Ouissame Mnie-Filali

Céline Faure

Laura Lambás-Señas

Mostafa El Mansari

Hassina Belblidia

Elise Gondard

Adeline Etievant

Helene Scarna

Anne Didier

Anne Berod

Adeline Etiévant

Hélène Scarna

Pierre Blier

Nasser Haddjeri
email: nasser.haddjeri@univ-lyon1.fr

Ph.D Haddjeri Nasser

Pharmacological blockade of 5-HT7 receptors as a putative fast acting antidepressant strategy

Keywords: Mood / Anxiety / Stress Disorders, Neuropharmacology, Serotonin, Receptor, Pharmacology, electrophysiology, 5-HT7 receptors, antidepressant, dorsal raphe, neurogenesis

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INTRODUCTION

There are extensive data demonstrating the involvement of the brain serotonin (5-HT) system in both the pathogenesis of depression and the action of antidepressant (AD) drugs. However, optimal therapeutic effectiveness is not achieved with current ADs, because most agents present major drawbacks in clinical use, such as a delayed onset of their action and a high percentage of non-responders. Selective 5-HT reuptake inhibitors (SSRIs) exert their actions by enhancing synaptic availability of 5-HT. However, a series of neuroadaptive changes must first occur for this to happen, and these changes are thought to underlie the prolonged therapeutic latency of these drugs [START_REF] Blier | Serotonin and drug-induced therapeutic responses in major depression, obsessive-compulsive and panic disorders[END_REF][START_REF] Duman | Regulation of adult neurogenesis by antidepressant treatment[END_REF][START_REF] Nestler | Neurobiology of depression[END_REF]. Examples of the above mentioned changes include a progressive desensitization of raphe 5-HT 1A autoreceptors and a stimulation of the cell proliferation in the dentate gyrus (DG) of the hippocampus following long-term treatment with SSRIs, with a time course consistent with their delayed AD effects [START_REF] Blier | Serotonin and drug-induced therapeutic responses in major depression, obsessive-compulsive and panic disorders[END_REF][START_REF] Hensler | Differential regulation of 5-HT1A receptor-G protein interactions in brain following chronic antidepressant administration[END_REF][START_REF] Santarelli | Requirement of hippocampal neurogenesis for the behavioral effects of antidepressants[END_REF][START_REF] Castrén | Neurotrophic effects of antidepressant drugs[END_REF][START_REF] Lanfumey | 5-HT1 receptors[END_REF]Faure et al., 2006a).

Recently, attention has been given to 5-HT 7 receptors because of their potential role in mood disorders including depression (for reviews, see [START_REF] Hedlund | The 5-HT7 receptor and disorders of the nervous system: an overview[END_REF][START_REF] Mnie-Filali | Therapeutic Potential of 5-HT(7) Receptors in Mood Disorders[END_REF]. The 5-HT 7 receptor is the most recently identified member of the 5-HT receptor family and has been cloned from several species including humans (Hedlund and Sutcliffe, 2004). 5-HT 7 receptors have been shown to be involved in various functions modulated by the 5-HT system such as circadian rhythms and thermoregulation [START_REF] Thomas | SB-656104-A, a novel selective 5-HT7 receptor antagonist, modulates REM sleep in rats[END_REF]Hedlund et al., 2004;[START_REF] Faure | Effects of the 5-HT7 receptor antagonist SB-269970 on rat hormonal and temperature responses to the 5-HT1A/7 receptor agonist 8-OH-DPAT[END_REF]. Moreover, 5-HT 7 receptors in the hippocampal formation also seem to be involved in interactions between 5-HT system and the hypothalamic-pituitary-adrenal axis [START_REF] Yau | Acute restraint stress increases 5-HT7 receptor mRNA expression in the rat hippocampus[END_REF][START_REF] Laplante | Serotonin regulates hippocampal glucocorticoid receptor expression via a 5-HT7 receptor[END_REF]. In addition, the localization of 5-HT 7 receptors in corticolimbic areas related to affective processes and their involvement in functions impaired in depressed patients suggest an important role of these receptors in mood disorders [START_REF] Ruat | Molecular cloning, characterization, and localization of a high-affinity serotonin receptor (5-HT7) activating cAMP formation[END_REF][START_REF] To | Characterization and distribution of putative 5-ht7 receptors in guinea-pig brain[END_REF]Mnie-Filali et al., 2007a;[START_REF] Hedlund | The 5-HT7 receptor and disorders of the nervous system: an overview[END_REF]. More direct evidence, such as down-regulation of 5-HT 7 receptors in hypothalamus after chronic AD treatments [START_REF] Sleight | Identification of 5hydroxytryptamine7 receptor binding sites in rat hypothalamus: sensitivity to chronic antidepressant treatment[END_REF][START_REF] Mullins | Effects of antidepressants on 5-HT7 receptor regulation in the rat hypothalamus[END_REF] and antidepressant-like behaviors produced by 5-HT 7 receptor knock-out mice [START_REF] Hedlund | 5-HT7 receptor inhibition and inactivation induce antidepressant-like behavior and sleep pattern[END_REF][START_REF] Guscott | Genetic knockout and pharmacological blockade studies of the 5-HT7 receptor suggest therapeutic potential in depression[END_REF] strengthen this hypothesis.

In this study, the AD potential of the selective 5-HT 7 receptor antagonist SB-269970 was first assessed using the forced swim test (FST) in rats. Then, we investigated whether only a one-week treatment with SB-269970 is able to elicit the same functional and cellular changes as those induced by classical ADs, including desensitization of DRN 5-HT 1A autoreceptors, enhanced inhibitory tone mediated by postsynaptic 5-HT 1A receptors in the hippocampus, and the stimulation of hippocampal cell proliferation. The effects of SB-269970 were compared with those induced by one-week treatment with the widely used SSRI fluoxetine. The action of SB-269970 and fluoxetine was then assessed in the olfactory bulbectomy (OBX) paradigm, considered as a "chronic" behavioural model of depression in which classical AD treatments require the administration for 2-3 weeks before any AD-like effects can be observed [START_REF] Song | The olfactory bulbectomised rat as a model of depression[END_REF]. Finally, the potential of SB-269970 to produce, like fluoxetine, anxious-and/or depressive-like effects following early life exposure was examined.

MATERIALS AND METHODS

Animals

Experiments were carried out in male Sprague-Dawley rats weighing 250 to 300g [Harlan (Gannat, France) or Charles River (Saint-Constant, QC, Canada)]. Animals were kept under standard laboratory conditions (12-h light:12-h dark cycle) with free access to food and water. They were habituated at least 1 week to the laboratory facility before each

Forced swim test (FST)

The FST was used as previously described [START_REF] Porsolt | Behavioral despair in mice: a primary screening test for antidepressants[END_REF] to evaluate potential AD effects. Briefly, rats experienced a pre-test session followed 24 h later by a test session.

For both the pre-test and the test sessions, conducted under low illumination (12 lux), the rats were placed in a plastic cylindrical tank (50 cm high by 20 cm in diameter) filled with water at 23±2 °C, with a depth of 40 cm, for which the hind limbs could not reach the tank floor. In all experiments, the pre-test was carried out for 15 min and the test for 6 min in the same tank. Vehicle (water) or SB-269970 (0.5 mg/kg, i.p.) were administered between these two sessions (23 h, 5 h, and 1 h prior to the test sessions).

Following either pre-test or test sessions, rats were dried with a towel and kept warm for 30 min before returning to their home cage. A camera coupled with a computer recorded the behavior of the animal online during the FST and the immobility duration was then assessed by image analysis through a specialized digital interface (Videotrack, ViewPoint, Lyon, France) as previously described by [START_REF] Haddjeri | In-vivo modulation of central 5-hydroxytryptamine (5-HT1A) receptor-mediated responses by the cholinergic system[END_REF]. Software

from ViewPoint permitted us to analyze data offline avoiding observer subjectivity.

Locomotor activity

These experiments were performed in order to ensure that behaviors in the FST were not secondary to a non-specific alteration in locomotor activity produced by the treatments.

In order to simulate the treatment procedure used in the FST, rats were injected with vehicle (water) or SB-269970 (0.5 mg/kg, i.p.) three times within 24 hours: 23 h, 5 h and 1 h before the test. They were then placed in activity cages in an actimeter (Imetronic, France) equipped with two infrared beams (one at the front and one at the back) positioned 4 cm above the floor. Locomotor activity was estimated by the determination of successive beam breaks at the front and at the back and vice versa (crossovers) during a 10-min session.

Head Twitch Response (HTR)

The global design of these experiments was chosen on the basis of previous work of [START_REF] Sánchez | R-citalopram inhibits functional and 5-HTP-evoked behavioural responses to the SSRI, escitalopram[END_REF]. Thirty minutes after drugs or vehicle (saline or polyethylene glycol 5% for AS19) injection, the rats were given L-5-HTP (87.3 mg/kg, s.c.). Each animal was then placed in an observation cage and the cumulative number of head twitches (which consists of a characteristic, rapid and rotational flick of the head, ears and neck) was counted during 10 min. Assessments were performed by observers blind to the drug condition of rats.

Extracellular unitary recordings of dorsal raphe nucleus (DRN) 5-HT neurons

Extracellular unitary recordings of DRN 5-HT neurons were performed in chloral hydrate anesthetized rats (400 mg/kg, i.p.), with single-barrelled glass micropipettes positioned 1 mm anterior to lambda on the midline [START_REF] Haddjeri | Pre-and post-synaptic effects of the 5-HT3 agonist 2-methyl-5-HT on the 5-HT system in the rat brain[END_REF]. Presumed DRN 5-HT neurons were identified using the criteria of [START_REF] Aghajanian | Intracellular recordings from serotonergic dorsal raphe neurons: pacemaker potentials and the effect of LSD[END_REF], i.e. a slow (0.5-2.5 Hz) and regular firing rate and long-duration (0.8-1.2 ms) positive action potentials. For drug administration, a baseline firing rate was established over 1-2 minutes and the 5-HT 7 agonist AS19 (dissolved in polyethylene glycol 5%) at dose of 2.5 to 10 mg/kg was intravenously injected in 1-2-min intervals until cell firing was completely suppressed with or without prior injection (2-3 minutes) of SB-269970 (0.1 mg/kg, i.v. dissolved in saline) as well as in rats pre-treated for 15 days with fluoxetine (10 mg/kg/day, i.p. dissolved in saline). In each rat, only one neuron was studied.

