Chronic citalopram administration desensitizes prefrontal cortex but not somatodendritic α2-adrenoceptors in rat brain

Chronic citalopram administration desensitizes prefrontal cortex but not somatodendritic α2-adrenoceptors in rat brain

Accepted Manuscript Chronic citalopram administration desensitizes prefrontal cortex but not somatodendritic α2-adrenoceptors in rat brain Begoña Fern...

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Accepted Manuscript Chronic citalopram administration desensitizes prefrontal cortex but not somatodendritic α2-adrenoceptors in rat brain Begoña Fernández-Pastor, Jorge E. Ortega, Laura Grandoso, Elena Castro, Luisa Ugedo, Ángel Pazos, J. Javier Meana PII:

S0028-3908(16)30541-X

DOI:

10.1016/j.neuropharm.2016.11.025

Reference:

NP 6521

To appear in:

Neuropharmacology

Received Date: 27 September 2016 Revised Date:

18 November 2016

Accepted Date: 26 November 2016

Please cite this article as: Fernández-Pastor, B., Ortega, J.E., Grandoso, L., Castro, E., Ugedo, L., Pazos, E., Meana, J.J., Chronic citalopram administration desensitizes prefrontal cortex but not somatodendritic α2-adrenoceptors in rat brain, Neuropharmacology (2016), doi: 10.1016/ j.neuropharm.2016.11.025. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Chronic citalopram administration desensitizes prefrontal cortex but not somatodendritic α2-adrenoceptors in rat brain

a,1

a

Luisa Ugedo , Ángel Pazos

, Jorge E. Ortega

b,c

a,b,d,*

, Laura Grandoso

and J. Javier Meana

a,2

, Elena Castro

b,c

,

a,b,d

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Begoña Fernández-Pastor

a

Department of Pharmacology, University of the Basque Country UPV/EHU, Leioa, Bizkaia, b

c

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Spain; Centro de Investigación Biomédica en Red de Salud Mental CIBERSAM, Spain;

Departament of Physiology and Pharmacology, Institute of Biomedicine & Biotechnology of

Cantabria (IBBTEC), University of Cantabria-CSIC-IDICAN, Santander, Cantabria, Spain; d

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BioCruces Health Research Institute, Bizkaia, Spain

Correspondence: Jorge E. Ortega, Department of Pharmacology, University of the Basque

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Country UPV/EHU, E-48940 Leioa, Bizkaia, Spain. Tel: +34-946015674, Fax: +34-946013220, Email: [email protected]

1

Present address: Laboratorios Dr. Esteve, Barcelona, Spain.

2

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Present address: Progenika Biopharma, Derio, Bizkaia, Spain.

Running title: Chronic citalopram and α2-adrenoceptors

ACCEPTED MANUSCRIPT Abstract Selective serotonin reuptake inhibitors (SSRIs) regulate brain noradrenergic neurotransmission both at somatodendritic and nerve terminal areas. Previous studies have demonstrated that noradrenaline (NA) reuptake inhibitors are able to desensitize

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α2-adrenoceptor-mediated responses. The present study was undertaken to elucidate the effects of repeated treatment with the SSRI citalopram on the α2-adrenoceptor

sensitivity in locus coeruleus (LC) and prefrontal cortex (PFC), by using in vivo

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microdialysis and electrophysiological techniques, and in vitro stimulation of

[35S]GTPγS binding autoradiography. Repeated, but not acute, treatment with

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citalopram (5 mg/kg, i.p., 14 days) increased extracellular NA concentration selectively in PFC. The α2-adrenoceptor agonist clonidine (0.3 mg/kg, i.p.), administered to salinetreated animals (1 ml/kg i.p., 14 days) induced NA decrease in LC (Emax=-44±4%; p<0.001) and in PFC (Emax=-61±5%¸ p<0.001). In citalopram chronically-treated rats,

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clonidine administration exerted a lower decrease of NA (Emax=-25±7%; p<0.001) in PFC whereas the effect in LC was not different to controls (Emax=-36±4%). Clonidine

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administration (0.625-20 µg/kg, i.v.) evoked a dose-dependent decrease of the firing activity of LC noradrenergic neurons in both citalopram- (ED50=3.2±0.4 µg/kg) and

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saline-treated groups (ED50=2.6±0.5 µg/kg). No significant differences between groups were found in ED50 values. The α2-adrenoceptor agonist UK14304 stimulated specific [35S]GTPγS binding in brain sections contaning LC (144±14%) and PFC (194±32%) of saline-treated animals. In citalopram-treated animals, this increase did not differ from controls in LC (146±22%) but was lower in PFC (141±8%; p<0.05). Taken together, long-term citalopram treatment induces a desensitization of α2-adrenoceptors acting as axon terminal autoreceptors in PFC without changes in somatodendritic α2adrenoceptor sensitivity.

