David Delotterie

Geoffrey Ruiz

Jacques Brocard

Annie Schweitzer

Corinne Roucard

Yann Roche

Marie-Franç Oise Suaud-Chagny

Karine Bressand

Annie Andrieux
email: <annie.andrieux@ujf-grenoble.fr

Synapcell Sas

Walsh D Fa

Ekelund J , Paunio T , Lonnqvist J , Peltonen L Kendler Ks

O Donovan ' Mc

Owen Mj

Maier W Wildenauer Db

Blouin Jl

Antonarakis Se

Mowry Bj

Silverman Jm

Crowe Rr

Cloninger Cr

Chronic administration of atypical antipsychotics improves behavioral and synaptic defects of STOP null mice

Keywords: Animals, Antipsychotic Agents, pharmacology, Behavior, Animal, drug effects, Clozapine, pharmacology, Drug Evaluation, Preclinical, methods, Hippocampus, drug effects, metabolism, Male, Mice, Mice, Knockout, Microtubule-Associated Proteins, genetics, Neuronal Plasticity, drug effects, Risperidone, pharmacology schizophrenia, STOP-null mice, cognitive defects, social investigation, antipsychotics '

Recent studies have suggested that schizophrenia is associated with alterations in the synaptic connectivity involving cytoskeletal proteins. The microtubule-associated protein STOP (Stable Tubule Only Polypeptide) plays a key-role in neuronal architecture and synaptic plasticity and it has been demonstrated that STOP gene deletion in mice leads to a phenotype mimicking aspects of positive and negative symptoms and cognitive deficits classically observed in schizophrenic patients. In STOP-null mice, behavioral defects are associated with synaptic plasticity abnormalities including defects in long-term potentiation. In these mice, long-term administration of typical antipsychotics has been shown to partially alleviate behavioral defects but, as in humans, such a treatment was poorly active on deficits related to negative symptoms and cognitive impairments. Here, we assessed the effects of risperidone and clozapine, two atypical antipsychotics, on STOP-null mice behavior and synaptic plasticity. Long-term administration of either drug results in alleviation of behavioral alterations mimicking some negative symptoms and partial amelioration of some cognitive defects in STOP-null mice. Interestingly, clozapine treatment also improves synaptic plasticity of the STOP-null animals by restoring long-term potentiation in the hippocampus. All together, the pharmacological reactivity of STOP-null mice to antipsychotics evokes the pharmacological response of humans to such drugs. Totally, our study suggests that STOP-null mice may provide a useful preclinical model to evaluate pharmacological properties of antipsychotic drugs.

Introduction

Schizophrenia is a chronic disease characterized by positive, negative or mood symptoms and cognitive deficits. Common negative symptoms include lack of motivation, anhedonia and social withdrawal. Cognitive dysfunctions encompass affected attention and perception, problem-solving, short-and long-term memory. Schizophrenia symptoms are sensitive to antipsychotics (APs) which can be classified as follows: typical APs known to be mainly efficient in improving positive symptoms ( ; Ereshefsky et al. 1990 Mueser and ), atypical AP drugs (e.g., risperidone and clozapine) known to partially act on negative symptoms and cognitive function ( McGurk 2004 ). Atypical agents have been found to increase cortical dopamine and acetylcholine release, and have some effects on the [START_REF] Meltzer | What s atypical about atypical antipsychotic drugs?[END_REF] glutamatergic system not shared by the typical agents (

). Effects on neuronal survival and plasticity, together with [START_REF] Coyle | NMDA receptor function, neuroplasticity, and the pathophysiology of schizophrenia[END_REF] decreased neurotoxicity, might also contribute to their clinical advantage over typical AP drugs (

). [START_REF] Meltzer | What s atypical about atypical antipsychotic drugs?[END_REF] However, there is consensus that none of the currently available APs completely alleviate the core symptoms and the debilitating cognitive dysfunction associated with schizophrenia ( ; ). Monitoring reactivity of the symptoms to [START_REF] Peuskens | Treatment of cognitive dysfunction in schizophrenia[END_REF][START_REF] Sergi | Social cognition in schizophrenia: relationships with neurocognition and negative symptoms[END_REF] AP treatment in a preclinical model exhibiting positive, negative and cognitive disorders may help developing drugs that better target each kind of symptoms and could ultimately lead to better outcome and quality of life for patients.

At the physiopathological level, several lines of evidence suggest that schizophrenia is associated with alterations of neuronal architecture, particularly in the synaptic connectivity ( ; ; ; ). [START_REF] Frankle | The synaptic hypothesis of schizophrenia[END_REF][START_REF] Mirnics | Analysis of complex brain disorders with gene expression microarrays: schizophrenia as a disease of the synapse[END_REF]Owen et al. 2005aOwen et al. 2005b Recently, the protein disrupted-in-schizophrenia1 (DISC1) has been demonstrated to bind to microtubules; its truncation in schizophrenic patients contributes to impair intraneuronal transport, neurite cytoarchitecture, and/or neuronal migration ( ; Duan et al. 2007 Mackie et al. ; ; ;

). Dysbindin, another protein linked to schizophrenia is also 2007 [START_REF] Matsuzaki | Molecular mechanism of schizophrenia with reference to disrupted-in-schizophrenia 1(DISC1)[END_REF][START_REF] Shinoda | DISC1 regulates neurotrophin-induced axon elongation via interaction with Grb2[END_REF][START_REF] Taya | DISC1 regulates the transport of the NUDEL/LIS1/14-3-3epsilon complex through kinesin-1[END_REF] able to bind and regulate microtubules (