For the chronic studies, in order to determine the possible changes of the spontaneous firing activity of dorsal raphe 5-HT neurons, rats were injected daily for one week with fluoxetine (10 mg/kg, i.p. dissolved in saline) or SB-269970 (2 mg/kg, i.p. dissolved in saline). Four to five electrode descents were carried out through this nucleus about one hour after the last i.p. injection. Finally, the responsiveness of 5-HT neurons to enhanced 5-HT levels was evaluated by using the SSRI paroxetine (0.5 mg/kg, i.v.) in control and treated rats for one week with SB-269970 (2 mg/kg, i.p.). The percentage of inhibition of cell firing (% inhibition) was calculated by comparison of the mean baseline firing obtained from a 1-2 min interval prior to the initial drug application and the mean cell firing rate obtained from a 1-2 min interval following each drug injection.

Recordings from dorsal hippocampus CA3 pyramidal neurons

Rats received a daily injection of vehicle (saline), fluoxetine (10 mg/kg, i.p.) or SB-269970 (2 mg/kg, i.p.) for one week. Recording and microiontophoresis were performed with five-barreled glass micropipettes broken back to 8-12 μM under microscopic control. The central barrel was filled with a 2 M NaCl solution and used for extracellular unitary recordings. Pyramidal neurons were identified by their large amplitude (0.5-1.2 mV) and long-duration (0.8-1.2 ms) simple spikes alternating with complex spike discharges [START_REF] Kandel | Electrophysiology of hippocampal neurons. II. After potentials and repetitive firing[END_REF]. The side barrels contained the following solutions: 5-HT creatinine sulfate (2 mM in 200 mM NaCl, pH 4), quisqualate (1.5 mM in 200 mM NaCl, pH 8), and 2 M NaCl used for automatic current balancing. The rats were mounted in a stereotaxic apparatus and the microelectrodes were lowered at 4.2 mm lateral and 4.2 mm anterior to lambda into the CA3 region of the dorsal hippocampus.

Since most hippocampus pyramidal neurons are not spontaneously active under chloral hydrate anesthesia, a leak or a small ejection current of quisqualate (+2 to -8 nA) was used to activate them within their physiological firing range (10-15 Hz;[START_REF] Ranck | Behavioral correlates and firing repertoires of neurons in the dorsal hippocampal formation and septum of unrestrained rats[END_REF]. A 10 or 20 nA ejection current of 5-HT was used, each ejection period lasting 50 s. In order to assess the degree of activation of the postsynaptic 5-HT 1A receptors exerting an inhibitory influence on the firing activity of CA3 pyramidal neurons, WAY 100,635 was administered intravenously to disinhibit the hippocampal neurons resulting in an increase of their firing activity. The saline injection corresponds to the 0 mg/kg dose. WAY 100,635 was administered in incremental doses of 25 μg/kg at time intervals of 2 min. To avoid residual drug effects, only one cell was studied in each rat. The change of firing activity was assessed by calculating the mean firing rate of neurons from about 1-2 min prior to and after (until a 'plateau') the i.v. administration of the drugs and the percent of change was calculated.

Cell proliferation in the subgranular and the subventricular zones.

As previously described (Mnie-Filali et al., 2007b), rat hippocampal cell proliferation was evaluated after 7 daily injections with saline, fluoxetine (10 mg/kg, i.p.) or SB-269970 (2 mg/kg, i.p.). A group of rats treated with SB-269970 received a daily administration of PCPA (400 mg/kg, i.p.) during the last three pretreatment days. On the sixth day, all the groups of rats received four injections of BrdU (50 mg/kg, i.p.) separated by 2 h intervals. Twenty-four hours after the last BrdU injection, animals were deeply anesthetized and transcardially perfused with NaCl 0.9% for 5 min followed by 4% cold formaldehyde in phosphate buffer (PB: 0.1 M, pH 7.4) for 17 min. The brains were then removed, post-fixed overnight in formaldehyde and stored at 4°C in 30%

sucrose. Thirty µm-coronal sections were cut through the olfactory bulb or the hippocampus (respectively from +2.28 to -0.48 mm and from -1.8 to -5.8 mm, posterior to Bregma, according to [START_REF] Paxinos | The Rat Brain in Stereotaxic Coordinates, Fourth Edition[END_REF] collected as a series of 12 serial sections and stored in phosphate buffer saline (PBS) containing 0.1% of sodium azide at 4°C. These free-floating sections were used in the determination of BrdU labeling. DNA denaturation was conducted by incubation for 2 h in 50% formamide/2×SSC (0.3M NaCl, 0.003M sodium citrate) at 65°C followed by two rinses in 2× SCC buffer. Sections were incubated for 30 min in HCl (2N) and then 10 min in borate buffer (0.1M, pH 8).

All incubations were followed by 3 rinses of 10 min in PBST (PBS + 0.1% Triton X-100) at room temperature. Sections were processed for 3 min on pepsin (0.45 U/ml in 0.1N HCl) and transferred for 15 min onto 3% H 2 O 2 to eliminate endogenous peroxidase activity. They were subsequently incubated overnight with anti-BrdU antibody (ABCys, Paris, France) diluted 1/200 in PBST-1% bovine serum albumin at room temperature.

Sections were then washed by 3 rinses in PBST and incubated in a biotinylated secondary rabbit anti-rat IgG (1/250, Vector Laboratories, Burlingame, CA) for 90 min followed by amplification with an avidin horseradish peroxidase diluted 1/200 in PBST (Vector Laboratories, Burlingame, CA). Visualization of bound peroxidase was achieved by reaction in a solution of 50 mM Tris-HCl (pH 7.6) containing 0.003% H 2 O 2 , 0.8% NiCl 2 and 0.02% diaminobenzidine. Every twelfth section throughout the hippocampus was processed for BrdU immunohistochemistry. All BrdU-labeled cells in the granule cell layer were counted in each section by an experimenter blinded to the identity of the sections and data were collected with the help of mapping software (Mercator Pro, Explora Nova, La Rochelle, France), coupled to a Zeiss microscope (Axioskop 20, Zeiss, Lena, Germany). A cell was counted as being in the subgranular zone (SGZ) of the DG if it was located in a 40 µm wide band immediately adjacent to the surface of the DG granule cell layer. The total number of BrdU-labeled cells per section was determined and multiplied by 12 to obtain the total estimated number of cells per dentate gyrus. Ten sections were examined per animal (n = 7 to 8 rats per group). We used the protocol of [START_REF] Kuhn | Neurogenesis in the dentate gyrus of the adult rat: age-related decrease of neuronal progenitor proliferation[END_REF] to assess cell proliferation in the subventricular zone (SVZ). BdrUlabeled cells were counted in the walls of the lateral ventricles on four sections of SVZ per brain taken between 9.4 and 8.8 mm from Bregma and the total number of BrdUlabeled cells was counted and expressed per SVZ and per section.

Olfactory bulbectomy (OBX) and assessment of hyperlocomotion

Bilateral OBX was performed in rats anaesthetized with chloral hydrate (400 mg/kg i.p)

and mounted in a stereotaxic frame. The head was shaved and a midline sagittal incision made. Bilateral burr holes (2 mm diameter) were drilled at 7 mm anterior to Bregma and ± 2.5 mm lateral from midline and olfactory bulbs were removed by suction with a vacuum pump attached to a Pasteur pipette. The cavities were then packed with haemostatic sponges (Bloxang, Bausch & Lomb-Laboratoires Chauvin, France) and the wound was closed with sterile suture. Sham-operated rats were treated similarly, with the exception that olfactory bulbs were not removed. After surgery, animals were injected 5 ml i.p. with 5% glucose. The animals were allowed to recover for 14 days following surgery during which they were handled daily to eliminate any aggressiveness that may have otherwise arisen [START_REF] Song | The olfactory bulbectomised rat as a model of depression[END_REF].

After recovery, rats were daily injected (between 8:30-10:30 a.m.) for 7 days with SB-269970 (2 mg/kg/day, i.p.), 7 or 21 days with fluoxetine (10 mg/kg/day, i.p.), or vehicle (water). A 5-min open field test was conducted 24h after the last drug injection (i.e., 22 and 36 days after bulbectomy, respectively). The open field chamber consisted of a square wooden box (85×85×70 cm 3 ). Since conditions of very high brightness are crucial to observe the hyperactivity in OBX-rats [START_REF] Mar | Antidepressants preferentially enhance habituation to novelty in the olfactory bulbectomized rat[END_REF][START_REF] Song | The olfactory bulbectomised rat as a model of depression[END_REF], the inner faces of the walls were covered with aluminium foil and illumination was provided by a 75 W bulb positioned 90 cm directly over the center of the open field (1700-2000 lux). Each rat was placed into the center of the open field and exploratory activity was monitored by a video camera fixed above the arena and relayed to a computer system. To estimate locomotor activity, the open field was virtually divided into 25 squares of 17x17 cm and the number of crossed squares was calculated.

Neonatal exposure to 5-HT agents

Female Sprague-Dawley rats with litters (standardized to ten male pups) of postnatal day 1 were purchased. From their arrival, litters were randomly assigned to receive either fluoxetine (10 mg/kg), SB-269970 (2 mg/kg), or AS19 (5 mg/kg). Corresponding to their group, rat pups were given i.p. injections from days P8 to P21 of either drug or an equivalent volume of saline twice daily. As previously described, behavioral testing in the FST and the open field was conducted at 2 months of age [START_REF] Hansen | Neonatal administration of the selective serotonin reuptake inhibitor Lu 10-134-C increases forced swimming-induced immobility in adult rats: a putative animal model of depression?[END_REF][START_REF] Ansorge | Early-life blockade of the 5-HT transporter alters emotional behavior in adult mice[END_REF].

Statistical analysis

Electrophysiological data were expressed as percentage of baseline values ± S.E.M.

Behavioural results were presented as mean ± S.E.M. In all experiments, statistical differences between the groups were analyzed with Student's t test or with one-way ANOVA or two-way ANOVA repeated measures followed by the post-hoc PLSD-Fisher test. Differences were considered to be statistically significant when P values were less than 0.05. was assessed using the FST. As previously shown (Wesolowska and Kowalska, 2008)

RESULTS

Effect

HT 1A receptors in the hippocampus

It has been demonstrated that chronic administration of SSRIs, such as fluoxetine or citalopram, induces a robust decrease of 5-HT neuronal activity that is followed by a full recovery, with a delay that corresponds to the onset of their therapeutic effects.

