Effects of Repeated Treatment with and , 5-HT Uptake Inhibitors, on 5-HT1A and 5-HT2 Receptors in the Rat Brain

Violetta Klimek, Ph.D., Joanna Zak-Knapik, M.Sc., Marzena Mackowiak, M.Sc. Institute of Pharmacology, Polish Academy of Sciences, 12 Smetna St., 31-343 Krakow, Poland Submitted: December 10, 1992 Accepted: July 9, 1993

Repeated treatment with fluoxetine and citalopram, which are potent 5-HT reuptake inhibitors, resulted in different regulation of 5-HTIA and 5-HT2 receptors in the rat brain. Their effects were compared with those ofother : , and levoprotiline. The density of 5-HTlA receptors, labelled with [3H]8-OH-DPAT, in the rat hippocampus was enhanced after citalopram, imipramine, mianserin and levoprotiline, but not altered after fluoxetine administra- tion. [3H]Ketanserin binding sites, which label 5-HT2 receptors, were increased after fluoxetine and levoprotiline, but decreased after citalopram, imipramine and mianserin in the rat cerebral cortex. Acute administration of fluoxetine, but not citalopram, resulted in a decreased density of 5-HTlA receptors. 5-HT2 receptors were not changed by acute administration of either fluoxetine or citalopram. The obtained results indicate that besides 5-HT reuptake inhibiting properties of both compounds, there may exist an additional mechanism(s) of their action, which leads to different regulation of 5-HT1A and 5-HT2 receptors.

Key Words: citalopram, fluoxetine, antidepressants, repeated treatment, binding, 5-HTIA, 5-HT2 receptors, rat

INTRODUCTION

For many years it has been assumed that the sants share the property of inhibition of 5-HT and noradren- system is involved in the pathogenesis and treatment of alin uptake and many of these compounds also antagonize depression. The deficiency of (5-HT) was postu- postsynaptic 5-HT receptors. These are able to aug- lated as a probable cause of depression. A vast body of ment 5-HT transmission by prolonging the residence time of evidence obtained in both pre-clinical and clinical studies 5-HT in the synaptic cleft (Caldecott-Hazard et al 1991; showed delayed effects of enhancement of 5-HT activity Wong et al 1990). The majority of drugs (after two to three weeks) observed across several classes of administered repeatedly to rats, decreased the number of antidepressants, including tricyclic mixed uptake inhibitors, 5-HT2 sites and their function measured as the monoamine oxidase inhibitors, electroconvulsive therapy head-twitch response (Goodwin et al 1984; Green 1987; and others (Coppen and Doogan 1988; Meltzer and Lowy Peroutka and Snyder 1980). In contrast to the data obtained 1987; Van Praag and Korf 1971). Most tricyclic antidepres- with administration of drugs, repeated electroconvulsive shock was found to increase the number and function of 5-HT2receptors (Green et al 1983; Kellaretal 1981; Vetulani Address reprint requests to: Violetta Klimek, Institute of Phar- macology, Polish Academy of Sciences, 12 Smetna St., 31-343 et al 1981). In tissue obtained at postmortem, Krak6w, Poland. the number of 5-HT2 receptors was increased in patients J Psychiatr Neurosci, VoL 19, No. 1, 1994 63 64 Journal ofPsychiatry & Neuroscience VoL 19,, No. 1, 1994