). Thus, cytoskeletal alterations in general and more specifically abnormal [START_REF] Talbot | Dysbindin-1 is a synaptic and microtubular protein that binds brain snapin[END_REF] microtubule dynamics may contribute to connectivity disorders observed in schizophrenia ( ; ; Blackwood et al. 2007Camargo et al. 2008 ; ; ;

). [START_REF] Chiba | Effect of antipsychotic drugson DISC1 and dysbindin expression in mouse frontal cortex and hippocampus[END_REF][START_REF] Clapcote | Behavioral phenotypes of Disc1 missense mutations in mice[END_REF][START_REF] Hikida | Dominant-negative DISC1 transgenic mice display schizophrenia-associated phenotypes detected by measures translatable to humans[END_REF][START_REF] Hikida | Dominant-negative DISC1 transgenic mice display schizophrenia-associated phenotypes detected by measures translatable to humans[END_REF] Consistent with this hypothesis, disruption of the MAP6 gene (

) encoding a microtubule-associated protein called [START_REF] Denarier | Genomic structure and chromosomal mapping of the mouse STOP gene (Mtap6)[END_REF] STOP (Stable Tubule Only Polypeptide) in mice generated a set of defects similar to those of schizophrenia disorders (Andrieux et al.

). Correlatively, the MAP6 gene -the human homologous of STOP gene -is localized on chromosome 11q14, a region highly 2002 associated with schizoid personality disorders (

). Interestingly, an association between single nucleotide polymorphism Lewis et al. 2003 markers of the MAP6 gene and schizophrenia has been found, together with a dysregulation of the isoform2 of MAP6 transcripts in the frontal cortex of schizophrenic patients (

). [START_REF] Shimizu | Genetic and expression analyses of the STOP (MAP6) gene in schizophrenia[END_REF] The STOP-null mice, at the behavioral level, have been shown to display behavioral abnormalities thought to simulate some aspects of positive symptoms classically observed in schizophrenic patients ( ; ). These mice exhibit increased Andrieux et al. 2002Brun et al. 2005 basal locomotor activity during the dark phase of the light/dark cycle associated with purposeless and disorganized activity (Andrieux et ;

). Additionally, STOP-null mice display a hyper-sensitive locomotor reaction to acutely stressful situations, al. 2002situations, al. Brun et al. 2005 such as a single saline injection or exposure to novelty and an increased locomotor stimulatory effect of psychotomimetic drugs such as amphetamine (

). Some of these locomotor activity defects are present in juvenile mice, whereas others appear only in ). Here, we tested the effect of atypical APs, namely clozapine and risperidone, administrated Andrieux et al. 2002 chronically, on several impaired behaviors exhibited by STOP-null mice. We found that atypical AP administration in STOP-null mice resulted in noticeable amelioration of the negative symptoms and in a partial alleviation of the cognitive defects. Correlatively, we showed that clozapine treatment was able to restore hippocampal long term synaptic plasticity in STOP-null mice.

Materials and methods

Animals

Male STOP-null mice (STOP / ) and their wild-type (WT) littermates (STOP / ) were generated as previously described (

Drugs and long-term treatments

Drugs used for long-term treatments in this study were either (i) risperidone purchased from Janssen-Cilag Ltd (Risperdal , Beerse, ® Belgium), dissolved in drinking water or (ii) clozapine provided by Tocris Bioscience (Bristol, United Kingdom), resuspended with a few drops of HCl before its volume was completed with NaCl (NaCl 0,9 sterile, Physiologica) and injected daily intra-peritoneally. We % carefully estimated water consumption of WT and STOP-null mice in the presence or in the absence of risperidone and found no differences between genotypes or treatments (average value for all groups: 15 of the body weight/day). Risperidone concentrations in the % drinking water were calculated to obtain the desired delivered doses (0.1mg/kg/day and 0.3 mg/kg/day).

When mice reached 3 months, they were submitted to a long-term treatment with either antipsychotic drug or vehicle during 4 weeks.

The treatment was then pursued during a 3 to 4-weeks behavioral testing period.

3 10 Drug-free and long-term APs-treated animals (4-to 5-month-old, 25 to 30 g) were investigated during the light phase, in a random order for comparison between genotypes and treatments. Experimenter was blind to genotype during testing. In all experiments, animals were allowed to habituate to the testing room 1 h before the test. The usual sequence of tests was as follows: Y-maze test, social investigation test, marble burying test, forced swim test and electrophysiological recordings (see below). All the devices used for testing were extensively washed with water between two animals to avoid olfactive cues. The luminosity of the testing room was controlled and fixed around 30 35 Lux.

-

Y-maze Test

Spontaneous alternation behavior and exploratory activity were recorded in a Y-maze. The apparatus was made of three equal arms (dimensions: 20 cm long, 8 cm wide, 15 cm high) made of white polyvinyl chloride. The starting arm and the two arms forming the Y were radiating at an angle of 120 from each other. Each mouse was placed in the starting arm of the maze and its position recorded during °a 5-min session of free roaming by a video tracking system placed above the maze (Smart , Panlab, Harvard Apparatus). The total ® distance traveled across the maze was automatically recorded. The sequence and number of arm entries were manually counted from videotrack recordings. It was considered that a mouse had entered an arm when all four paws were positioned in the arm runway. A spontaneous alternation was defined as entries into all three arms on 3 consecutive choices. The alternation score ( ) for each mouse was % defined as the ratio of the actual number of alternations to the total possible number of alternations over the testing period: Alternation % = 100 (Number of alternations)/(Total arm entries-2) . Mice that completed less than eight arm entries within 5 min were excluded from

× [ ] further analysis ( ). [START_REF] Sakaguchi | Effects of systemic administration of beta-casomorphin-5 on learning and memory in mice[END_REF]

Social investigation test

The social behavior of STOP and WT mice was assessed towards an encaged WT NMRI male mouse (Janvier, France) used as intruder. Without any former isolation, a tested mouse was placed in an open field (50 cm long, 50 cm wide, 20 cm high) containing a wire mesh cylinder in a corner (diameter: 12 cm; height: 14 cm; mesh dimensions: 6 mm) and covered with a lid. After habituation of the animal to its new environment for 5 min, the intruder was introduced in the cylinder and the number of contacts and contact duration were measured during a 10-min session. A contact was defined as a direct sniffing and full use of vibrissae performed by the tested animal towards the intruder.