Analysis of DRN 5-HT spontaneous firing showed a significant effect of treatment (F 2,119 =16.81, p<0.0001, one-way ANOVA). As expected, a one-week regimen of the SSRI fluoxetine (10 mg/kg/day, i.p.) significantly decreased by 63% the spontaneous firing activity of 5-HT neurons (p<0.001, PLSD Fisher test, Fig. 4A). This neuronal activity was not, however, modified by a one-week regimen of SB-269970 (2 mg/kg/day) in comparison to controls (Fig 4A). It is assumed that the recovery of the firing rate of 5-HT neurons after chronic SSRIs is due to 5-HT 1A receptor desensitization [START_REF] Chaput | In vivo electrophysiological evidence for the regulatory role of autoreceptors on serotonergic terminals[END_REF][START_REF] Poul | Early desensitization of somato-dendritic 5-HT1A autoreceptors in rats treated with fluoxetine or paroxetine[END_REF][START_REF] Czachura | Effects of acute and chronic administration of fluoxetine on the activity of serotonergic neurons in the dorsal raphe nucleus of the rat[END_REF]. In order to assess the sensitivity of presynaptic 5-HT 1A autoreceptors after a one-week regimen of SB-269970, the capacity of the SSRI paroxetine (0.5 mg/kg, i.v.) to inhibit DRN 5-HT neurons firing activity was examined in vehicle-and SB-269970-treated rats. This probe was deemed optimal to assess the responsiveness of 5-HT 1A autoreceptors because it is devoid of 5-HT 7 affinity and its inhibitory effect is not dependent on 5-HT 1A receptors on a corticalraphe feedback loop involved in inhibiting 5-HT neuron firing [START_REF] Blier | Modification of 5-HT neuron properties by sustained administration of the 5-HT1A agonist gepirone: electrophysiological studies in the rat brain[END_REF]. Paroxetine (0.5 mg/kg, i.v.) completely suppressed the firing activity of DRN 5-HT neurons in control rats, while it reduced this parameter by only 29% in rats preinjected with SB-269970 (n=5 rats per group, Student's t-test; t 8 =3.2; p<0.05). These results suggest that a one-week regimen of SB-269970 produced an early desensitization of 5-HT 1A and/or 5-HT 7 receptors. In addition, it has been previously reported that clinically effective ADs induced an augmentation of the tonic stimulation of inhibitory post-synaptic 5-HT 1A receptors (Haddjeri et al., 1998a,b). Because most CA3 hippocampal pyramidal neurons are not spontaneously active under chloral hydrate anesthesia, an ejection current of quisqualate (0 to -2 nA) is necessary to activate them (Haddjeri et al., 1998a(Haddjeri et al., ,b, 2000;;[START_REF] El Mansari | Effects of acute and long-term administration of escitalopram and citalopram on serotonin neurotransmission: an in vivo electrophysiological study in rat brain[END_REF][START_REF] Lucas | Serotonin(4) (5-HT(4)) receptor agonists are putative antidepressants with a rapid onset of action[END_REF]. The effect of WAY 100,635 was then assessed in control and drug-treated rats (Fig. 4B). WAY 100,635 slightly increased the quisqualate-activated firing activity of CA3 pyramidal neurons within a dose range of 50-100 µg/kg (i.v.) in controls but more potently in drugtreated rats. A two-way ANOVA with repeated measures revealed a significant effect of treatment (F (2,14) =4.34; p<0.05), WAY 100,635 doses (F (3,42) =13.06; p<0.0001), and treatment X WAY 100,635 dose interaction (F (6,42) =3.33; p<0.01). Post-hoc PLSD Fisher tests revealed that dose-response curves for both SB-269970 and fluoxetine treated rats were significantly different from that of the control rats (p<0.05, Fig. 4B). Hence, a oneweek treatment with SB-269970 and fluoxetine induced a facilitation of 5-HT neurotransmission, as correlated by an enhanced 5-HT 1A -mediated inhibitory tone on CA3 pyramidal neurons.

A one-week regimen of SB-269970, but not of fluoxetine, promoted rat hippocampal cell proliferation

It is now widely accepted that, despite the variety of their pharmacological profiles, a common feature of ADs is their ability to enhance hippocampal cell proliferation. Hence, it has been suggested that assessing an increase of this parameter might be a useful paradigm to screen compounds with AD activity. Thus, the ability of SB-269970 to enhance such cell proliferation was investigated. The examination of hippocampal sections showed that BrdU-positive nuclei in both control and drug-treated rats were restricted in the subgranular cell layer (SGZ) of the dentate gyrus (DG) at the border between the granule cell layer and hilus, and appeared in irregular shapes as clusters of 2-3 cells (Fig. 5A). Analysis of data obtained in the SGZ of control and treated rats revealed a significant effect of treatment (F 2,19 =3.79; p<0.05, one-way ANOVA).

Interestingly, as illustrated in figure 5B, a one-week regimen of SB-269970 (2 mg/kg/day, i.p.) significantly enhanced the number of BrdU-positive cells in the SGZ of the DG by 41% when compared to controls (p<0.05, PLSD-Fisher test), whereas no significant effect was observed after a 7-day regimen of the SSRI fluoxetine (10 mg/kg/day, i.p.). Moreover, this enhancement was specific to hippocampal cell proliferation since neither SB-269970 nor fluoxetine affected the number of newborn cells in the subventricular zone (SVZ) of the olfactory bulb (Fig. 5C; F 2,17 =0.53; p>0.05, one-way ANOVA). Altogether, these results showed that the 5-HT 7 receptor antagonist promoted cell proliferation selectively in the SGZ of the DG of the hippocampus, more promptly than with SSRIs. In order to determine that the effect of SB-269970 on cell proliferation was mediated through the 5-HT system, the inhibitor of 5-HT synthesis, parachlorophenylalanine (PCPA), was administered to control and SB-269970 pre-treated rats. As illustrated in Figure 5D, a statistical difference was found when groups were compared using one-way ANOVA (F 2,18 =4.41; p<0.05). Data obtained for the SGZ of rats exposed to PCPA alone (100 mg/kg/day for 4 days, i.p.) showed that the number of newborn cells in the SGZ was decreased by 54% compared to controls (p<0.05, PLSD-Fisher test). Interestingly, in rats receiving regimen of SB-269970 for one week, the enhancement of cell proliferation in the SGZ previously obtained was no longer present after PCPA, as evidenced by the significant 42% decrease compared to controls (p<0.05, PLSD-Fisher test). These results suggest that a functional 5-HT system is necessary for SB-269970 to exert its effect on hippocampal cell proliferation.

Effects of treatment with SB-269970 or fluoxetine in OBX rats

It had previously been reported that OBX rats exhibit locomotor hyperactivity that is suppressed after long-term antidepressant treatment. The effect of SB-269970 was thus investigated on this behavioral parameter. Analysis of data illustrated in Figure 6A revealed a significant effect of treatment (F 2,15 =10.55; p<0.01, one-way ANOVA). As expected, OBX rats that received vehicle for 7 days displayed a strong increase (+135%)

of

Neonatal exposure to SB-269970 or AS19 had no detectable effects in adulthood

In neonatal rodents, sustained administration of 5-HT ADs (including SSRIs and tricyclic antidepressants) during the early life period from postnatal 8 (PN8) to PN21 results in a pattern of maladaptive behaviors that are evident and persistent into adulthood [START_REF] Mirmiran | Suppression of active sleep by chronic treatment with chlorimipramine during early postnatal development: effects upon adult sleep and behavior in the rat[END_REF][START_REF] Maudhuit | Electrophysiological activity of raphe dorsalis serotoninergic neurones in a possible model of endogenous depression[END_REF][START_REF] Hansen | Neonatal administration of the selective serotonin reuptake inhibitor Lu 10-134-C increases forced swimming-induced immobility in adult rats: a putative animal model of depression?[END_REF][START_REF] Ansorge | Early-life blockade of the 5-HT transporter alters emotional behavior in adult mice[END_REF].

These behavioral changes include alterations in locomotor activity and increased immobility in the FST [START_REF] Mirmiran | Suppression of active sleep by chronic treatment with chlorimipramine during early postnatal development: effects upon adult sleep and behavior in the rat[END_REF][START_REF] Hilakivi | Increased adult behavioral 'despair' in rats neonatally exposed to desipramine or zimeldine: an animal model of depression?[END_REF][START_REF] Alexandre | Early life blockade of 5-hydroxytryptamine 1A receptors normalizes sleep and depression-like behavior in adult knock-out mice lacking the serotonin transporter[END_REF][START_REF] Hansen | Neonatal administration of the selective serotonin reuptake inhibitor Lu 10-134-C increases forced swimming-induced immobility in adult rats: a putative animal model of depression?[END_REF]. The effect of post-natal exposure to fluoxetine (10 mg/kg/day for 14 days, i.p.), SB-269970 (2 mg/kg/day for 14 days, i.p.) or with AS19 (5 mg/kg/day for 14 days, i.p.) was examined in adulthood (Fig. 7). A significant effect of treatment (F 3,40 =3.01; p<0.05, one-way ANOVA) was obtained. In comparison to control animals, rats neonatally exposed to the SSRI fluoxetine exhibited a 58% decrease in the number of visits to the center of the open field apparatus (p<0.05, PLSD-Fisher test). No significant difference was observed after neonatal exposure to SB-269970 or AS19 (Fig. 7A). These results showed an increase in anxiety-like behavior by post-natal exposure to the SSRI fluoxetine, but not with the 5-HT 7 receptor agonist or the antagonist. In the FST, a significant effect of treatment was observed (F 3,34 =5.35; p<0.01, one-way ANOVA). The immobility duration was significantly enhanced by 32% in fluoxetineexposed rats (p<0.05, PLSD-Fisher test) during the neonatal period, but not in AS19-or SB-269970-exposed animals (Fig. 7B). Taken together, these results showed that neonatal exposure to the SSRI fluoxetine, but not to AS19 or SB-269970, produced anxiety-and depression-like behaviors in early adulthood of the rats.

