View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Digital.CSIC Desensitization of 5-HT1A Autoreceptors by a Low Chronic Fluoxetine Dose Effect of the Concurrent Administration of WAY-100635 Ildefonso Hervás, Ph.D., M. Teresa Vilaró, Ph.D., Luz Romero, Ph.D., M. Cecilia Scorza, M.D., Guadalupe Mengod, Ph.D., and Francesc Artigas, Ph.D. Using microdialysis, receptor autoradiography and in situ autoreceptors, an effect prevented by the concurrent WAY- hybridization, we examined the effects of fluoxetine alone or 100635 administration. However, WAY-100635 (alone or with WAY-100635 on: (a) extracellular 5-HT in frontal with fluoxetine) did not change 5-HT1A autoreceptor cortex; and (b) density and sensitivity of 5-HT1A sensitivity. The density of 5-HT1A receptors and its encoding autoreceptors in rat brain. WAY-100635 (0.3 mg/kg, s.c.) mRNA, was unaffected by these treatments. These results doubled the increase in extracellular 5-HT produced by suggest that prolonged blockade of 5-HT1A receptors in vivo fluoxetine (3 mg/kg, i.p.) in frontal cortex. Two-week prevents the autoreceptor desensitization induced by minipump treatments with these daily doses significantly fluoxetine but does not result in receptor sensitization. raised extracellular 5-HT to 275 Ϯ 33% (fluoxetine) and [Neuropsychopharmacology 24:11–20, 2001] © 2000 245 Ϯ 10% (fluoxetine plus WAY-100635) of controls. American College of Neuropsychopharmacology. Published Fluoxetine 3 mg/kg·day desensitized dorsal raphe 5-HT1A by Elsevier Science Inc. KEY WORDS: 5-hydroxytryptamine uptake; 5-HT1A receptors; anism with 5-HT1A receptor antagonists augments the Dorsal raphe nucleus; Frontal cortex; Microdialysis; neurochemical and behavioral effects of SSRIs (Gartside Selective serotonin reuptake inhibitors (SSRI) et al. 1995; Artigas et al. 1996; Hashimoto et al. 1997; Selective serotonin (5-hydroxytryptamine, 5-HT) re- Mitchell and Redfern 1997; Grignaschi et al. 1998; Trillat ␤ uptake inhibitors (SSRIs) are extensively used in the et al. 1998). At the clinical level, the -adrenoceptor/ treatment of major depression. The increase in fore- 5-HT1A receptor antagonist pindolol accelerates the an- brain extracellular 5-HT elicited by SSRIs is limited by a tidepressant effects of SSRIs in open-label and placebo- negative feed-back involving raphe autoreceptors (Arti- controlled trials (Artigas et al. 1994; Blier and Bergeron gas et al. 1996). The prevention of this inhibitory mech- 1995; Pérez et al. 1997; Zanardi et al. 1997, 1998; Bordet et al. 1998). However, its effectiveness to potentiate an- tidepressant response in chronically ill or treatment- From the Department of Neurochemistry, Instituto de Investiga- resistant patients is still controversial (Maes et al. 1996, ciones Biomédicas de Barcelona (CSIC), IDIBAPS, Barcelona, Spain. 1999; Berman et al. 1997; Moreno et al. 1997; Pérez et al. M. Cecilia Scorza permanent address: Instituto Clemente Estable, Montevideo, Uruguay. 1999). Based on this rationale, selective 5-HT1A receptor Address correspondence to: Francesc Artigas, Department of Neu- antagonists (for use with SSRIs; add-on strategy) and rochemistry, Instituto de Investigaciones Biomédicas de Barcelona dual action compounds (5-HT reuptake inhibitor ϩ 5-HT (CSIC), IDIBAPS, Rosselló, 161, 6th floor, 08036 Barcelona, Spain. 1A Received 13 April 2000; revised 19 May 2000; accepted 30 June antagonist) are being developed for use in the treat- 2000. ment of major depression. NEUROPSYCHOPHARMACOLOGY 2001–VOL. 24, NO. 1 © 2000 American College of Neuropsychopharmacology Published by Elsevier Science Inc. 0893-133X/01/$–see front matter 655 Avenue of the Americas, New York, NY 10010 PII S0893-133X(00)00175-5 12 I. Hervás et al. NEUROPSYCHOPHARMACOLOGY 2001–VOL. 24, NO. 1 The prolonged administration of SSRIs has been re- Dialysate samples of 5 ␮l were collected at 20-min in- ported to desensitize raphe 5-HT1A autoreceptors, as as- tervals into polypropylene microcentrifuge vials. After sessed by single unit recordings and brain microdialy- an initial 1-h sample of dialysate was discarded, four to sis (Blier and de Montigny 1994; Invernizzi et al. 1994; six fractions were collected to obtain basal values before Arborelius et al. 1995; Le Poul et al. 1995). This reduces drug administration. At the end of the experiments, rats the efficacy of the above negative feedback and in- were killed by an overdose of sodium pentobarbital and creases extracellular 5-HT (Bel and Artigas 1993; Inv- the placement of the dialysis probes was checked by ernizzi et al. 1994; Rutter et al. 1994; Arborelius et al. perfusing Fast Green dye and examination of the probe 1996). Other studies, however, have failed to observe track after cutting the brain at the appropriate level. such effects even using large doses of SSRIs (Hjorth and 5-HT was analyzed by a modification of a high per- Auerbach 1994a; Auerbach and Hjorth 1995; Bosker et formance liquid chromatography method previously al. 1995; Invernizzi et al. 1995). described (updated in Adell and Artigas 1998). 