Marble burying test

The procedure for marble burying was adapted with minor modifications from that described by (

). Briefly, mice [START_REF] Millan | S33005, a novel ligand at both serotonin and norepinephrine transporters: II. Behavioral profile in comparison with venlafaxine, reboxetine, citalopram, and clomipramine[END_REF] were individually placed in transparent polysulfone cages (dimensions: 36 X 21 X 14 cm) containing a 4-cm layer of sawdust and 12 glass marbles (diameter: 1.6 cm) evenly spaced over the cage (3 rows of 4 marbles). In order to prevent escape, the cage was covered with a filtering lid. Thirty minutes later, the animals were removed from the cages and the number of marbles buried more than two-thirds in the sawdust was scored.

Forced Swim Test

Studies were carried out on mice according to the slightly modified method of Porsolt and co-workers (

). Animals [START_REF] Porsolt | Behavioral despair in mice: a primary screening test for antidepressants[END_REF] were placed individually into polypropylene beakers (height 20 cm, diameter 16 cm) containing 2.5 liters of water (12-cm high) maintained at 20 22 C. The animals were left in the beaker for 6 min. After the first 2 min, total duration of immobility was measured by a -°well-trained experimenter during a 4-min session. Visually, the mouse was judged to be immobile when it remained floating passively, performing slow motion to keep its head above the water.

Electrophysiological recordings

For preparation of hippocampal slices, adult mice (5 month-old) were deeply anesthetized with ketamine. Brain slices (300 m thick) μ were cut in a cold cutting artificial cerebrospinal fluid (ACSF) of the following composition (in mM): NaCl (0), KCl (2.5), MgCl2 (7),

" " CaCl2 (0.5), D-Glucose (10), NaHCO3 (26), NaH2PO4 (1.25), Sucrose (230). Slices were then submerged in a waiting chamber in "

recording ACSF equilibrated with 95 O2 and 5 CO2 of the following composition (in mM): NaCl (124), KCl (5), MgCl2 (2), CaCl2

" % %

(2), D-Glucose (11), NaHCO3 (26), NaH2PO4 (1.25) and maintained at room temperature for at least one hour before recording.

Recordings were done using microelectrode array (MEA) technology allowing stimulation and recording of bioelectrical activity from different areas of the slice. For recordings, slices were placed within the recording chamber of a MEA probe (64 64 planar × microelectrodes for Med64 apparatus, Panasonic) with perfusion of recording ACSF equilibrated with 95 O2 and 5 CO2 and following the tetanus and iii) less than 20 variation during the post-tetanus recording were kept for further analysis. Electrophysiological % data were plotted as a function of time and comparisons were performed between pooled data recorded 30 to 40 minutes after occurrence of the LTP protocol as previously described ( ).

Andrieux et al. 2002

Statistical analysis

All statistical analyses were performed using Prism 4.0 (GraphPad Software, La Jolla, CA) and data were represented as boxed medians with 25th and 75th percentiles and error bars corresponding to the lowest and the highest data points. For all tests, results were compared between WT and KO placebo groups first, using unpaired non-parametric Mann-Whitney tests. To assay APs effect, comparisons were then separately performed for each genotype (WT: placebo, dose 1, dose 2 and KO: placebo, dose 1, dose2) using non-parametric Kruskals-Wallis ANOVA tests followed by Dunns post-hoc comparisons.

'

Results

We monitored the effect of long-term treatments with atypical antipsychotics (APs) on STOP-null mice, using several behavioral tasks. Mice of WT and STOP KO genotypes were submitted to a chronic treatment with risperidone, clozapine or vehicle during 4 weeks.

The treatment was then pursued during the 3 to 4-weeks behavioral testing period. Since risperidone was administrated in the drinking water while clozapine was injected daily, placebo groups can not be directly compared and each treated group will be compared to the corresponding placebo.

Social investigation test

Social investigation has been addressed by measuring the number of contacts and contact duration between an immobilized intruder mouse (male NMRI) and a freely-moving mouse to be tested. Results are presented in . In drug free animals, as compared to WT, Fig. 1 STOP-null mice exhibited a reduction of social investigation affecting both the number of contacts with the intruder 9.5 vs 20 for KO and We then assessed social behavior of STOP-null mice after long-term administration of risperidone or clozapine.