DISCUSSION

The present results show that behavioral, electrophysiological and neuroanatomical changes that usually correlate with the onset of beneficial action of long-term treatment with SSRIs are already observed after only 7 days of treatment with the 5-HT 7 receptor antagonist SB-269970.

The first results showed that SB-269970 reduced immobility in the FST without modifying locomotor activity. These data confirm previous results showing an antidepressant-like action of SB-269970 in the same test, with doses ranging from 1.25 -2.5 mg/kg (Wesolowska and Kowalska, 2008). This group reported that an intrahippocampal injection of SB-269970 also reduces immobility time, suggesting that the hippocampus is one of the brain structures involved in the beneficial action of 5-HT 7 antagonism [START_REF] Wesolowska | Potential anxiolytic and antidepressant effects of the selective 5-HT7 receptor antagonist SB 269970 after intrahippocampal administration to rats[END_REF]. It has also been reported that both pharmacological inhibition and genetic inactivation of 5-HT 7 receptors induce antidepressant-like behaviour in the mouse tail suspension test [START_REF] Hedlund | 5-HT7 receptor inhibition and inactivation induce antidepressant-like behavior and sleep pattern[END_REF][START_REF] Bonaventure | Selective blockade of 5-hydroxytryptamine (5-HT)7 receptors enhances 5-HT transmission, antidepressant-like behavior, and rapid eye movement sleep suppression induced by citalopram in rodents[END_REF][START_REF] Sarkisyan | The 5-HT(7) receptor as a mediator and modulator of antidepressant-like behavior[END_REF], as well as an anti-anxiety effect in the Vogel drinking test in rats, the elevated plus maze in rats, and in the four-plate test in mice [START_REF] Wesolowska | Potential anxiolytic and antidepressant effects of the selective 5-HT7 receptor antagonist SB 269970 after intrahippocampal administration to rats[END_REF]. Interestingly, a dose of 2 mg/kg of SB-269970 had no detectable effect in the present study by itself, suggesting a lack of modification of 5-HT release as previously shown [START_REF] Bonaventure | Selective blockade of 5-hydroxytryptamine (5-HT)7 receptors enhances 5-HT transmission, antidepressant-like behavior, and rapid eye movement sleep suppression induced by citalopram in rodents[END_REF], it counteracted the anxiogeniclike effect of acute administration of fluoxetine in the illuminated open field (Fig. 1).

Previous reports have shown that the SSRIs fluoxetine and citalopram display anxiogenic behaviors via the activation of 5-HT 2C receptors since their effects were prevented by a 5-HT 2C antagonist [START_REF] Burghardt | Acute selective serotonin reuptake inhibitors increase conditioned fear expression: blockade with a 5-HT(2C) receptor antagonist[END_REF][START_REF] Dekeyne | Citalopram reduces social interaction in rats by activation of serotonin (5-HT)(2C) receptors[END_REF][START_REF] Greenwood | Anxiety-like behaviors produced by acute fluoxetine administration in male Fischer 344 rats are prevented by prior exercise[END_REF].

Hence, it can be suggested that an enhanced activation of 5-HT 2C , but also of 5-HT 7 , receptors might be a mechanism for the anxiogenic effects of SSRIs observed initially during treatment, based on the present results. These findings are of particular interest since several atypical antipsychotics, such as amisulpride and aripiprazole, which are potent 5-HT 7 antagonist, are used in the treatment of mood disorders [START_REF] Smeraldi | Amisulpride versus fluoxetine in patients with dysthymia or major depression in partial remission: a double-blind, comparative study[END_REF][START_REF] Montgomery | Dopaminergic deficit and the role of amisulpride in the treatment of mood disorders[END_REF][START_REF] Na | Novel quinazolinone derivatives as 5-HT7 receptor ligands[END_REF][START_REF] Berman | Aripiprazole augmentation in major depressive disorder: a double-blind, placebo-controlled study in patients with inadequate response to antidepressants[END_REF]. Additionally, [START_REF] Abbas | Amisulpride is a potent 5-HT7 antagonist: relevance for antidepressant actions in vivo[END_REF] demonstrated that, in contrast to their wild-type littermates, 5-HT 7 receptor knockout mice did not respond to amisulpride in the FST and the tail suspension test. This indicates that 5-HT 7 receptor antagonism may underly, at least in part, the antidepressantlike actions of antipsychotics such as amisulpride and aripiprazole [START_REF] Abbas | Amisulpride is a potent 5-HT7 antagonist: relevance for antidepressant actions in vivo[END_REF][START_REF] Sarkisyan | The 5-HT(7) receptor as a mediator and modulator of antidepressant-like behavior[END_REF]. Taken together, these data suggest that 5-HT 7 receptor antagonists are of potential interest for the treatment of both depression and anxiety.

Another acute action of 5-HT 7 receptor manipulation was tested in the 5-HTPinduced HTR paradigm. The increase of monoamine contents in brain, especially 5-HT, is known to cause several abnormal behaviors in animals, such as HTR and head-weaving [START_REF] Kim | NMDA receptor antagonists enhance 5-HT2 receptor-mediated behavior, head-twitch response, in mice[END_REF]. It is also reported that HTR is induced by 5-HT 2A receptor stimulation in the prefrontal cortex [START_REF] Willins | Direct injection of 5-HT2A receptor agonists into the medial prefrontal cortex produces a head-twitch response in rats[END_REF][START_REF] González-Maeso | Transcriptome fingerprints distinguish hallucinogenic and nonhallucinogenic 5-hydroxytryptamine 2A receptor agonist effects in mouse somatosensory cortex[END_REF][START_REF] Nakagawasai | Monoamine oxidase and head-twitch response in mice. Mechanisms of alpha-methylated substrate derivatives[END_REF][START_REF] Moya | Functional selectivity of hallucinogenic phenethylamine and phenylisopropylamine derivatives at human 5-hydroxytryptamine (5-HT)2A and 5-HT2C receptors[END_REF]. It was documented for the first time that 5-HT 7 receptor stimulation is also able to produce HTR (Fig. 2). This effect was, however, smaller in amplitude than that produced by fluoxetine, suggesting that the increase of cerebral 5-HT level induced by fluoxetine and 5-HTP activates a larger number of 5-HT receptors to induce HTR. Similarly, several studies have previously shown that ADs including SSRIs (citalopram, escitalopram and fluoxetine) and monoamine oxidase inhibitors (tranylcypromine, pargyline) potentiate the 5-HTP-induced behavioral syndrome [START_REF] Ortmann | The effects of 5-HT uptake-and MAO-inhibitors on L-5-HTP-induced excitation in rats[END_REF][START_REF] Shimomura | Backward walking induced by L-5hydroxytryptophan in mice[END_REF][START_REF] Hyttel | The pharmacological effect of citalopram residues in the (S)-(+)-enantiomer[END_REF][START_REF] Stórustovu | R-citalopram functionally antagonises escitalopram in vivo and in vitro: evidence for kinetic interaction at the serotonin transporter[END_REF][START_REF] Sánchez | R-citalopram inhibits functional and 5-HTP-evoked behavioural responses to the SSRI, escitalopram[END_REF]. Notably, the 5-HT 7 receptor antagonist SB-269970 did not modify the 5-HTP-induced HTR but prevented both the AS19-and fluoxetine-stimulating effects. Interestingly, various drugs with 5-HT 7 antagonistic activity, including atypical antipsychotics olanzapine and risperidone, antagonize HTR induced by 5-HTP or tryptamine [START_REF] Kitaichi | Effects of risperidone on phencyclidine-induced behaviors: comparison with haloperidol and ritanserin[END_REF][START_REF] Fu | Behavioral characteristics of olanzapine: an atypical neuroleptic[END_REF][START_REF] Van Oekelen | Role of 5-HT(2) receptors in the tryptamine-induced 5-HT syndrome in rats[END_REF].

