RESULTS (Hyperfilm MP, Amersham) for 20 days at -70'~. The specificity of in situ hybridization was assessed The typical autoradiogram showing the do- by pretreatment of some tissue sections with parnine D2 receptor mRNA is presented in the Fig. 1. RNAase A (20 yglml) for 40 min at 30°c, which The effect of lithium on the level of mRNA cod- completely eliminated the hybridization signal ing for dopaminergic D2 receptor in the rat STR and with the cDNA probe. Moreover, when the hybridi- NAS core and shell is presented in Table I. Single zation was carried out in the presence of a 100-fold dose of lithium already produced an increase in the excess of the unlabelled probe, the signal also dis- levels of mRNA coding for D2 receptor but the ef- appeared. fect was statistically significant only in the core re- Optical density measurements were made from gion of the NAS. Further administration of lithium the autoradiograms corresponding to the sections of up to 7 or 14 days resulted in the statistically signi- the NAS and STR, using an image analysing system ficant increase in the levels of mRNA coding for D2 (Java: Jandel, Corte Madera CA, USA). The aver- receptor in all brain regions examined and the effect age optical density values were calculated after sub- was the strongest in the shell region of the NAS. traction of the film background density. The mean The increase in the levels of mRNA coding for D2 optical density values were obtained by averaging receptors strongly depended on time after the last out the measurements from autoradiograms of the dose of the drug; i.e. the effect were the most pro- sections obtained from 5-6 animals per group. nounced at 24 h after the administration of lithium.

Statistical analysis DISCUSSION

The results were statistically assessed by one- In the present study we determined the time way analysis of variance (ANOVA), and inter- course of changes in the levels of mRNA coding for

Fig. 1. A, example photomicrograph of brain section hybridised with probe against D2 receptor mRNA. B, diagrammatic rep- resentation of the areas used in optical density analysis of specific brain regions. 32 M. Dziedzicka-Wasylewska and K. Wedzony

TABLE I

The effect of lithium administration on the level of mRNA (optical density arbitrary units) coding for dopamine D2 receptors in the striatum (STR) and the nucleus accumbens (NAS) shell and core of the rat

Treatment (days) STR NAS core NAS shell

1 day, 3 h 7 days, 3 h 7 days 24 h 14 days 3 h 14 days 24 h

LiCl (6 mEq/kg, p.0.) was administered as a single dose or once daily ror 7 or 14 days. The rat brains were removed at 3 or 24 h after the last administration of the drug. The mean optical density values were obtained by averaging uot the measure- ments from autoradiograms of the sections obtained from 5-6 animals.ANOVA followed by Duncan's test; *P<0.01 vs. the control level. dopamine D2 receptors in the rat striatum (STR) and Bernard et al. 1991, Chen et al. 1991, 1993, Jaber the nucleus accumbens (NAS) shell and core upon et al. 1992, 1994) that dopaminergic transmission the prolonged administration of lithium. Since the indeed regulates the level of mRNA coding for D2 literature concerning the effect of lithium on the do- DA receptor, by exerting tonic inhibition on do- paminergic system is full of discrepancies which pamine D2 mRNA. Unfortunately up to the present might result from different experimental para- it was not possible to show unequivocally any ef- digms, it seemed interesting to see whether any al- fects of lithium, at "therapeutic" doses, on the par- terations develop in dependence on various periods ameters characteristic for binding to DA of drug administration as well as on time after the receptors. It must be stressed, however, that in ma- last dose of the drug. jority of studies the radioligands of antagonistic na- The increase in the levels of rnRNA coding for ture towards the DA receptors have been used, such dopaminergic D2 receptor in both examined brain as ['H] or ['H]. DA D2 (as well structures was observed in the present study upon as Dl) receptor exists in two different conforma- the repeated administration of lithium, and the ef- tional states of the same which can be re- fect was also more pronounced in the NAS. Al- vealed by competition curves, but not by though the mechanism of from using antagonistic ligands, which have equal af- synaptic cleft to the nuclear compartments respon- finity for the two states of the receptor so that any sible for regulation of synthesis of D2 receptors are alterations in the high- and low-affinity sites of D2 not known at present, it is tempting to postulate the receptor elicited by chronic lithium treatment may enhancement of biosynthesis of postsynaptic dopa- not be revealed by the examination of antagonistic minergic D2 receptor as the adaptive change to the binding. However, as has been recently shown by weaken DA release from terminals, the effect which Carli et al. (1994), the affinity of the DA receptors we have shown recently (Dziedzicka-Wasylewska for DA was lower after lithium treatment. The et al. 1995). A neurochemical transsynaptic mech- changes in the affinities were proposed to be due anism which would enhance dopaminergic com- rather to the conformational change at the pensation involves an increase in postsynaptic DA level. The increase in the biosynthesis of D2 recep- receptors. It has been already shown (Coirini et al. tors observed in the present study the most probably 1990, Srivastava et al. 1990, Angulo et al. 1991, is the adaptive compensatory mechanism and may Lithium and dopamine receptor D2 mRNA 33 well result from the complex events taking place in Bunney W.E.Jr., Brodie H.K.H., Murphy D.L., Goodwin F.K. the dopaminergic synaptic cleft following pro- (1971) Studies of alpha-methyl-para-tyrosine, L-DOPA and L-tryptophan in depression and mania. Am. J. Psy- longed lithium administration, i.e.: attenuated DA chiat. 127: 872-881. release from terminals and diminished coupling ef- Bunney W.E., Garland B.L. (1983) Possible receptor effects ficacy between the DA receptors and the guanine of chronic lithium administration. Neuropharmacology nucleotide regulatory protein (possibly due to the 22: 367-372. changes in for various G Carli M., Anand-Srivastava M.B., Molina-Holgado E., Dewar K.M., Reader T. (1995) Effects of chronic lithium treat- following treatment with lithium (Li et al. 1991) but ments on central dopaminergic receptor systems: G pro- also to the alterations in the organization of neuro- teins as a possible targets. Neurochem. Int. 24: 13-22. nal plasmatic membranes, as has been shown by Chen J.F., Aloyo V.J., Weiss B. (1993) Continuous treatment Lopez-Corcuera et al. (1988) - the events which are with the D2 dopamine receptor agonist de- postulated as the mechanism responsible for anti- creases D2 dopamine receptors, D2 dopamine receptor messenger RNA and proenkephalin messenger RNA, and manic action of lithium. On the other hand, the increases mu opioid receptors in mouse striatum. Neuros- changes observed at the level of mRNA coding for cience 54: 669-680. D2 receptor are at present interpreted as an adaptive Chen J.F., Qin Z.H., Szele F., Bai G., Weiss B. (1991) Neur- change to the weaken dopamine release. However, onal localization and modulation of the D2 dopamine re- further studies, possibly using radioligands of agon- ceptor mRNA in brain of normal mice and mice lesioned with 6-hydroxydopamine. Neuropharmacology 30: 927- istic nature towards D2 dopaminergic receptors, 941. might reveal new aspects of the role of D2 receptor Coirini H., Schumacher M., Angulo J., McEwen B. (1990) In- itself in the mechanism of therapeutic action of li- crease in striatal dopamine D2 receptor mRNA after le- thium. sions or haloperidol treatment. Eur. J. Pharmacol. 186: 369-371. ACKNOWLEDGEMENT Dziedzicka-Wasylewska M., MaCkowiak M., Fijal K., Wed- zony K. 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