5-HT To our knowledge, there are no published reports on was separated on a 3 ␮m ODS 2 column (7.5 cm ϫ 0.46 the effects of prolonged treatments with combinations cm; Beckman, San Ramon, CA) and detected ampero- of SSRIs and 5-HT1A receptor antagonists on these ex- metrically with a Hewlett Packard 1049 detector set at perimental paradigms, except in abstract form (Dawson the potential of ϩ0.6V. Retention time was 3.5–4 min. et al. 1998). As with pindolol, future selective 5-HT1A re- The detection limit for 5-HT was typically 0.5–1 fmol/ ceptor antagonists would be administered for a limited sample. Dialysate 5-HT values were calculated by refer- period of time. Should prolonged blockade of 5-HT1A ence to standard curves run daily. receptor result in receptor sensitization, the withdrawal of the antagonist would increase the efficacy of the Treatments above negative feed-back and reduce the ability of the SSRI to increase extracellular 5-HT, thus increasing the possi- Four different experiments were conducted. In the first bility of a clinical relapse. Since this is crucial for the one, we examined the effects on extracellular 5-HT in success of this therapeutic strategy, we examined the ef- frontal cortex of single doses of fluoxetine (3 mg/kg, i.p.) fects of two-week treatments with fluoxetine, WAY- plus (20 min later) saline or WAY-100635 [N-(2-(4-(2- 100635 and their combination on the labeling and sensi- methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridyl) и tivity of 5-HT1A autoreceptors in rat brain . cyclohexanecarboxamide 3HCl] (RBI, Natick, MA) (0.3 mg/kg, s.c.). A second experiment assessed the effect of two-week minipump treatments with fluoxetine (3 mg/ METHODS kg·day) and fluoxetine plus WAY-100635 (0.3 mg/ kg·day) on the basal extracellular 5-HT concentration. In Microdialysis Procedures this group, microdialysis experiments were conducted on Male Wistar rats (280–300 g; Iffa-Credo, Lyon, France) the 14th day of treatment, with the minipump on board. were used. Animal care followed the European Union In a third experiment, the same treatments (fluoxe- regulations (O.J. of E.C. L358/1 18/12/1986). A de- tine 3 mg/kg·day and fluoxetine 3 mg/kg·day plus tailed description of the microdialysis procedures can WAY-100635 0.3 mg/kg·day) were given but minipumps be found in Adell and Artigas (1998). Briefly, anesthe- were removed under light anesthesia on day 14th and tized rats (pentobarbital, 60 mg/kg, i.p.) were placed in microdialysis experiments were conducted two days a David Kopf (Tujunga, CA) stereotaxic frame. Concen- later to examine the labeling and sensitivity of 5-HT1A tric dialysis probes (4.0 mm long) were implanted in receptors. A complete washout of fluoxetine was ex- frontal cortex and secured to the skull with anchor pected at this time, as judged from its complete elimina- screws and dental cement. Dialysis membranes were tion in two days from the brain compartment after 21- made from hollow Cuprophan fibers with 252 ␮m OD, day treatments with higher doses (10–30 mg/kg·day) 220 ␮m ID, and 5000-dalton molecular weight cutoff (Gardier et al. 1993). In these experimental groups, dial- (GFE09; Gambro, Lund, Sweden). ysis probes were perfused with an artificial CSF contain- The stereotaxic coordinates (in mm; AP ϩ3.4, DV ing 1 ␮M citalopram. In this experimental condition, the Ϫ6.0, L Ϫ2.5) were taken from bregma and dura mater systemic administration of SSRIs reduces extracellular according to the rat brain atlas of Paxinos and Watson 5-HT due to the activation of somatodendritic 5-HT1A (1986). Rats were allowed to recover from anesthesia in autoreceptors (see Discussion). With the same time the dialysis cages (cubic, 40 cm each side) and 20–24 h schedule (two-week treatment plus two-day washout) later the probes were perfused with artificial CSF and experimental conditions, a fourth experiment exam- (aCSF; composition: NaCl 125 mM, KCl 2.5 mM, MgCl2 ined the effect of WAY-100635 (0.3 mg/kg·day) alone on ␮ 1.18 mM, and CaCl2 1.26 mM; pH 6.5–7.0) at 0.25 l/ the sensitivity of raphe 5-HT1A receptors. Control rats in min. In some experiments, the aCSF was supplemented all groups received the corresponding vehicle for the with 1 ␮M of the SSRI citalopram. same time periods. NEUROPSYCHOPHARMACOLOGY 2001–VOL. 24, NO. 1 Fluoxetine, WAY-100635, and 5-HT1A Receptors 13 In chronic experiments, rats were implanted s.c. with Data Treatment Alzet 2002 minipumps filled to deliver vehicle (water/ Dialysate 5-HT concentrations are expressed as fmol/ dimethyl sulfoxide 50/50%), fluoxetine 3 mg/kg·day fraction and represented in some figures as percentage (Eli Lilly and Co., Indianapolis, IN), WAY-100635 0.3 of baseline (average of four pre-drug fractions).