The present study also showed that raphe-hippocampus 5-HT transmission can be markedly modified by 5-HT 7 receptor blockade. Indeed, acute activation of 5-HT 7 receptors by AS19 suppressed the firing activity of all 5-HT neurons recorded in DRN and SB-269970 prevented this effect (Fig. 3), suggesting that 5-HT firing activity is under negative 5-HT 7 receptor control. This uniform responsiveness of 5-HT neurons stands in contrast with the observation that 5-HT 4 receptor agonists, which have been proposed to be fast acting ADs, activate only a subpopulation of 5-HT neurons [START_REF] Lucas | Serotonin(4) (5-HT(4)) receptor agonists are putative antidepressants with a rapid onset of action[END_REF]. In addition, the present data showed that the 5-HT 7 receptor antagonists SB-269970 and DR-4365 prevented the inhibition of DRN 5-HT firing activity caused by the SSRIs paroxetine and fluvoxamine, respectively (see Figure S1 in the Supplemental Material). Such a result fits well with the potentiating action of SB-269970 on 5-HT release, the antidepressant-like behavior, and REM sleep suppression induced by the SSRI citalopram [START_REF] Bonaventure | Selective blockade of 5-hydroxytryptamine (5-HT)7 receptors enhances 5-HT transmission, antidepressant-like behavior, and rapid eye movement sleep suppression induced by citalopram in rodents[END_REF]. The control of DRN neuronal activity by 5-HT 7 receptors seems to be indirect and it has been suggested that 5-HT 7 receptors are not be localized directly on 5-HT neurons but rather on GABAergic and/or glutamatergic neurons [START_REF] Harsing | A 5-HT7 heteroreceptor-mediated inhibition of [3H]serotonin release in raphe nuclei slices of the rat: evidence for a serotonergic-glutamatergic interaction[END_REF][START_REF] Monti | The serotonin 5-HT7 receptor agonist LP-44 microinjected into the dorsal raphe nucleus suppresses REM sleep in the rat[END_REF]. On the other hand, in rats chronically treated with the SSRI fluoxetine, the selective 5-HT 7 agonist AS-19 no longer suppressed 5-HT firing activity, suggesting a desensitization of 5-HT 7 receptors. As previously shown [START_REF] Czachura | Effects of acute and chronic administration of fluoxetine on the activity of serotonergic neurons in the dorsal raphe nucleus of the rat[END_REF], a 7-day treatment with fluoxetine decreases DRN 5-HT firing activity, in agreement with previous studies using different SSRIs [START_REF] Chaput | In vivo electrophysiological evidence for the regulatory role of autoreceptors on serotonergic terminals[END_REF][START_REF] De Montigny | Modification of serotonergic neuron properties by long-term treatment with serotonin reuptake blockers[END_REF]. This decrease is attributable to an increased level of extracellular 5-HT [START_REF] Bel | Fluvoxamine preferentially increases extracellular 5hydroxytryptamine in the raphe nuclei: an in vivo microdialysis study[END_REF][START_REF] Yoshioka | Changes in the regulation 5-hydroxytryptamine release by alpha2-adrenoceptors in the rat hippocampus after long-term desipramine treatment[END_REF][START_REF] Bonaventure | Selective blockade of 5-hydroxytryptamine (5-HT)7 receptors enhances 5-HT transmission, antidepressant-like behavior, and rapid eye movement sleep suppression induced by citalopram in rodents[END_REF] that leads to a greater activation of 5-HT 1A autoreceptors [START_REF] Blier | Effects of the two antidepressant drugs mianserin and indalpine on the serotonergic system: single-cell studies in the rat[END_REF][START_REF] Chaput | In vivo electrophysiological evidence for the regulatory role of autoreceptors on serotonergic terminals[END_REF][START_REF] Poul | Early desensitization of somato-dendritic 5-HT1A autoreceptors in rats treated with fluoxetine or paroxetine[END_REF]Haddjeri et al., 1998a). A full recovery is usually reported after about 2 or 3 weeks treatment with classical SSRIs [START_REF] Chaput | In vivo electrophysiological evidence for the regulatory role of autoreceptors on serotonergic terminals[END_REF][START_REF] De Montigny | Modification of serotonergic neuron properties by long-term treatment with serotonin reuptake blockers[END_REF][START_REF] Czachura | Effects of acute and chronic administration of fluoxetine on the activity of serotonergic neurons in the dorsal raphe nucleus of the rat[END_REF], a time course consistent with their delayed therapeutic effect [START_REF] Blier | Serotonin and drug-induced therapeutic responses in major depression, obsessive-compulsive and panic disorders[END_REF][START_REF] Lanfumey | 5-HT1 receptors[END_REF]. The recovery of the firing rate is believed to be due to a desensitization of the somatodendritic 5-HT 1A autoreceptors of 5-HT neurons in DRN [START_REF] Blier | Current advances and trends in the treatment of depression[END_REF]. Importantly, 5-HT firing activity was not modified after 7 days of SB-269970 administration, but acute injection of paroxetine failed to induce a complete suppression of the 5-HT firing activity, indicating a desensitization of these 5-HT 1A autoreceptors. Hence, a reduced 5-HT 1A and/or 5-HT 7 receptor responsiveness in the DRN after 5-HT 7 receptor blockade takes place in a faster manner than most ADs. This desensitization may be secondary to an early increase of the extracellular 5-HT levels after only a 7-day treatment with SB-269970. This would be consistent with an enhancement of 5-HT transmission in projection areas such as the dorsal hippocampus after one week of SB 269970 administration. In fact, this enhancement was shown by the manifestation of an enhanced inhibitory tone on postsynaptic 5-HT 1A receptors, since the selective antagonist WAY 100,635 was able to increase CA3 pyramidal neuron activity (Fig. 4). Such an effect was reported using classical ADs after 2 to 3 weeks of treatment (Haddjeri et al., 1998b;[START_REF] Besson | Effects of the combination of mirtazapine and paroxetine on the 5-HT neurotransmission[END_REF][START_REF] El Mansari | Effects of acute and long-term administration of escitalopram and citalopram on serotonin neurotransmission: an in vivo electrophysiological study in rat brain[END_REF][START_REF] Lucas | Serotonin(4) (5-HT(4)) receptor agonists are putative antidepressants with a rapid onset of action[END_REF]. However, it was observed that fluoxetine was also effective after a one-week treatment. Hence, a sub-acute treatment with this SSRI may be sufficient to induce a disinhibition of CA3 pyramidal neurons in dorsal hippocampus, but not a recovery of 5-HT neuronal activity in DRN. Then, both desensitization of 5-HT 1A autoreceptors and increased tonus on hippocampal postsynaptic 5-HT receptors may be necessary to induce detectable AD effects.

The current findings on 5-HT 1A neurotransmission are in line with the fact that a one-week treatment with SB-269970, but not fluoxetine, promoted specifically the proliferation rate in the dentate gyrus of hippocampus (Fig. 5). This result brings additional support to the idea that 5-HT 7 receptor antagonists display a fast antidepressant-like profile. In fact, such progenic effect was previously observed after only long-term (2 to 3 weeks) treatment with various ADs including fluoxetine [START_REF] Malberg | Chronic antidepressant treatment increases neurogenesis in adult rat hippocampus[END_REF][START_REF] Santarelli | Requirement of hippocampal neurogenesis for the behavioral effects of antidepressants[END_REF][START_REF] Malberg | Cell proliferation in adult hippocampus is decreased by inescapable stress: reversal by fluoxetine treatment[END_REF]Mnie-filali et al., 2007b).

To our knowledge, only non selective 5-HT 7 receptors antagonists (such as atypical neuroleptics risperidone or clozapine) were used to assess the effects on hippocampal cell proliferation but the results were not conclusive [START_REF] Halim | Effects of chronic haloperidol and clozapine treatment on neurogenesis in the adult rat hippocampus[END_REF][START_REF] Kodama | Chronic olanzapine or fluoxetine administration increases cell proliferation in hippocampus and prefrontal cortex of adult rat[END_REF][START_REF] Schmitt | Hippocampal volume and cell proliferation after acute and chronic clozapine or haloperidol treatment[END_REF]. Also, [START_REF] Sarkisyan | The 5-HT7 receptor is involved in allocentric spatial memory information processing[END_REF] reported that the rate of cell proliferation in the dentate gyrus was identical in 5-HT 7 receptor wild-type and knock-out mice even if such mice present antidepressant-like phenotypes [START_REF] Guscott | Genetic knockout and pharmacological blockade studies of the 5-HT7 receptor suggest therapeutic potential in depression[END_REF][START_REF] Hedlund | 5-HT7 receptor inhibition and inactivation induce antidepressant-like behavior and sleep pattern[END_REF]. As it has been recently proposed [START_REF] Bessa | The mood-improving actions of antidepressants do not depend on neurogenesis but are associated with neuronal remodeling[END_REF], neuronal plasticity (dendritic remodeling and synaptic contacts) in the hippocampus, rather than neurogenesis itself, could be responsible for the latter behavioural phenotypes of 5-HT 7 mutant mice. Interestingly, the stimulation of hippocampal cell proliferation induced by SB-269970 was prevented by PCPA exposure, in line with previous studies showing a robust decline in hippocampal proliferation rate following PCPA exposure [START_REF] Banasr | Serotonin mediates oestrogen stimulation of cell proliferation in the adult dentate gyrus[END_REF][START_REF] Jha | Selective serotonin depletion does not regulate hippocampal neurogenesis in the adult rat brain: differential effects of pchlorophenylalanine and 5,7-dihydroxytryptamine[END_REF]. Hence, the present results suggest that the stimulating effect of 5-HT 7 receptor antagonism on cell proliferation in hippocampus necessitates an intact 5-HT system, and is not mediated by a direct action of SB-269970.

As previously discussed, SB-269970 produced an AD effect in the FST. Despite the fact that FST is a useful, high-throughput, and reliable test, classical ADs such as fluoxetine produce a response after only 2 days of treatment which is inconsistent with their delayed therapeutic action. It is therefore difficult to determine if SB-269970 has a more rapid onset of action in comparison to these ADs based on this test. To address this issue, we used OBX rat, which require long-term (2-3 weeks) treatment to observe an AD-like response [START_REF] Mcgrath | The effect of venlafaxine treatment on the behavioural and neurochemical changes in the olfactory bulbectomised rat[END_REF][START_REF] Song | The olfactory bulbectomised rat as a model of depression[END_REF]). An exaggeration of locomotor activity in OBX animals was observed compared to sham rats when placed in a high illuminated open-field (Fig. 6). This is consistent with previous reports and is believed to be due to a deficit of habituation to a new stressful environment [START_REF] Cairncross | The effect of psychoactive drugs on plasma corticosterone levels and behaviour in the bulbectomized rat[END_REF][START_REF] Van Riezen | Effects of psychotropic drugs on the behavior and neurochemistry of olfactory bulbectomized rats[END_REF][START_REF] Mcnish | Olfactory bulbectomy enhances sensitization of the acoustic startle reflex produced by acute or repeated stress[END_REF][START_REF] Song | The olfactory bulbectomised rat as a model of depression[END_REF][START_REF] Romeas | Simultaneous anhedonia and exaggerated locomotor activation in an animal model of depression[END_REF]. Importantly, while fluoxetine was ineffective after a 7-day treatment (as expected for an SSRI; [START_REF] Lucas | Serotonin(4) (5-HT(4)) receptor agonists are putative antidepressants with a rapid onset of action[END_REF][START_REF] Breuer | Long-term behavioral changes after cessation of chronic antidepressant treatment in olfactory bulbectomized rats[END_REF], SB-269970 reversed the OBX-induced hyperactivity. This is the first report of a rapid effect of 5-HT 7 receptor antagonist in a "chronic" behavioral model of depression. The faster effect of SB-269970 in the OBX paradigm is an important indication of the 5-HT 7 receptor antagonists as putative fast-acting ADs.

