European Neuropsychopharmacology (]]]]) ], ]]]–]]]
www.elsevier.com/locate/euroneuro
5-HT2A receptors are involved in cognitive but not antidepressant effects of fluoxetine Anna Castañéa,b,c,n, Lucila Kargiemana,d,e, Pau Celadaa,b,c, Analía Bortolozzia,b,c, Francesc Artigasa,b,c a
Department of Neurochemistry and Neuropharmacology, CSIC-Institut d’Investigacions Biomèdiques de Barcelona (IIBB), Barcelona, Spain b Centro de Investigación Biomédica en Red de Salud Mental (CIBERSAM), ISCIII, Madrid, Spain c Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain d Laboratory of Experimental Psychology & Neuroscience, Institute of Cognitive Neurology, Favaloro University, Buenos Aires, Argentina e UDP-INECO Foundation Core on Neuroscience, Diego Portales University, Santiago, Chile Received 15 October 2014; received in revised form 24 March 2015; accepted 1 April 2015
KEYWORDS
Abstract
5-HT2A receptors; Antidepressant drugs; Prefrontal cortex; Novel object recognition; Tail suspension test; Working memory
The prefrontal cortex (PFC) plays a crucial role in cognitive and affective functions. It contains a rich serotonergic (serotonin, 5-HT) innervation and a high density of 5-HT receptors. Endogenous 5-HT exerts robust actions on the activity of pyramidal neurons in medial PFC (mPFC) via excitatory 5-HT2A and inhibitory 5-HT1A receptors, suggesting the involvement of 5-HT neurotransmission in cortical functions. However, the underlying mechanisms must be elucidated. Here we examine the role of 5HT2A receptors in the processing of emotional and cognitive signals evoked by increasing the 5-HT tone after acute blockade of the 5-HT transporter. Fluoxetine (5–20 mg/kg i.p.) dose-dependently reduced the immobility time in the tail-suspension test in wild-type (WT) and 5-HT2A knockout (KO2A) mice, with non-significant differences between genotypes. Fluoxetine (10 mg/kg i.p.) significantly impaired mice performance in the novel object recognition test 24 h post-administration in WT, but not in KO2A mice. The comparable effect of fluoxetine on extracellular 5-HT in the mPFC of both genotypes suggests that presynaptic differences are not accountable. In contrast, single unit recordings of mPFC putative pyramidal neurons showed that fluoxetine (1.8–7.2 mg/kg i.v.) significantly increased neuronal discharge in KO2A but not in WT mice. This effect is possibly mediated by an altered excitatory/ inhibitory balance in the PFC in KO2A mice. Overall, the present results suggest that 5-HT2A receptors play a detrimental role in long-term memory deficits mediated by an excess 5-HT in PFC. & 2015 Elsevier B.V. and ECNP. All rights reserved.
n Corresponding author at: Department of Neurochemistry and Neuropharmacology, CSIC-Institut d’Investigacions Biomèdiques de Barcelona (IIBB), Barcelona, Spain. Tel.: +34 933638321; fax: +34 933638301. E-mail address: [email protected] (A. Castañé).
http://dx.doi.org/10.1016/j.euroneuro.2015.04.006 0924-977X/& 2015 Elsevier B.V. and ECNP. All rights reserved.
Please cite this article as: Castañé, A., et al., 5-HT2A receptors are involved in cognitive but not antidepressant effects of fluoxetine. European Neuropsychopharmacology (2015), http://dx.doi.org/10.1016/j.euroneuro.2015.04.006
2
A. Castañé et al.
Introduction The prefrontal cortex (PFC) is a critical area in mediating higher-order executive tasks such as working memory, inhibitory control and behavioural flexibility among others, under a strict temporal pattern (Fuster, 2001). Moreover, it plays an important role in emotional responses (Euston et al., 2012). In fact, the PFC has been recently identified as a core region for the interaction between cognition and emotional processing (Ray and Zald, 2012). The PFC receives a moderate to dense serotonergic innervation from the raphe nuclei (Azmitia and Segal, 1978; Blue et al., 1988) and contains several serotonin (5-hydroxytryptamine, 5-HT) receptor subtypes, with a particular high density of 5-HT1A and 5-HT2A receptors (Pompeiano et al., 1992, 1994) which may represent major modulators of PFC tasks. Previous studies in rats and monkeys showed that 5-HT transmission in the orbitofrontal cortex (OFC) is involved in flexible behaviours. Specifically, 5-HT depletion in the OFC as well as the lesion of this area induced reversal learning deficits (Boulougouris et al., 2007; Clarke et al., 2007). 5-HT2A receptors have been shown to modulate this type of behavioural flexibility, since the selective 5-HT2A receptor antagonist M100907 impaired reversal performance in rats (Boulougouris et al., 2008). Likewise, local prefrontal M100907 application reduced persistent neuronal activity during working memory tasks in monkeys (Williams et al., 2002) and a recent computational study indicated that 5HT2A receptors contribute to spatial working memory tasks (Cano-Colino et al., 2014). However, evidence on the role of serotonergic transmission through 5-HT2A receptors in cognitive domains remains scarce. Prefrontal cognitive dysfunction is a core feature in many psychiatric disorders including depression and schizophrenia. 5HT2A receptors have been proposed as possible targets to improve cognition and depressive-like effects in mental illnesses (Mestre et al., 2013). It has been recently described that the chronic intermittent cold (CIC) stress model of depression is associated with reduced serotonergic transmission in the OFC and induces reversal learning deficits in rats, that are sensitive to selective serotonin reuptake inhibitors (SSRIs) treatment (Danet et al., 2010) through a 5-HT2A receptor mechanism (Furr et al., 2012). In contrast to the above data, atypical antipsychotics, which exert their therapeutic action, at least in part, by blocking 5-HT2A-mediated responses (Nyberg et al., 1999) procured some advantages over classic antipsychotics for treating cognitive impairment in schizophrenia (Meltzer et al., 2012; Meltzer, 2013). On the other hand, clinical studies showed that atypical antipsychotics increased the efficacy of SSRIs in treatmentresistant depression, possibly through 5-HT2A receptor blockade (Ostroff and Nelson, 1999; Nelson and Papakostas, 2009). Preclinical data also suggest that selective blockade of 5-HT2A receptors may have a synergistic effect with SSRIs antidepressant treatment (Marek et al., 2005). Thus, Marek and collaborators described a synergistic antidepressant-like effect of a low dose of M100907 in rats using the DRL 72-s test. However, the neurobiological bases of this improvement remain poorly understood. Here we investigate the role of 5-HT2A receptors in the processing of emotional and cognitive responses mediated
by the mPFC, evoked by increasing the serotonergic tone after acute blockade of the serotonin transporter (SERT) with the SSRI fluoxetine in mice.
Experimental procedures Subjects We used 9–12-week old male 5-HT2A receptor knockout (5-HT2AR KO, KO2A) and wild-type mice (WT). Mice with the targeted deletion of 5-HT2AR were developed as reported (Fiorica-Howells et al., 2002). From an initial source, a stable colony of KO2A and WT mice were grown in our animal facilities. Animals were kept in a controlled environment (12 h light–dark cycle and 2272 1C room temperature) with food and water provided ad-libitum. All experimental procedures were in strict accordance with European Union regulations (Official Journal of the European Communities L358/1, 18 December 1986), and were approved by the Institutional Animal Care and Use Committee.
Drugs For behavioural and neurochemical studies, fluoxetine hydrochloride (Tocris, USA) was dissolved in distilled water and injected by intraperitoneal route (i.p) at a volume of 10 ml/kg. For the electrophysiological studies, fluoxetine hydrochloride was dissolved in saline and injected intravenously (i.v.) at a volume of 1–2 ml/kg. Fluoxetine doses are expressed as free base.
Tail suspension test (TST) TST was conducted essentially as previously described (Ferrés-Coy et al., 2013). Briefly, mice were suspended 30 cm above the floor by adhesive tape placed approximately 1 cm from the tip of the tail and were observed on a monitor through a video camera system (Smart, Panlab). The time mice spent immobile was recorded during 6 min. Pharmacological treatments were administered 1 h before the test.
Novel object recognition test (NOR) On day one, mice were habituated for 10 min to the maze where the task was performed. On the second day, mice were put back in the maze for 10 min where two identical objects were presented (Trial 1, T1), and the time the animals spent exploring each object was recorded. Three or twenty-four hour later, mice were put again in the maze for 10 min, where one of the familiar objects was replaced by a novel object (Trial 2, T2), and the total time spent exploring each of the two objects (novel, N and familiar, F) was computed. Object exploration was defined as the orientation of the nose to the object at a distanceo1 cm. A recognition index (RI) was calculated as the percentage of the time exploring the novel object divided by the total time exploring the two objects (novel and familiar) as described [RI =N/(F+N)*100]. RI450% is considered to reflect greater memory retention for the familiar object. Pharmacological treatments were administered 1 h before T1.
Intracerebral microdialysis Extracellular serotonin was measured by in vivo microdialysis as previously described (Castañé et al., 2008). Briefly, one concentric dialysis probe equipped with a Cuprophan membrane (2-mm long) was implanted in the medial PFC (mPFC) at coordinates (in mm): AP
Please cite this article as: Castañé, A., et al., 5-HT2A receptors are involved in cognitive but not antidepressant effects of fluoxetine. European Neuropsychopharmacology (2015), http://dx.doi.org/10.1016/j.euroneuro.2015.04.006
5-HT2A receptors are involved in cognitive +2.2; ML-0.2; DV-3.4 (Franklin and Paxinos, 1997), of anaesthetized mice (pentobarbital, 40 mg/kg, i.p.). Microdialysis experiments were performed 20–24 h after surgery. The aCSF was pumped at 2.0 μl/min (WPI model sp220i) and dialysates were collected every 30 min. Serotonin concentration was analysed by HPLC with amperometric detection (Hewlett Packard 1049) at +0.6 V, with a detection limit of 1.5 fmol/sample. Baseline serotonin levels were calculated as the average of the four pre-drug samples. Following sample collection, brains were removed and sectioned to ensure proper probe placement after cresyl violet staining.
Electrophysiological studies Single unit recordings: We assessed the effects of the i.v. administration of fluoxetine on the firing rate of putative mPFC pyramidal neurons in WT and KO2A mice. Recordings were performed as described previously (Lladó-Pelfort et al., 2012; Puig et al., 2005). Briefly, mice were anesthetized with chloral hydrate (400 mg/kg, i.p.) and positioned in a David Kopf stereotaxic frame. Additional doses of chloral hydrate (80 mg/kg) were administered i. v. through the femoral vein. Body temperature was maintained at 37 1C through the experiment with a heating pad. Putative pyramidal neurons were recorded extracellularly with glass micropipettes filled with 2 M NaCl. Impedance was between 6 and 10 MΩ. Single unit recordings were amplified with a Neurodata IR283 (Cygnus Technology Inc., Delaware Water Gap, PA), post-amplified and filtered with a Cibertec amplifier (Cibertec, Madrid, Spain) and computed on-line using a DAT 1401plus interface system Spike2 software (Cambridge Electronic Design, Cambridge, UK). Putative pyramidal neurons in the mPFC were recorded at AP +2.2 to +2.4; L-0.2 to 0.5; DV-1.1 to 2.6 mm below the brain surface and identified according to previously described electrophysiological characteristics: 1) duration of the depolarization phase of action potential (average of 10 spikes) and 2) basal discharge rate, as previously reported (Lladó-Pelfort et al., 2012; Wilson et al., 1994). Basal firing rate was recorded for at least 5 min and then, cumulative doses of fluoxetine (1.8 to 7.2 mg/kg, i.v.) were administered. At the end of the experiments, mice were killed by an anaesthetic overdose. Changes in discharge rate were quantified by averaging the values in the third minute after each dose injection.