In WT mice, risperidone treatment did not affect the number of contacts 20 (placebo), 20 (0.1mg/kg/day), 19.5 (0.3mg/kg/day); significant improvement of STOP-null mice treated with risperidone 0.3 mg/kg/day as compared to placebo ( < 0.01). p

In WT mice, clozapine treatment did not affect the number of contacts 12.5 (placebo), 12 (1mg/kg/day), 10.5 (3mg/kg/day); 0.924

[ p =
or time spent in investigation 30.5 sec (placebo), 28 sec (1mg/kg/day), 32 sec (3mg/kg/day); 0.909 . STOP-null mice groups also

] [ p =
] displayed significant differences in number of contacts 4.5 (placebo), 7 (1mg/kg/day), 8 (3 mg/kg/day); 0.030 and time spent in

Marble burying test

In rodents, digging of bedding and burying of objects considered as dangerous for the animal are natural tendencies that do not depend on the nature of their environment (laboratory or nature). The marble burying assay has been predominantly used for the screening of anxiolytic compounds ( ; ). [START_REF] Nicolas | A combined marble burying-locomotor activity test in mice: a practical screening test with sensitivity to different classes of anxiolytics and antidepressants[END_REF][START_REF] Young | TDIQ (5,6,7,8-tetrahydro-1,3-dioxolo 4,5-g isoquinoline) exhibits anxiolytic-like activity in a marble-burying assay in [ ] mice[END_REF] In the marble burying test, each mouse was introduced in a cage containing 12 marbles and left free to roam for 30 minutes; the number of buried marbles was then counted. Results are presented in . Fig. 2 In drug free animals, as compared to WT, STOP-null mice buried less marbles 0 vs 9 for KO and WT placebo mice in risperidone [ groups ( 0.042) and 0 vs 7 for KO and WT placebo mice in clozapine groups ( 0.017) . p

= p = ]
The ability to bury marbles was then investigated in risperidone or clozapine long-term-treated animals.

In WT mice, risperidone treatment did not affect the number of buried marbles 9 (placebo), 9 (0.1mg/kg/day), 9 (0.3mg/kg/day);

[ p = 0.887 while STOP-null mice groups showed nearly significant increases in number of buried marbles 0 (placebo), 6.5 (0.1mg/kg/day), 6

] [ (0.3mg/kg/day); 0.

. p = ]

In WT mice, clozapine treatment did not affect the number of buried marbles 7 (placebo), 7 (0.1mg/kg/day), 8 (0.3mg/kg/day); [ p = 0.920 while STOP-null mice groups showed non-significant increases in number of buried marbles 0 (placebo), 0 (0.1mg/kg/day), 5

] [ (0.3mg/kg/day); 0.329 . p = ] These results indicate that although both long-term treatments increased STOP-null mice ability to bury marbles, none did so to a significant level.

Forced swim test

Resignation/despair was assessed in a forced swim test. Mice were placed in a glass beaker containing water for 6 minutes and immobility was measured during the last 4 minutes. Results are presented in . Fig. 3 In drug free animals, as compared to WT, STOP-null mice stayed immobile longer 127.5 vs 73.5 sec for KO and WT placebo mice in [ risperidone groups (p 0.114) and 95.5 vs 49.5 sec for KO and WT placebo mice in clozapine groups (p 0.019) .

= = ]

We then assessed resignation of STOP-null mice after long-term administration of risperidone or clozapine.

Total immobility of WT animals was not modified by risperidone treatment 73.5 sec (placebo), 73 sec (0.1mg/kg/day), 68.5 sec Altogether, these results indicate increased resignation in STOP-null mice which is ameliorated by AP long-term treatments.

Y-maze test

Spatial working memory was assessed by testing the spontaneous alternation performance of mice in a Y-maze during a 5-min session of free roaming. Exploratory activity was estimated by the total distance traveled across the maze. The alternation score for each mouse was defined as the ratio of the actual number of alternations to the total possible number of alternations. Results are presented in . Fig. 4 In drug-free animals, as compared to WT, STOP-null mice displayed the same exploratory activity measured from the total distance traveled (data not shown) but displayed significantly less spontaneous alternation performance 52.5 vs 63.6 for KO and WT placebo ). Quantitative analysis showed that after clozapine treatment, STOP-null mice groups Fig. 5a exhibited significant differences in LTP values measured between 30 and 40 minutes post-tetanus 127.9 (placebo), 136.3

[ % %
(3mg/kg/day), 171.1 (10 mg/kg/day); normalized field EPSP amplitudes, 0.027 ( ). Post-hoc analysis showed significant

% p = ]

Discussion

The goal of the present work was to assay the sensitivity to a long-term atypical AP treatment, namely clozapine and risperidone, on behavioural and synaptic impairments exhibited by STOP-null mice. The present study extended the behavioural phenotype of STOP-null mice revealing that STOP (MAP6) gene deletion in mice leads to multiple behavioural abnormalities relevant to negative symptoms and cognitive impairments of schizophrenia.

In schizophrenia, negative symptoms are close to most of depression symptoms (

). Social interactions measured in Barnes et al. 1989 rodents are directly comparable to social interaction measured in humans. It has been previously described that STOP-null mice show severe social withdrawal both in aggressive and non-aggressive components (Andrieux et al.2002; ). In this study, using B gou et al. 2008 The behavioural despair of the animal, placed in an unusual and inescapable environment ( ; ) and [START_REF] Porsolt | Behavioural despair in rats: a new model sensitive to antidepressant treatments[END_REF][START_REF] Porsolt | Behavioral despair in mice: a primary screening test for antidepressants[END_REF] revealed through increased immobility in the forced swim test, could be notably due to a loss of motivation ( ; [START_REF] Noda | Effects of antidepressants on phencyclidine-induced enhancement of immobility in a forced swimming test in mice[END_REF] and so relevant to the negative symptomatology in animal models of schizophrenia. In this study, STOP-null mice spent more time 1995 floating with minimum movements than WT mice. This altered behaviour of STOP-null mice may potentially be the consequence of an intrinsic lack of motivation as already suspected in a previous study (

). Comparable deficits have been observed in B gou et al. 2008 é transgenic mice expressing dominant negative copies of DISC1, another genetic model for schizophrenia (

). Chronic [START_REF] Hikida | Dominant-negative DISC1 transgenic mice display schizophrenia-associated phenotypes detected by measures translatable to humans[END_REF] treatments with either clozapine or risperidone alleviate behavioural despair in STOP-null mice, potentially by improving their motivational ability.