Finally, the profile of 5-HT 7 receptor antagonists was tested on neonatal AD exposure paradigm. Neonatal administration of ADs (such as clomipramine, citalopram or fluoxetine) to rodents during the early life period (from PN8 to PN21) was shown to produce a constellation of maladaptive behaviors, possibly related to anxiety and depression, that persist in adulthood [START_REF] Vogel | Animal depression model by neonatal clomipramine: reduction of shock induced aggression[END_REF][START_REF] Andersen | Differences in behavior and monoamine laterality following neonatal clomipramine treatment[END_REF][START_REF] Ansorge | Early-life blockade of the 5-HT transporter alters emotional behavior in adult mice[END_REF][START_REF] Maciag | Neonatal citalopram exposure produces lasting changes in behavior which are reversed by adult imipramine treatment[END_REF][START_REF] Popa | Lasting syndrome of depression produced by reduction in serotonin uptake during postnatal development: evidence from sleep, stress, and behavior[END_REF]. In this study, postnatal exposure to the SSRI fluoxetine significantly decreased visits to an open field center in adulthood while increasing total immobility time in the FST, in agreement with previous reports [START_REF] Ansorge | Early-life blockade of the 5-HT transporter alters emotional behavior in adult mice[END_REF][START_REF] Karpova | Long-lasting behavioural and molecular alterations induced by early postnatal fluoxetine exposure are restored by chronic fluoxetine treatment in adult mice[END_REF][START_REF] Popa | A longitudinal study of 5-HT outflow during chronic fluoxetine treatment using a new technique of chronic microdialysis in a highly emotional mouse strain[END_REF]. 5-HT 1A receptors have been proposed to play a crucial role in the mediation of such a deleterious action of SERT blockade [START_REF] Alexandre | Early life blockade of 5-hydroxytryptamine 1A receptors normalizes sleep and depression-like behavior in adult knock-out mice lacking the serotonin transporter[END_REF][START_REF] Popa | Lasting syndrome of depression produced by reduction in serotonin uptake during postnatal development: evidence from sleep, stress, and behavior[END_REF]. The use of preferential 5-HT receptor agonists in this paradigm also indicated the involvement of other 5-HT receptor subtypes. Indeed, early life stimulation of 5-HT 1B , 5-HT 2A , 5-HT 2C , and 5-HT 4 receptors induced a significant decrease in the number of visits in the open-field center (see Figure S2 in the Supplemental Material). In contrast, such behavioral disturbances were not observed after a neonatal administration of AS19 or SB-269970.

Overall, the multiple experimental approaches used herein provided important support for the hypothesis that 5-HT 7 receptor antagonists may act as AD agents with a rapid onset of action. Thus far, only one 5-HT 7 receptor antagonist has been assessed in a

  experiment. Experiments were in accordance to the European Communities Council Directives 86/609, OJ L 358,1, Dec 12, 1987, for the care and use of laboratory animals. Also, the electrophysiological experiments performed in Canada were approved by the local Animal Care Committee and were in accordance with the guidelines set by the Canadian Council for Animal Care. Drugs SB-269970 ((R)-3-(2-(2-(4-methyl-piperidin-1-yl)ethyl)-pyrrolidine-1-sulphonyl)phenol)), AS19 ((2S)-(+)-5-(1,3,5-trimethylpyrazol-4-yl)-2-(dimethylamino)tetralin), and paroxetine were obtained from TOCRIS (Illkirch, France). L-5-HTP (L-5-Hydroxytryptophan), PCPA (parachlorophenylalanine), WAY-100635 (N-{2-[4(2methoxyphenyl)-1-piperazinyl] ethyl} -N-(2-pyridinyl) cyclohexanecarboxamide trihydrochloride), 8-OH-DPAT (8-hydroxy-2-(di-n-propylamino)tetralin), and BrdU (5-Bromo-3'deoxyuridine) were purchased from Sigma Aldrich (St-Quentin Fallavier, France). Fluoxetine was obtained from LKT Laboratories Inc. (St Paul, MN, USA). Open field test Open field was performed in a white wooden chamber (100×100×40 cm). Testing took place under bright ambient light conditions (400 lux) to increase the anxiety component of the center area of the open field. Rats were intraperitoneally injected with vehicle (water) (vehicle and SB-269970 groups) or fluoxetine (10 mg/kg) (fluoxetine and fluoxetine+SB-269970 groups) 1 hour prior to the test and then with vehicle (vehicle and fluoxetine groups) or SB-269970 (2 mg/kg) (SB-269970 and fluoxetine+SB-269970 groups) thirty minutes later. Each rat was then placed in a corner of the open field and allowed to explore freely for 5 min. Exploratory activity was monitored by a video camera fixed above the arena and relayed to a computer system. Anxiety-like behaviour was assessed as the number of visits to the center area of the open field (defined as the central of 60 × 60 cm portion) over a 5 min period.

  of 5-HT 7 receptor antagonist in the open-field test and the FST In a first set of experiments, the illuminated open-field test was used to assess the effect of 5-HT 7 receptor blockade on the anxiogenic-like effect of fluoxetine. Analysis of data illustrated in figure 1 revealed a significant effect of treatment (F 3,28 =4.87, p<0.01, one-way ANOVA). When compared to vehicle-treated animals, acute administration of fluoxetine induced a 80% decrease in the number of visits to the center of the open-field apparatus (p<0.01, PLSD Fisher test), whereas no significant change was observed after acute administration with SB-269970. Interestingly, administration of SB-269970 after fluoxetine counteracted the decrease of the number of visits to the center induced by fluoxetine (p<0.05, PLSD Fisher test). These results demonstrated that fluoxetine, acutely administered, induced an anxiogenic-like effect that was prevented by the 5-HT 7 receptor antagonist SB-269970. In a second set of experiments, the AD potential of SB-269970

Effect of 5 -

 5 Fig 3C). Typical examples showing the reduction of the spontaneous firing activity of 5-

  locomotor activity when placed in the open field in comparison to the sham-operated animals (p<0.001, PLSD-Fisher test). Interestingly, a one-week treatment with SB-269970 (2 mg/kg/day, i.p.) significantly reduced this hyperlocomotion (p<0.05, PLSD-Fisher test). In contrast, the OBX-fluoxetine treated rats (7 days, 10 mg/kg/day, i.p.) still exhibited a strong hyperactivity compared to the sham-operated animals [+121%; p<0.05; PLSD-Fisher test performed after a global one-way ANOVA (F 2,15 =5.84; p<0.05), Fig. 6B]. Only a three-week treatment with fluoxetine was able to reverse the OBX-induced hyperactivity [p<0.001; PLSD-Fisher test performed after a global one-way ANOVA (F 2,15 =11.25; p<0.01), Fig. 6C)]. This suggests that SB-269970 had a faster antidepressant-like action than fluoxetine.

Figure 1 .

 1 Figure 1. Effect of the selective 5-HT 7 antagonist SB-269970 (2 mg/kg, i.p.) and of the

Figure 2 .

 2 Figure 2. Number of head-twitches elicited in 10 min by SB-269970 (2 mg/kg, s.c.)

Figure 3 .

 3 Figure 3. Examples of integrated firing histograms of dorsal raphe 5-HT neurons

Figure 4 .

 4 Figure 4. A: Histograms representing the spontaneous firing rate of dorsal raphe 5-HT

Figure 5 .

 5 Figure 5. Effect of SB-269970 (2 mg/kg/day, 7 days) and fluoxetine (10 mg/kg/day, 7

Figure 6 .

 6 Figure 6. Effect of SB-269970 (2 mg/kg/day i.p., 7 days) (A) and fluoxetine (10

Figure 7 .

 7 Figure 7. Effect of early life repeated administrations of fluoxetine, SB-269970 and

Acknowledgements

The authors wish to acknowledge the kindness and the helpful advices of Dr. Guy Debonnel, deceased November 04, 2006. The technical assistance from Wilfried Lebon and Davy Vidon-Buthion was greatly appreciated. This research was supported by Claude Bernard University-Lyon 1 and the CNRS.

Merck, Labopharm, Servier and Eli Lilly and Dr. Haddjeri has received research grants from Lundbeck and Solvay. The others authors declare no conflict of interest.


M.D. Raffaella Santi
email: raffaella.santi@yahoo.it

Valentina Cetica

Alessandro Franchi

Monica Pepi

Anna Maria Cesinaro

Clelia Miracco

Milena Paglierani

Vincenzo De Giorgi

Chiara Delfino

Elisa Margherita Difonzo

Nicola Pimpinelli

Simonetta Bianchi

Iacopo Sardi

Marco Santucci

Daniela Massi

TUMOR SUPPRESSOR GENE TP53 MUTATIONS IN ATYPICAL VASCULAR LESIONS OF THE BREAST SKIN FOLLOWING RADIOTHERAPY

Keywords: Atypical vascular lesions, Angiosarcoma, Radiation therapy, TP53, Atypical vascular lesions, angiosarcoma, radiation therapy, TP53; breast skin pt, Not Italic, pt, Not Italic, pt, Not Italic, pt, Not Italic pt, Not Italic, pt, Not Italic, 12 pt, Not Italic, Auto, French (Canada Auto, French (Canada Auto, French (Canada

published or not. The documents may come

MATERIALS AND METHODS

Selection of cases

Twenty patients classified as either radiation-associated cutaneous AVL (12 cases) or AS (8 cases) were identified from the files of the Division of Pathological Anatomy, F o r P e e r R e v i e w 7 (CC1). Endogenous biotin was blocked with the relevant kit. Tissue sections were incubated with the anti-human p53 antibody for 32 minutes at 37°C in BenchMark® XT autostainer and revealed with the iVIEW DAB detection kit, yielding a brown reaction product. After the staining run was complete, the slides were removed from the autostainer, the sections lightly counterstained with Mayer's haematoxylin, dehydrated, and mounted with permanent mounting medium. As negative control, the primary antibody was substituted with a Ventana dispenser, filled with a non-immune mouse serum at the same concentration.

Microdissection

One paraffin block was selected to represent the tumour from each patient. Six to ten serial sections (4 µm thickness) were obtained from the paraffin-embedded tissue blocks. The first and last sections were stained with haematoxylin and eosin to confirm that the intervening blank slides contained the lesional material. Tissue fragments were microdissected using stereomicroscopic visualisation from blank, deparaffinised slides. Areas that contained large amounts of blood, necrosis, or contaminating normal tissue were avoided in microdissection. Post-microdissected slides were evaluated to confirm accurate sample microdissection.

TP53 mutation analysis

TP53 mutation analysis was performed in 12 AVL and 8 AS. DNA was extracted from microdissected paraffin-embedded tissues, using a manual protocol 17 .

PCR primers for exons 4-9, encoding the DNA binding domain of TP53 gene, were designed to obtain fragments of no more than 300 bp. Amplification reactions were performed with 60 ng of DNA, 10 ng of each primer, 200 µM dNTPs, 1x PCR reaction

In silico analysis

Unknown mutations were tested by bioinformatics. We used two dedicated softwares: SIFT (Sorting Intolerant From Tolerant: http://blocks.fhcrc.org/sift/SIFT.html) and POLYPHEN (prediction of functional effect of human nsSNPs: http://genetics.bwh.harvard.edu/pph/).