Statistical analysis
3 interaction between the two factors [F(3,63) = 1.220, n.s.]. Post-hoc analysis revealed a significant decrease of immobility time in both wild-type and KO2A mice after 10 and 20 mg/kg of fluoxetine (po0.01, all cases) compared to controls of the same genotype (Figure 1).
Effects of acute fluoxetine in the novel object recognition test in WT and KO2A mice Statistical analysis revealed a non-significant effect of treatment [F(1,26) = 0.903, n.s.], genotype [F(1,26) = 1.292, n.s.], and interaction between treatment and genotype [F(1,26) = 0.8523, n.s.] when object recognition memory was assessed at 3 h (Figure 2a). Acute fluoxetine treatment significantly impaired object recognition memory in WT but not KO2A mice when tested at 24 h (Figure 2b). Two-way ANOVA showed a significant effect of treatment [F(1,32) = 4.179, po0.05], genotype [F(1,32) = 7.201, po0.05] and the interaction between treatment and genotype [F(1,32) = 7.346, po0.05]. Subsequent post-hoc analysis revealed a significant effect of fluoxetine treatment in WT mice compared to controls (po0.01) and a significant difference between WT and KO2A mice treated with fluoxetine (po0.01) (n= 7–9/group).
Effect of fluoxetine on extracellular 5-HT Basal extracellular concentrations of 5-HT in dialysate samples of mPFC were, WT: 3.070.5 (n=17); KO2A: 2.770.5 (n=17) expressed as fmol/60 μl (n.s., Student's t-test). The systemic administration of fluoxetine (10 mg/kg, i.p.) induced a similar increase of extracellular 5-HT concentrations in mPFC of WT and KO2A mice compared to vehicle-treated animals (Figure 3). Thus, three-way ANOVA showed a main effect of treatment [F(1,29) =23.7021, po0.001], time [F(11,319) =11.8423, po0.001] and the interaction between treatment and time [F(11,319) =9.0806, po0.001], but not of genotype or interaction between treatment and genotype. Subsequent post-hoc analysis revealed a significant difference
Behavioural data were analysed using a two-way ANOVA (genotype and treatment as between factors of variation). Microdialysis data were analysed by a three-way ANOVA with repeated measures (genotype and treatment as between factors and fraction as a within factor of variation). Electrophysiological data were analysed by the Student-t-test, one-way or two-way ANOVA, as appropriate. Significant main effects of genotype, treatment, fraction or interactions between these factors were further explored through posthoc Duncan comparisons to establish simple effects. Statistical significance was set at the 95% confidence level.
Results Antidepressant-like responses of fluoxetine in WT and KO2A mice Acute fluoxetine treatment (5, 10 and 20 mg/kg i.p.) induced antidepressant-like responses in the TST in both WT and KO2A mice (n= 8–13/group). Two-way ANOVA showed a significant effect of treatment [F(3,63) =29.143, po0.001] but not of genotype ([F(1,63) = 2.862, n.s.], nor the
Figure 1 Effects of acute fluoxetine (FLX) administration (5, 10, 20 mg/kg, i.p) in wild-type (WT) and 5-HT2A receptor knockout (KO2A) mice in the TST. The treatment was administered 1 h before the test. Data are expressed as mean7SEM of immobility time in seconds (s). Duncan post-hoc, **po0.01 vs controls (VEH).
Please cite this article as: Castañé, A., et al., 5-HT2A receptors are involved in cognitive but not antidepressant effects of fluoxetine. European Neuropsychopharmacology (2015), http://dx.doi.org/10.1016/j.euroneuro.2015.04.006
4
A. Castañé et al.
Figure 2 Effects of acute fluoxetine administration in the novel object recognition test in wild-type (WT) and 5-HT2A receptor knockout (KO2A) mice. Fluoxetine (FLX, 10 mg/kg, i.p) or vehicle (VEH) treatments were administered 1 h before the trial 1, in WT and KO2A mice. Test was performed at 3 h (a) or 24 h (b). Data are expressed as mean7SEM of recognition index expressed in percentage (%). Duncan post-hoc, **po0.01 vs VEH, po0.01 vs WT.
Figure 3 Effects of systemic administration of fluoxetine (10 mg/kg) on dialysate 5-HT concentrations in mPFC of wildtype (WT) and 5-HT2A receptor knockout mice (KO2A). Data are expressed as mean7SEM of percentages of pre-treatment values. Duncan post-hoc, **po0.01, fluoxetine-treated (FLX) vs vehicle-treated (VEH) mice.
between fluoxetine- and vehicle-treated mice in both genotypes (po0.01).
Electrophysiological studies Firing characteristics of putative pyramidal neurons in mice mPFC A total of 16 and 12 putative pyramidal neurons (one per mouse) were recorded in untreated WT and KO2A mice, respectively. The baseline firing rate was 1.6370.50 spikes/ s and 0.9670.29 spikes/s for WT and KO2A mice, respectively (p= 0.30, Student's t-test). Action potential duration was also similar in both groups: 0.7570.04 ms and 0.7970.03 ms for WT and KO2A mice, respectively. Effects of fluoxetine on mPFC putative pyramidal neuron firing The administration of fluoxetine (1.8–7.2 mg/kg, i.v.) to WT mice did not modify the firing rate of putative pyramidal neurons in the mPFC (1.4470.42, 1.6870.50, 1.5570.42,
1.0170.29 spikes/s in basal conditions and after the administration of 1.8, 3.6 and 7.2 mg/kg fluoxetine, respectively; n =13) (Figure. 4a and c). In contrast, the administration of FLX (1.8–7.2 mg/kg, i.v.) to KO2A mice increased the firing rate of putative pyramidal neurons in the mPFC (0.9670.38, 1.1470.40, 1.8470.80, 2.2270.77 in basal conditions and after the administration of 1.8, 3.6 and 7.2 mg/kg fluoxetine, respectively; n= 9) (Figure. 4b and c). Thus, two-way ANOVA revealed a significant treatment x genotype interaction [F(3,60) =3.42, po0.03] with significant post-hoc differences between baseline and fluoxetine 7.2 mg/kg in the KO2A mice and between WT and KO2A mice after treatment with fluoxetine 7.2 mg/kg (Figure 4c).
Discussion The present study shows that the increase in serotonergic tone produced by SERT blockade with fluoxetine evoked similar antidepressant-like responses in the TST in WT mice and in KO2A mice. In contrast, fluoxetine impaired declarative memory in WT (but not KO2A) mice 24 h after administration, as assessed by the NOR test, indicating that i) an acute increase of the serotonergic tone in forebrain— possibly in the PFC—impairs long-term memory, and ii) this impairment is mediated—at least in part—by the activation of 5-HT2A receptors. Early studies using antipsychotic medications suggested a 5HT2A receptor mechanism in increasing the efficacy of selective serotonin reuptake inhibitors (SSRIs) in treatmentresistant depression (Ostroff and Nelson, 1999; Nelson and Papakostas, 2009). Likewise, Marek et al. (2005) reported on a synergistic antidepressant-like effect when combining a low dose of the selective 5-HT2A receptor antagonist M100907 with fluoxetine in the differential-reinforcement-of low rate 72-s schedule of reinforcement (DRL 72-s) paradigm. The present data are partly at variance with these observations since we did not find an enhancement of fluoxetine-induced antidepressant-like responses in the
Please cite this article as: Castañé, A., et al., 5-HT2A receptors are involved in cognitive but not antidepressant effects of fluoxetine. European Neuropsychopharmacology (2015), http://dx.doi.org/10.1016/j.euroneuro.2015.04.006
5-HT2A receptors are involved in cognitive
Figure 4 (a, b) Integrated firing rate histogram showing the effect of the i.v. administration of FLX (mg/kg; vertical arrows) on the activity of mPFC pyramidal neurons. (c) Bar histogram showing the average effect produced by FLX in WT and KO2A mice. Duncan post-hoc, *po0.05 vs basal, po0.05 vs WT.
TST in the KO2A mice. Differences in the species used (mice vs rats) and/or the differential contribution of serotonergic neurotransmission to the behaviours involved in both tests may be accountable. In regards to the TST, a high 5-HT tone in cortical and limbic structures (in particular PFC; Warden et al., 2012) is required to reduce behavioural despair during stressful situations. In agreement, the reduction of immobility is associated with an increase of 5-HT release in mouse PFC during the exposure to TST (Ferrés-Coy et al., 2013). However, in the DRL 72-s test, rats must learn to control their impulsivity to press a lever during a prolonged
5 period of time (72 s) in order to obtain a food reward. Thus, whereas the TST is mainly related to an emotional behaviour, the DRL 72-s test involves more complex responses requiring increased attention, impulse control and time perception. This suggests that different PFC areas and possibly different 5-HT receptors are involved in both tests. The comparable enhancement of extracellular 5-HT produced by fluoxetine in WT and KO2A mice observed in microdialysis experiments is in accordance with the inability of M100907 to enhance fluoxetine effect on extracellular 5HT (Marek et al., 2005). It also shows that the observed differences in the NOR test between WT and KO2A mice are not due to a differential enhancement of presynaptic 5-HT function in both genotypes during acquisition and/or memory consolidation (o 4 h). Indeed, the [3H]-citalopram binding site density in the dorsal raphe nucleus was similar for both genotypes (unpublished observations). On the contrary, electrophysiological experiments revealed the existence of genotype differences in the postsynaptic response to fluoxetine administration. Unlike in previous studies from this laboratory in rats (Lladó-Pelfort et al., 2012; Puig et al., 2005) we did not identify pyramidal neurons in mouse mPFC by antidromic activation due to the inherent difficulties to perform this procedure in mouse brain. However, the recorded neurons are most likely pyramidal neurons in view of their slower discharge rate and longer duration of the action potential, as compared with GABAergic interneurons. Hence, GABAergic neurons have a mean action potential duration below 0.5 ms (Lladó-Pelfort et al., 2012; Tierney et al., 2004) whereas the corresponding values in the present study were 0.7570.04 ms and 0.7970.03 ms for WT and KO2A mice, respectively. Likewise, discharge rates (1.6370.50 spikes/s and 0.9670.29 spikes/s for WT and KO2A mice, respectively) were well below those of fast-spiking GABAergic interneurons (Lladó-Pelfort et al., 2012; Tierney et al., 2004). As previously reported in the rat (Ceci et al., 1994; Gronier and Rasmussen, 2003), acute fluoxetine treatment did not alter the discharge rate of PFC neurons in WT mice. However, fluoxetine increased dose-dependently the discharge rate of putative pyramidal neurons in the mPFC of KO2A mice. The lack of effect of fluoxetine on neuronal discharge in WT mice may be attributed to the concurrent activation of excitatory and inhibitory receptors in cortical microcircuits involving pyramidal neurons and GABAergic interneurons. The main 5-HT receptors in mPFC are 5-HT1A and 5-HT2A receptors, which are expressed by a high percentage of pyramidal neurons and GABAergic interneurons in the rat PFC (Santana et al., 2004) where they mediate excitatory (5-HT2A) and inhibitory (5-HT1A) actions of 5-HT (Araneda and Andrade, 1991; Celada et al., 2013; Puig et al., 2005). Interestingly, these receptors are highly co-expressed in the same PFC neurons in rats and mice (Amargós-Bosch et al., 2004) and are possibly segregated into different neuronal compartments. Thus, 5-HT1A receptors have been identified in the axon hillock of pyramidal neurons (de Felipe et al., 2001) or in the somatodendritic compartment (Riad et al., 2000), whereas 5-HT2A receptors are predominantly localized in the cell body and apical dendrites (Jakab and Goldman-Rakic, 1998; Martín-Ruiz et al., 2001). The
Please cite this article as: Castañé, A., et al., 5-HT2A receptors are involved in cognitive but not antidepressant effects of fluoxetine. European Neuropsychopharmacology (2015), http://dx.doi.org/10.1016/j.euroneuro.2015.04.006