Whereas marble burying is more closely related to impulsive behaviour than anxious states, this test has been predominantly used as a screening model of detection of new anxiolytic compounds ( ; ). STOP-null mice bury a substantially [START_REF] Nicolas | A combined marble burying-locomotor activity test in mice: a practical screening test with sensitivity to different classes of anxiolytics and antidepressants[END_REF][START_REF] Young | TDIQ (5,6,7,8-tetrahydro-1,3-dioxolo 4,5-g isoquinoline) exhibits anxiolytic-like activity in a marble-burying assay in [ ] mice[END_REF] lower number of marbles than WT mice. Such behaviour could rely on low anxious state or low motivation for physical environment. Low ). The poor effect of atypical APs treatment on cognitive functions in STOP-null mice is compatible with et al. 2009[START_REF] Volavka | Clozapine, olanzapine, risperidone, and haloperidol in the treatment of patients with chronic schizophrenia and schizoaffective disorder[END_REF] the effects of atypical APs in humans. Typical APs have been shown to be deleterious on cognitive abilities in humans whereas atypical APs do not induce these defects. In total, we detected the same pharmacological reactivity of typical/atypical APs on STOP-null mice behaviour. The beneficial effect of atypical APs on negative and cognitive impairments over typical drugs is thought to be linked to their pharmacological profile, atypical APs exhibiting higher affinity for 5-HT-2 receptors than typical APs ( ). [START_REF] Duncan | Mechanisms of typical and atypical antipsychotic drug action in relation to dopamine and NMDA receptor hypofunction hypotheses of schizophrenia[END_REF] A growing body of evidence involves glutamatergic transmission in the aetiology of schizophrenia and drugs directly targeting glutamatergic receptors have been shown to exhibit antipsychotic properties (

). Accordingly, STOP-null mice display [START_REF] Patil | Activation of mGlu2/3 receptors as a new approach to treat schizophrenia: a randomized Phase 2 clinical trial[END_REF] abnormal glutamatergic neurotransmission revealed by impaired long-term potentiation (

). Some beneficial effects of Andrieux et al. 2002 clozapine are thought to occur through glutamatergic transmission ( ; ) and interestingly, long-term [START_REF] Coyle | NMDA receptor function, neuroplasticity, and the pathophysiology of schizophrenia[END_REF]Tsai 2004 Frankle et al. 2003 treatment of STOP-null mice with clozapine restored LTP whereas risperidone did not. These results indicate that LTP measurements in STOP-null mice may discriminate between APs with or without glutamatergic profiles.

A translational preclinical model of human psychiatric disorder is all the more relevant that it respects construct, face and predictive validity ( ; ). As outlined in the introduction STOP-null mouse model fulfils the Ellenbroek and Cools 1990 Miczek and de Wit 2008 construct and face validity: it is constructed from the neurodevelopmental hypothesis of schizophrenia, with synaptic impairment. Indeed, an association between single nucleotide polymorphism markers of the MAP6 gene and schizophrenia has been found [START_REF] Shimizu | Genetic and expression analyses of the STOP (MAP6) gene in schizophrenia[END_REF] ) as well as a deregulation of STOP expression inprefrontal cortex of schizophrenic patients (

). STOP-null Martins- [START_REF] Martins-De-Souza | Prefrontal cortex shotgun proteome analysis reveals altered calcium homeostasis and immune system imbalance in schizophrenia[END_REF] mice have been shown to display behavioral abnormalities thought to simulate aspects of positive symptoms classically observed in schizophrenic patients. The present data, added to all previous ones, give a comprehensive phenotype of STOP null mice according to the behavioral defects related to the core symptoms of schizophrenia, namely negative symptoms and cognitive deficits, which are known to be resistant to current treatments. The pharmacological results of the present study referring to the possibility to mimic the benefit effects of APs in humans are relevant for the predictive validity of STOP-null mice. However, knowing that current atypical AP treatment in humans displayed limited effects, the predictive validity of STOP null mice need to be further supported by studies showing behavioral alterations resistant to APs. Altogether, we suggest that STOP null mice could be a very useful tool to develop AP drugs by monitoring pharmacological effects both at the behavioural and biological levels.

  at 29 30 C. Stimulation was applied on Schaeffer collaterals every 30 sec during baseline (10 min) and after tetanus (TB, 100 -°Hz, 1 sec) (60 min) and recordings of field EPSPs were made within Ammon s Horn 1. Normalized field EPSP amplitudes were calculated ' and only slices showing i) less than 10 variation during the first 10 min of recording before tetanus, ii) more than 10 variation directly % %

[

  WT placebo mice in risperidone groups ( 0.006) and 4.5 vs 12.5 for KO and WT placebo mice in clozapine groups ( 0.200) and the p = p = ] time spent in social investigation 9 vs 29 sec for KO and WT placebo mice in risperidone groups ( 0.006) and 6.5 vs 30.5 sec for KO [

  or time spent in investigation 29 sec (placebo), 33 sec (0.1mg/kg/day), 33 sec (0.3mg/kg/day); 0.316 . STOP-null mice groups in number of contacts 9.5 (placebo), 17 (0.1mg/kg/day), 23 (0.3mg/kg/day); 0.012 and time spent in [ (placebo), 18 sec (0.1mg/kg/day), 22 (0.3 mg/kg/day); 0.005 . In both sets of observations, post-hoc analyses show [

  5 sec (placebo), 14 sec (1mg/kg/day), 25.5 sec (3 mg/kg/day); 0.015 . Again, post-hoc analyses show significant [ p = ] improvement of STOP-null mice treated with clozapine 3 mg/kg/day as compared to placebo ( < 0.05). p Altogether these results indicate that long term treatment with APs risperidone or clozapine alleviate social behavioral impairment in STOP-null mice.