RESULTS

Clinical findings

Clinical data are summarised in Table 1. Patients with AVL were all female, ranging from 47 to 69 years of age. Eleven patients underwent conservative surgery for breast carcinoma. In an additional case, a radical bilateral mastectomy was performed for metachronous bilateral breast carcinoma. All patients received postoperative external radiotherapy with doses varying from 44 to 60 Gy. Lesions were clinically described as solitary or multiple erythematous, skin-coloured or transparent papules, vesicles or discrete nodules/plaques, appearing within the prior irradiation field with a latency interval from 19 to 192 months after radiotherapy (mean 80.7, median 73). No oedema or ecchymoses were described. Other skin lesions at the site of radiotherapy have not been detected. In ten cases, lesions were completely removed by local excision, whereas in two cases an incision biopsy was performed. Patients with AS were all female, ranging from 53 to 90 years of age. They all had an antecedent history of radiotherapy following conservative/radical surgery for ipsilateral breast carcinoma. Most of the lesions were described as multiple purpleerythematous plaques or nodules on mammary irradiated skin. One patient had a purple-erythematous ring-shaped patch, whereas in another case AS presented as a diffuse breast oedema (peau d'orange). The latent period between radiation therapy for breast carcinoma and the diagnosis of AS ranged from 72 to 168 months (mean 115.2, median 96) after radiotherapy for the five patients whose anamnestic data were available. Patients were treated with radical mastectomy in four cases; in three patients initial treatment consisted in incomplete excision. Follow-up data were available in 7 patients. Four are alive 12 to 41 months after diagnosis (mean 25.2, median 24). Three patients died of metastatic breast carcinoma from 12 to 16 months after the diagnosis of cutaneous AS.

Histopathological findings

At low power, AVL appeared as relatively well-circumscribed, wedge-shaped proliferations of markedly dilated irregular vascular spaces located in the dermis (Figures 1,2). Vascular proliferations often projected the overlying normal epidermis upwards, thus explaining the elevated clinical appearance of the lesion (Figure 3). In other cases, lesions were flat, with a plaque-like morphology. Vascular spaces were lined by a single layer of endothelial cells with plump-oval or flattened nuclei (Figure 4A-B). Endothelial cells occasionally protruded into the lumen in a hobnail pattern 4C). Numerous small projections of stroma covered by a single layer of endothelium were a constant finding (Figures 4D). The papillae were either attached to the internal surface of the vessels or apparently lying free in the lumen. Other lesions descended into the deep dermis. Vascular spaces became smaller and their lumina more irregular, angulated or slit-like. Dissection of dermal collagen was only focally and rarely encountered. Mitoses were found in one case. Lumina were empty or filled with a small amount of proteinaceous material. Erythrocytes were seen within vascular spaces in a small number of lesions, whereas extravasated red blood cells or "blood lakes" were not observed. The stroma contained a lymphohistiocytic infiltrate with a variable number of plasma cells that varied from mild to dense.

AS were multifocal or diffuse vascular proliferations showing infiltration into the subcutis. Some cases also showed infiltration into the underlying breast parenchyma.

Tumours were all high-grade and were composed of solid aggregates of epithelioid or spindle-cells with slit-like spaces containing intraluminal and extravasated red blood cells. In all cases, tumour cells displayed prominent nuclei, brisk mitotic activity and an infiltrative growth pattern with prominent dissection between dermal collagen bundles. In three AS, areas consistent with AVL were observed at the periphery of the malignant tumour (Figures 56).

Immunohistochemical findings

p53 immunostaining was evaluated in 10/12 AVL, with variable expression and heterogeneous distribution within single cases (Table 2, Figure 7). In 4/10 (40%) cases p53 expression was detected in less than 20% of positive cells, whereas in another 4/10 (40%) cases, p53 was expressed by a variable percentage between 20% and 50% of cells. In one case (10%), no p53 stained cell was detectable, whereas in another case (10%), the percentage of stained cells was more than 50%. p53 protein was expressed in 7/8 (87.5%) AS. In four cases (50%) immunostaining was less than 20% of the neoplastic cells. In two cases (25%), the percentage of positive cells was comprised between 20% and 50%, whereas in one case (12.5%) positive cells constituted more than 50% of the lesion.

AS with peripheral AVL, resembling areas showed no difference in p53 protein expression.

TP53 mutation analysis

Overall, we identified some TP53 variation in 10/12 (83.3%) AVL and in 7/8 (87.5%) AS. Detailed results of TP53 mutational analysis are summarised in Table 2.

The most common variation in both categories was the P72R polymorphism in exon 4, found in 6/12 (50%) AVL and in 5/8 (62.5%) AS. The F54L variation in exon 4 was detected in 5/12 (42%) AVL but in none of the AS. Interestingly, one AS sample carried a pathogenetically relevant disruptive mutation c.592delG, a frameshift deletion in exon 6, causing a premature stop codon (Figure 8). The other unknown mutations were analysed in silico with two different bioinformatic tools, and seemed to be non-damaging (non-disruptive) mutations, except for K319N in exon 9, which was found in 2/12 (16.6%) AVL and 2/8 (25%) AS (Table 3).

DISCUSSION

TP53 mutation analysis has never previously been applied to the evaluation of AVL molecular profiles. In this study, we found TP53 variations in the majority (83%)

F o r P e e r R e v i e w 12
of AVL and in a similar percentage (87.5%) of post-radiation cutaneous AS. The most common variation in both categories was the P72R polymorphism in both AVL and AS, and the F54L variation in exon 4 detected in AVL. Interestingly, in one angiosarcoma sample, but in none of the AVL samples, we found a pathogenetically relevant disruptive mutation c.592delG in exon 6, a frameshift deletion causing a premature stop codon, resulting in a truncated p53 protein. Although the recognition of AVL as a morphological precursor to AS is still a controversial issue, the common mutational pathway suggested by our data provides preliminary results in support of this hypothesis, and also information relevant to the pathogenesis of atypical and malignant vascular proliferations of irradiated breast skin. It is conceivable that the TP53 mutations occur early in the tumorigenesis of these atypical vascular proliferations, and may be responsible in part for the initiation or potentiation of endothelial cell growth. Subsequently, the angiosarcoma lesions could be exposed to additional stimuli, giving rise to the frankly malignant phenotype.

The TP53 proline 72 arginine (P72R) polymorphism was a common mutation harboured in AVL and AS categories. TP53 mutations causing a malfunction of the p53 pathway are frequently encountered in many different types of human cancers 19 .

The core domain of p53 is highly conserved and confers DNA-binding specificity to the protein. Variations at this site make up 98% of the transforming mutations in p53 18 . Conversely, polymorphic sequence variations are expected to have modest or no cancer-related biological effect 19 . The TP53 codon 72 has been extensively studied because it affects the gene coding sequence, generating polymorphic variants with different biochemical and biological activities. The P72R variant has been associated with cancer susceptibility and worse prognosis 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 . In addition, codon 72 polymorphism has been shown to modulate TP53- Nevertheless, meta-analyses based on several studies investigating the susceptibility to lung and cervical cancer found no association between TP53 codon 72 polymorphism and cancer risk 30, 31 . Methodological issues could have contributed to heterogeneity among previously reported results 30, 31 .

In agreement with previous studies, we found that p53 immunohistochemistry did not always correlate with the mutational status of the TP53 gene 32 . p53 immunohistochemistry in AVL and AS revealed similar staining characteristics in both pattern and quantity. This further supports the biological and molecular similarities between these apparently distinct entities. Nuclear p53 immunostaining was variable in cases harbouring TP53 mutations, suggesting that TP53 mutations may be only one of the factors resulting in an altered p53 signal system. Very little has been done at the molecular level to increase our understanding of the genetic alterations involved in malignant transformation of endothelial cells.

Transgenic mice that were deficient in p53 demonstrated a relatively high incidence (23%) of cutaneous AS, suggesting that alterations in the p53 gene may be pathogenetically important 10 . In human tissues, Naka et al 13 analysed p53 mutations in paraffin-embedded specimens from 33 patients with AS by PCR single-stranded polymorphism, followed by direct sequencing, and found 17 cases with p53 mutations, concluding that p53 mutations are a major pathway for the development of human AS. At the protein level, overexpression of p53 and MDM-2 protein has been found to correlate with the increased VEGF expression that is found in nearly 80% of AS 33 . Another study also supported the hypothesis that VEGF and the VEGF receptor system play a role in proliferation and growth of AS by showing that tumour AVL described in the current study displayed a fully benign clinical course, in agreement with the vast majority of cases so far described. 1-5, 7-8, 35 Histopathologically, our cases met the diagnostic criteria proposed by Fineberg and Rosen 1 . In 1999, Diaz-Cascajo et al. first proposed the term "benign lymphangiomatous papules (BLAP)" on the basis of the clinical and histological findings together with the absence of recurrences or distant metastases in their series and in previously reported cases 2 . Requena et al suggested the consideration of BLAP separated from more atypical vascular proliferations resembling benign lymphangioendothelioma (acquired progressive lymphangioma), patch-stage Kaposi's sarcoma, and well-differentiated AS 8 . In 2008, Patton et al 5 reinforced the observation and subclassified AVL in to lymphatic type (LT AVL) and vascular type (VT AVL). LT AVL were described as circumscribed proliferations of ectatic thinwalled vessels confined to the dermis; the vessels were often back to back and showed hobnail endothelial cells that protruded into the lumina 5 . In contrast, VT AVL showed round to linear capillary vessels randomly arranged throughout the dermis, and 40% of these lesions displayed significant cytological atypia 5 . Interestingly, VT AVLs were considered to have a higher risk of association with AS than with LT AVL 5 . In our series, 2 cases could be classified as LT AVL, and 3 VT AVL cases displayed a vascular pattern, but in the majority of cases features of both patterns were almost equally represented, in support of the AVL morphological heterogeneity.