6
A. Castañé et al.
existence of simultaneous responses mediated by both receptors in the same neuronal populations is shown by biphasic responses to endogenous 5-HT (Puig et al., 2005) and by the emergence of 5-HT2A receptor-mediated excitations, after the blockade of 5-HT1A receptor-mediated inhibitions (Amargós-Bosch et al., 2004). Therefore, the excess 5-HT produced by SERT blockade with fluoxetine may simultaneously activate 5-HT1A and 5HT2A receptors in mPFC, leading to a compensation of inhibitory and excitatory responses in the same neurons or microcircuits. However, this possibility is at variance with the predominant inhibitory action of 5-HT on pyramidal neuron activity, -mediated by 5-HT1A receptors- after the stimulation of the dorsal and median raphe nuclei at physiological rates (Puig et al., 2005). Moreover, the duplication of the stimulation frequency (to enhance 5-HT release) increased the duration of inhibitory responses and turned some excitations into inhibitions (Puig et al., 2005), indicating a predominant role of 5-HT1A receptors in the control of pyramidal neuron activity in mPFC. One possibility to reconcile these two observations (e.g., lack of effect of fluoxetine vs predominant inhibitory responses of endogenous 5-HT) is that the excess 5-HT produced in both experimental situations may occur in
different neuronal micro-compartments and/or with a different temporal pattern (Figure 5). Hence, pyramidal inhibitions evoked by raphe stimulation are phasic and typically occur in the 20–200 ms scale, indicating that 5HT is released—intra- or extras-synaptically—, but in the close vicinity of 5-HT1A receptors. The possible location of 5-HT1A receptors in the axon hillock of pyramidal neurons (de Felipe et al., 2001), may facilitate inhibitory responses, making 5-HT1A receptors to act as neuronal switches such as GABAA receptors, also located in the axon hillock (de Felipe et al., 2001). On the other hand, SERT blockade in the very dense network of 5-HT axon terminals (4106 varicosities/mm3 in rat neocortex; Beaudet and Descarries, 1976) is likely to produce a tonic and overall increase of 5-HT in the extracellular brain space, leading to an activation of 5-HT receptors different from that resulting from the phasic release after raphe stimulation. Interestingly, in vitro experiments in PFC slices indicate that bath applied 5-HT preferentially activates 5-HT2 receptors in fast-spiking interneurons and decreases the firing rate of pyramidal neurons in a concentration-dependent manner (Zhong and Yan, 2011). However, bath application of fluoxetine—while increasing the firing rate of fast spiking interneurons—did
Figure 5 Schematic representation of the differential activation of 5-HT receptors in mPFC after midbrain stimulation and during SERT blockade with fluoxetine (FLX). (a) The electrical stimulation of DR/MnR excites and inhibits pyramidal neurons by stimulating 5-HT2A and 5-HT1A receptors, respectively, after the phasic release of 5-HT following the arrival of action potentials at nerve terminals (green circles). The duration of these effects is limited by 5-HT reuptake. Pyramidal 5-HT1A receptors are localized in the axon hillock, together with GABAA receptors (De Felipe et al., 2001) or in the somatodendritic compartment (Riad et al., 2000) whereas 5-HT2A receptors are predominantly localized in apical dendrites. Given the abundant co-expression of both receptors (Amargós-Bosch et al., 2004) the occurrence of excitations or inhibitions may depend on a precise topology between certain 5-HT neurons or neuronal clusters in the DR/MnR and 5-HT1A- or 5-HT2A-receptor-rich compartments according to the association between 5-HT axons and such receptor-rich areas (De Felipe et al., 2001; Jansson et al., 2001), as shown in the figure. (b) The blockade of SERT by fluoxetine produces a tonic increase of the 5-HT concentration in the extracellular space of PFC, as observed in microdialysis studies which may evoke a generalized activation of 5-HT receptors in pyramidal and GABAergic neurons. The present results suggest that 5-HT2A receptors in GABAergic neurons play a predominant role in the control of pyramidal neuron activity in conditions of SERT blockade by fluoxetine.
Please cite this article as: Castañé, A., et al., 5-HT2A receptors are involved in cognitive but not antidepressant effects of fluoxetine. European Neuropsychopharmacology (2015), http://dx.doi.org/10.1016/j.euroneuro.2015.04.006
5-HT2A receptors are involved in cognitive not simultaneously reduce the firing rate of pyramidal neurons (Zhong and Yan, 2011). Our in vivo observations are in agreement with this view, since fluoxetine did not alter the firing rate of putative pyramidal neurons in WT mice but increased it in KO2A mice. A preferential activation of 5-HT2A receptors in pyramidal neurons by fluoxetine would have led to the reduction of the firing rate in KO2A mice, whereas the opposite effect was observed. Given the lack of 5-HT2A receptors in all neuronal populations in KO2A mice, the increase in pyramidal discharge cannot be ascribed to the activation of 5-HT2A receptors in the recorded neurons. Although the participation of other excitatory 5-HT receptors (e.g., 5-HT2C, 5-HT4, 5-HT7) cannot be discarded, the increase in pyramidal discharge may actually be a disinhibition (Figure 5), after the stimulation of 5-HT1A receptors in fast-spiking interneurons by the excess 5-HT, as observed for 5-HT1A agonists (Lladó-Pelfort et al., 2012). A key process in cognitive functions mediated by the PFC is working/short-term memory. Working memory is associated to persistent neuronal activity in the dorsolateral PFC (prelimbic mPFC in the rat/mouse) in tasks involving delayed responses (Curtis and D’Esposito, 2003) such as the NOR test. Several neurotransmitters have been related to working memory, in particular the catecholamines (Vijayraghavan et al., 2007; Williams and Goldman-Rakic, 1995) whereas much less is known about 5-HT. The present observations suggest that 5-HT2A receptors might play a detrimental role in short-term memory and/or in memory consolidation. Hence while differences between WT and KO2A in the NOR test after a 3 h delay did not reach statistical significance, they were of greater magnitude and statistically significant at 24 h, suggesting that the indirect activation of 5-HT2A receptors by fluoxetine do impair long-term memories. Further studies are needed to elucidate whether acquisition, consolidation and/or memory retrieval are compromised after fluoxetine treatment. In humans, cognitive side effects of initial treatment with fluoxetine have been described (Bangs et al., 1994; Joss et al., 2003), although their underlying mechanism it is currently unknown. Here we propose that the increased discharge rate of putative pyramidal PFC neurons in KO2A mice may facilitate the persistent neuronal activity required for consolidation of memory processes (Fuster, 2001). However, the role of 5-HT2A receptors in learning and memory is still scarce and deserves further research (Meneses, 2007; Zhang et al., 2013). In summary, the present study shows that the blockade or absence of 5-HT2A receptors do not increase fluoxetineinduced antidepressant-like responses in the TST whereas it has a beneficial role in long-term memory, as assessed in the NOR test. These results may help to improve our understanding on the role of 5-HT in PFC and particularly on cognitive functions.
Role of funding source Funding for this study was provided by Grants SAF 2012-35183, the Ministry of Economy and Competitiveness, co-financed by European Regional Development Fund (ERDF) and by the Grants PI12/00156, PI13/01390 and PI10/00290 (PN de I +D+I 2008-2011, ISCIIISubdirección General de Evaluación y Fomento de la Investigación
7 cofinanced by the European Regional Development Fund. “Una manera de hacer Europa”), and Centro de Investigación Biomédica en Red de Salud Mental, CIBERSAM. AC was supported by a postdoctoral contract from the ISCIII (CD05/00234). None of the financing Institutions had further role in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication.
Contributors Author AC designed the study and wrote the protocol, managed the literature searches and analyses and undertook the statistical analysis. Authors AC and LK performed the behavioural experiments. Authors LK and PC performed electrophysiology experiments. Authors AC and AB performed microdialysis experiments. Authors AC and FA wrote the manuscript. All authors contributed to and have approved the final manuscript.
Conflict of interest FA has received consulting and educational honoraria on antidepressant drugs from Lundbeck and he is PI of a grant from Lundbeck. He is also member of the advisory board of Neurolixis. The rest of authors declare no conflict of interest.
Acknowledgements We thank Leticia Campa for her skilful maintenance of HPLC equipment and analyses of dialysate samples. We also thank Dr. Jay Gingrich for the generous supply of 5-HT2A receptor knockout mice.
References Amargós-Bosch, M., Bortolozzi, A., Puig, M.V., Serrats, J., Adell, A., Celada, P., Toth, M., Mengod, G., Artigas, F., 2004. Coexpression and in vivo interaction of serotonin1A and serotonin2A receptors in pyramidal neurons of prefrontal cortex. Cereb. Cortex 14, 281–299. Araneda, R., Andrade, R., 1991. 5-Hydroxytryptamine2 and 5hydroxytryptamine 1A receptors mediate opposing responses on membrane excitability in rat association cortex. Neuroscience 40, 399–412. Azmitia, E.C., Segal, M., 1978. An autoradiographic analysis of the differential ascending projections of the dorsal and median raphe nuclei in the rat. J. Comp. Neurol. 179, 641–667. Bangs, M.E., Petti, T.A., Janus, M.D., 1994. Fluoxetine-induced memory impairment in an adolescent. J. Am. Acad. Child Adolesc. Psychiatry 33, 1303–1306. Beaudet, A., Descarries, L., 1976. Quantitative data on serotonin nerve terminals in adult rat neocortex. Brain Res. 111, 301–309. Blue, M.E., Yagaloff, K.A., Mamounas, L.A., Hartig, P.R., Molliver, M.E., 1988. Correspondence between 5-HT2 receptors and serotonergic axons in rat neocortex. Brain Res. 453, 315–328. Boulougouris, V., Dalley, J.W., Robbins, T.W., 2007. Effects of orbitofrontal, infralimbic and prelimbic cortical lesions on serial spatial reversal learning in the rat. Behav. Brain Res. 179, 219–228. Boulougouris, V., Glennon, J.C., Robbins, T.W., 2008. Dissociable effects of selective 5-HT2A and 5-HT2C receptor antagonists on serial spatial reversal learning in rats. Neuropsychopharmacology 33, 2007–2019. Cano-Colino, M., Almeida, R., Gomez-Cabrero, D., Artigas, F., Compte, A., 2014. Serotonin regulates performance nonmonotonically in a spatial working memory network. Cereb. Cortex 24, 2449–2463.