[

  (0.3mg/kg/day); 0.362 . STOP-null mice groups displayed significant differences in the time of immobility 127.5 sec (placebo), 93.5 p = ] [ sec (0.1mg/kg/day) and 70.5 sec (0.3mg/kg/day); 0.005 . Post-hoc analysis showed significant improvement of STOP-null mice treated p = ] with risperidone 0.3 mg/kg/day as compared to placebo (p < 0.01).Total immobility of WT animals was not modified by clozapine treatment 49.5 sec (placebo), 68 sec (1mg/kg/day), 60 sec [ (3mg/kg/day); 0.551 . STOP-null mice groups displayed significant differences in the time of immobility 95.5 sec (placebo), 88 1mg/kg/day), 52 sec (0.3mg/kg/day); 0.028 . Post-hoc analyzes showed significant improvement of STOP-null mice treated with p = ] risperidone 0.3 mg/kg/day as compared to placebo (p < 0.05).

  untreated STOP-null mice only showed slight differences in spontaneous alternation 52.5 (placebo), 64.6 1mg/kg/day), 60.7 (0.3mg/kg/day); 0.219 . These results indicate impairment of working memory in STOP-null mice and a limited mice have been shown to display severe defects in long-term synaptic plasticity with highly reduced Long-Term Potentiation (LTP) in the CA1 region of the hippocampus (). Here, we studied the effect of chronic treatment withAndrieux et al. 2002 risperidone or clozapine on LTP. In the clozapine group (daily injection of either clozapine or vehicle), STOP-null mice exhibited reduced LTP as compared to wild type 127.9 vs 165.5 (normalized field EPSP amplitudes measured between 30 and 40 minutes post-tetanus) [ % % for KO and WT placebo mice ( 0.006) ( ). Synaptic potentiation was obviously improved by clozapine treatments at early time p = ] Fig. 5b points and persisted for at least 1 hour (

  Fig.5bimprovement of STOP-null mice LTP with clozapine 10 mg/kg/day as compared to placebo (p < 0.05). Risperidone treatment did not alleviate LTP defects of STOP-null mice (data not shown).

é

  a new paradigm, we found a strong decrease in number of contacts and time spent sniffing in STOP-null mice, indicating a marked social deficit. Interestingly, both clozapine and risperidone long-term treatments strongly improved social interactions in STOP-null mice whereas typical AP long-term treatments have been shown to be less active ().B gou et al. 2008 é

  anxious state is also observed in the elevated plus maze test, as STOP-null mice spent more time in the open arms than WT animals (D. D. unpublished observations) and low motivation for physical environment has already been suggested from STOP-null mice unable to perform object recognition tests ( ). In any case, long-term treatment with either clozapine or risperidone increased the Andrieux et al. 2002 number of buried marbles, but never to significant levels. Better suited tests for anxiety versus motivation should be used in the future. It has been previously demonstrated that STOP-null mice present cognitive deficits related to the episodic ( ), Andrieux et al. 2002 social, spatial and working memories ( ). Here, we actually show that chronic treatment with risperidone does not restore B gou et al. 2008 é impairment in spatial short-term working memory of STOP-null mice in the spontaneous alternation task. Altogether these results clearly indicate that STOP-null mice exhibit strong deficits related to negative symptoms and cognitive impairments of schizophrenia. Atypical APs significantly improve deficits related to negative symptoms in STOP-null mice while typical APs only slightly alleviate them ( ). In parallel, atypical APs display a slight benefit effect on the cognitive impairments B gou et al. 2008 é in STOP-null mice, known to be resistant to typical treatment with haloperidol ( ). B gou et al. 2008 é Typical versus atypical APs sensitivity of STOP-null mice are in accordance with clinical data showing that clozapine and risperidone have a trend to a positive action on negative impairments equivalent or superior to the typical APs, particularly versus haloperidol (Leucht ;

Fig. 1 Fig. 2

 12 Fig. 1 Social investigation test. Number (a and b) and total duration (c and d) of contacts with the intruder in placebo and long-term risperidone (a and c) or clozapine (b and d) -treated WT and STOP-null mice. STOP-null mice treated with placebo present a social withdrawal when compared with their WT littermates , , : >0.05, <0.05, <0.01, respectively, KO vs WT placebo . Chronic treatment with risperidone [ns * ** p p p ] or clozapine significantly improved social behaviour in STOP-null mice , : <0.05, <0.01, respectively, between KO groups, with [# ## p p A different from , <0.01for risperidone (a and c) and <0.05 for clozapine (b and d) long-term treatments . Note that clozapine and C p p ] risperidone are not administrated the same way (injection and drinking water, respectively) preventing direct comparisons between the two groups

Fig. 3

 3 Fig. 3 Forced swim test. Total time of immobility in placebo and long-term risperidone (a) or clozapine (b) -treated WT and STOP-null mice. Immobility time was longer in STOP-null mice treated with placebo compared with their WT littermates , : >0.05, <0.05, [ns * p p respectively, KO vs WT placebo . Administration of a chronic treatment with either risperidone or clozapine produced a significant decrease ] in immobility time in STOP-null mice , : <0.05, <0.01, respectively, between KO groups, with different from , <0.01 for [# ## p p A C p risperidone (a) and <0.05 for clozapine (b) long-term treatments p ]

10

10

10

10

10

10

Ackowledgements:

We thank C. Dumont and D. Proietto for technical help.