The morphological profile of AVL showing progression to AS is yet to be clearly defined. In previous studies, AVL showing progression to AS were described showing prominent nucleoli, occasional infiltration of the subcutis, mitotic figures, and moderate to significant cytological atypia 3, 4, 5 . As previously emphasised 5 , we agree that histopathology in the context of post-radiation vascular proliferation of the breast skin should be interpreted with extreme caution, especially when specimens represent only partial sampling of the lesion. Clinical features may be diagnostically helpful since all the patients who experienced an adverse outcome after an initial diagnosis of AVL were older, had a shorter interval from radiations as compared to those who showed no recurrence or progression, and were associated with erythema, induration, inflammatory changes, discolouration and telangiectasias 4 . In any event, these latter morphological observations, coupled with our preliminary findings, suggest an initial common mutational pathway involving p53 alterations, which favours the hypothesis that indeed AVL and AS may represent a morphologic continuum, implying that AVL could, at least in some cases, be the actual precursor of AS or incipient AS. However, the risk of progression to malignancy is likely to be quite small given the limited number of AVL showing progression to AS described so far.

In conclusion, as this is the first report on the involvement of p53 in AVL, replication of these findings in other studies is encouraged. Advances in the search for molecular changes of radiation-induced atypical vascular proliferations may increase our understanding of the relationship between AVL and AS, and lead to improved diagnosis and treatment choices for affected patients. ... [START_REF] Fineberg | Cutaneous angiosarcoma and atypical vascular lesions of the skin and breast after radiation therapy for breast carcinoma[END_REF] ... [START_REF] Cho | Crystal structure of a p53 tumor suppressor-DNA complex: understanding tumorigenic mutations[END_REF] ... [START_REF] Donehower | Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours[END_REF] ... [26] ... [START_REF] Hashimoto | Expression of vascular endothelial growth factor and its receptor mRNA in angiosarcoma[END_REF] ... [START_REF] Patton | Atypical vascular lesions after surgery and radiation of the breast: a clinicopathologic study of 32 cases analyzing histologic heterogeneity and association with angiosarcoma[END_REF] ... [20] ... [START_REF] Naka | Mutations of p53 tumor-suppressor gene in angiosarcoma[END_REF] ... [START_REF] Boltze | Homozygous proline at codon 72 of p53 as a potential risk factor favoring the development of undifferentiated thyroid carcinoma[END_REF] ... [START_REF] Soini | p53 mutations in primary hepatic angiosarcomas not associated with vinyl chloride exposure[END_REF] ... [START_REF] Uchin | Radiotherapy-associated atypical vascular lesions of the breast[END_REF] ... [START_REF] Toyama | Association of TP53 codon 72 polymorphism and the outcome of adjuvant therapy in breast cancer patients[END_REF] ... [START_REF] Requena | Benign vascular proliferations in irradiated skin[END_REF] ... [START_REF] Klug | TP53 codon 72 polymorphism and cervical cancer: a pooled analysis of individual data from 49 studies[END_REF] ...

... [START_REF] Rosso | Acquired progressive lymphangioma of the skin following radiotherapy for breast carcinoma[END_REF] ... [START_REF] Matakidou | TP53 polymorphisms and lung cancer risk: a systematic review and meta-analysis[END_REF] ... [START_REF] Coombs | Optimisation of DNA and RNA extraction from archival formalin-fixed tissue[END_REF] ... [START_REF] Taylor | Immunohistochemical detection of p53 protein accumulation in head and neck cancer: correlation with p53 gene alterations[END_REF] ... [25] ... [START_REF] Hollstein | p53 mutations at A:T base pairs in angiosarcomas of vinyl chloride-exposed factory workers[END_REF] .

  collecting evidence in support of a molecular link between AVL and AS.

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9 Follow

 9 -up data were available for all AVL patients. None of the patients with AVL developed an AS or recurrent lesions or metastases, and they all remained well 3 to 146 months after diagnosis (mean 42.6, median 34).

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  No underline, Font color: Auto Formatted: No underline, Font color: Auto Formatted: No underline, Font color: Auto, Not Highlight Formatted: No underline, Font color: Auto Deleted: in part and therefore, modify sensitivity to chemotherapeutic agents 29 .

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  haemangiosarcoma cell line ISO-HAS contain a p53 mutation and express the mRNA of VEGF and its 2 receptors, flt-1 and KDR 34 .

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  A, Thomas M, Kalita A, et al. Role of a p53 polymorphism in the development of human papillomavirus-associated cancer. Nature 1998;393:229-34. 21. Sourvinos G, Rizos E, Spandidos DA. p53 Codon 72 polymorphism is linked to the development and not the progression of benign and malignant laryngeal tumours. Oral Oncol 2001;37:572-8. 22. Cortezzi SS, Provazzi PJ, Sobrinho JS, et al. Analysis of human papillomavirus prevalence and TP53 polymorphism in head and neck squamous cell carcinomas. Cancer Genet Cytogenet 2004;150:44-9. 23. Siddique MM, Balram C, Fiszer-Maliszewska L, et al. Evidence for selective expression of the p53 codon 72 polymorphism implications in cancer development. Cancer Epidemiol Biomarkers Prev 2005;14:2245-52. 24. Nelson HH, Wilkojmen M, Marsit CJ, Kelsey KT. TP53 mutation, allelism and survival in non-small cell lung cancer. Carcinogenesis 2005;26:1770-3. 25. Bond GL, Levine AJ. A single nucleotide polymorphism in the p53 pathway interacts with gender, environmental stresses and tumor genetics to influence cancer in humans. Oncogene 2007;26:1317-23.

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  No underline, Font color: Auto Formatted: No underline, Font color: Auto Formatted: Not Highlight Formatted: No underline, Font color: Auto, Italian (Italy) Formatted: No underline, Font color: Auto Formatted: No underline, Font color: Auto, French (Canada) Formatted: No underline, Font color: Auto Formatted: Not Highlight Formatted: No underline, Font color: Auto MC, Jost CA, Brooks LA, et al. A common polymorphism acts as an intragenic modifier of mutant p53 behaviour. Nat Genet 2000;25:47-54. C, Rössle M, Haas C, et al. MDM-2 oncoprotein overexpression, p53 gene mutation, and VEGF up-regulation in angiosarcomas. Am J Pathol 1998;153:1425-33. 34. Amo Y, Masuzawa M, Hamada Y, Katsuoka K. Expression of vascular endothelial growth factor in a human hemangiosarcoma cell line (ISO-HAS). Arch Dermatol Res 2001;293:296-301. 35. Gengler C, Coindre JM, Leroux A, et al. Vascular proliferations of the skin after radiation therapy for breast cancer: clinicopathologic analysis of a series in favor of a benign process: a study from the French Sarcoma Group. Cancer 2007;109:1584-98.

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Figure 1 Atypical

 1 Figure 1 Atypical vascular lesion showing dilated vascular spaces in the upper dermis surrounded by a moderate chronic inflammatory infiltrate (original magnification x20).

Figure 2 Atypical

 2 Figure 2 Atypical vascular lesion characterised by more irregular, anastomosing, markedlydilated vascular spaces in the superficial dermis (original magnification x10).

Figure 3 Atypical

 3 Figure 3 Atypical vascular lesion, vascular channels proliferation in the upper dermis results in an upward projection of the overlying epidermis clinically featuring an exophytic papule (original magnification x10).

Figure 4 Atypical

 4 Figure 4 Atypical vascular lesion, A) vascular spaces are lined by a single layer of endothelial cells with plump-oval or flattened nuclei; B) empty vascular lumens, surrounded by a brisk mononuclear inflammatory infiltrate; C) endothelial cells occasionally protrude into the lumen in a hobnail pattern; D) small stromal projections covered by a single layer of endothelium are detected within the vascular lumen (original magnification x40).

Figure 5 Formatted:Formatted 29 AFigure 6 A

 5296 Figure 5

Figure 7 Immunohistochemical

 7 Figure 7 Immunohistochemical p53 staining pattern in endothelial cell nuclei of atypical vascular lesions (A-B, upper row) and angiosarcomas (C-D, upper row) is heterogeneous even in the same lesion (original magnification x40).

Formatted 30 Figure 8 Figure 8

 3088 Figure 8 Identification of the c.592delG mutation in exon 6 of the TP53 gene by direct DNA sequencing in one angiosarcoma sample.

TABLE 1

 1 Clinical features of the 12 atypical vascular lesions and 8 angiosarcomas developed in the field of irradiation of breast skin

	Age/	Primary	Surgery	Latency	Clinical presentation	Treatment	Follow-Up (months)
	Sex	disease		interval*
				(months)
	67/F	Bilateral BC	Radical, bilateral	192	Multiple papules and vesicles	CE. Other lesions	Alive NRM, (29)
					with clear fluid on the left breast	are still present.
	64/F	Ipsilateral BC	Conservative	96	Single erythematous nodule	CE	Alive NRM, (45)
	47/F	Ipsilateral BC	Conservative	19	Single erythematous plaque	CE	Alive NRM, (57)
	69/F	Ipsilateral BC	Conservative	74	Erythematous papule and	CE	Alive NRM, (18)
					nodule
	49/F	Ipsilateral BC	Conservative	75	Erythematous plaque/nodule	CE	Alive NRM, (15)
	58/F	Ipsilateral BC	Conservative	141	Nodular lesion	IB	Alive NRM, (15)
	63/F	Ipsilateral BC	Conservative	60	Erythematous papule	IB	Alive NRM, (64)
	66/F	Ipsilateral BC	Conservative	48	Erythematous papule	CE	Alive NRM, (74)
	61/F	Ipsilateral BC	Conservative	57	Nodular lesion	CE	Alive NRM, (6)
	65/F	Ipsilateral BC	Conservative	72	Erythematous papule	CE	Alive NRM, (3)
	47/F	Ipsilateral BC	Conservative	96	Erythematous itchy nodular	CE	Alive NRM, (39)
					lesion
	56/F	Ipsilateral BC	Conservative	39	Whitish nodular lesion	CE	Alive NRM, (146)
	55/F	Ipsilateral BC	Conservative	96	Multiple erythematous plaques	RS	NA
	76/F	Ipsilateral BC	Conservative	144	Multiple erythematous plaques	RS	Alive (36)
	81/F	Ipsilateral BC	Conservative	168	Erythematous ring-shaped patch RS	Alive (41)
	80/F	Ipsilateral BC	Conservative	NA	Erythematous plaque/nodular	Incomplete excision Dead of metastatic BC (16)
					lesion
	79/F	Ipsilateral BC	Conservative	96	Erythematous plaque/nodular	RS	Dead of metastatic BC (12)

TABLE 3

 3 In silico analysis of TP53 mutations by two different bioinformatic tools

	.. [33]

Published on behalf of the British Division of the International Academy of Pathology Histopathology

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