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α2

(-) Citalopram: ↑ 5-HT

↑ NA

α2

Postsynaptic neuron

Desensitized α2-adrenoceptor

α2

Prefrontal cortex

NA

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(-)

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NA

(-)

Non-desensitized α2-adrenoceptor

NA

5-HT

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Locus coeruleus

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Raphe nuclei

Serotonin transporter inhibition

5-HT

5-HT

Serotonin transporter inhibition

ACCEPTED MANUSCRIPT Keywords Microdialysis, locus coeruleus, prefrontal cortex, noradrenaline, α2-adenoceptor,

Chemical compounds

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citalopram

Citalopram hydrobromide (PubChem CID 77995); clonidine hydrocloride (PubChem CID 20179); UK14304 (PubChem CID 2435); RX821002 (PubChem CID 108094),

Abbreviations LC: Locus coeruleus NA: noradrenaline NARI: NA reuptake inhibitor

PFC: prefrontal cortex

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NET: NA transporter

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chloral hydrate (PubChem CID 2707).

SERT: serotonin transporter

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SSRI: selective serotonin reuptake inhibitor

ACCEPTED MANUSCRIPT 1. Introduction Most antidepressant drugs exert their pharmacological activity through modulation of monoaminergic systems. Brainstem noradrenergic neurons of locus coeruleus (LC), the main source of noradrenergic innervation in the brain, are thought to be involved in the

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pathophysiology of depression (Ressler and Nemeroff, 1999). In the LC, α2-

adrenoceptors exert an inhibitory role on somatodendritic noradrenaline (NA) release (Callado and Stamford, 1999; Mateo et al, 1998). In terminal noradrenergic areas,

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concentration of synaptic NA is under negative regulation of two different α2-

adrenoceptor populations, the α2-adrenoceptors located in the LC controlling as

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autoreceptors the noradrenergic firing activity (Mateo et al, 1998; Van Gaalen et al, 1997) and the α2-adrenoceptors located in terminals where they modulate NA release (Dalley and Stanford, 1995; Van Veldhuizen et al, 1993). In this sense, it has been previously described that local administration into the LC of the NA reuptake inhibitor

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(NARI) antidepressant desipramine increases NA in the area and induces a decrease of noradrenergic firing activity trough α2-adrenoceptor activation, leading to a subsequent

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decrease of NA release in the prefrontal cortex (PFC) (Mateo et al, 1998).

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Early concepts suggested that the selective regulation of serotonergic function accounts for the antidepressant effect of selective serotonin reuptake inhibitors (SSRIs). However, reciprocal anatomo-physiological interactions between noradrenergic and serotonergic systems make possible that even if their pharmacological activity is mediated by inhibition of serotonin reuptake, SSRIs might also act through modulation of the brain noradrenergic system. Thus, it has been described a confluence between noradrenergic and serotonergic systems in the LC (Cedarbaum and Aghajanian, 1978; Maeda et al, 1991) and in terminal areas, such as the PFC. It has been previously

ACCEPTED MANUSCRIPT described that endogenous serotonin release in the LC is able to regulate NA in the area (Singewald and Philippu, 1998) and, subsequently, modulates noradrenergic firing activity (Mateo et al, 2000). Acute administration of the SSRI citalopram induces a dose-dependent increase of serotonin synaptic concentrations in both LC and PFC areas

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(Millan et al, 1999; Ortega et al, 2013). In accordance, acute administration of the SSRIs citalopram and paroxetine enhances NA in the LC but decreases the firing

activity of LC neurons and the NA release in terminal areas (Fernández-Pastor et al,

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2013; Mateo et al, 2000; Szabo et al, 1999). This inhibitory effect of SSRIs involves α2adrenoceptors located on LC neurons (Mateo et al, 2000) in a similar way to the

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inhibition induced by acute administration of the NARI desipramine (Mateo et al, 1998). Therefore, the acute in vivo inhibitory effect of antidepressant drugs on brain noradrenergic neurons seems to be independent of their intrinsic pharmacological in

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vitro mechanism (NA or serotonin reuptake inhibition).

A drawback of all marketed antidepressants, regardless of their mechanisms of action, is the long delay necessary to achieve therapeutic efficacy. This lag time is believed to

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reflect, to a large extent, the time required for desensitization of the inhibitory autoreceptors regulating monoamine release (Artigas et al, 1996). Consistent with this

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hypothesis, several studies have demonstrated α2-adrenoceptor up-regulation and supersensitivity in postmortem brain of depressed subjects and in platelets of depressed patients (for a review, see Cottingham and Wang, 2012). Interestingly, antidepressant treatment induces α2-adrenoceptor down-regulation that results in reduced functionality (García-Sevilla et al, 1990; Rivero et al, 2014; reviewed in Cottingham and Wang, 2012). In agreement with these findings in humans, α2-adrenoceptor down-regulation and desensitization in the rat central nervous system are common responses to the

ACCEPTED MANUSCRIPT chronic treatment with antidepressant drugs that increase synaptic NA (reviewed in Cottingham and Wang, 2012). In fact, the sustained increase of extracellular NA following chronic administration of the NARIs desipramine, reboxetine or the MAO inhibitor clorgyline has been proposed as the mechanism of α2-adrenoceptor

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desensitization observed by in vivo microdialysis (Invernizzi et al, 2001; Mateo et al, 2001; Page and Lucki, 2002; Parini et al, 2005; Sacchetti et al, 2001).