Abbreviations


Vincent Le Cam
email: vincent.le-cam@lcpc.fr

Laurent Gaillet

Marianne Perrin

Christian Tessier'

Louis- Marie Cottineau

A smart and wireless sensors network for cable health monitoring

Keywords: Cables, diagnostic, WiFi, monitoring, acoustic

In the collection of possible non destructive techniques used for bridges, acoustic emission is a favourite non-destructive solution towards health monitoring of cables of suspended bridges. A first generation of such an acoustic system made of a supervisor and a distribution of wired sensors fixed onto bridge cables have been developed and deployed on several bridges in France. The aim of this article is to present the new generation of this acoustic system redeveloped by implementing algorithms on a distribution of smart and wireless sensors. Each sensor of the platform consists of a small electronic layer including a TCP/IP stack and an embedded µClinux Operating System. Sensors are able to detect and to record the full shape of acoustic waves due to wire breaks while they are kept synchronized by means of a GPS method.

Résumé

Parmi les différentes techniques de contrôle non destructifs possibles appliquées pour les ponts, l'émission acoustique est la solution favorite pour la surveillance des câbles. Une première génération d'un tel système acoustique composé d'un superviseur et d'une distribution de capteurs fixés sur les câbles a été développée et déployée sur plusieurs ponts en France. L'objectif de cet article est de présenter la nouvelle génération de ce système acoustique développée par l'implémentation d'algorithmes sur une distribution de capteurs sans fils et intelligents. Chaque capteur consiste en une carte électronique intégrant le protocole TCP/IP et un système d'exploitation embarqué µClinux. Ces capteurs sont capables de détecter et d'enregistrer la forme d'onde complète provenant de la rupture d'un fil et sont synchronisés par une méthode GPS.

Introduction

For safety, structure durability and cost efficiency reasons, monitoring civil engineering structures and especially cable stayed and suspension bridges is sometimes necessary. The installation of an acoustic monitoring system allows the detection and the monitoring of the degradation of civil engineering cables by the recording of acoustic events mainly associated with severe breaks of wire. It should be noted that this technique allows the testing of the whole cable and not only of the external part as in the case of a visual inspection of the structure. Acoustic emission is a usual non destructive technique used for cable monitoring which consist of detecting and localizing individual wire-breaks that could occur in cables from various types of bridges [START_REF] Fricker | Site installation and testing of a continuous acoustic monitoring[END_REF][START_REF] Shigeishi | Acoustic emission to assess and monitor the integrity of bridges[END_REF][START_REF] Watson | Acoustic emission monitoring applications for civil structures[END_REF][START_REF] Ramadan | Detection of stress corrosion cracking of high-strength steel used in prestressed concrete structures by acoustic emission technique[END_REF]. By knowing the number of wire-breaks and their localization on cables, this system delivers to structure's manager knowledge on its health [START_REF] Watson | Acoustic emission monitoring applications for civil structures[END_REF][START_REF] Woodward | Detecting fractures in steel cables[END_REF][START_REF] Cullington | continuous acoustic monitoring of grouted post-tensioned concrete bridges[END_REF][START_REF] Robert | Surveilance acoustique des câbles[END_REF] This confirmation could lead structure's managers to repair cables [START_REF] Yuyama | Detection and evaluation of failures in high-strength tendon of prestressed concrete bridges by acoustic emission[END_REF], to stop traffic etc. To reach such a diagnostic tool, specific instrumentation must be thinked, this article underlines how accurate wireless sensors must be (up to few microsecond) and how reliable wireless communications have to be. Those technical challenges that have been solved pave the ground of promising future developments in terms of wireless and smart sensors developped in LCPC laboratories.

Acoustic emission for wire-breaks detection

The principle of the system is based on the detection of an acoustic wave which is emitted on a wire break in the two opposite directions of the corresponding cable.

Figure 1. a) Wire-break occurrence in a cable b) Wave detection by sensors

This acoustic wave propagation is detected by a set of acoustic sensors along the monitored cable [START_REF] Holford | The non-destructive testing of wire ropes by acoustic emission[END_REF]. In this application, the sensitive element selected for each sensor is an accelerometer. Each sensor is assumed to be an electronic entity that embeds a processor. It is programmed to permanently digitize electric signals from the accelerometer and to compare its amplitude to a predefined threshold (an acceleration threshold defined around few 'g'). This threshold is different from zero due to the fact that there is always a minimum of acoustic activity in cables. If the amplitude of a recorded sample from the signal exceeds the threshold, this value is recorded and time stamped. Then, the sensor sends that information to a remote supervisor as shown in figure 1. The rupture localization is performed by a supervision system from sensor relative locations and occurrence times (time-stamped signals). When wire break signal is detected by 3 sensors (at least), a real wave propagation rate can be computed. Due to environmental conditions existing on bridges, some uncorrelated signals can be computed by the supervisor and criteria based on acoustic wave velocity and the number of sensors which have detected break signal are applied.

Implementation on sensors network. Toward a new instrumentation

First generation

This Structural Health Monitoring (SHM) application has first been implemented through a system based on smart and wired sensors (Motorola 68HC11 as embedded processor). The wired communication bus technology used the serial RS485 link. Several bridges of different type (suspension, prestressed, cable stayed) have been instrumented and monitored with success by such this system [START_REF] Yuyama | Detection and evaluation of failures in high-strength tendon of prestressed concrete bridges by acoustic emission[END_REF][START_REF] Holford | The non-destructive testing of wire ropes by acoustic emission[END_REF][START_REF] Taylor | The Acoustic Emission of Steel Wire Ropes[END_REF]. Even if the system delivered clear-cuts results from laboratory configurations and under real conditions, its massive deployment as well as is actual maintenance makes it expensive and subject to technological improvements.