Please cite this article as: Castañé, A., et al., 5-HT2A receptors are involved in cognitive but not antidepressant effects of fluoxetine. European Neuropsychopharmacology (2015), http://dx.doi.org/10.1016/j.euroneuro.2015.04.006
8
A. Castañé et al.
Castañé, A., Artigas, F., Bortolozzi, A., 2008. The absence of 5-HT (1A) receptors has minor effects on dopamine but not serotonin release evoked by MK-801 in mice prefrontal cortex. Psychopharmacology 200, 281–290. Ceci, A., Fodritto, F., Borsini, F., 1994. Repeated treatment with fluoxetine decreases the number of spontaneously active cells per track in frontal cortex. Eur. J. Pharmacol. 271, 231–234. Celada, P., Puig, M.V., Artigas, F., 2013. Serotonin modulation of cortical neurons and networks. Front. Integr. Neurosci. 7, 1–20 article 25. Clarke, H.F., Walker, S.C., Dalley, J.W., Robbins, T.W., Roberts, A. C., 2007. Cognitive inflexibility after prefrontal serotonin depletion is behaviorally and neurochemically specific. Cereb. Cortex 17, 18–27. Curtis, C.E., D'Esposito, M., 2003. Persistent activity in the prefrontal cortex during working memory. Trends Cogn. Sci. 7, 415–423. Danet, M., Lapiz-Bluhm, S., Morilak, D.A., 2010. A cognitive deficit induced in rats by chronic intermittent cold stress is reversed by chronic antidepressant treatment. Int. J. Neuropsychopharmacol. 13, 997–1009. De Felipe, J., Arellano, J.I., Gómez, A., Azmitia, E.C., Muñoz, A., 2001. Pyramidal cell axons show a local specialization for GABA and 5-HT inputs in monkey and human cerebral cortex. J. Comp. Neurol. 433, 148–155. Euston, D.R., Gruber, A.J., McNaughton, B.L., 2012. The role of medial prefrontal cortex in memory and decision making. Neuron 76, 1057–1070. Ferrés-Coy, A., Santana, N., Castañé, A., Cortés, R., Carmona, M. C., Toth, M., Montefeltro, A., Artigas, F., Bortolozzi, A., 2013. Acute 5-HT1A autoreceptor knockdown increases antidepressant responses and serotonin release in stressful conditions. Psychopharmacology 225, 61–74. Fiorica-Howells, E., Hen, R., Gingrich, J., Li, Z., Gershon, M.D., 2002. 5-HT(2A) receptors: location and functional analysis in intestines of wild-type and 5-HT(2A) knockout mice. Am. J. Physiol. Gastrointest. Liver Physiol. 282, G877–G893. Franklin, K., Paxinos, G., 1997. The mouse brain in stereotaxic coordinates. Academic, San Diego, California. Furr, A., Lapiz-Bluhm, M.D., Morilak, D.A., 2012. 5-HT2A receptors in the orbitofrontal cortex facilitate reversal learning and contribute to the beneficial cognitive effects of chronic citalopram treatment in rats. Int. J. Neuropsychopharmacol. 15, 1295–1305. Fuster, J.M., 2001. The prefrontal cortex-an update: time is of the essence. Neuron 30, 319–333. Gronier, B.S., Rasmussen, K., 2003. Electrophysiological effects of acute and chronic olanzapine and fluoxetine in the rat prefrontal cortex. Neurosci. Lett. 349, 196–200. Jakab, R.L., Goldman-Rakic, P.S., 1998. 5-Hydroxytryptamine2A serotonin receptors in the primate cerebral cortex: possible site of action of hallucinogenic and antipsychotic drugs in pyramidal cell apical dendrites. Proc. Natl. Acad. Sci. USA 95, 735–740. Jansson, A., Tinner, B., Bancila, M., Vergé, D., Steinbusch, H.W., Agnati, L.F., Fuxe, K., 2001. Relationships of 5-hydroxytryptamine immunoreactive terminal-like varicosities to 5-hydroxytryptamine2A receptor-immunoreactive neuronal processes in the rat forebrain. J. Chem. Neuroanat. 22, 185–203. Joss, J.D., Burton, R.M., Keller, C.A., 2003. Memory loss in a patient treated with fluoxetine. Ann. Pharmacother. 37, 1800–1803. Lladó-Pelfort, L., Santana, N., Ghisi, V., Artigas, F., Celada, P., 2012. 5-HT1A receptor agonists enhance pyramidal cell firing in prefrontal cortex through a preferential action on GABA interneurons. Cereb. Cortex 22, 1487–1497. Marek, G.J., Martin-Ruiz, R., Abo, A., Artigas, F., 2005. The selective 5-HT2A receptor antagonist M100907 enhances antidepressant-like behavioural effects of the SSRI fluoxetine. Neuropsychopharmacology 30, 2205–2215.
Martín-Ruiz, R., Puig, M.V., Celada, P., Shapiro, D.A., Roth, B.L., Mengod, G., Artigas, F., 2001. Control of serotonergic function in medial prefrontal cortex by serotonin-2A receptors through a glutamate-dependent mechanism. J. Neurosci. 21, 9856–9866. Meltzer, H.Y., 2013. Update on typical and atypical antipsychotic drugs. Annu. Rev. Med. 64, 393–406. Meltzer, H.Y., Massey, B.W., Horiguchi, M., 2012. Serotonin receptors as targets for drugs useful to treat psychosis and cognitive impairment in schizophrenia. Curr. Pharm. Biotechnol. 13, 1572–1586. Meneses, A., 2007. Stimulation of 5-HT1A, 5-HT1B, 5-HT2A/2C, 5HT3 and 5-HT4 receptors or 5-HT uptake inhibition: short- and long-term memory. Behav. Brain Res. 184, 81–90. Mestre, T.A., Zurowski, M., Fox, S.H., 2013. 5-Hydroxytryptamine 2A receptor antagonists as potential treatment for psychiatric disorders. Expert Opin. Investig. Drugs 22, 411–421. Nelson, J.C., Papakostas, G.I., 2009. Atypical antipsychotic augmentation in major depressive disorder: a meta-analysis of placebo-controlled randomized trials. Am. J. Psychiatry 166, 980–991. Nyberg, S., Eriksson, B., Oxenstierna, G., Halldin, C., Farde, L., 1999. Suggested minimal effective dose of risperidone based on PET-measured D2 and 5-HT2A receptor occupancy in schizophrenic patients. Am. J. Psychiatry 156, 869–875. Ostroff, R.B., Nelson, J.C., 1999. Risperidone augmentation of selective serotonin reuptake inhibitors in major depression. J. Clin. Psychiatry 60, 256–259. Pompeiano, M., Palacios, J.M., Mengod, G., 1992. Distribution and cellular localization of mRNA coding for 5-HT1A receptor in the rat brain: correlation with receptor binding. J. Neurosci. 12, 440–453. Pompeiano, M., Palacios, J.M., Mengod, G., 1994. Distribution of the serotonin 5-HT2 receptor family mRNAs: comparison between 5-HT2A and 5-HT2C receptors. Brain Res. Mol. Brain Res. 23, 163–178. Puig, M.V., Artigas, F., Celada, P., 2005. Modulation of the activity of pyramidal neurons in rat prefrontal cortex by raphe stimulation in vivo: involvement of serotonin and GABA. Cereb. Cortex 15, 1–14. Ray, R.D., Zald, D.H., 2012. Anatomical insights into the interaction of emotion and cognition in the prefrontal cortex. Neurosci. Biobehav. Rev. 36, 479–501. Riad, M., Garcia, S., Watkins, K.C., Jodoin, N., Doucet, E., Langlois, X., el Mestikawy, S., Hamon, M., Descarries, L., 2000. Somatodendritic localization of 5-HT1A and preterminal axonal localization of 5-HT1B serotonin receptors in adult rat brain. J. Comp. Neurol. 417, 181–194. Santana, N., Bortolozzi, A., Serrats, J., Mengod, G., Artigas, F., 2004. Expression of serotonin1A and serotonin2A receptors in pyramidal and GABAergic neurons of the rat prefrontal cortex. Cereb. Cortex 14, 1100–1109. Tierney, P.L., Dégenètais, E., Thierry, A.M., Glowinski, J., Gioanni, Y., 2004. Influence of the hippocampus on interneurons of the rat prefrontal cortex. Eur. J. Neurosci. 20, 514–524. Vijayraghavan, S., Wang, M., Birnbaum, S.G., Williams, G.V., Arnsten, A.F., 2007. Inverted-U dopamine D1 receptor actions on prefrontal neurons engaged in working memory. Nat. Neurosci. 10, 376–384. Warden, M.R., Selimbeyoglu, A., Mirzabekov, J.J., Lo, M., Thompson, K.R., Kim, S.Y., Adhikari, A., Tye, K.M., Frank, L.M., Deisseroth, K., 2012. A prefrontal cortex-brainstem neuronal projection that controls response to behavioural challenge. Nature 492, 428–432. Williams, G.V., Goldman-Rakic, P.S., 1995. Modulation of memory fields by dopamine D1 receptors in prefrontal cortex. Nature 376, 572–575.
Please cite this article as: Castañé, A., et al., 5-HT2A receptors are involved in cognitive but not antidepressant effects of fluoxetine. European Neuropsychopharmacology (2015), http://dx.doi.org/10.1016/j.euroneuro.2015.04.006
5-HT2A receptors are involved in cognitive Williams, G.V., Rao, S.G., Goldman-Rakic, P.S., 2002. The physiological role of 5-HT2A receptors in working memory. J. Neurosci. 22, 2843–2854. Wilson, F.A., O’Scalaidhe, S.P., Goldman-Rakic, P.S., 1994. Functional synergism between putative gamma-aminobutyratecontaining neurons and pyramidal neurons in prefrontal cortex. Proc. Natl. Acad. Sci. USA 91, 4009–4013.
9 Zhang, G., Ásgeirsdóttir, H.N., Cohen, S.J., Munchow, A.H., Barrera, M.P., Stackman Jr, R.W., 2013. Stimulation of serotonin 2A receptors facilitates consolidation and extinction of fear memory in C57BL/6J mice. Neuropharmacology 64, 403–413. Zhong, P., Yan, Z., 2011. Differential regulation of the excitability of prefrontal cortical fast-spiking interneurons and pyramidal neurons by serotonin and fluoxetine. PLoS One 6, e16970.