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Currently, it is not clear whether antidepressant response to SSRIs involves the

regulation of α2-adrenoceptor-mediated functions. The aim of the present study was to

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evaluate the effect of chronic citalopram treatment on α2-adrenoceptor sensitivity by using in vivo microdialysis and electrophysiological assays and by in vitro [35S]GTPγS binding autoradiography. These approaches evaluate the contribution of somatodendritic and terminal α2-adrenoceptor subpopulations to the long-term

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citalopram modulation of noradrenergic transmission.

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2. Materials and methods

2.1. Animals and treatments

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Experiments were performed on male Sprague-Dawley rats (SGIker facilities, University of the Basque Country, UPV/EHU, Spain). Animals were housed 4/5 per cage in a 12 h light-dark cycle at room temperature (22ºC) with food and water ad libitum. Animal care and experimental protocols were in agreement with European Union regulations and approved by the UPV/EHU Ethical Board for Animal Welfare (CEEA).

ACCEPTED MANUSCRIPT Animals weighed 175-200 g at the start of treatment and 275-310 g when the experiments were carried out. Rats were chronically treated with citalopram 5 mg/kg i.p., every 24 h for 14 days (diluted in saline 0.9%, 5 mg/ml). Control group received

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saline under similar conditions (0.9%, 1 ml/kg i.p., every 24 h for 14 days).

2.2. Drugs and reagents

Citalopram HBr, 5-bromo-6-(2-imidazolin-2-ylamino)quinoxaline (UK14304) and 2-

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methoxyidazoxan (RX821002) were provided by Tocris Cookson Ltd. (Bristol, UK);

clonidine HCl was purchased from Sigma Co. (St. Louis, MO, USA); chloral hydrate

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was provided by Fluka Chemie AG (Buchs, Switzerland). [35S]GTPγS was purchased from DuPont NEN (Brussels, Belgium). All reagents were of the highest purity available and were obtained in the standard commercial sources.

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2.3. Microdialysis

Two sets of experiments were carried out. In the first one, two dialysis probes were implanted (13th day) in the rat brain under anesthesia (chloral hydrate 400 mg/kg i.p.).

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Twenty four hours after implantation, a challenge dose of citalopram (5 mg/kg i.p.) was administered (14th day) both to citalopram- and saline-pretreated groups. Basal NA

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concentrations were evaluated between 35 min and 140 min (three 35 µl dialysate fractions) and 48 h (three 35 µl dialysate fractions) after the last dose administration. A challenge dose of saline vehicle was also performed in a third group of rats that was used as injection control.

In the second set of experiments, the functional sensitivity of α2-adrenoceptors after chronic citalopram or saline treatments was assessed by clonidine systemic

ACCEPTED MANUSCRIPT administration after a 48 h washout period. This time has been described as a suitable period to avoid residual effects of different antidepressants (Mateo et al, 2001; Muguruza et al, 2014; Sacchetti et al, 2001). Clonidine was selected as α2-adrenoceptor agonist because of the existence of previous validated data of its activity by in vivo

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microdialysis (Horrillo et al, 2016; Mateo et al, 2001) and its good solubility in saline solution. For this purpose, rats were anesthetized with chloral hydrate (400 mg/kg i.p.) on the 15th day for microdialysis probe implantation and clonidine (0.3 mg/kg, i.p.) or

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2.3.1. Probe implantation and microdialysis.

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vehicle (saline 1 ml/kg, i.p.) were injected on the 16th day.

Microdialysis experiments were carried out as described (Ortega et al, 2010). Two concentric Cuprophan microdialysis probes were stereotaxically implanted choosing coordinates according to Paxinos and Watson Atlas (1986). One (exposed tip 2.0 mm x

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0.25 mm) was implanted in the vicinity of the right LC (AP–3.7, L+1.3, V–8.2, taken from λ suture point and the incisor bar lowered to a 15º angle) and the other (exposed

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bregma).

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tip 4.0 mm x 0.25 mm) in the ipsilateral PFC (AP+2.8, L+1.0, V–5, taken from

Microdialysis experiments were performed one day after surgery to allow rats to recover from surgery. Animals were placed in a freely awake animal system (CMA/Microdialysis AB, Sweden). The probes were perfused with modified CSF solution (148 mM NaCl, 2.7 mM KCl, 1.2 mM CaCl2 and 0.85 mM MgCl2, pH 7.4) at 1 µl/min flow rate. Dialysates were collected every 35 min in vials containing 5 µl perchloric acid 0.1 M. Drugs were administered by intraperitoneal injection after at least three stable baseline samples collection. Further, animals were killed and the brains

ACCEPTED MANUSCRIPT were dissected to verify the correct implantation of the probes. In vitro recovery for NA was in 10-15% range.

2.3.2. Analysis of extracellular NA concentrations.

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Immediately after sample collection, NA (injection volume, 37 µl) was analyzed, as

reported (Ortega et al, 2012), using HPLC with electrochemical detection fitted to +650 mV (Hewlett-Packard 1049A). Separation was carried out at 25ºC on a Chrospher

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100RP-18, 5-µm particle size, 125 mm x 4 mm column. The mobile phase composition was 12 mM citric acid, 1 mM EDTA, 1.2 mM octyl sodium sulphate (pH 5.0) and 12%

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(vol/vol) methanol, and was delivered at a 0.8 ml/min flow rate by a Hewlett-Packard 1100 pump. NA concentration in dialysates was estimated with reference to standards. The detection limit was 20-25 fmol per sample.