LCPC generic wireless sensor platform

After active technological survey specific developments in Wireless Sensors Network (WSN) [START_REF] Culler; Estrin | Overview of Wireless Sensor Networks[END_REF] applications, LCPC took the decision to develop its own generic wireless platform. LCPC goal is to develop a WSN platform for which simplest (or none) hardware and software modifications can cover a large range of applications. This modularity takes form in a sensor made of a "mother board" that integrates redundant needs (computation, communication, data storage) and a pluggable "daughter board" that integrates application specific needs such as the sensitive element (accelerometer, temperature, pressure,...) or its specific conditioning design.

Figure 2. a) Mother board b) Daughter board c) both plugged one to the other

The mother board is presumed designed for good and provides the platform cost advantage. Thanks to its exact reproducibility, its cost rapidly decreases. The daughter board design is the only entity that must be designed according to the application specifics needs. This combination allows user to make low-cost and heterogeneous networks of sensors by designing as many various daughter boards as required by the application.

Implementation of Operating System in embedded world is a quite difficult task. The sensor core processor -Analog Device BlackFin BF537-has been produced able to run such an OS. The "ucLinux" is one of those derived from the Linux family that fits the embedded world. That implementation offers two main advantages. First, a very high level of abstraction between hardware peripherals and source code is obtained. As any OS does, ucLinux OS includes many drivers that allow users to program wireless sensors in the standard ANSI C language, sensor's algorithms become fully independent from hardware. Secondly, as a majority of OS, "ucLinux" fully deals with the IP protocol. Even if IP protocol has often been seen as an heavyweight protocol for wireless develoment, its recents evolutions make it available for WSN implementations [START_REF] Dunkels | Swedish Institute of Computer Science "Making TCP/IP Viable for Wireless Sensor Networks[END_REF].Thus, porting ucLinux on LCPC mother board where Wifi is the implementation of IP makes the sensor fully accessible from any remote PC linked to a Wifi access point.

Combining embedded OS to Wifi implementation delivers a real breakthrough in structural health monitoring by means of wireless sensors. LCPC wireless sensors can fully and remotely be updated without any expensive removing operation from the structure. The result is the following: any sensor, Access Point or supervision PC of the LCPC wireless system could be seen as an IP entity such it would be in a traditionnal network of computer under Linux operating system. Each sensor can be adressed, ping and remotly accessible via Telnet command.

Most of instrumentation systems require data able to be time correlated. Thus, time synchronization is an essential sub-function to solve in a WSN. LCPC wireless platform provides an accurate and absolute time-synchronisation mechanism using GPS algorithms [START_REF] Elson | Time Synchronization for Wireless Sensor Networks[END_REF]. The synchronisation mechanism implemented in LCPC platform uses the ability of a small GPS componant (welded on the mother board) to deliver a PPS signal that is a common and absolutly synchronised (G.M.T.) signal that rises up every 1 sec at an accuracy of 20 nanoseconds. By connecting the µS time counter reset input of the platform to the PPS signal, it could deliver an event time-stamping up to a few µS. Moreover, by an real-time algorithm able to take into account temperature (that influence on quartz drift) and natural quartz error, LCPC platform is able to time-stamp any event at an accuracy of less than 4 µS. This level of accuracy fits the need in cases of wave propagation where typical velocities reach ~ 5000 m/s and wished localization are about few centimenterss.

Acoustic system implementation on the new LCPC WSN

The wire-break in cables monitoring system have first been implemented on this new LCPC WSN platform as its capacities fit the desired needs: signal algorithms are performed and sensors are able to deliver the full shape of the signal on threshold event. Absolute time-stamping -thanks to the GPS method-performs events localization and simplify data management. The use of a the worldwide Internet Protocol for sensors communication (implemented through the WiFi 802.11g wireless standard) radically simplify system supervision. From now, it consists of a binary executable file running on a traditional PC. LCPC is testing this new acoustic system by means of 20 sensors on a suspended bridge where the previous generation is already set. Because this system is recent, sensors are still wired but only for energy (8 to 24V) so instrumentation cost is already cheaper. Thanks to active works in terms of energy harvesting and solar cell recovery, LCPC wireless platform will be fully autonomous on 2009.

Conclusion

This acoustic monitoring system has been developed (3rd generation) and has been applied for several decades in France by LCPC [START_REF] Gaillet | Acoustic monitoring of prestressed concrete bridge and other civil structures with cables[END_REF][START_REF] Kretz | Haute surveillance et évaluation de l'aptitude au service du pont suspendu d'Aquitaine[END_REF]. It already made possible to safeguard several bridge threatened of destruction. These last years, this method has been extended to the monitoring of prestressed concrete bridges with cables in cement grout; the instrumentation of the Pont Neuf Bridge of Foix has validated the concept.

New design of sensors embedding "ucLinux" as the Operating System and TCP/IP for communications is a breakthrough in thinking instrumentation. New sensors can fully be remotely reconfigured without any physical removing from the bridge. Even if basic wires are still necessary for long term monitoring period, LCPC platform is actually subject of converging efforts in order to make sensors fully wireless. Acoustic emission method requires further studies in wave propagation and characterisation when applied to cables and especially for wire-breaks detection but existing LCPC method has already proved a certain efficiency. Its implementation on the LCPC wireless sensor platform should offer soon a new, simplest and less expensive tool for bridge managers.

Figure 3 .

 3 Figure 3. a) Both systems deployed b) Full signal shape recorded.