Please cite this article as: Castañé, A., et al., 5-HT2A receptors are involved in cognitive but not antidepressant effects of fluoxetine. European Neuropsychopharmacology (2015), http://dx.doi.org/10.1016/j.euroneuro.2015.04.006
www.elsevier.com/locate/euroneuro
5-HT2A receptors are involved in cognitive but not antidepressant effects of fluoxetine Anna Castañéa,b,c,n, Lucila Kargiemana,d,e, Pau Celadaa,b,c, Analía Bortolozzia,b,c, Francesc Artigasa,b,c a
Department of Neurochemistry and Neuropharmacology, CSIC-Institut d’Investigacions Biomèdiques de Barcelona (IIBB), Barcelona, Spain b Centro de Investigación Biomédica en Red de Salud Mental (CIBERSAM), ISCIII, Madrid, Spain c Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain d Laboratory of Experimental Psychology & Neuroscience, Institute of Cognitive Neurology, Favaloro University, Buenos Aires, Argentina e UDP-INECO Foundation Core on Neuroscience, Diego Portales University, Santiago, Chile Received 15 October 2014; received in revised form 24 March 2015; accepted 1 April 2015
KEYWORDS
Abstract
5-HT2A receptors; Antidepressant drugs; Prefrontal cortex; Novel object recognition; Tail suspension test; Working memory
The prefrontal cortex (PFC) plays a crucial role in cognitive and affective functions. It contains a rich serotonergic (serotonin, 5-HT) innervation and a high density of 5-HT receptors. Endogenous 5-HT exerts robust actions on the activity of pyramidal neurons in medial PFC (mPFC) via excitatory 5-HT2A and inhibitory 5-HT1A receptors, suggesting the involvement of 5-HT neurotransmission in cortical functions. However, the underlying mechanisms must be elucidated. Here we examine the role of 5HT2A receptors in the processing of emotional and cognitive signals evoked by increasing the 5-HT tone after acute blockade of the 5-HT transporter. Fluoxetine (5–20 mg/kg i.p.) dose-dependently reduced the immobility time in the tail-suspension test in wild-type (WT) and 5-HT2A knockout (KO2A) mice, with non-significant differences between genotypes. Fluoxetine (10 mg/kg i.p.) significantly impaired mice performance in the novel object recognition test 24 h post-administration in WT, but not in KO2A mice. The comparable effect of fluoxetine on extracellular 5-HT in the mPFC of both genotypes suggests that presynaptic differences are not accountable. In contrast, single unit recordings of mPFC putative pyramidal neurons showed that fluoxetine (1.8–7.2 mg/kg i.v.) significantly increased neuronal discharge in KO2A but not in WT mice. This effect is possibly mediated by an altered excitatory/ inhibitory balance in the PFC in KO2A mice. Overall, the present results suggest that 5-HT2A receptors play a detrimental role in long-term memory deficits mediated by an excess 5-HT in PFC. & 2015 Elsevier B.V. and ECNP. All rights reserved.
n Corresponding author at: Department of Neurochemistry and Neuropharmacology, CSIC-Institut d’Investigacions Biomèdiques de Barcelona (IIBB), Barcelona, Spain. Tel.: +34 933638321; fax: +34 933638301. E-mail address: [email protected] (A. Castañé).
http://dx.doi.org/10.1016/j.euroneuro.2015.04.006 0924-977X/& 2015 Elsevier B.V. and ECNP. All rights reserved.
Please cite this article as: Castañé, A., et al., 5-HT2A receptors are involved in cognitive but not antidepressant effects of fluoxetine. European Neuropsychopharmacology (2015), http://dx.doi.org/10.1016/j.euroneuro.2015.04.006
2
A. Castañé et al.
Introduction The prefrontal cortex (PFC) is a critical area in mediating higher-order executive tasks such as working memory, inhibitory control and behavioural flexibility among others, under a strict temporal pattern (Fuster, 2001). Moreover, it plays an important role in emotional responses (Euston et al., 2012). In fact, the PFC has been recently identified as a core region for the interaction between cognition and emotional processing (Ray and Zald, 2012). The PFC receives a moderate to dense serotonergic innervation from the raphe nuclei (Azmitia and Segal, 1978; Blue et al., 1988) and contains several serotonin (5-hydroxytryptamine, 5-HT) receptor subtypes, with a particular high density of 5-HT1A and 5-HT2A receptors (Pompeiano et al., 1992, 1994) which may represent major modulators of PFC tasks. Previous studies in rats and monkeys showed that 5-HT transmission in the orbitofrontal cortex (OFC) is involved in flexible behaviours. Specifically, 5-HT depletion in the OFC as well as the lesion of this area induced reversal learning deficits (Boulougouris et al., 2007; Clarke et al., 2007). 5-HT2A receptors have been shown to modulate this type of behavioural flexibility, since the selective 5-HT2A receptor antagonist M100907 impaired reversal performance in rats (Boulougouris et al., 2008). Likewise, local prefrontal M100907 application reduced persistent neuronal activity during working memory tasks in monkeys (Williams et al., 2002) and a recent computational study indicated that 5HT2A receptors contribute to spatial working memory tasks (Cano-Colino et al., 2014). However, evidence on the role of serotonergic transmission through 5-HT2A receptors in cognitive domains remains scarce. Prefrontal cognitive dysfunction is a core feature in many psychiatric disorders including depression and schizophrenia. 5HT2A receptors have been proposed as possible targets to improve cognition and depressive-like effects in mental illnesses (Mestre et al., 2013). It has been recently described that the chronic intermittent cold (CIC) stress model of depression is associated with reduced serotonergic transmission in the OFC and induces reversal learning deficits in rats, that are sensitive to selective serotonin reuptake inhibitors (SSRIs) treatment (Danet et al., 2010) through a 5-HT2A receptor mechanism (Furr et al., 2012). In contrast to the above data, atypical antipsychotics, which exert their therapeutic action, at least in part, by blocking 5-HT2A-mediated responses (Nyberg et al., 1999) procured some advantages over classic antipsychotics for treating cognitive impairment in schizophrenia (Meltzer et al., 2012; Meltzer, 2013). On the other hand, clinical studies showed that atypical antipsychotics increased the efficacy of SSRIs in treatmentresistant depression, possibly through 5-HT2A receptor blockade (Ostroff and Nelson, 1999; Nelson and Papakostas, 2009). Preclinical data also suggest that selective blockade of 5-HT2A receptors may have a synergistic effect with SSRIs antidepressant treatment (Marek et al., 2005). Thus, Marek and collaborators described a synergistic antidepressant-like effect of a low dose of M100907 in rats using the DRL 72-s test. However, the neurobiological bases of this improvement remain poorly understood. Here we investigate the role of 5-HT2A receptors in the processing of emotional and cognitive responses mediated
by the mPFC, evoked by increasing the serotonergic tone after acute blockade of the serotonin transporter (SERT) with the SSRI fluoxetine in mice.
Experimental procedures Subjects We used 9–12-week old male 5-HT2A receptor knockout (5-HT2AR KO, KO2A) and wild-type mice (WT). Mice with the targeted deletion of 5-HT2AR were developed as reported (Fiorica-Howells et al., 2002). From an initial source, a stable colony of KO2A and WT mice were grown in our animal facilities. Animals were kept in a controlled environment (12 h light–dark cycle and 2272 1C room temperature) with food and water provided ad-libitum. All experimental procedures were in strict accordance with European Union regulations (Official Journal of the European Communities L358/1, 18 December 1986), and were approved by the Institutional Animal Care and Use Committee.
Drugs For behavioural and neurochemical studies, fluoxetine hydrochloride (Tocris, USA) was dissolved in distilled water and injected by intraperitoneal route (i.p) at a volume of 10 ml/kg. For the electrophysiological studies, fluoxetine hydrochloride was dissolved in saline and injected intravenously (i.v.) at a volume of 1–2 ml/kg. Fluoxetine doses are expressed as free base.
Tail suspension test (TST) TST was conducted essentially as previously described (Ferrés-Coy et al., 2013). Briefly, mice were suspended 30 cm above the floor by adhesive tape placed approximately 1 cm from the tip of the tail and were observed on a monitor through a video camera system (Smart, Panlab). The time mice spent immobile was recorded during 6 min. Pharmacological treatments were administered 1 h before the test.
Novel object recognition test (NOR) On day one, mice were habituated for 10 min to the maze where the task was performed. On the second day, mice were put back in the maze for 10 min where two identical objects were presented (Trial 1, T1), and the time the animals spent exploring each object was recorded. Three or twenty-four hour later, mice were put again in the maze for 10 min, where one of the familiar objects was replaced by a novel object (Trial 2, T2), and the total time spent exploring each of the two objects (novel, N and familiar, F) was computed. Object exploration was defined as the orientation of the nose to the object at a distanceo1 cm. A recognition index (RI) was calculated as the percentage of the time exploring the novel object divided by the total time exploring the two objects (novel and familiar) as described [RI =N/(F+N)*100]. RI450% is considered to reflect greater memory retention for the familiar object. Pharmacological treatments were administered 1 h before T1.
Intracerebral microdialysis Extracellular serotonin was measured by in vivo microdialysis as previously described (Castañé et al., 2008). Briefly, one concentric dialysis probe equipped with a Cuprophan membrane (2-mm long) was implanted in the medial PFC (mPFC) at coordinates (in mm): AP
Please cite this article as: Castañé, A., et al., 5-HT2A receptors are involved in cognitive but not antidepressant effects of fluoxetine. European Neuropsychopharmacology (2015), http://dx.doi.org/10.1016/j.euroneuro.2015.04.006
5-HT2A receptors are involved in cognitive +2.2; ML-0.2; DV-3.4 (Franklin and Paxinos, 1997), of anaesthetized mice (pentobarbital, 40 mg/kg, i.p.). Microdialysis experiments were performed 20–24 h after surgery. The aCSF was pumped at 2.0 μl/min (WPI model sp220i) and dialysates were collected every 30 min. Serotonin concentration was analysed by HPLC with amperometric detection (Hewlett Packard 1049) at +0.6 V, with a detection limit of 1.5 fmol/sample. Baseline serotonin levels were calculated as the average of the four pre-drug samples. Following sample collection, brains were removed and sectioned to ensure proper probe placement after cresyl violet staining.
Electrophysiological studies Single unit recordings: We assessed the effects of the i.v. administration of fluoxetine on the firing rate of putative mPFC pyramidal neurons in WT and KO2A mice. Recordings were performed as described previously (Lladó-Pelfort et al., 2012; Puig et al., 2005). Briefly, mice were anesthetized with chloral hydrate (400 mg/kg, i.p.) and positioned in a David Kopf stereotaxic frame. Additional doses of chloral hydrate (80 mg/kg) were administered i. v. through the femoral vein. Body temperature was maintained at 37 1C through the experiment with a heating pad. Putative pyramidal neurons were recorded extracellularly with glass micropipettes filled with 2 M NaCl. Impedance was between 6 and 10 MΩ. Single unit recordings were amplified with a Neurodata IR283 (Cygnus Technology Inc., Delaware Water Gap, PA), post-amplified and filtered with a Cibertec amplifier (Cibertec, Madrid, Spain) and computed on-line using a DAT 1401plus interface system Spike2 software (Cambridge Electronic Design, Cambridge, UK). Putative pyramidal neurons in the mPFC were recorded at AP +2.2 to +2.4; L-0.2 to 0.5; DV-1.1 to 2.6 mm below the brain surface and identified according to previously described electrophysiological characteristics: 1) duration of the depolarization phase of action potential (average of 10 spikes) and 2) basal discharge rate, as previously reported (Lladó-Pelfort et al., 2012; Wilson et al., 1994). Basal firing rate was recorded for at least 5 min and then, cumulative doses of fluoxetine (1.8 to 7.2 mg/kg, i.v.) were administered. At the end of the experiments, mice were killed by an anaesthetic overdose. Changes in discharge rate were quantified by averaging the values in the third minute after each dose injection.