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2.4. In vivo electrophysiology

Electrophysiological experiments were carried out to record the single-unit extracellular firing rate from LC noradrenergic neurons in rats anesthetized with chloral hydrate (400

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mg/kg i.p.) as previously described (Mateo et al, 2000). Rats chronically treated with

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citalopram (5 mg/kg i.p., every 24 h for 14 days) or saline (1 ml/kg i.p., every 24 h for 14 days) were studied. The experiments were performed 48 h after the last administration of citalopram or saline. Firing rates were recorded before (basal) and after clonidine (0.625-20 µg/kg, i.v.) or vehicle (saline) administration.

The recording electrode was an Omegadot glass micropipette filled with a 2% solution Pontamine Sky Blue in 0.5% sodium acetate and broken back to a tip diameter of 1-2 µm. The electrode was stereotaxically implanted into the LC (AP–3.7, L–1.1, V–5.5 to

ACCEPTED MANUSCRIPT –6.5, related to the λ suture point with the incisor bar lowered to 15º angle, nose down). Extracellular signal from the recording electrode was amplified and monitored on an oscilloscope and an audiomonitor. The signal was processed using computer software (Spike 2 software, Cambridge Electronic Design UK). Only one cell was used in each

2.5. [35S]GTPγγS binding autoradiographic assays

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Aghajanian (1976).

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animal. LC cells were identified by criteria previously described by Cedarbaum and

Animals were killed by decapitation after 48 h washout period of chronic

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citalopram/saline treatments. Brains were immediately removed and coronal sections (20 µm) were obtained. [35S]GTPγS autoradiography was performed as previously described (Rodriguez-Puertas et al, 2000).

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Slide-mounted sections were pre-incubated for 30 min at room temperature in a buffer containing 50 mM Tris-HCl, 0.2 mM EGTA, 3 mM MgCl2, 100 mM NaCl, 1 mM dldithiothreitol and 2 mM GDP at pH 7.7. Slides were subsequently incubated, for 2 h, in

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the same buffer containing adenosine deaminase (3 mU/ml) with 0.04 nM [35S]GTPγS

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in the absence (basal binding), presence of the α2-adrenoceptor agonist UK14304 (10 µM) (stimulated binding), presence of the α2-adrenoceptor antagonist RX821002 (10 µM) + UK14304 (10 µM) or presence of 10 µM cold GTPγS (non-specific binding). For these experiments, the full α2-adrenoceptor agonist UK14304 was selected instead of the partial α2-adrenoceptor agonist clonidine. After the incubation, the sections were washed twice for 15 min in cold 50 mM Tris-HCl buffer (pH 7.4) at 4°C, rinsed in distilled cold water and then dried under a cold air stream. Sections were exposed to film BioMax MR (Carestream) together with 14C microscales at 4oC for 2 days.

ACCEPTED MANUSCRIPT [35S]GTPγS Autoradiographic densities in LC and PFC were determined by densitometry using the Scion Image software (Scion Corporation, Frederick, MD, USA). Relative optical density values were averaged over two consecutive sections per rat (bilateral readings) and converted to nCi/g of tissue. [35S]GTPγS binding

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stimulations induced by UK14304 are presented as percentage of basal binding (100%).

2.6. Statistical Analysis

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The mean value of NA concentrations in the three initial dialysate samples was taken as 100% value. NA values are expressed as percentages of this baseline concentration.

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Basal values were compared by one-way ANOVA followed by post-hoc Dunnet’s test. Clonidine effect was compared by two-way ANOVA of repeated measures (time) followed by Bonferroni’s test. In these analyses all the experimental points, including basal values, were considered. F values were expressed as Ftr (treatment; between-

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groups), Ft (time; within-groups) or Fi (treatment x time; interaction). The maximal effects of clonidine/vehicle (Emax) were obtained. Complementary, the areas under the curve (AUC) were also calculated as the summation effect of the percentage changes

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under the baseline during the full period after clonidine/vehicle administration. One-

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way ANOVA followed by Bonferroni’s test was used to compare Emax and AUC of the different treatment groups. Results are expressed as mean±S.E.M. values.

For electrophysiological experiments, dose-effect curves of clonidine were constructed for chronic citalopram- and saline-treated rats by the best non-linear fitting to the logistic equation: E=Emax / [(1+ED50n / [A]n)], were [A] is the dose of clonidine, E is the effect on the firing rate induced by clonidine, Emax is the maximal effect, ED50 is the

ACCEPTED MANUSCRIPT effective concentration for eliciting 50% of the Emax and n is the slope factor of doseeffect curve. ED50, Emax and n were estimated by this analysis.

Differences between groups in basal firing rate, ED50, basal [35S]GTPγS binding and

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UK14304-stimulated [35S]GTPγS binding values were assessed by Student t-test. The statistical significance was chosen at a p<0.05 value. Statistical procedures were

performed using GrahPad PrismTM (GraphPad Sofware, San Diego, CA, USA) and

3. Results 3.1. Microdialysis experiments

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InVivoStat softwares.