Table 1 Effect of citalopram and fluoxetine on: (A) [3H]-8-OH-DPAT binding to the rat hippocampal membranes and (B) [3H]-ketanserin to the rat cerebral cortex Acute Repeated Treatment Bma KD n Bmax KD n A Vehicle 11.17 ±1.39 1.66 ±0.11 5 11.18 ±0.52 1.57 ± 0.15 5 Citalopram 12.15± 1.25 1.88 ± 0.31 5 14.09 ± 0.85a 1.68 ± 0.14 5 Fluoxetine 8.55 ± 0.69a 1.75 ± 0.12 5 10.63 ± 0.35 2.00 ± 0.22 5 B Vehicle 10.99 ± 0.91 0.55 ± 0.05 7 12.47 ± 0.70 0.52 ± 0.04 7 Citalopram 9.36 ± 0.65 0.53 ± 0.05 5 9.80 ± 0.75a 0.38 ± 0.04 5 Fluoxetine 10.11 ± 0.61 0.36 ± 0.02 7 15.25 ± 0.41a 0.47 ± 0.04 7 The number of binding sites, Bmax (pmol/g tissue) and the binding affinity constants, KD (nM), were calculated by Scatchard analysis, n = number of Scatchard plots. Data represent mean ± SEM (analysis of variance followed by Dunnett's procedure): a p < 0.05 suffering from major depression, but was normal in patients 24 hours after administration of the antidepressant drugs, taking tricyclics (Mann et al 1989; Yates et al 1990). their brains were rapidly removed, dissected and frozen. A possibility of inducing an antidepressant effect via [3H]8-OH-DPAT binding to 5-HT1A receptors was 5-HT1A receptors has recently been suggested (Cervo and performed in the rat hippocampus according to the method Samanin 1987; Kennett et al 1987; Tatarczynska and of Schlegel and Peroutka (1986). Briefly, the hippocampal Chojnacka-Wojcik 1989). Electrophysiological evidence tissue was homogenized in 20 volume of 50 mM Tris-HCl suggests enhanced postsynaptic 5-HTIA receptor function buffer (ph = 7.7 at 25°C) using an Ultra-Turrax T25, and was after chronic administration of antidepressants (Blier et al then centrifuged at 32 000 g for ten minutes. This step was 1987). The number of5-HT1A binding sites was higher in the repeated twice. Before the third centrifugation, the samples hippocampus of antidepressant-treated suicides in compari- were incubated at 37°C for ten minutes, to reduce endoge- son with -free suicides (Cheetham 1990). nous 5-HT. The final pellet was resuspended in 50 volume. The present study was aimed at determining whether or of Tris-HCl buffer containing 10 lM , 4 mM not the 5-HT uptake inhibitors citalopram and fluoxetine, calcium chloride and 0.1% ascorbic acid. One milliliter ofthe given repeatedly and acutely, can influence the binding of tissue suspension (5 mg of wet weight), 100 p1 of 10 tM [3H]8-OH-DPAT to 5-HTIA receptors in rat hippocampus, serotonin (for non-specific binding) and 100p1 of [3H]8-OH- which represent the postsynaptic sites, and of [3H]ketanser- in total volume of 1.2 ml were incubated at 370C for ine to 5-HT2 receptors in comparison with such effects of -DPAT; other antidepressant drugs. These are imipramine, a classical 15 minutes. The incubation was terminated by a rapid , mianserin, a 5-HT antagonist having vacuum filtration through Whatman GF/B filters, which were also noradrenolytic properties (Mogilnicka and Klimek then washed three times with 5 ml of ice-cold buffer using a 1979), and levoprotiline with absence ofany influence on the Brandel-cell harvester, MB-48. concentrations system (Wendt 1988). ranging from 0.1 nM to 8.5 nM were used for the Scatchard plots. METHODS [3H]Ketanserin binding was performed in the rat neocortex according to the slightly modified method of Male Wistar rats (200 g to 250 g) were kept under standard Leysen et al (1982). The cerebral cortex tissue was homoge- laboratory conditions on a constant 12-hour light/dark cycle. nized in 20 volume of50 mM TrisHCl buffer (pH 7.7 at 25°C) Citalopram and fluoxetine were administered repeatedly in a and centrifuged at 32 000 g for 20 minutes. The final pellet dose of 10 mg/kg po, twice a day for 14 days. In acute was resuspend in 50 volume of the same buffer. The non- experiments either compound was administered in a daily specific binding was defined in the presence of 1 FM methy- dose of 20 mg/kg po. Other antidepressant drugs, imipra- sergide. Samples (total volume of 1.2 ml) were incubated at mine, mianserin and levoprotiline were used as reference 37°C for 20 minutes followed by a rapid vacuum filtration compounds and were given to rats repeatedly in a dose of through Whatman GF/B glass filters, and were then washed 10 mg/kg po, twice a day for 14 days. Rats were sacrificed three times with 5 ml portions of ice-cold Tris-HCl buffer. January 1994 Effects ofrepeated treatment in the rat brain 65