Statistical analysis
3 interaction between the two factors [F(3,63) = 1.220, n.s.]. Post-hoc analysis revealed a significant decrease of immobility time in both wild-type and KO2A mice after 10 and 20 mg/kg of fluoxetine (po0.01, all cases) compared to controls of the same genotype (Figure 1).
Effects of acute fluoxetine in the novel object recognition test in WT and KO2A mice Statistical analysis revealed a non-significant effect of treatment [F(1,26) = 0.903, n.s.], genotype [F(1,26) = 1.292, n.s.], and interaction between treatment and genotype [F(1,26) = 0.8523, n.s.] when object recognition memory was assessed at 3 h (Figure 2a). Acute fluoxetine treatment significantly impaired object recognition memory in WT but not KO2A mice when tested at 24 h (Figure 2b). Two-way ANOVA showed a significant effect of treatment [F(1,32) = 4.179, po0.05], genotype [F(1,32) = 7.201, po0.05] and the interaction between treatment and genotype [F(1,32) = 7.346, po0.05]. Subsequent post-hoc analysis revealed a significant effect of fluoxetine treatment in WT mice compared to controls (po0.01) and a significant difference between WT and KO2A mice treated with fluoxetine (po0.01) (n= 7–9/group).
Effect of fluoxetine on extracellular 5-HT Basal extracellular concentrations of 5-HT in dialysate samples of mPFC were, WT: 3.070.5 (n=17); KO2A: 2.770.5 (n=17) expressed as fmol/60 μl (n.s., Student's t-test). The systemic administration of fluoxetine (10 mg/kg, i.p.) induced a similar increase of extracellular 5-HT concentrations in mPFC of WT and KO2A mice compared to vehicle-treated animals (Figure 3). Thus, three-way ANOVA showed a main effect of treatment [F(1,29) =23.7021, po0.001], time [F(11,319) =11.8423, po0.001] and the interaction between treatment and time [F(11,319) =9.0806, po0.001], but not of genotype or interaction between treatment and genotype. Subsequent post-hoc analysis revealed a significant difference
Behavioural data were analysed using a two-way ANOVA (genotype and treatment as between factors of variation). Microdialysis data were analysed by a three-way ANOVA with repeated measures (genotype and treatment as between factors and fraction as a within factor of variation). Electrophysiological data were analysed by the Student-t-test, one-way or two-way ANOVA, as appropriate. Significant main effects of genotype, treatment, fraction or interactions between these factors were further explored through posthoc Duncan comparisons to establish simple effects. Statistical significance was set at the 95% confidence level.
Results Antidepressant-like responses of fluoxetine in WT and KO2A mice Acute fluoxetine treatment (5, 10 and 20 mg/kg i.p.) induced antidepressant-like responses in the TST in both WT and KO2A mice (n= 8–13/group). Two-way ANOVA showed a significant effect of treatment [F(3,63) =29.143, po0.001] but not of genotype ([F(1,63) = 2.862, n.s.], nor the
Figure 1 Effects of acute fluoxetine (FLX) administration (5, 10, 20 mg/kg, i.p) in wild-type (WT) and 5-HT2A receptor knockout (KO2A) mice in the TST. The treatment was administered 1 h before the test. Data are expressed as mean7SEM of immobility time in seconds (s). Duncan post-hoc, **po0.01 vs controls (VEH).
Please cite this article as: Castañé, A., et al., 5-HT2A receptors are involved in cognitive but not antidepressant effects of fluoxetine. European Neuropsychopharmacology (2015), http://dx.doi.org/10.1016/j.euroneuro.2015.04.006
4
A. Castañé et al.
Figure 2 Effects of acute fluoxetine administration in the novel object recognition test in wild-type (WT) and 5-HT2A receptor knockout (KO2A) mice. Fluoxetine (FLX, 10 mg/kg, i.p) or vehicle (VEH) treatments were administered 1 h before the trial 1, in WT and KO2A mice. Test was performed at 3 h (a) or 24 h (b). Data are expressed as mean7SEM of recognition index expressed in percentage (%). Duncan post-hoc, **po0.01 vs VEH, po0.01 vs WT.
Figure 3 Effects of systemic administration of fluoxetine (10 mg/kg) on dialysate 5-HT concentrations in mPFC of wildtype (WT) and 5-HT2A receptor knockout mice (KO2A). Data are expressed as mean7SEM of percentages of pre-treatment values. Duncan post-hoc, **po0.01, fluoxetine-treated (FLX) vs vehicle-treated (VEH) mice.
between fluoxetine- and vehicle-treated mice in both genotypes (po0.01).
Electrophysiological studies Firing characteristics of putative pyramidal neurons in mice mPFC A total of 16 and 12 putative pyramidal neurons (one per mouse) were recorded in untreated WT and KO2A mice, respectively. The baseline firing rate was 1.6370.50 spikes/ s and 0.9670.29 spikes/s for WT and KO2A mice, respectively (p= 0.30, Student's t-test). Action potential duration was also similar in both groups: 0.7570.04 ms and 0.7970.03 ms for WT and KO2A mice, respectively. Effects of fluoxetine on mPFC putative pyramidal neuron firing The administration of fluoxetine (1.8–7.2 mg/kg, i.v.) to WT mice did not modify the firing rate of putative pyramidal neurons in the mPFC (1.4470.42, 1.6870.50, 1.5570.42,
1.0170.29 spikes/s in basal conditions and after the administration of 1.8, 3.6 and 7.2 mg/kg fluoxetine, respectively; n =13) (Figure. 4a and c). In contrast, the administration of FLX (1.8–7.2 mg/kg, i.v.) to KO2A mice increased the firing rate of putative pyramidal neurons in the mPFC (0.9670.38, 1.1470.40, 1.8470.80, 2.2270.77 in basal conditions and after the administration of 1.8, 3.6 and 7.2 mg/kg fluoxetine, respectively; n= 9) (Figure. 4b and c). Thus, two-way ANOVA revealed a significant treatment x genotype interaction [F(3,60) =3.42, po0.03] with significant post-hoc differences between baseline and fluoxetine 7.2 mg/kg in the KO2A mice and between WT and KO2A mice after treatment with fluoxetine 7.2 mg/kg (Figure 4c).
Discussion The present study shows that the increase in serotonergic tone produced by SERT blockade with fluoxetine evoked similar antidepressant-like responses in the TST in WT mice and in KO2A mice. In contrast, fluoxetine impaired declarative memory in WT (but not KO2A) mice 24 h after administration, as assessed by the NOR test, indicating that i) an acute increase of the serotonergic tone in forebrain— possibly in the PFC—impairs long-term memory, and ii) this impairment is mediated—at least in part—by the activation of 5-HT2A receptors. Early studies using antipsychotic medications suggested a 5HT2A receptor mechanism in increasing the efficacy of selective serotonin reuptake inhibitors (SSRIs) in treatmentresistant depression (Ostroff and Nelson, 1999; Nelson and Papakostas, 2009). Likewise, Marek et al. (2005) reported on a synergistic antidepressant-like effect when combining a low dose of the selective 5-HT2A receptor antagonist M100907 with fluoxetine in the differential-reinforcement-of low rate 72-s schedule of reinforcement (DRL 72-s) paradigm. The present data are partly at variance with these observations since we did not find an enhancement of fluoxetine-induced antidepressant-like responses in the
Please cite this article as: Castañé, A., et al., 5-HT2A receptors are involved in cognitive but not antidepressant effects of fluoxetine. European Neuropsychopharmacology (2015), http://dx.doi.org/10.1016/j.euroneuro.2015.04.006
5-HT2A receptors are involved in cognitive
Figure 4 (a, b) Integrated firing rate histogram showing the effect of the i.v. administration of FLX (mg/kg; vertical arrows) on the activity of mPFC pyramidal neurons. (c) Bar histogram showing the average effect produced by FLX in WT and KO2A mice. Duncan post-hoc, *po0.05 vs basal, po0.05 vs WT.
TST in the KO2A mice. Differences in the species used (mice vs rats) and/or the differential contribution of serotonergic neurotransmission to the behaviours involved in both tests may be accountable. In regards to the TST, a high 5-HT tone in cortical and limbic structures (in particular PFC; Warden et al., 2012) is required to reduce behavioural despair during stressful situations. In agreement, the reduction of immobility is associated with an increase of 5-HT release in mouse PFC during the exposure to TST (Ferrés-Coy et al., 2013). However, in the DRL 72-s test, rats must learn to control their impulsivity to press a lever during a prolonged
5 period of time (72 s) in order to obtain a food reward. Thus, whereas the TST is mainly related to an emotional behaviour, the DRL 72-s test involves more complex responses requiring increased attention, impulse control and time perception. This suggests that different PFC areas and possibly different 5-HT receptors are involved in both tests. The comparable enhancement of extracellular 5-HT produced by fluoxetine in WT and KO2A mice observed in microdialysis experiments is in accordance with the inability of M100907 to enhance fluoxetine effect on extracellular 5HT (Marek et al., 2005). It also shows that the observed differences in the NOR test between WT and KO2A mice are not due to a differential enhancement of presynaptic 5-HT function in both genotypes during acquisition and/or memory consolidation (o 4 h). Indeed, the [3H]-citalopram binding site density in the dorsal raphe nucleus was similar for both genotypes (unpublished observations). On the contrary, electrophysiological experiments revealed the existence of genotype differences in the postsynaptic response to fluoxetine administration. Unlike in previous studies from this laboratory in rats (Lladó-Pelfort et al., 2012; Puig et al., 2005) we did not identify pyramidal neurons in mouse mPFC by antidromic activation due to the inherent difficulties to perform this procedure in mouse brain. However, the recorded neurons are most likely pyramidal neurons in view of their slower discharge rate and longer duration of the action potential, as compared with GABAergic interneurons. Hence, GABAergic neurons have a mean action potential duration below 0.5 ms (Lladó-Pelfort et al., 2012; Tierney et al., 2004) whereas the corresponding values in the present study were 0.7570.04 ms and 0.7970.03 ms for WT and KO2A mice, respectively. Likewise, discharge rates (1.6370.50 spikes/s and 0.9670.29 spikes/s for WT and KO2A mice, respectively) were well below those of fast-spiking GABAergic interneurons (Lladó-Pelfort et al., 2012; Tierney et al., 2004). As previously reported in the rat (Ceci et al., 1994; Gronier and Rasmussen, 2003), acute fluoxetine treatment did not alter the discharge rate of PFC neurons in WT mice. However, fluoxetine increased dose-dependently the discharge rate of putative pyramidal neurons in the mPFC of KO2A mice. The lack of effect of fluoxetine on neuronal discharge in WT mice may be attributed to the concurrent activation of excitatory and inhibitory receptors in cortical microcircuits involving pyramidal neurons and GABAergic interneurons. The main 5-HT receptors in mPFC are 5-HT1A and 5-HT2A receptors, which are expressed by a high percentage of pyramidal neurons and GABAergic interneurons in the rat PFC (Santana et al., 2004) where they mediate excitatory (5-HT2A) and inhibitory (5-HT1A) actions of 5-HT (Araneda and Andrade, 1991; Celada et al., 2013; Puig et al., 2005). Interestingly, these receptors are highly co-expressed in the same PFC neurons in rats and mice (Amargós-Bosch et al., 2004) and are possibly segregated into different neuronal compartments. Thus, 5-HT1A receptors have been identified in the axon hillock of pyramidal neurons (de Felipe et al., 2001) or in the somatodendritic compartment (Riad et al., 2000), whereas 5-HT2A receptors are predominantly localized in the cell body and apical dendrites (Jakab and Goldman-Rakic, 1998; Martín-Ruiz et al., 2001). The
Please cite this article as: Castañé, A., et al., 5-HT2A receptors are involved in cognitive but not antidepressant effects of fluoxetine. European Neuropsychopharmacology (2015), http://dx.doi.org/10.1016/j.euroneuro.2015.04.006