3.1.1. Effect of acute or chronic citalopram administration on NA evaluated in LC and PFC.

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The effect of a challenge dose of citalopram (5 mg/kg i.p., administered the 14th day) on extracellular NA in LC and PFC in groups previously treated with saline (1 ml/kg i.p., every 24 h for 13 days) or citalopram (5 mg/kg i.p., every 24 h for 13 days) was

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evaluated immediately (35-140 min) or 48 h following the challenge citalopram

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administration. A control group with a single saline administration (1 ml/kg i.p.) was also performed.

In LC, no differences in NA concentrations were found between the three groups evaluated immediately after injection (F[2,38]=0.04; p>0.05) (Table 1). A 48 h washout period did not modify NA in the LC. However, in PFC an enhancement of NA was observed after citalopram challenge administration in the group chronically pre-treated with citalopram (F[2,41]=23,47; p<0.001) but not in the group chronically pre-treated

ACCEPTED MANUSCRIPT with saline, equivalent to acute citalopram conditions (Table 1). NA concentrations in PFC returned to control values 48 h after the last administration of citalopram (Table 1). The finding demonstrated that citalopram is able to increase NA in PFC after chronic, but not in acute conditions. Before 48 h, the enhanced NA concentration in PFC

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returned to basal values, supporting this interval as a suitable washout period for further experiments.

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3.1.2. Modulation of NA release by α2-adrenoceptors in LC and PFC of chronicallytreated rats with citalopram.

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The functional sensitivity of α2-adrenoceptors that modulate NA release in LC and PFC was evaluated in rats chronically treated with saline or citalopram. For that purpose, the α2-adrenoceptor agonist clonidine (0.3 mg/kg i.p.) was administered 48 h after the last administration of chronic saline or chronic citalopram. Under chronic saline treatment

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conditions, clonidine induced a significant decrease of NA in LC (Emax=-44±4%; p<0.001) (Ftr[1,10]=61.92, p<0.0001; Ft[7,70]=4.86, p<0.001; Fi[7,70]=7.88, p<0.0001,

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n=12) and in PFC (Emax=-61±5%; p<0.001) (Ftr[1,8]=40.65, p<0.001; Ft[7,56]=7.98, p<0.0001; Fi[7,56]=8.33, p<0.0001, n=10) when compared with the control group

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(vehicle administration) (Figure 1). In the group chronically treated with citalopram, clonidine decreased NA in LC (Emax=-36±4%; p<0.001) (Ftr[1,8]=11.34, p<0.001; Ft[7,56]=3.03, p<0.001; Fi[7,56]=4.13, p<0.0001, n=10) and in PFC (Emax=-25±7%; p<0.01) (Ftr[1,8]=11.07, p<0.05; Ft[7,56]=1.75, p>0.05; Fi[7,56]=3.56, p<0.01, n=10) when compared with the control group (vehicle administration) (Figure 1). The finding demonstrates that systemic clonidine induces a decrease of extracellular NA both in LC and PFC.

ACCEPTED MANUSCRIPT Significant differences in the response to clonidine between chronic citalopram-, and chronic saline-treated groups were found in PFC (Ftr[1,12]=27.50, p<0.001; Ft[7,84]=24.36, p<0.0001; Fi[7,84]=7.31, p<0.0001, n=14) but not in LC (Ftr[1,14]=0.83, p>0.05; Ft[7,98]=30.98, p<0.0001; Fi[7,98]=1.21, p>0.05, n=16)

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(Figure 1). Therefore, chronic citalopram seems to reduce sensitivity of NA release to clonidine in PFC but not in LC.

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3.2. Electrophysiology experiments

The spontaneous firing activity of LC noradrenergic neurons was recorded after 48 h

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washout in rats chronically treated with saline or citalopram. Basal firing rate was 2.0±0.3 Hz (n=18) in saline-treated rats and 2.3±0.2 Hz (n=24) in citalopram-treated animals with no significant differences between groups (t=0.57, p>0.05).

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3.2.1. Modulation of LC noradrenergic cell firing activity by α2-adrenoceptors in chronically-treated rats with citalopram.

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The functional sensitivity of α2-adrenoceptors that modulate firing activity of noradrenergic LC neurons was assessed in rats chronically treated with saline or

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citalopram. Increasing cumulative doses of clonidine were systemically administered in both groups (0.650-20 µg/kg, i.v.). Clonidine reduced the spontaneous activity of LC neurons in a dose-dependent manner (Figure 2). Complete inhibition was achieved in all cells tested, and the mean ED50 values estimated from the dose-effect curves were 2.6±0.5 µg/kg (n=5) in the chronic saline group and 3.2±0.4 µg/kg (n=7) in the chronic citalopram group. These ED50 values did not shown significant differences between the two experimental groups (Figure 2). The data reveal that sensitivity to clonidine of

ACCEPTED MANUSCRIPT somatodendritic α2-adrenoceptors in the LC is not altered by chronic citalopram treatment.