Table 2 Effect of repeated administration of antidepressant drugs on [3H]-8-OH-DPAT and [3H]-ketanserin binding sites in the rat hippocampus and cerebral cortex, respectively. Treatment Bmax KD n Bmax KD n Vehicle 10.61 ± 0.40 1.85 ± 0.36 5 12.98 ± 0.60 0.58 ± 0.03 8 Imipramine 12.84 ± 0.29a 1.48 ± 0.16 5 10.20 ± 0.73b 0.57 ± 0.04 5 Mianserifc 12.61 ± 0.49a 1.45 ± 0.17 6 8.63 ± 0.42b 0.58 ± 0.03 4 Levoprotiline 14.95 ± 0.71b 1.52 ± 0.13 5 15.77 ± 0.79a 0.57 ± 0.05 5 The number of binding sites, Bma, (pmol/g tissue) and binding affinity constants, KD (nM) were calculated by the Scatchard analysis, n = number ofScatchard plots. Data represent means ± S.E.M. (analysis ofvariance followed by Dunnett's procedure); ap < 0.05; bp < 0.001; Cmiansenn treated rats were sacrificed 48 hours after the last dose. [3H]Ketanserin concentrations, ranging from 0.01 nM to citalopram, a short half-life of about three hours in rats was 4 nM, were used for the Scatchard plots. found (Arnt et al 1984) whereas fluoxetine has a rather long Data were analyzed using an AccuFitTM Saturation half-life of about 24 hours (Wong et al 1990). It is possible program (Beckman). An analysis of variance (ANOVA), that the decreased number of 5-HTIA receptors after the followed by Dunnett's test, were applied for determination single dose of fluoxetine is due to its long-lasting effect on of the statistical significance. 5-HT uptake, which may increase 5-HT content at the synapse (Wong et al 1990). Since 5-HTIA receptors have an RESULTS nM affinity for 5-HT, and the 5-HT2 receptors show an nM affinity for it, it is rather likely that the former receptors are Table 1 shows the density (Bm.) and the affinity (KD) of the first to be stimulated and then desensitized (Conn and 5-HTIA (A) and 5-HT2 (B) receptors after the acute and Sanders-Bush 1987; Leysen 1992). However, after repeated repeated administration of citalopram and fluoxetine. The doses of fluoxetine, 5-HTIA receptors were no longer density of 5-HT1A sites, labelled by [3H]8-OH-DPAT, was decreased. The most likely interpretation for this effect is that decreased by the acute dose of fluoxetine, and not changed reuptake blockade, by increasing the intrasynaptic availabil- by the acute dose of citalopram. After two weeks of ity of 5-HT, results in a greater activation of terminal citalopram administration, the density of 5-HTIA receptors autoreceptors, which in turn, reduce the amount of released increased, yet after fluoxetine it did not differ from that of 5-HT. Autoradiographic studies showed that repeated doses the vehicle-treated rats. However, when repeated treatment of fluoxetine did not change the density of 5-HT1A receptors with fluoxetine is compared with acute fluoxetine adminis- in the rat hippocampus, but increased the tration, a significant difference is observed: there is a 24% number ofthose sites (Welner et al 1989). The present results increase in the number of [3H]8-OH-DPAT binding sites. show an increase in the 5-HTIA receptor density in the Acute administration of either citalopram or fluoxetine hippocampus, which represents post-synaptic binding sites did not affect the density or affinity of5-HT2 receptors. After (Hamon et al 1987), after repeated treatment with most ofthe repeated treatment with either compound, the [3H]ketanserin tested compounds. These data are consistent with behavioral binding sites were decreased by citalopram and increased by and electrophysiological reports on the increased neuronal fluoxetine administration (Table IB). responsiveness to 5-HT (De Montigny and Aghajanian 1978; Effects of other antidepressant drugs given repeatedly on Willner 1985). the binding parameters of 5-HTlA and 5-HT2 receptors are summarized in Table 2. Imipramine, mianserin and The widely reported 5-HT2 receptor down-regulation levoprotiline raised the density of5-HT1A receptors in the rat produced by tricyclic antidepressants, which is related to the hippocampus. [3H]ketanserin binding sites in the rat cerebral therapeutic action of the compounds (Peroutka and Snyder cortex were decreased after imipramine and mianserin, and 1980; Willner 1985), has been regarded as a consequence of increased after levoprotiline administration. The density and 5-HT uptake-inhibiting effects of these compounds or their affmity of either 5-HTIA or 5-HT2 receptors after acute 5-HT2 receptorblocking properties. The 5-HT2 receptors can administration of antidepressant drugs listed in Table 2 did be desensitized rapidly, both after chronic treatment with not differ from the data of vehicle-treated rats (data not 5-HT-agonists with a high affinity for 5-HT2 receptors and shown). after chronic treatment with 5-HT2 antagonists (Leysen 1990 1992). Besides, when administered chronically also, mixed DISCUSSION 5-HT1A agonists can cause the 5-HT2 receptor down- regulation (Leysen 1992). Our results also show that such The 5-HT uptake inhibitors investigated are both potent, different compounds as imipramine, mianserin and however, they act with different half-lives. In the case of citalopram can reduce the number of [3H]ketanserin binding 66 Journal ofPsychiatry & Neuroscience VoL 19, No. 1, 1994

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