6
A. Castañé et al.
existence of simultaneous responses mediated by both receptors in the same neuronal populations is shown by biphasic responses to endogenous 5-HT (Puig et al., 2005) and by the emergence of 5-HT2A receptor-mediated excitations, after the blockade of 5-HT1A receptor-mediated inhibitions (Amargós-Bosch et al., 2004). Therefore, the excess 5-HT produced by SERT blockade with fluoxetine may simultaneously activate 5-HT1A and 5HT2A receptors in mPFC, leading to a compensation of inhibitory and excitatory responses in the same neurons or microcircuits. However, this possibility is at variance with the predominant inhibitory action of 5-HT on pyramidal neuron activity, -mediated by 5-HT1A receptors- after the stimulation of the dorsal and median raphe nuclei at physiological rates (Puig et al., 2005). Moreover, the duplication of the stimulation frequency (to enhance 5-HT release) increased the duration of inhibitory responses and turned some excitations into inhibitions (Puig et al., 2005), indicating a predominant role of 5-HT1A receptors in the control of pyramidal neuron activity in mPFC. One possibility to reconcile these two observations (e.g., lack of effect of fluoxetine vs predominant inhibitory responses of endogenous 5-HT) is that the excess 5-HT produced in both experimental situations may occur in
different neuronal micro-compartments and/or with a different temporal pattern (Figure 5). Hence, pyramidal inhibitions evoked by raphe stimulation are phasic and typically occur in the 20–200 ms scale, indicating that 5HT is released—intra- or extras-synaptically—, but in the close vicinity of 5-HT1A receptors. The possible location of 5-HT1A receptors in the axon hillock of pyramidal neurons (de Felipe et al., 2001), may facilitate inhibitory responses, making 5-HT1A receptors to act as neuronal switches such as GABAA receptors, also located in the axon hillock (de Felipe et al., 2001). On the other hand, SERT blockade in the very dense network of 5-HT axon terminals (4106 varicosities/mm3 in rat neocortex; Beaudet and Descarries, 1976) is likely to produce a tonic and overall increase of 5-HT in the extracellular brain space, leading to an activation of 5-HT receptors different from that resulting from the phasic release after raphe stimulation. Interestingly, in vitro experiments in PFC slices indicate that bath applied 5-HT preferentially activates 5-HT2 receptors in fast-spiking interneurons and decreases the firing rate of pyramidal neurons in a concentration-dependent manner (Zhong and Yan, 2011). However, bath application of fluoxetine—while increasing the firing rate of fast spiking interneurons—did
Figure 5 Schematic representation of the differential activation of 5-HT receptors in mPFC after midbrain stimulation and during SERT blockade with fluoxetine (FLX). (a) The electrical stimulation of DR/MnR excites and inhibits pyramidal neurons by stimulating 5-HT2A and 5-HT1A receptors, respectively, after the phasic release of 5-HT following the arrival of action potentials at nerve terminals (green circles). The duration of these effects is limited by 5-HT reuptake. Pyramidal 5-HT1A receptors are localized in the axon hillock, together with GABAA receptors (De Felipe et al., 2001) or in the somatodendritic compartment (Riad et al., 2000) whereas 5-HT2A receptors are predominantly localized in apical dendrites. Given the abundant co-expression of both receptors (Amargós-Bosch et al., 2004) the occurrence of excitations or inhibitions may depend on a precise topology between certain 5-HT neurons or neuronal clusters in the DR/MnR and 5-HT1A- or 5-HT2A-receptor-rich compartments according to the association between 5-HT axons and such receptor-rich areas (De Felipe et al., 2001; Jansson et al., 2001), as shown in the figure. (b) The blockade of SERT by fluoxetine produces a tonic increase of the 5-HT concentration in the extracellular space of PFC, as observed in microdialysis studies which may evoke a generalized activation of 5-HT receptors in pyramidal and GABAergic neurons. The present results suggest that 5-HT2A receptors in GABAergic neurons play a predominant role in the control of pyramidal neuron activity in conditions of SERT blockade by fluoxetine.
Please cite this article as: Castañé, A., et al., 5-HT2A receptors are involved in cognitive but not antidepressant effects of fluoxetine. European Neuropsychopharmacology (2015), http://dx.doi.org/10.1016/j.euroneuro.2015.04.006
5-HT2A receptors are involved in cognitive not simultaneously reduce the firing rate of pyramidal neurons (Zhong and Yan, 2011). Our in vivo observations are in agreement with this view, since fluoxetine did not alter the firing rate of putative pyramidal neurons in WT mice but increased it in KO2A mice. A preferential activation of 5-HT2A receptors in pyramidal neurons by fluoxetine would have led to the reduction of the firing rate in KO2A mice, whereas the opposite effect was observed. Given the lack of 5-HT2A receptors in all neuronal populations in KO2A mice, the increase in pyramidal discharge cannot be ascribed to the activation of 5-HT2A receptors in the recorded neurons. Although the participation of other excitatory 5-HT receptors (e.g., 5-HT2C, 5-HT4, 5-HT7) cannot be discarded, the increase in pyramidal discharge may actually be a disinhibition (Figure 5), after the stimulation of 5-HT1A receptors in fast-spiking interneurons by the excess 5-HT, as observed for 5-HT1A agonists (Lladó-Pelfort et al., 2012). A key process in cognitive functions mediated by the PFC is working/short-term memory. Working memory is associated to persistent neuronal activity in the dorsolateral PFC (prelimbic mPFC in the rat/mouse) in tasks involving delayed responses (Curtis and D’Esposito, 2003) such as the NOR test. Several neurotransmitters have been related to working memory, in particular the catecholamines (Vijayraghavan et al., 2007; Williams and Goldman-Rakic, 1995) whereas much less is known about 5-HT. The present observations suggest that 5-HT2A receptors might play a detrimental role in short-term memory and/or in memory consolidation. Hence while differences between WT and KO2A in the NOR test after a 3 h delay did not reach statistical significance, they were of greater magnitude and statistically significant at 24 h, suggesting that the indirect activation of 5-HT2A receptors by fluoxetine do impair long-term memories. Further studies are needed to elucidate whether acquisition, consolidation and/or memory retrieval are compromised after fluoxetine treatment. In humans, cognitive side effects of initial treatment with fluoxetine have been described (Bangs et al., 1994; Joss et al., 2003), although their underlying mechanism it is currently unknown. Here we propose that the increased discharge rate of putative pyramidal PFC neurons in KO2A mice may facilitate the persistent neuronal activity required for consolidation of memory processes (Fuster, 2001). However, the role of 5-HT2A receptors in learning and memory is still scarce and deserves further research (Meneses, 2007; Zhang et al., 2013). In summary, the present study shows that the blockade or absence of 5-HT2A receptors do not increase fluoxetineinduced antidepressant-like responses in the TST whereas it has a beneficial role in long-term memory, as assessed in the NOR test. These results may help to improve our understanding on the role of 5-HT in PFC and particularly on cognitive functions.
Role of funding source Funding for this study was provided by Grants SAF 2012-35183, the Ministry of Economy and Competitiveness, co-financed by European Regional Development Fund (ERDF) and by the Grants PI12/00156, PI13/01390 and PI10/00290 (PN de I +D+I 2008-2011, ISCIIISubdirección General de Evaluación y Fomento de la Investigación
7 cofinanced by the European Regional Development Fund. “Una manera de hacer Europa”), and Centro de Investigación Biomédica en Red de Salud Mental, CIBERSAM. AC was supported by a postdoctoral contract from the ISCIII (CD05/00234). None of the financing Institutions had further role in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication.
Contributors Author AC designed the study and wrote the protocol, managed the literature searches and analyses and undertook the statistical analysis. Authors AC and LK performed the behavioural experiments. Authors LK and PC performed electrophysiology experiments. Authors AC and AB performed microdialysis experiments. Authors AC and FA wrote the manuscript. All authors contributed to and have approved the final manuscript.
Conflict of interest FA has received consulting and educational honoraria on antidepressant drugs from Lundbeck and he is PI of a grant from Lundbeck. He is also member of the advisory board of Neurolixis. The rest of authors declare no conflict of interest.
Acknowledgements We thank Leticia Campa for her skilful maintenance of HPLC equipment and analyses of dialysate samples. We also thank Dr. Jay Gingrich for the generous supply of 5-HT2A receptor knockout mice.
References Amargós-Bosch, M., Bortolozzi, A., Puig, M.V., Serrats, J., Adell, A., Celada, P., Toth, M., Mengod, G., Artigas, F., 2004. Coexpression and in vivo interaction of serotonin1A and serotonin2A receptors in pyramidal neurons of prefrontal cortex. Cereb. Cortex 14, 281–299. Araneda, R., Andrade, R., 1991. 5-Hydroxytryptamine2 and 5hydroxytryptamine 1A receptors mediate opposing responses on membrane excitability in rat association cortex. Neuroscience 40, 399–412. Azmitia, E.C., Segal, M., 1978. An autoradiographic analysis of the differential ascending projections of the dorsal and median raphe nuclei in the rat. J. Comp. Neurol. 179, 641–667. Bangs, M.E., Petti, T.A., Janus, M.D., 1994. Fluoxetine-induced memory impairment in an adolescent. J. Am. Acad. Child Adolesc. Psychiatry 33, 1303–1306. Beaudet, A., Descarries, L., 1976. Quantitative data on serotonin nerve terminals in adult rat neocortex. Brain Res. 111, 301–309. Blue, M.E., Yagaloff, K.A., Mamounas, L.A., Hartig, P.R., Molliver, M.E., 1988. Correspondence between 5-HT2 receptors and serotonergic axons in rat neocortex. Brain Res. 453, 315–328. Boulougouris, V., Dalley, J.W., Robbins, T.W., 2007. Effects of orbitofrontal, infralimbic and prelimbic cortical lesions on serial spatial reversal learning in the rat. Behav. Brain Res. 179, 219–228. Boulougouris, V., Glennon, J.C., Robbins, T.W., 2008. Dissociable effects of selective 5-HT2A and 5-HT2C receptor antagonists on serial spatial reversal learning in rats. Neuropsychopharmacology 33, 2007–2019. Cano-Colino, M., Almeida, R., Gomez-Cabrero, D., Artigas, F., Compte, A., 2014. Serotonin regulates performance nonmonotonically in a spatial working memory network. Cereb. Cortex 24, 2449–2463.