3.3. [35S]GTPγγS binding autoradiography assays

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Basal [35S]GTPγS binding values in LC were 319±24 nCi/g in chronic saline-treated (n=6) and 313±24 nCi/g in chronic citalopram-treated (n=7) rats without statistical

differences between groups. In PFC, basal [35S]GTPγS binding were 153±3 nCi/g in

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chronic saline- (n=5), and 128±5 nCi/g in chronic citalopram-treated (n=8) groups.

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Statistical analysis showed differences of [35S]GTPγS binding values between these two groups (t=3.7; p<0.01).

3.3.1. Modulation of [35S]GTPγS binding stimulation by α2-adrenoceptors in LC and PFC of chronically-treated rats with citalopram.

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The functional sensitivity of α2-adrenoceptors that contribute to stimulate [35S]GTPγS binding was evaluated in rats chronically treated with saline or citalopram. The α2-

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adrenoceptor agonist UK14304 (10 µM) was added to brain sections containing LC or PFC. The UK14304-induced stimulation of specific [35S]GTPγS binding in the LC area

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reached similar effects in chronically saline-treated (144±14%, n=6) than in chronically citalopram-treated animals (146±22%, n=6) (t=0.06, p>0.05) (Figure 3a). However, the PFC stimulation of specific [35S]GTPγS binding by UK14304 reached a higher effect in the chronic saline- (194±32%, n=5) than in the chronic citalopram-treated group (141±8%, n=9) (t=2.1, p<0.05) (Figures 3b and 4). The selective α2-adrenoceptor antagonist RX821002 (10 µM) blocked the effect of UK14304 in both LC and PFC confirming that stimulation of [35S]GTPγS binding was a selective α2-adrenoceptor-

ACCEPTED MANUSCRIPT mediated effect (Figure 5). These results suggest that chronic citalopram administration reduces activation of G-proteins by α2-adrenoceptors in PFC but not in LC.

4. Discussion

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The present study demonstrates that chronic treatment with the SSRI citalopram induces α2-adrenoceptor desensitization in rat PFC contributing to positively modulate NA

release in the area, whereas the α2-adrenoceptor subpopulations that control local NA

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release and noradrenergic firing activity of LC remain unaltered. Reduction in the

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receptor-coupling to G-proteins, the first step of the α2-adrenoceptor signaling, seems to be the mechanism involved in this desensitization. Since an enhanced density and sensitivity of α2-adrenoceptors is an established finding in brain and platelets of depressed subjects (Cottingham and Wang, 2012), it is conceivable that desensitization

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of these receptors could contribute to the pharmacological activity of SSRIs.

Although it has been proposed hypothetical relationship between specific

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neurotransmitter alterations and depression symptoms, antidepressants with noradrenergic and serotonergic profile appear to be equally effective and might act

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through a common mechanism, resulting in similar patterns of symptomatic response (Nelson et al, 2005). Several reports indicate that SSRIs modulate noradrenergic transmission in vivo (Bymaster et al, 2002; David et al, 2003; Gobert et al, 1997; Kaneko et al, 2016; Millan et al, 2000, 2001). However, controversial results have been published depending on doses and/or SSRIs. Citalopram represents the most selective SSRI (Sánchez and Hyttel, 1999). At a 5 mg/kg i.p., the dose herein tested, citalopram increases brain serotonin concentrations (Millan et al, 1999) whereas does not modify NA under acute administration (Ortega et al, 2010). Higher doses of citalopram produce

ACCEPTED MANUSCRIPT robust decreases of NA in PFC (Fernández-Pastor et al, 2013; Kaneko et al, 2016; Ortega et al, 2010), which is difficult to reconcile with an endogenous NA-mediated desensitization of α2-adrenoceptors in the area as previously seen with NARIs (Invernizzi et al, 2001; Mateo et al, 2001; Page and Lucki, 2002; Parini et al, 2005;

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Sacchetti et al, 2001). In order to interpret this paradox, it must be taken into account

that a maintained increase of synaptic serotonin by long-term citalopram presence could exert progressive adaptations on putative serotonin targets that mediate the

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physiological inhibition induced by the serotonergic system on the central noradrenergic activity. The resultant serotonergic desensitization led to enhance the noradrenergic

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activity. This hypothesis agrees with studies showing that chronic, but not acute, paroxetine and sertraline enhances NA in terminal areas (Hajós-Korcsok et al, 2000; Thomas et al, 1998). However, fluoxetine, paroxetine and sertraline selectivity for the serotonin transporter (SERT) versus the NA transporter (NET) is modest compared with

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citalopram (Cryan et al, 2004; Sanchez and Hyttell, 1999). By this reason, direct inhibition of NET by these SSRI antidepressant drugs represents a possible explanation for the enhanced NA availability. Moreover, a moderate blockade of NET by the SSRI

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escitalopram has been suggested in mutant mice lacking the SERT (Nguyen et al, 2013).

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Nevertheless, in the present study, the absence of significant action of chronic citalopram on NA in LC, a brain area with high NET expression, suggests that this SSRI does not interact directly with NET. Therefore, on the basis of this information and the failure of citalopram to modulate noradrenergic activity after pretreatment with the serotonin inhibitor p-chlorophenylalanine (Mateo et al, 2000), an alternative desensitization of serotonin receptor targets might be hypothesized. Here, evidence was obtained that somatodendritic NA and firing activity remained unchanged in LC after citalopram treatment, indicating that this hypothetical adaptive serotonergic mechanism

ACCEPTED MANUSCRIPT is selectively manifested in PFC. Serotonin 5HT1A and 5HT2A receptor-mediated desensitization of noradrenergic activity following chronic citalopram has already been demonstrated (Szabo et al, 2000). These previous electrophysiological studies focused on the modulation of LC activity, whereas potential changes in terminal noradrenergic

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areas remain still to be elucidated. In this context, administration of α2-adrenoceptor antagonists together with SSRIs or NARIs has been proposed as a pharmacological strategy to overcome the delay in obtaining lesser α2-adrenoceptor-dependent tonic

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inhibition in order to potentiate antidepressant effects (Ortega et al, 2010; Yanpallewar

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et al, 2010).

Since α2-adrenoceptor desensitization is a common mechanism for antidepressant treatments that increase synaptic NA (Cottingham and Wang, 2012), the responses to systemic administration of the α2-adrenoceptor agonist clonidine were studied.

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Extracellular NA in LC and PFC were simultaneously evaluated in saline- and citalopram-treated rats. Clonidine was administered 48 h after the last dose of citalopram, when brain concentrations of citalopram are residual (Muguruza et al, 2014)

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and, therefore, unable to increase serotonergic input to noradrenergic neurons, allowing

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NA to return to basal values. This washout approach makes it easier the study of α2adrenoceptor function due to the absence of competitive effects between the endogenous neurotransmitter and the α2-adrenoceptor agonist. Under these conditions, in citalopram group a lower response of NA release to clonidine in PFC but not in LC was observed. The finding indicates that locally increased NA is necessary to promote the down-regulation of α2-adrenoceptors that inhibit neurotransmitter release. Since clonidine was injected systemically, subsensitive NA release inhibition in PFC might be due to modulation of somatodendritic α2-adrenoceptors that control firing activity

ACCEPTED MANUSCRIPT or/and to desensitization of α2-adrenoceptors that regulate release in noradrenergic terminals. This question was addressed by measuring the basal firing rate of LC neurons and the sensitivity of α2-adrenoceptors that inhibits their firing activity. Basal firing rate was similar between saline- and citalopram-treated rats. Moreover, the dose-effect

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curves to clonidine were also similar in both groups, arguing a normal functionality of α2-adrenoceptors that inhibit the firing activity of LC neurons. In contrast to the present results, it has been reported a reduction of LC firing activity after long-term treatment

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with the SSRIs paroxetine and citalopram (Szabo and Blier, 2001; Szabo et al, 1999)

through a mechanism that involve serotonin 5-HT1A receptors located on glutamatergic

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terminals and serotonin 5-HT2 receptors on GABAergic terminals in the LC area (Haddjeri et al, 1997). However, the experiments were performed with the minipumps in place, not allowing the recovery of NA neurons from the effect due to residual presence of drugs, as it was carried out in the present work. In any case, the enhancing

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effect of the α2-adrenoceptor antagonist idazoxan on LC NA neurons was similar in the group treated with antidepressants and in their respective controls (Szabo and Blier,

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firing activity.

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2001), arguing the idea of non altered functionality of α2-adrenoceptors that control LC

Thus, electrophysiological and microdialysis experiments strongly suggested that cortical α2-adrenoceptor function is selectively attenuated after long-term citalopram administration. This fact was confirmed by [35S]GTPγS autoradiography, showing that a lower G-protein activation was induced by the α2-adrenoceptor full agonist UK14304 in chronic citalopram-treated animals.

ACCEPTED MANUSCRIPT In vivo desensitization of α2-adrenoceptors that inhibit NA release in terminal areas with unaltered somatodendritic α2-adrenoceptors that control LC firing activity is a repeated finding after chronic NARI treatment (Mateo et al, 2001; Parini et al, 2005). In agreement with the present results, similar responses to control conditions have been

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obtained after chronic citalopram both for α2-adrenoceptor modulation of LC

somatodendritic NA release (Kawahara et al, 2007) and for α2-adrenoceptor-mediated inhibition of LC firing activity (Grandoso et al, 2005). As a general rule, the adjustment

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of the function of monoaminergic pathways that imply a final increase of NA and/or

serotonin in terminal areas seem to be a long-term operating mode of antidepressants.

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Afterwards, changes in the activation of different postsynaptic receptors involving an alteration of terminal neurocircuitry seem to be the most plausible mechanisms for the final antidepressant response. Identification of such a mechanism is still a challenge for

5. Conclusions

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the understanding of antidepressant long-term mechanisms.