Please cite this article as: Castañé, A., et al., 5-HT2A receptors are involved in cognitive but not antidepressant effects of fluoxetine. European Neuropsychopharmacology (2015), http://dx.doi.org/10.1016/j.euroneuro.2015.04.006
8
A. Castañé et al.
Castañé, A., Artigas, F., Bortolozzi, A., 2008. The absence of 5-HT (1A) receptors has minor effects on dopamine but not serotonin release evoked by MK-801 in mice prefrontal cortex. Psychopharmacology 200, 281–290. Ceci, A., Fodritto, F., Borsini, F., 1994. Repeated treatment with fluoxetine decreases the number of spontaneously active cells per track in frontal cortex. Eur. J. Pharmacol. 271, 231–234. Celada, P., Puig, M.V., Artigas, F., 2013. Serotonin modulation of cortical neurons and networks. Front. Integr. Neurosci. 7, 1–20 article 25. Clarke, H.F., Walker, S.C., Dalley, J.W., Robbins, T.W., Roberts, A. C., 2007. Cognitive inflexibility after prefrontal serotonin depletion is behaviorally and neurochemically specific. Cereb. Cortex 17, 18–27. Curtis, C.E., D'Esposito, M., 2003. Persistent activity in the prefrontal cortex during working memory. Trends Cogn. Sci. 7, 415–423. Danet, M., Lapiz-Bluhm, S., Morilak, D.A., 2010. A cognitive deficit induced in rats by chronic intermittent cold stress is reversed by chronic antidepressant treatment. Int. J. Neuropsychopharmacol. 13, 997–1009. De Felipe, J., Arellano, J.I., Gómez, A., Azmitia, E.C., Muñoz, A., 2001. Pyramidal cell axons show a local specialization for GABA and 5-HT inputs in monkey and human cerebral cortex. J. Comp. Neurol. 433, 148–155. Euston, D.R., Gruber, A.J., McNaughton, B.L., 2012. The role of medial prefrontal cortex in memory and decision making. Neuron 76, 1057–1070. Ferrés-Coy, A., Santana, N., Castañé, A., Cortés, R., Carmona, M. C., Toth, M., Montefeltro, A., Artigas, F., Bortolozzi, A., 2013. Acute 5-HT1A autoreceptor knockdown increases antidepressant responses and serotonin release in stressful conditions. Psychopharmacology 225, 61–74. Fiorica-Howells, E., Hen, R., Gingrich, J., Li, Z., Gershon, M.D., 2002. 5-HT(2A) receptors: location and functional analysis in intestines of wild-type and 5-HT(2A) knockout mice. Am. J. Physiol. Gastrointest. Liver Physiol. 282, G877–G893. Franklin, K., Paxinos, G., 1997. The mouse brain in stereotaxic coordinates. Academic, San Diego, California. Furr, A., Lapiz-Bluhm, M.D., Morilak, D.A., 2012. 5-HT2A receptors in the orbitofrontal cortex facilitate reversal learning and contribute to the beneficial cognitive effects of chronic citalopram treatment in rats. Int. J. Neuropsychopharmacol. 15, 1295–1305. Fuster, J.M., 2001. The prefrontal cortex-an update: time is of the essence. Neuron 30, 319–333. Gronier, B.S., Rasmussen, K., 2003. Electrophysiological effects of acute and chronic olanzapine and fluoxetine in the rat prefrontal cortex. Neurosci. Lett. 349, 196–200. Jakab, R.L., Goldman-Rakic, P.S., 1998. 5-Hydroxytryptamine2A serotonin receptors in the primate cerebral cortex: possible site of action of hallucinogenic and antipsychotic drugs in pyramidal cell apical dendrites. Proc. Natl. Acad. Sci. USA 95, 735–740. Jansson, A., Tinner, B., Bancila, M., Vergé, D., Steinbusch, H.W., Agnati, L.F., Fuxe, K., 2001. Relationships of 5-hydroxytryptamine immunoreactive terminal-like varicosities to 5-hydroxytryptamine2A receptor-immunoreactive neuronal processes in the rat forebrain. J. Chem. Neuroanat. 22, 185–203. Joss, J.D., Burton, R.M., Keller, C.A., 2003. Memory loss in a patient treated with fluoxetine. Ann. Pharmacother. 37, 1800–1803. Lladó-Pelfort, L., Santana, N., Ghisi, V., Artigas, F., Celada, P., 2012. 5-HT1A receptor agonists enhance pyramidal cell firing in prefrontal cortex through a preferential action on GABA interneurons. Cereb. Cortex 22, 1487–1497. Marek, G.J., Martin-Ruiz, R., Abo, A., Artigas, F., 2005. The selective 5-HT2A receptor antagonist M100907 enhances antidepressant-like behavioural effects of the SSRI fluoxetine. Neuropsychopharmacology 30, 2205–2215.
Martín-Ruiz, R., Puig, M.V., Celada, P., Shapiro, D.A., Roth, B.L., Mengod, G., Artigas, F., 2001. Control of serotonergic function in medial prefrontal cortex by serotonin-2A receptors through a glutamate-dependent mechanism. J. Neurosci. 21, 9856–9866. Meltzer, H.Y., 2013. Update on typical and atypical antipsychotic drugs. Annu. Rev. Med. 64, 393–406. Meltzer, H.Y., Massey, B.W., Horiguchi, M., 2012. Serotonin receptors as targets for drugs useful to treat psychosis and cognitive impairment in schizophrenia. Curr. Pharm. Biotechnol. 13, 1572–1586. Meneses, A., 2007. Stimulation of 5-HT1A, 5-HT1B, 5-HT2A/2C, 5HT3 and 5-HT4 receptors or 5-HT uptake inhibition: short- and long-term memory. Behav. Brain Res. 184, 81–90. Mestre, T.A., Zurowski, M., Fox, S.H., 2013. 5-Hydroxytryptamine 2A receptor antagonists as potential treatment for psychiatric disorders. Expert Opin. Investig. Drugs 22, 411–421. Nelson, J.C., Papakostas, G.I., 2009. Atypical antipsychotic augmentation in major depressive disorder: a meta-analysis of placebo-controlled randomized trials. Am. J. Psychiatry 166, 980–991. Nyberg, S., Eriksson, B., Oxenstierna, G., Halldin, C., Farde, L., 1999. Suggested minimal effective dose of risperidone based on PET-measured D2 and 5-HT2A receptor occupancy in schizophrenic patients. Am. J. Psychiatry 156, 869–875. Ostroff, R.B., Nelson, J.C., 1999. Risperidone augmentation of selective serotonin reuptake inhibitors in major depression. J. Clin. Psychiatry 60, 256–259. Pompeiano, M., Palacios, J.M., Mengod, G., 1992. Distribution and cellular localization of mRNA coding for 5-HT1A receptor in the rat brain: correlation with receptor binding. J. Neurosci. 12, 440–453. Pompeiano, M., Palacios, J.M., Mengod, G., 1994. Distribution of the serotonin 5-HT2 receptor family mRNAs: comparison between 5-HT2A and 5-HT2C receptors. Brain Res. Mol. Brain Res. 23, 163–178. Puig, M.V., Artigas, F., Celada, P., 2005. Modulation of the activity of pyramidal neurons in rat prefrontal cortex by raphe stimulation in vivo: involvement of serotonin and GABA. Cereb. Cortex 15, 1–14. Ray, R.D., Zald, D.H., 2012. Anatomical insights into the interaction of emotion and cognition in the prefrontal cortex. Neurosci. Biobehav. Rev. 36, 479–501. Riad, M., Garcia, S., Watkins, K.C., Jodoin, N., Doucet, E., Langlois, X., el Mestikawy, S., Hamon, M., Descarries, L., 2000. Somatodendritic localization of 5-HT1A and preterminal axonal localization of 5-HT1B serotonin receptors in adult rat brain. J. Comp. Neurol. 417, 181–194. Santana, N., Bortolozzi, A., Serrats, J., Mengod, G., Artigas, F., 2004. Expression of serotonin1A and serotonin2A receptors in pyramidal and GABAergic neurons of the rat prefrontal cortex. Cereb. Cortex 14, 1100–1109. Tierney, P.L., Dégenètais, E., Thierry, A.M., Glowinski, J., Gioanni, Y., 2004. Influence of the hippocampus on interneurons of the rat prefrontal cortex. Eur. J. Neurosci. 20, 514–524. Vijayraghavan, S., Wang, M., Birnbaum, S.G., Williams, G.V., Arnsten, A.F., 2007. Inverted-U dopamine D1 receptor actions on prefrontal neurons engaged in working memory. Nat. Neurosci. 10, 376–384. Warden, M.R., Selimbeyoglu, A., Mirzabekov, J.J., Lo, M., Thompson, K.R., Kim, S.Y., Adhikari, A., Tye, K.M., Frank, L.M., Deisseroth, K., 2012. A prefrontal cortex-brainstem neuronal projection that controls response to behavioural challenge. Nature 492, 428–432. Williams, G.V., Goldman-Rakic, P.S., 1995. Modulation of memory fields by dopamine D1 receptors in prefrontal cortex. Nature 376, 572–575.
Please cite this article as: Castañé, A., et al., 5-HT2A receptors are involved in cognitive but not antidepressant effects of fluoxetine. European Neuropsychopharmacology (2015), http://dx.doi.org/10.1016/j.euroneuro.2015.04.006
5-HT2A receptors are involved in cognitive Williams, G.V., Rao, S.G., Goldman-Rakic, P.S., 2002. The physiological role of 5-HT2A receptors in working memory. J. Neurosci. 22, 2843–2854. Wilson, F.A., O’Scalaidhe, S.P., Goldman-Rakic, P.S., 1994. Functional synergism between putative gamma-aminobutyratecontaining neurons and pyramidal neurons in prefrontal cortex. Proc. Natl. Acad. Sci. USA 91, 4009–4013.
9 Zhang, G., Ásgeirsdóttir, H.N., Cohen, S.J., Munchow, A.H., Barrera, M.P., Stackman Jr, R.W., 2013. Stimulation of serotonin 2A receptors facilitates consolidation and extinction of fear memory in C57BL/6J mice. Neuropharmacology 64, 403–413. Zhong, P., Yan, Z., 2011. Differential regulation of the excitability of prefrontal cortical fast-spiking interneurons and pyramidal neurons by serotonin and fluoxetine. PLoS One 6, e16970.
Please cite this article as: Castañé, A., et al., 5-HT2A receptors are involved in cognitive but not antidepressant effects of fluoxetine. European Neuropsychopharmacology (2015), http://dx.doi.org/10.1016/j.euroneuro.2015.04.006