Long-term antidepressant treatment induces in vivo desensitization of cortical α2-

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adrenoceptors regulating the local release of NA, whereas α2-adrenoceptors that

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modulate the noradrenergic firing activity remains unaltered. The mechanism seems to be common to NARI, MAO inhibitor and SSRI antidepressants, independently of their primary mechanism of action. Accordingly, it has been postulated that the time necessary to achieve antidepressant therapeutic efficacy reflects the time inhibitory α2adrenoceptors require to become desensitized, and subsequently, to promote an increased NA release by nerve terminals (Cottingham and Wang, 2012; Mavroidis et al, 1984). In this context, adjunctive antidepressant treatment with α2-adrenoceptor antagonists has been found to increase clinical response and generate a more rapid onset

ACCEPTED MANUSCRIPT of action (Hannan et al, 2007; Sanacora et al, 2004). Likewise, this facilitatory effect of α2-adrenoceptor antagonism could be a contributing pharmacological mechanism to the enhancement of antidepressant response induced by atypical antipsychotic drugs (Cruz

Funding and disclosure

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The authors declare no conflict of interest

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et al, 2010; Zhou et al, 2015 and references therein).

Acknowledgments

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This work was supported by Spanish MCyT and MINECO (grant numbers SAF01/0553; SAF2009/08460 and SAF2013/48586R), Basque Government (grant

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number IT616-13) and the ERD Funds.

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ACCEPTED MANUSCRIPT Table 1 Effect of acute and chronic citalopram administration on extracellular NA concentration in the LC area and the PFC of rats

Locus coeruleus (LC)

n

1.99±0.17

3

2.01±0.03

5

1.98±0.12

5

Citalopram challenge on chronic saline

Citalopram challenge on chronic saline

Citalopram challenge on chronic citalopram treatment and no washout

Citalopram challenge on chronic citalopram treatment and 48 h washout

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treatment and 48 h washout

n

1.88±0.08

3

2.05±0.02

5

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treatment and no washout

NA (nM)

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Saline challenge and no washout

NA (nM)

Prefrontal cortex (PFC)

1.82±0.06

5

2.02±0.06

5

2.34±0.05***

6

2.11±0.16

5

1.96±0.05

6

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The data represent the extracellular NA concentration of three fractions collected inmediately (35-140 min) and after 48 h saline or citalopram challenge administration. Values are mean±SEM of n separate

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animals. *** p<0.001; one-way ANOVA followed by Dunnet’s test (versus saline) was performed.

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PFC (c). Points are mean±S.E.M. values of 3 (vehicle), 7-9 (chronic saline + clonidine) and 7 (chronic citalopram + clonidine) experiments and are expressed as percentage of the corresponding basal values. The arrow represents administration of clonidine (0.3

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mg/kg i.p.) or vehicle (1 ml/kg i.p.). (b,d) Bars representation (mean±S.E.M.) of the areas under the 105-280-min time-course curves. ns: non-significant difference;

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***p<0.001; one-way ANOVA followed by Bonferroni’s post-hoc test.

Figure 2 Effect of chronic citalopram administration on the sensitivity of α2adrenoceptors in LC. Cumulative dose-effect curves for the inhibition of the firing rate

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of LC neurons by clonidine (0.625-20 µg/kg, i.v.) in chronic saline-treated rats (1 ml/kg i.p. every 24 h for 14 days; Ο) and in chronic citalopram-treated rats (5 mg/kg every 24 ). Points are mean±S.E.M. values of 5 (chronic saline) and 8

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h i.p., for 14 days;

(chronic citalopram) experiments and are expressed as inhibition percentage over the

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corresponding basal firing value.

Figure 3 Effect of chronic citalopram administration on α2-adrenoceptor sensitivity in LC (a) and PFC (b) evaluated by stimulation of [35S]GTPγS binding with UK14304 (10 µM). Bars representation (mean±S.E.M.) of 5-9 individual values. *p<0.05; Student’s ttest.

ACCEPTED MANUSCRIPT Figure 4 Effect of chronic citalopram administration on the sensitivity of α2adrenoceptor in rat PFC. Representative autoradiograms of basal [35S]GTPγS binding (A, A´), non-specific [35S]GTPγS binding (B, B´) and UK14304-stimulated [35S]GTPγS

rats. Bar: 5 mm. PFC: prefrontal cortex.

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binding (C, C´) in chronic saline- (A, B, C) and chronic citalopram-treated (A´, B´, C´)

Figure 5 Representative autoradiograms of UK14304-induced stimulation of

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[35S]GTPγS binding in sections containing the PFC (A, B, C, D) and the LC (A´, B´, C´, D´) of rats chronically treated with citalopram (5 mg/kg i.p. every 24 hours for 14 days).

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A, A´: Basal [35S]GTPγS binding; B, B´: non-specific [35S]GTPγS binding; C, C´: UK14304-mediated (10 µM) stimulation of [35S]GTPγS binding and D, D´: blockade by the antagonist RX821002 (10 µM) of UK14304-mediated (10 µM) [35S]GTPγS binding

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LC location.

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stimulation. Bar: 5 mm. PFC: prefrontal cortex; LC: locus coeruleus. Arrow indicates

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ACCEPTED MANUSCRIPT Highlights Chronic SSRI citalopram treatment increases NA concentration selectively in PFC. Chronic citalopram does not change somatodendritic α2-adrenoceptor sensitivity.

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Chronic citalopram induces selective α2-adrenoceptor desensitization in PFC. Reduction of α2-adrenoceptor-coupling to G-proteins is the mechanism involved.

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Addition of α2-antagonists to SSRIs could increase antidepressant response.